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2The Dynamic Discipline of SpeciesDelimitation Progress TowardEffectively Recognizing SpeciesBoundaries in Natural PopulationsSteven D Leavitt Corrie S Moreau and H Thorsten Lumbsch
Contents
21 Introduction Whatrsquos in a Name TheImportance of Accurate SpeciesDelimitations 12
211 Species Concepts and Criteria 12212 Species in Lichenized Fungi Cases
of Cryptic Diversity Polymorphic Lineagesand Striking Biogeographic Patterns 13
22 A Practical Guide to ContemporarySpecies Delimitation 16
221 Corroborating Traditional Taxonomyand Discovering Cryptic Species UsingSingle-Locus Data 17
222 Sampling Across the Genome MultilocusSequence Data and Genome-Wide Markers 26
23 Can We Make Species Delimitationin Lichen-Forming Fungi TrulyIntegrative 30
231 Selecting the Appropriate Data 32
24 Conclusions What About Taxonomy 34
References 35
Abstract
Species represent a fundamental unit in evolu-tionary biology and provide a valuable contextfor organizing evaluating and communicatingimportant biological concepts and principlesEmpirical species delimitation is a dynamicdiscipline with ongoing methodological andbioinformatical developments Novel analyticalmethods increasing availability of geneticgeno-mic data increasing computational power reas-sessments of morphological and chemicalcharacters and improved availability of distribu-tional and ecological records offer excitingavenues for empirically exploring species delim-itation and evolutionary relationships amongspecies-level lineages In this chapter we aimto contribute a contemporary perspective ondelimiting species including a brief discussionon species concepts and practical direction forempirical species delimitation studies Usinglichen-forming fungi as an example we illustratethe importance and difficulties in documentingand describing species-level biodiversity
Keywords
Barcoding Coalescence DNA taxonomy Fungi Gene tree Genomics Lichens Species circumscription Species concept Species treeSD Leavitt (amp)
Committee on Evolutionary BiologyUniversity of Chicago Chicago IL USAe-mail sleavittfieldmuseumorg
SD Leavitt CS Moreau H Thorsten LumbschDepartment of Science and Education Field Museumof Natural History Chicago IL USA
copy Springer India 2015DK Upreti et al (eds) Recent Advances in LichenologyDOI 101007978-81-322-2235-4_2
11
21 Introduction Whatrsquosin a Name The Importanceof Accurate SpeciesDelimitations
Although there are over 15 million species for-mally named by scientists current estimates ofthe number of species alive on the planet todayrange from approximately two million to overone hundred million (Caley et al 2014) Docu-menting describing and naming this diversity isparamount for conservation human health foodsecurity and recreation (Tewksbury et al 2014)In a broad sense species delimitation is theprocess of identifying how individuals andpopulations fit into natural species-level clustersand not simply constructs of classification(Carstens et al 2013) Empirical species delimi-tation is a dynamic discipline with ongoingmethodological and bioinformatical develop-ments due to a growing interest in empiricalspecies delimitations Novel analytical methodsincreasing availability of geneticgenomic dataincreasing computation power reassessments ofmorphological and chemical characters andimproved availability of distributional and eco-logical records offer exciting avenues for empir-ically exploring species delimitation andevolutionary relationships in species all over theworld In this chapter we aim to contribute acontemporary perspective on delimiting speciesand offer practical direction for empirical speciesdelimitation studies
To illustrate the importance and difficulties indocumenting and describing biodiversity we willuse the lichens as an example Lichens describe amutualism between a fungus (mycobiont) and aphotosynthetic partner (photobiont) which can beeither a green algae andor cyanobacteriumLichens are ubiquitous components of most ter-restrial ecosystems playing important ecologicalroles and contributing to global biogeochemicalcycles (Porada et al 2014 Bonan and Shugart1989 Longton 1997) Due to the fact that manylichens live and grow continuously for decades oreven hundreds of years showing cumulativeresponses to changes in atmospheric pollutionlevels landmanagement practices and fluctuation
climate these are commonly used as bioindicatorsto monitor the impacts of air pollution forest agesoil quality and climate change (McCune 2000)As iconic examples of symbiosis lichens alsoprovide crucial insight into general patterns andprocesses in symbiotic systems Central tounderstanding the dynamic roles of lichens is ourability to accurately delimit and recognize speciesboundaries Increased accuracy in recognizingspecies boundaries in lichenized fungi has majorimplications for enhancing our perspective onbiological diversity evolution ecology symbi-otic interactions biomonitoring research andconservation policy
211 Species Concepts and Criteria
Species serve as a central unit for categorizingbiological diversity Humans including bird-watchers doctors fisherman gardeners politi-cians scientists and others rely to varyingdegrees on recognizing species for distinguishingdifferent kinds of organisms and effective com-munication In the biological sciences speciesrepresent one of the most fundamental units andprovide a valuable context for organizing eval-uating and communicating important biologicalconcepts and principles (Coyne and Orr 2004Mayr 1963 Darwin 1859) Due to the fact thatbiological information is commonly providedwith reference to a species unit accurate speciescircumscriptions are integral to interpreting bio-logical patterns and processes across a widerange of subdisciplines in biology (eg anatomybehavior ecology evolution physiology etc)
In spite of the underlying importance of spe-cies in biology the conceptualization of the termldquospeciesrdquo remains somewhat contentious (deQueiroz 2007 Hausdorf 2011 Coyne and Orr2004 Mayden 1997 Simpson 1951 Mayr1963) Most biologists agree that biological nat-ure is aggregated into discrete evolutionarilyindependent entities ie ldquospeciesrdquo (Coyne andOrr 2004) although theorists and empiricistsalike continue to debate over an all-encompass-ing species concept and appropriate operationalcriteria for delimiting species (Hausdorf 2011
12 SD Leavitt et al
de Queiroz 2007 Hey 2006 Donoghue andGauthier 2004 Cracraft 1983 Mishler andBrandon 1987 Mayr 1970) Over two-dozendifferent and at least partially incompatible spe-cies concepts have been proposed each based ondistinct biological properties eg differences ingenetic or morphological features adaptivezones or ecological niches mate recognitionsystems reproductive compatibility monophylyetc (de Queiroz 2007 Mayden 1997) Hausdorf(2011) argues that most species concepts areuseable but acceptance of a specific conceptrequires an appropriate adaptation of the termldquospeciesrdquo and of species delimitation In contrastde Queiroz (1998) and Mayden (1997) argue thatdistinct species concepts emphasize differentproperties of species rather than fundamentalconceptual differences and all modern speciesconcepts share an important commonalitymdashequating species with segments of metapopula-tion lineages This ldquogeneral lineage conceptrdquo(GLC de Queiroz 1999) highlights that no singleproperty should be regarded as defining for therecognition of species apart from forming lin-eages (Simpson 1951) and segments of meta-population lineages (ie ldquospeciesrdquo) may existregardless of our ability to empirically delimitthem (Camargo and Sites 2013)
We concur that the GLC provides a practicalsolution to the species concept impasse and ourdiscussion of species delimitation is based on theGLC Arguably the major implication of the GLCis that most of the earlier species concepts shouldbe regarded as secondary species ldquocriteriardquo ratherthan ldquoconceptsrdquo that can provide evidence oflineages separation (Sites and Marshall 2003Camargo and Sites 2013 Mayden 1997 de Que-iroz 2007) This pivotal distinction disentanglesthe conceptual issues of defining ldquospeciesrdquo frommethodological issues of delimiting speciesboundaries (Camargo and Sites 2013) The GLCallows researchers to delimit species using differ-ent empirical properties and facilitates the devel-opment of new methods to test hypotheses oflineage separation (de Queiroz 2007) Althoughdifferent datasets and operational criteria may giveconflicting or ambiguous results due to multipleevolutionary processes occurring within and
between populations (eg Miralles and Vences2013 Satler et al 2013) the use of several inde-pendent suites of characters such as genetic datamorphology geographic range host preferencechemistry and cross-validation using inferencesfrom multiple empirical operational criteria canprovide robust hypotheses of species boundaries(Carstens et al 2013 Fujita et al 2012)
212 Species in Lichenized FungiCases of Cryptic DiversityPolymorphic Lineagesand Striking BiogeographicPatterns
Similar to most major biological groups includ-ing birds (McKay et al 2013) fish (Wagner et al2013) plants (Griffin and Hoffmann 2014)arthropods (Schlick-Steiner et al 2010 Moreau2009) and many others finding and applying theappropriate character sets and analytical tools isone of the greatest challenges with empiricalspecies delimitation in lichen-forming fungi(Lumbsch and Leavitt 2011) Understanding thedifferences between morphological variationwithin a species and among closely related groupsis central to identifying diagnostic charactersrequired for establishing accurate phenotype-based taxonomic boundaries However in prac-tice a clear demarcation between intraspecificand interspecific variation is commonly subject toobservational bias and individual interpretation
Traditionally differences in morphologicalchemical and ecological features have been thepredominant source of diagnostic taxonomiccharacters for circumscribing lichen-formingfungal species (Printzen 2009) However lichensgenerally display few taxonomically useful char-acters relative to other groups (eg vascularplants vertebrates and arthropods) (Printzen2009) and varying levels of intraspecific variationamong different species groups may confoundaccurate taxonomic circumscriptions While somespecies may have little variation high levels ofintraspecific phenotypic variation are well docu-mented in some lichen-forming fungi (eg Xant-hoparmelia Hale 1990) Therefore molecular
2 The Dynamic Discipline of Species Delimitation hellip 13
genetic data now play a prominent role in delim-iting fungal species and understanding evolu-tionary relationships in lichens (Lumbsch andLeavitt 2011 Printzen 2009)
Arguably the use of molecular data has led toan improved perspective on the taxonomic valueof many phenotypic characters in lichenized fungiand species delimitation in general Cryptic spe-cies-level lineages are commonly identified usingmolecular data and in some cases these previ-ously unrecognized lineages are corroborated byformerly overlooked phenotypic characters (Pino-Bodas et al 2012a Spribille et al 2011 Divakaret al 2010 Arguumlello et al 2007) Other studieshave revealed the fact that some species-levellineages are likely comprised of chemically andmorphologically polymorphic individuals whichare conventionally considered as separate species(eg Leavitt et al 2011a Pino-Bodas et al 2011Velmala et al 2009) While these studies high-light the limitations of using traditional taxo-nomic characters for distinguishing naturalgroups in lichen-forming fungi they also providea valuable perspective on the importance ofongoing research in even the best-studied lichengroups Furthermore an improved perspective ofspecies boundaries has led to a strikingimprovement in understanding diversification anddistribution in many groups of lichenized fungi
Traditional phenotype-based approaches tospecies recognition appear to vastly underesti-mate diversity in lichen-forming fungi Whilemolecular research has corroborated traditionalphenotype-based hypotheses of species bound-aries in a number of cases studies repeatedlydemonstrate that our current interpretation ofmorphological and chemical characters oftenfails to accurately characterize species-leveldiversity (reviewed in Lumbsch and Leavitt2011) A growing body of evidence reveals that asignificant proportion of unknown diversityestimated for fungi including lichen-formingfungi is hidden under names of supposedlycommon and widespread species For exampleapproximately 80 unrecognized species-levellineages are estimated to occur in Parmeliaceae(Crespo and Lumbsch 2010) Even higher levels
of unrecognized species diversity are estimatedto occur in other families such as Graphidaceae(Rivas Plata and Luumlcking 2013 Rivas Plata andLumbsch 2011 Luumlcking 2012)
The topic of cryptic species cases wheretwo or more distinct species-level lineages areerroneously classified (and hidden) under onenominal taxon (Bickford et al 2007) has beenfrequently reviewed for lichen-forming fungi(Lumbsch and Leavitt 2011 Crespo and Lum-bsch 2010 Crespo and Peacuterez-Ortega 2009Printzen 2009) Although novel diagnostic phe-notypic characters may potentially be identifiedcorroborating ldquocrypticrdquo species these previouslyunrecognized lineages generally remain difficultto classify within a traditional phenotype-basedtaxonomy (Leavitt et al 2013c d) In somecases it appears that similar phenotypic charac-ters may arise in parallel at local or regionalscales but may not be correlated with naturalgroups or genetic isolation (Muggia et al 2014Rivas Plata and Lumbsch 2011 Grube andHawksworth 2007) For example in the cosmo-politan species Tephromela atra (Fig 21) up to15 independent lineages were identified usingphylogenetic analyses of molecular sequencedata However the continuum of morphologicaland chemical variability in the T atra complexcurrently prevents the description of new speciesusing traditional phenotype-based characters(Muggia et al 2014)
Recent research on the genus Cladonia(Cladoniaceae) highlights a fitting example of thecomplexities associated within using phenotypiccharacters for delimiting species in lichen-forming fungi (Fig 21 Pino-Bodas et al 20112012a b 2013a b) In the Cladonia gracilisgroup most currently accepted species were notrecovered as monophyletic clades and traditionaldiagnostic morphological characters were shownto be highly homoplasious (Pino-Bodas et al2011) Similarly C iberica and C subturgidahave been shown to constitute a single morpho-logically and chemically polymorphic species(Pino-Bodas et al 2012b) Similar patterns ofhigh degrees of morphological and chemicalpolymorphisms have also been observed in the
14 SD Leavitt et al
C cariosa group (Pino-Bodas et al 2012a) andC humilis species complex (Pino-Bodas et al2013a) High levels of intraspecific morphologi-cal and chemical polymorphisms are not restric-ted to Cladonia A clear demarcation betweenintraspecific and interspecific morphological andor chemical variation does not exist for a largeproportion of Xanthoparmelia species in westernNorth America and high intraspecific morpho-logical and chemical variation are common for anumber of species-level genetic groups (Fig 21
Leavitt et al 2011b c 2013e) In some cases asmany as eight traditionally circumscribed Xant-hoparmelia species were recovered in a singlespecies-level genetic group (Leavitt et al 2011c)High levels of intraspecific phenotypic variationhave been observed in a number of other generain Parmeliaceae including Bryoria (Velmalaet al 2009) Cetraria (Peacuterez-Ortega et al 2012)Vulpicida (Mark et al 2012) and others
The importance of biogeography in lichen-forming fungal evolution has remained somewhat
Fig 21 Examples of common lichens in which tradi-tional morphology-based species circumscriptions fail toreflect natural species-level fungal lineages a Cladoniagracilis photographed in the Clearwater Valley BritishColumbia Canada (see Pino-Bodas et al 2011) (photo-graph creditCurtis Bjoumlrk) bR shushanii a member of theRhizoplaca melanophthalma species group from the
Aquarius Plateau Utah USA (see Leavitt et al 2011a)(photograph credit S Leavitt) c Xanthoparmelia affwyomingica occurring in Coloradorsquos Front Range USA(see Leavitt et al 2011b c Leavitt et al 2013e) (photo-graph credit S Leavitt) d Tephromela atra sensu latofound in the Santa Monica Mountains California USA(see Muggia et al 2014) (photograph credit J Hollinger)
2 The Dynamic Discipline of Species Delimitation hellip 15
ambiguous due to the occurrence of phenotypi-cally similar lichens occurring across broadintercontinental distributions and uncertainty ofappropriate species circumscriptions While anumber of lichen-forming fungal species havebeen found to be truly widespread (eg Lindblomand Soslashchting 2008 Fernaacutendez-Mendoza et al2011 Ahti and Hawksworth 2005 Del-Pradoet al 2013) improved recognition of speciesboundaries has provided insight into importantbiogeographical patterns in lichens previouslyassumed to have cosmopolitan distribution pat-terns (Leavitt et al 2013d Del-Prado et al 2013Amo de Paz et al 2012 Seacuterusiaux et al 2011Otaacutelora et al 2010 Elix et al 2009) Crypticdiversity and complex biogeographic patterns arehighlighted in the Rhizoplaca melanophthalmaspecies group (Fig 21 Leavitt et al 2013d)Analyses of R melanophthalma sensu lato col-lected from five continents supported the presenceof cryptic species within this complex Two ofthese lineages were found to have broad inter-continental distributions while the other four werelimited to western North America (Leavitt et al2013d) Most strikingly of the six lineages fivewere found on a single mountain in the westernUSA and the sixth occurred no more than 200 kmaway from this mountain A recent study ofPseudocyphellaria (Lobariaceae) sensu lato inHawaii revealed a surprising number of previouslyunrecognized species hidden within nominal taxawith putative broad geographic distributionssuggesting a high degree of endemism in Hawaii(Moncada et al 2014) These studies providecrucial impetus to reevaluate species boundaries inorder to improve our understanding of diversitydistributions and evolution in lichenized fungi
22 A Practical Guideto Contemporary SpeciesDelimitation
As it has become clear that conventional phe-notype-based criteria for species circumscriptionsare often problematic (Bickford et al 2007)molecular data are thereby particularly valuablefor assessing traditional species boundaries and
for species delimitation in general (Lumbsch andLeavitt 2011 Fujita et al 2012) Below weprovide an overview of relevant operationalspecies delimitation methods the majority ofwhich are reliant on molecular data coupled withmolecular phylogenetic and population geneticanalyses Assuming that species do in fact rep-resent ldquosegments of metapopulation lineagesrdquo (deQueiroz 1998) direct genetic evidence of lineagestatus is particularly relevant to species delimi-tation studies when analyzed within a rigorousstatistical framework regardless of whether lin-eages differ in phenotypic characters that areapparent to human observers (Fujita et al 2012)This perspective should not be taken as supportfor disregarding phenotypic characters in speciesdelimitation studies (Edwards and Knowles2014 Fujita et al 2012 Yeates et al 2011)Rather hypotheses of species boundaries shouldbe considered more robust with increasing cor-roboration from independent data sources (iemolecular chemistry morphology ecologyetc) and the integration of independent data forempirical species delimitation studies should be amajor focus of species delimitation research(Fujita et al 2012)
Themajority of molecular phylogenetic studiesof lichenized fungi focus on generating sequencedata from a number of specimens representing thefocal group inferring a gene tree from the geneticdata matrix and assessing the monophyly of thesampled taxa This approach has provided valu-able acumen into evolutionary relationships thevalue of morphological characters for taxonomyand insight into diversity of lichenized fungi(Thell et al 2009 Reese Naeligsborg et al 2007Westberg et al 2007 Martiacuten et al 2003 Arup andGrube 2000 Lohtander et al 2000 Stenroos andDePriest 1998) However simply assessing themonophyly of traditional phenotype-based spe-cies often offers an incomplete perspective onspecies boundaries Studies explicitly designed forempirically delimiting species are pivotal toadvancing our understanding of speciation andspecies diversity in lichens For example althougha nominal species may be recovered as mono-phyletic in a gene tree intraspecific phylogeneticsubstructure may correspond to evolutionarily
16 SD Leavitt et al
independent lineages (eg Leavitt et al 2011a)As a construct of taxonomy recognizing amonophyletic clade comprised of multiple mor-phological indistinguishable species-level lin-eages as a single species may hold some appealhowever failing to formally recognize this diver-sity can have far-reaching implications in ourbiological (eg ecology evolution reproduction)interpretation of the group Alternatively well-supported intraspecific phylogenetic substructuremay be the result of stochastic evolutionary pro-cesses uniparental inheritance etc rather thanevolutionary independence Interpreting this typeof phylogenetic substructure as species-leveldiversity can introduce detrimental bias
Empirical species delimitation methods havebroadly focused on four main areas detectingputative species individual specimen assignmentto a species group or operational taxonomic unit(OTU) validation of candidate species or OTUsas evolutionarily distinct lineages and inferringspecies relationships (ie species tree inference)Ideally operational species delimitation criteriashould be based on explicit statistical protocolsin order to objectively assess species boundariesand minimize the need of subjective interpreta-tions or taxonomic expertise In this section weprovide a brief overview on a number of empir-ical species delimitation methods that to varyingdegrees fit these criteria For convenience wedivide these methods into three general catego-ries (i) species discovery methods for assigningsamples to populations without a priori infor-mation (ii) species validation approaches andcoestimating individual assignments and speciestrees and (iii) species delimitation using geno-mic data (Table 21)
Empirical species delimitation has receivedincreasing attention including ongoing develop-ment of bioinformatical approaches and themethods and programs provided in this chapterare by no means intended to be a comprehensivelist of all available analytical approaches Ratherthe methods provided here have been shown tobe useful in a number of previous studies orshow particular promise for future speciesdelimitation studies Our aim is that these can
serve as a starting point when designing studiesto assess species limits Not surprisingly variousapproaches to species delimitation may yielddifferent estimates of species boundaries and theresearcher may be required to make some degreeof subjective interpretation of the most biologi-cally appropriate species boundaries Comple-mentarily recently developed metrics forquantifying the congruence between two taxo-nomies (Ctax) and the potential for an approach tocapture a high number of species boundaries(Rtax) provide a means to objectively assess dis-crepancies among species delimitation methods(Miralles and Vences 2013) More sophisticatedapproaches including selection of speciesdelimitation models using approximate Bayesiancomputing (Camargo et al 2012 Fan andKubatko 2011 Beaumont et al 2010) anddesigning and conducting a simulation study thatmatches the characteristics of the empirical study(Carstens et al 2013 Camargo et al 2012) canbe used to more objectively evaluate competinghypotheses of species boundaries In most con-texts it is likely better to fail to delimit speciesthan it is to falsely circumscribe entities that donot represent actual species and therefore theinferences drawn from species delimitation stud-ies should generally be conservative (Carstenset al 2013 Miralles and Vences 2013)
221 Corroborating TraditionalTaxonomy and DiscoveringCryptic Species Using Single-Locus Data
Objectively defining a threshold separatingintraspecific population substructure from inter-specific divergence is the general aim of speciesdelimitation studies using single-marker datasetsMost species delimitation methods based onsingle-locus sequence data fall under two generalcategories either genetic distance or tree-basedapproaches (Sites and Marshall 2004) Distance-based approaches attempt to detect a differencebetween intra- and interspecific distances (ieldquobarcode gaprdquo) where the pairwise genetic
2 The Dynamic Discipline of Species Delimitation hellip 17
Table
21
Somemetho
dsused
forspeciesdelim
itatio
ninclud
ingspeciesdiscov
erymetho
dsforassign
ingsamples
topo
pulatio
nswith
outapriori
inform
ation(BAPS
Gaussianclustering
Guillo
trsquosUnified
Mod
elS
TRUCTURES
TRUCTURAMA)genetic
distance-based
metho
dforsortingsequ
encesinto
hypo
theticalspecies(A
BGD)tree-
basedspeciesdiscov
erymetho
ds(bGMYCG
MYCb
PTP
PTP
ldquoSpecies
Delim
itatio
nrdquoplug
-inforGeneiou
s)and
jointdiscov
eryandvalid
ationmetho
ds(BPamp
PBrownie
DISSE
CTSp
eDeStem)
Metho
dDescriptio
nInpu
tdata
BAPS
mdashpo
pulatio
nassignmentusing
Bayesianclustering
Aprogram
forBayesianinferenceof
thegenetic
structurein
apopulatio
nclustering
molecular
data
andperformingadmixture
analysesGenetic
mixture
analyses
canbe
performed
atbo
thgroupandindividu
allevelsusingeither
ano
n-spatialo
rspatialm
odel
BAPS
treatsboth
theallele
frequenciesof
themolecular
markers
(ornucleotid
efrequenciesforDNA
sequ
ence
data)andthenu
mberof
genetically
diverged
groups
inpopulatio
nas
random
variablesIn
theldquoclusteringwith
linkedlocirdquomod
elagenetic
mixture
analysiscanbe
done
usinghaploidsequ
ence
data
orotherlin
kedgenetic
markersA
nalysesandmodel
comparisons
canalso
beperformed
usingafixednu
mber
ofgenetically
diverged
grou
psor
prespecified
populatio
nstructures
Limita
tions
Tem
poraldivergence
andrelatio
nships
amongputativ
egroups
arenot
explicitlyestim
atedE
quivalence
togenetic
clusters
tospecies-levelg
roupsisuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
Availablefrom
httpwwwhelsinkifi
bsgsoftwareBAPS
describedin
Coran-
deret
al(200
420
0620
08)CoranderandMarttinen20
06)
Genotyp
icdatahaploidsequence
dataor
linkedmarkers
(AFL
Por
SNPs)
Gaussianclusteringmdashpo
pulatio
nassignment
usingGaussianclustering
Aprogram
forBayesianinferenceof
thegenetic
structurein
apopulatio
nModelgroups
sampleinto
popu
latio
nsusinggeno
typicdata
bysearchingforclusters
that
canbe
attributed
tobeingmixturesof
norm
alallelefrequencydistributio
nsG
aussianclustering
requires
adatasetwhere
thecasesaredefinedby
variable
ofmetricscaleandhasbeen
used
with
geneticenvironmentalandmorphologicaldatasetsindividuallyinadditio
nto
integrated
datasets
Limita
tions
Tem
poraldivergence
andrelatio
nships
amongputativ
egroups
arenot
explicitlyestim
atedE
quivalence
togenetic
clusters
tospecies-levelg
roupsisuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
Implem
entedin
Rusingtheprabclus
(HausdorfandHennig20
10)andmclust
packages
(FraleyandRaftery
2007)
Genotyp
icdata
(flexible)
(con
tinued)
18 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
Guillo
trsquosUnified
Mod
elmdashpo
pulatio
nassignmentusingBayesianclustering
Thisapproach
provides
astatistical
model
that
allowsoneto
analyzegenetic
and
phenotyp
ticdata
with
inaun
ified
mod
elandinferencefram
eworkandop
tionally
incorporateinform
ationaboutthespatialdistributio
nof
samplesA
Bayesianclustering
algorithm
assumes
that
each
clusterin
ageographical
domaincanbe
approxim
ated
bypo
lygons
that
arecentered
around
pointsgeneratedby
aPo
issonprocessGuillo
trsquos
Unified
Mod
elisflexiblein
term
sof
thegenetic
data
that
itcanutilize
andcapableof
accurately
delim
iting
species
Limita
tions
Genetic
andphenotypic
data
cantracedifferentevolutionary
historiesfor
instancephylogenetic
divergence
forneutralgenetic
markers
andadaptatio
nfora
morphological
structure
Source
Availableas
anextensionof
theRGENELAND
package(G
uillo
tet
al20
05
2012)(http
www2im
mdtudk
gigu
Geneland)
Genotyp
icandno
n-genetic
(eg
phenotypicalecolog
icalgeograph
ical
behavioral)data
STRUCTUREmdash
populatio
nassignment
usingBayesianclustering
Amodel-based
clustering
methodusingmultilocus
genotype
data
toinferpopulatio
nstructureandassign
individualsto
populatio
nsIndividualsin
thesampleareassigned
probabilistically
topopulatio
nso
rjointly
totwoor
morepopulatio
nsiftheirgenotypes
indicatethatthey
areadmixedT
hemodeldoes
notassum
eaparticular
mutationprocess
anditcanbe
appliedto
mosto
fthecommonly
used
genetic
markersp
rovidedthat
they
areno
tcloselylin
ked
The
methodcanproducehigh
lyaccurate
assignmentsusing
modestn
umbersof
loci(Pritchard
etal2
000)T
hemostappropriatelevelo
fpopulatio
nstructurecanbe
inferred
byassessinglik
elihoodscores
orthead
hocΔKstatistic
(Evann
oet
al20
05)
Limita
tions
Identifying
themostappropriatenumberof
genetic
clusters
ischallenging
clusters
produced
byST
RUCTUREcanbe
strongly
influenced
byvariationin
sample
sizeclusters
createdby
STRUCTUREmay
notbe
consistent
with
theevolutionary
historyof
thepopulatio
nswhentherearerelativ
elylong
divergence
times
with
inevolutionary
lineagesTem
poraldivergence
andrelatio
nships
amongputativ
egroups
isnotexplicitlyestim
atedEquivalence
togenetic
clusters
tospecies-levelgroups
isuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
httppritchardlabstanfordedustructurehtm
ldescribedin
Falush
etal(200
3)andPritchard
etal(200
0)
Genotyp
icdata
(con
tinued)
2 The Dynamic Discipline of Species Delimitation hellip 19
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
Structuram
amdashpo
pulatio
nassignmentusing
Bayesianclustering
Implem
entstheclustering
algorithm
used
inST
RUCTURE(see
above)
that
clusters
samples
into
populatio
nsby
minim
izingHardyndashWeinb
ergdisequ
ilibrium
foragiven
partitioninglevelHow
everStructuram
aalso
includes
theadditio
nof
reversible-jum
pMCMCto
identifytheop
timalpartition
inglevelNearlyanytype
ofgenetic
datacanbe
inpu
tinto
Structuram
aandtheprog
ram
canassign
individu
alsto
populatio
nwith
orwith
outtheadmixture
Limita
tions
Tem
poraldivergence
andrelatio
nships
amongputativ
egroups
isnot
explicitlyestim
atedE
quivalence
togenetic
clusters
tospecies-levelg
roupsisuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
httpctegberkeleyedu
structuram
aindexhtmldescribedin
Huelsenbeck
etal(201
1)
Genotyp
icdata
ABGDmdashbarcodegapusinggenetic
distances
ldquoAutom
aticBarcode
Gap
Discoveryrdquosortssequencesinto
hypotheticalspeciesbasedon
thebarcodegap
The
methoduses
arecursiveapproach
topartition
thedata
andtestfor
sign
ificant
gapsA
BGDisfastsim
plemethodto
split
asequence
alignm
entd
atasetinto
candidatespeciesthat
should
becomplem
entedwith
otherevidence
inan
integrative
taxono
mic
approach
Limita
tions
Single-locus
data
aloneshould
only
beused
toprovideaprelim
inary
perspectiveof
speciesboundaries
andnotas
thesole
evidence
inspecies
circum
scriptions
Source
httpwwwabisnv
jussieufrpublicabg
ddescribedin
Puillandreet
al(201
2)
Single-locus
sequence
alignm
ent
GMYCampbG
MYCmdashgene
tree
The
GMYC
approach
combinesacoalescent
model
ofintraspecificbranchingwith
aYulemod
elforinterspecificbranching
which
isthen
fitto
aninferred
sing
le-gene
topology
toestim
atespeciesboundaries
andastatistical
measure
ofconfi
denceforthe
inferred
boundariesA
san
inputtheGMYCapproach
requires
anultram
etricgene
tree
andrecent
refinementscanaccountforuncertaintyin
phylogenetic
relatio
nships
and
parametersof
theGMYCmod
elT
heGMYCisgenerally
stable
across
awiderangeof
circum
stancesincludingvariousmethods
ofphylogeneticreconstructio
nthepresence
ofahigh
numbersingletonshigh
numbers
ofsampled
speciesandgaps
inintraspecific
sampling
theaccuracy
oftheGMYCismostsign
ificantly
affected
bythemean
populatio
nsize
relativ
eto
divergence
times
betweenthem
Limita
tions
The
GMYCmay
delim
itwell-supportedclades
ofhaplotypes
asindependent
lineagesandas
such
may
beproneto
over-delim
itatio
nSingle-locus
data
aloneshould
only
beused
toprov
ideaprelim
inaryperspectiveof
speciesbo
undaries
andno
tas
the
sole
evidence
inspeciescircum
scriptions
Source
(http
r-forger-projecto
rgprojectssplitshttpssitesgooglecom
site
noahmreidhom
esoftware)describedin
Monaghanet
al(200
9)Fu
jisaw
aandBarrac-
loug
h(201
3)andPo
nset
al(200
6)
Sing
le-locus
ultram
etricgene
tree (con
tinued)
20 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
PTPamp
bPTPmdash
gene
tree
The
Poissontree
processes(PTP)
modelcanbe
used
toinferputativ
especiesboundaries
onagivenphylogenetic
inputtreePT
Pcaninferputativ
especiesboundaries
that
are
consistent
with
thePS
CAnim
portantadvantageof
thismethodisthat
itmod
els
speciatio
nin
term
sof
thenumberof
substitutionsthereby
circum
ventingthepotentially
error-proneandcompute-intensive
processof
generatin
gultram
etrictreeswhich
are
required
asan
inpu
tfor
GMYCmod
el(see
abov
e)F
urthermoreitappearsthatthePT
Pmodel
may
outperform
theGMYCandotherOTU-picking
methods
whenevolutionary
distancesaresm
all
Limita
tions
Single-locus
data
aloneshould
only
beused
toprovideaprelim
inary
perspectiveof
speciesboundaries
andnotas
thesole
evidence
inspecies
circum
scriptions
Source
httpscoh-itsorgexelix
iswebsoftwarePT
Pindexhtmldescribedin
Zhang
etal(201
3)
Sing
le-locus
gene
tree
GeneiousSp
eciesDelim
itatio
nplug
-inmdash
gene
tree
Aplug-into
theGeneioussoftwareprovides
anexploratorytool
allowingtheuser
toassess
phylog
enetic
supportanddiagno
sabilityof
speciesdefinedas
user-selected
collections
oftaxa
onuser-sup
pliedtreesThe
plug
-incompu
tesstatisticsrelatin
gto
the
probability
oftheob
served
mon
ophyly
orexclusivity
having
occurred
bychance
ina
coalescent
processandassesses
thewith
in-andbetween-speciesgenetic
distancesto
infertheprobability
with
which
mem
bers
ofaputativ
especiesmight
beidentifi
edsuccessfully
with
tree-based
methods
Limita
tions
The
plug-insummarizes
measuresof
phylogenetic
supportand
diagnosabilityof
speciesdefinedas
user-selectedcollections
oftaxabutitdoes
not
providedefinitiv
esupportforspeciesgroupsItassumes
speciesaremonophyletic
Source
Implem
entedas
aplug-into
Geneious(geneiouscom)describedin
Mastersetal
(201
1)
Sing
le-locus
gene
tree
BPamp
Pmdashmultispecies
coalescent
model
for
speciesvalid
ation
Thisapproach
tospeciesdelim
itatio
nuses
aBayesianmodelingapproach
togeneratethe
posteriorprobabilitiesof
speciesassignmentstaking
accountof
uncertaintiesdueto
unknow
ngene
treesandtheancestralcoalescent
processThe
methodrelieson
auser-
specified
guidetreeimplem
entin
gareversible-jum
pMarkovchainMonte
Carlo
search
ofparameter
spacethatincludes
θpopulatio
ndivergenceand
estim
ated
distributio
nsof
gene
treesfrom
multip
leloci
Limita
tions
Misspecificatio
nsof
priorsandthegu
idetree
canresultin
inflatedspeciatio
nprobabilitiesitassumes
norecombinatio
nandcomputatio
nallim
itatio
nsrestrict
itsutility
with
next-generationdatasetswith
100s
ofloci
Source
httpabacusgeneuclacuksoftwarehtmldescribedin
YangandRannala
(201
0)andRannala
andYang(200
3)
Multilocus
sequence
alignm
entsandgroup
mem
bership
(con
tinued)
2 The Dynamic Discipline of Species Delimitation hellip 21
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
DISSE
CTmdashmultispecies
coalescent
model
forspeciesdelim
itatio
nDISSE
CTexplores
thefullspaceof
possible
clusteringsof
individualsandspeciestree
topologies
inaBayesianfram
ework
Toavoidtheneed
forreversible-jum
pMCMCit
uses
anapproxim
ationin
theform
ofapriorthatisamodificatio
nof
thebirthndashdeathprior
forthespeciestreeItisim
plem
entedas
partof
BEAST
andrequires
only
afewchanges
from
astandard
BEAST
analysisA
nalysesof
simulated
andem
piricald
atasuggestthat
themethodisshow
nto
beinsensitive
tothedegree
ofapproxim
ation
butq
uitesensitive
tootherparameters
Limita
tions
Recently
describedmethodlackingathorough
theoretical
andem
pirical
evaluatio
nItappearsthat
alargenu
mberof
sequencesarerequ
ired
todraw
firm
conclusion
sSo
urce
httpcodegooglecompbeast-m
cmcamphttpwwwin
driid
com
dissectinbeast
htmldescribedin
JonesandOxelm
an(201
4)
Multilocus
sequence
data
SpeD
eSTEMmdashmultispecies
coalescent
mod
elfordiscov
ery
valid
ation
andjoint
estim
ation
Thismaxim
umlik
elihoo
dandorinform
ationtheory-based
methodwas
developedto
test
speciesboundaries
inasystem
with
existin
gsubspecies
taxonomy(CarstensandDew
ey20
10)andcomputestheprobability
ofthegene
treesgiventhespeciestree
forall
hierarchical
perm
utations
oflin
eage
grou
pingSp
eciesbo
undaries
arecomparedusing
Akaikeinform
ationcriteria
andphylogenetic
uncertaintyin
thespeciestree
topologies
does
notaffect
speciesdelim
itatio
nsLimita
tions
Accuracyisdependenton
quality
ofthegene
tree
estim
ates
Source
(http
carstenslaborgohio-stateedusoftwarehtml)criteria
describedin
Ence
andCarstens(201
1)
Multilocus
sequence
alignm
entsandgroup
mem
bership
Browniemdash
multispecies
coalescent
model
forspeciesdelim
itatio
nThe
nonp
aram
etricheuristic
speciesdelim
itatio
napproach
implem
entedin
theprog
ram
Brownie(O
rsquoMeara
2010)jointly
sortsanonym
oussamples
into
speciesandinfers
aspeciestree
from
inputg
enetreesfrom
differentlociassumingthatforaspeciatio
neven
thecorrespondingnodeson
gene
treeswill
bemoreconsistent
with
each
than
the
divergenceswith
inspecies
Limita
tions
Findingboth
theoptim
umspeciestree
andspeciesboundaries
remains
compu
tatio
nally
challenging
andBrowniehasbeen
show
nto
frequently
yieldincorrect
resultsT
heaccuracy
ofthemethodislik
elycorrelated
with
nodalsupportvalues
inthe
individu
algene
topo
logies
Source
httpwwwbrianom
earain
fobrownie
describedin
OrsquoM
eara
(201
0)
Individual
gene
trees
(con
tinued)
22 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
BFD
mdashmultispecies
coalescent
model
for
speciesdelim
itatio
nThe
recently
developedmethod
Bayes
factor
delim
itatio
n(with
genomicdataB
FD)
combinesady
namic
programmingalgorithm
forestim
atingspeciestreesthat
bypasses
thecompu
tatio
nally
intensiveMCMCintegrationov
ergene
treesto
prov
idearigorous
techniqueforspeciesdelim
itatio
nstudiesusinggenome-wideSN
PdataCom
petin
gspeciesdelim
itatio
nmodelsarecomparedusingBayes
factorsanditappearsthat
this
approach
isrobustto
samplesizes(iefew
loci
andlim
itedsamples
perspecies)
and
misspecificatio
nof
thepriorforpo
pulatio
nsize
(θ)
Limita
tions
Recently
describedmethodlackingathorough
theoretical
andem
pirical
evaluatio
nSo
urce
httpwwwbeast2orgwikiindexphpBFD
describedin
Leacheacuteet
al(201
4)
Genom
e-wideSN
Pdata
2 The Dynamic Discipline of Species Delimitation hellip 23
distances among organisms belonging to thesame species are smaller than distances amongorganisms from different species (Puillandreet al 2012 Hebert et al 2003) Genetic distanceapproaches hold particular promise as an identi-fication tool shortcutting the difficulties of mor-phology-based identification (Hebert et al 2004)although in practice a barcode gap may not existfor many groups (Wiemers and Fiedler 2007)Furthermore the role of distance-based approa-ches using a single genetic marker for speciesdiscovery remains more controversial (Rubinoff2006) and without other corroborating evidenceOTUs inferred from single-locus dataset shouldonly be considered ldquocandidaterdquo species Tree-based methods aim to detect monophyletic cladescorresponding to species-level diversity bydetecting discontinuities associated within inter-and intraspecific branching patterns (Fujisawaand Barraclough 2013 Zhang et al 2013Monaghan et al 2009 Pons et al 2006) Tree-based species delimitation methods can also beused on the basis of other properties related tophylogenetic tree topologies (monophyly con-cordance with geography etc (Sites and Marshall2003 2004) Both distance- and tree-basedmethods have been applied for assessing speciesboundaries in lichen-forming fungi (Leavitt et al2012c 2014 Parnmen et al 2012 Del-Pradoet al 2010 2011 Molina et al 2011)
A number of tree-based methods partiallyautomate the species delimitation process withspecific bioinformatical analyses (Table 21)including the general mixed Yule coalescent(GMYC) approach (Fujisawa and Barraclough2013 Monaghan et al 2009 Pons et al 2006)and the Poisson tree processes (PTP) model(Zhang et al 2013) These methods provide rel-atively straightforward and objective pipelinesfor delimiting putative species-level lineagesfrom inferred gene trees by fitting within- andbetween-species branching models to an inferredsingle-locus topology A number of other tree-based methods for species delimitation areeffectively summarized in Sites and Marshall(2004)
When only single-locus data are available theGMYC has been shown to be a relatively robust
tool for species delimitation The GMYCapproach combines a coalescent model of intra-specific branching with a Yule model for inter-specific branching which is then fit to an inferredsingle-gene topology to estimate species bound-aries and a statistical measure of confidence for theinferred boundaries As an input the GMYCapproach requires an ultrametric gene tree andrecent refinements can account for uncertainty inphylogenetic relationships and parameters of theGMYC model (Fujisawa and Barraclough 2013Reid and Carstens 2012) Regardless of theseimprovements it may be difficult to accuratelyinfer an adequate ultrametric tree for large data-sets Although it appears that the GMYC isgenerally stable across a wide range of circum-stances including various methods of phyloge-netic reconstruction the presence of a highnumber singletons high numbers of sampledspecies and gaps in intraspecific sampling theaccuracy of the GMYC is most significantlyaffected by the mean population size relative todivergence times between them (Fujisawa andBarraclough 2013 Talavera et al 2013) Fur-thermore research suggests that the so-calledsingle-threshold version of the GMYC methodlikely outperforms the multiple-thresholdapproach (Fujisawa and Barraclough 2013Monaghan et al 2009) However other studiessuggest that theGMYCmethodmay often providehigher estimates for the total number ofOTUs thanother molecular species delimitation methods(Hamilton et al 2014 Miralles and Vences 2013Talavera et al 2013) warranting a cautiousinterpretation of results from GMYC analyses
Compared to the GMYC approach therecently introduced PTP method for speciesdelimitation has been suggested to be moreaccurate for preliminary species delimitation(Zhang et al 2013) Relative to the GMYC PTPoffers a more straightforward implementationrequiring a simple phylogenetic tree rather thanan ultrametric chronogram However moreresearch is required to assess the general perfor-mance of PTP across wide range of empirical andsimulated species delimitation studies At thispoint data support the use of the GMYC andPTP methods as objective and reasonable starting
24 SD Leavitt et al
points for species delimitation using single-genetopologies
The ldquoSpecies Delimitationrdquo plug-in to theGeneious software provides statistical approachesfor exploring species boundaries in single-genetopologies (Masters et al 2011) Using a priorispecimen assignments to putative species theldquoSpecies Delimitationrdquo plug-in computes statis-tics relating to the probability of the observedmonophyly or exclusivity having occurred bychance in a coalescent process and assesses thewithin- and between-species genetic distances inorder to infer the probability with which membersof a putative species might be identified suc-cessfully with tree-based methods An importantcontribution of the ldquoSpecies Delimitationrdquo plug-in is that it provides an objective means for usersto assess putative species within an empiricalstatistic-based framework rather than qualitativeevaluations of what level of hierarchy constitutesa species in a phylogeny
In contrast to simple sequence similaritythresholds (OTU-picking) for delimiting putativeevolutionarily significant units the AutomaticBarcode Gap Discovery (ABGD) method is anautomated procedure that sorts sequences intohypothetical species based on the existence of abarcode gap observed whenever intraspecificgenetic distances are less than those amongorganisms from different species (Puillandreet al 2012) Ultimately ABGD is a fast simplemethod that can be used to group individualsrepresented in a single-locus sequence alignmentinto candidate species that should be comple-mented with other lines of evidence in an inte-grative taxonomic approach (Kekkonen andHebert 2014 Puillandre et al 2012)
In general the ABGD GMYC and PTP ana-lytical protocols using single-locus data arerepeatable and computationally relatively fastproviding a valuable starting point for a pre-liminary perspective into species boundaries inunderstudied groups that can be validated withsubsequent studies (Kekkonen and Hebert 2014)Similarly analyses of single-locus data can beused to corroborate traditional phenotype-basedspecies boundaries and identify candidate speciesthat have previously been hidden within nominal
species However single-locus species delimita-tionmethodsmaybeparticularly prone to failure inrecognizing significant proportions of species-level biodiversity due to the strict criterion forreciprocal monophyly or alternatively providespurious inflations of estimated species diversitybased on genetic differences that donot correspondto species-level lineages For example recentestimates suggest that the incidence of species-level gene tree paraphyly is approximately 20 (Ross 2014) suggesting that analyses of single-locus datasets would likely fail to accurately deli-mit species in one in every five cases In contrastrecent empirical studies suggest that in some casesthe GMYC provides a striking overestimate ofspecies diversity (Miralles and Vences 2013)
The internal transcribed spacer region (ITS)has played a central role in molecular phyloge-netic studies of lichenized fungi and has recentlybeen adopted as the official barcoding marker forfungi (Schoch et al 2012) In many cases the ITSis sufficiently variable to resolve species bound-aries for lichenized fungi (Schoch et al 2012Kelly et al 2011) although accurate specimenidentification using sequence-based identificationapproaches requires a thoroughly curated refer-ence database (Leavitt et al 2014 Orock et al2012 Kelly et al 2011) In spite of the generalutility of the ITS marker a number of issues maypotentially limit its effective use in speciesdelimitation studies including the potential forintragenomic variation of the nuclear ribosomaltandem repeat and difficulties in aligning ITSsequences from divergent taxa (Kiss 2012)Because the ITS has been formally adopted as theofficial barcoding marker for fungi we recom-mend that species delimitation studies attempt toinclude this region for consistency across studiesHowever we recognize that in some taxonomicgroups the inclusion of the ITS may be prob-lematic and therefore suggest a careful screeningof other markers to identify appropriate loci forresolving species-level relationships
Ultimately the success of any single-locusspecies delimitation method largely depends onthe evolutionary history of the species groupunder study and the variability of the selectedmarker (Fig 22) Species delimitation will be
2 The Dynamic Discipline of Species Delimitation hellip 25
more difficult in more recently diverged lineagesand in cases with some level of interspecific geneflow relative to older well-diverged lineages(eg Leavitt et al 2012c 2013e) In the end wereemphasize that single-locus data alone shouldonly be used to provide a preliminary perspectiveof species boundaries and not as the sole evi-dence in species circumscriptions
222 Sampling Across the GenomeMultilocus Sequence Dataand Genome-Wide Markers
Although single-locus methods can provide anefficient approach for preliminary large-scaleassessments of species diversity there are
significant limitations particularly in recentlydiverged species groups where retention ofancestral polymorphisms and incomplete lineagesorting will likely result in different neutral locihaving unique gene topologies that do not mirrorthe speciation process (Knowles and Carstens2007 Heled and Drummond 2010 Taylor et al2000) In contrast to single-locus and strictlyphenotype-based approaches for species delimi-tation analyses of genetic data collected fromindependent genomic regions can provide robusthypotheses of species boundaries with increasingconfidence (Satler et al 2013 Zhang et al 2011Yang and Rannala 2010) Sequences from mul-tiple independent loci provide an importantsource of data for species delimitation studiesincluding recently developed models that
Fig 22 A simplified diagram illustrating the process ofspeciation through time in a single gene history andresulting gene topologies sampled at two points in thespeciation history (modified from Leliaert et al 2014)aEach dot represents a distinct gene copy and each row onenon-overlapping generation with lines connecting genecopies to their ancestors in the previous generation (onerow below) dashed diagonal lines represent reproductivebarriers two hypothetical sampling intervals at differentpoints in the speciation history are shown Tx and Ty
species delimitation methods using single-locus data areeffective only when species are reciprocally monophyleticin the sampled gene tree genetic clustering and coalescent-based species delimitation methods can circumscribespecies when species may not be monophyletic in sampledgenetic loci b Gene topology representing sampledhaplotypes at time Tx (shown in panel a) hypotheticalspecies are reciprocally monophyletic c Gene topologyrepresenting sampled haplotypes at time Ty (shown in panela) hypothetical species are para- and polyphyletic
26 SD Leavitt et al
combine individual gene genealogies and speciesphylogenies via modeling the coalescent historyof markers (Yang and Rannala 2010 Edwards2009 Knowles and Carstens 2007) As aresponse to advances in sequencing technologiesbioinformatical approaches for multimarker spe-cies delimitation analyses continue to be devel-oped (Table 21 Camargo and Sites 2013)
Long-term reproductive isolation of candidatespecies can be assessed with multilocus sequencedata by evaluating genealogical concordance ofunlinked markers (Baum and Shaw 1995 Aviseand Ball 1990) Within species the mixingeffects of recombination cause unlinked loci tohave distinct genealogical histories but geneticdrift and long-term divergence leads to concor-dant genealogical histories at loci across thegenome Relationships of individuals belongingto distinct candidate species can be evaluatedusing gene genealogies (eg haplotype networksor single-gene topologies) to identify lineagesthat exhibit genealogical exclusivity acrossunlinked neutral loci (Hudson and Coyne 2002Dettman et al 2003b Avise and Ball 1990) Thepresence of the same clades in the majority ofsingle-locus genealogies is taken as evidence thatthe clades represent reproductively isolated lin-eages (Dettman et al 2003a Pringle et al 2005)In practice the criteria of reciprocal monophylyand genealogical concordance of unlinked lociprovide a conservative approach for assessingspecies boundaries due to the fact that a sub-stantial amount of time is required after the initialdivergence of species until ancestral polymor-phisms have fully sorted (Knowles and Carstens2007 Hudson and Coyne 2002) Consequentlygroups with recent divergence histories willlikely go undiscovered under a genealogicalconcordance criterion due to the fact that speciesboundaries likely are not reflected in genegenealogies For example it would take morethan 1 million years after speciation before spe-cies would be delimited if 15 loci were sampledin species with an effective population size (Ne)of 100000 assuming one generation a yearunder a strict reciprocal monophyly criterion(Knowles and Carstens 2007) The amount oftime required for a species to be recognized using
a criterion of reciprocal monophyly increasesproportionally with increasing population sizes(Hudson and Coyne 2002) In a number ofstudies of lichen-forming fungi a genealogicalconcordance criterion has been used to bothdelimit previously unrecognized species-levellineages and validate some conventional pheno-type-based species (Leavitt et al 2012a c2013b Molina et al 2011 Kroken and Taylor2001)
Although analyses of molecular sequence datarelying on reciprocal monophyly or fixed char-acter differences for species delimitation canserve as important criteria for identifying speciesthese criteria are not commonly met acrossmultiple loci particularly in recent speciationhistories (Fig 22) To accommodate theobserved conflict among genealogies from mul-tiple loci with the underlying speciation historythe recent merge of coalescent theory with phy-logenetics has driven a major paradigm shift inspecies delimitation and molecular systematics ingeneral (Fujita et al 2012 Edwards 2009) Themultispecies coalescent model can be applied togenealogical histories from multiple independentloci to assemble separate coalescent processesoccurring in populations into a species tree(Degnan and Rosenberg 2009 Rannala andYang 2003) Within this coalescent-basedframework multiple individuals can be assignedto a single speciespopulation and the speciationhistory of ancestral and descendant species-levellineages can be inferred as a ldquospecies treerdquo incontrast to estimating gene genealogies fromindividual samples (Degnan and Rosenberg2006 2009 Rannala and Yang 2003)
A number of multispecies coalescent-basedspecies delimitation methods have recently beenintroduced offering an exciting framework forempirically assessing species boundaries byselecting the best species tree model from a set ofalternative models representing differenthypotheses of species boundaries (Table 21)For example SpeDeSTEM (Ence and Carstens2011) finds the maximum likelihood values for aspecies tree assuming all putative species areseparate lineages and for alternative species treeswhere two or more species are collapsed into a
2 The Dynamic Discipline of Species Delimitation hellip 27
single lineage The best fitting model is thenselected using the Akaike information criterionunder the assumption of constant populationsizes and fixed gene topologies across the speciestree The species delimitation program BayesianPhylogenetic and Phylogeography (BPampPYangand Rannala 2010) accommodates gene treeuncertainty and variable population sizes andsamples from the Bayesian posterior distributionof species delimitation models using reversible-jump Markov chain Monte Carlo (rjMCMC)sampling BPampP requires a user-provided guidetree with resolution finer than the species leveland evaluates alternative modes derived from allpossible subtrees that are generated by collapsingor splitting nodes on the guide tree (Yang andRannala 2010 Rannala and Yang 2003) Theproportion of time spent on each model is pro-portional to the posterior probability of themodel ie ldquospeciation probabilitiesrdquo WhileBPampP ranks among the most popular coalescent-based species delimitation programs (Fujita et al2012) misspecifications of the guide tree andorthe prior distribution for population size (θ) canresult in strong support for models containing aninflated number of species (Leacheacute and Fujita2010 Zhang et al 2011) The nonparametricheuristic species delimitation approach imple-mented in the program Brownie (OrsquoMeara 2010)jointly sorts anonymous samples into species andinfers a species tree from input gene trees fromdifferent loci assuming that for a speciationevent the corresponding nodes on gene trees willbe more consistent with each than the diver-gences within species However finding both theoptimum species tree and species boundariesremains computationally challenging andBrownie has been shown to frequently yieldincorrect results (OrsquoMeara 2010)
Most recently available coalescent-basedspecies delimitation methods require individualassignments to a species or population a priori Ina number of scenarios correct assignments ofsamples to species may be difficult includingpresence of cryptic species incongruencebetween conventional species and moleculardata or simply the fact that accurate specimenidentification in complex groups with subtle or
difficult to discern diagnostic characters is anontrivial task (Leavitt et al 2013e) Statisticalmethods for assessing individual assignment andspecies detection prior to coalescent-based spe-cies delimitation and species tree reconstructionand species provide a more objective approachfor understanding species boundaries
Population assignment tests using a variety ofclustering algorithms using genetic data offer auseful approach for grouping individuals intoputative reproductively isolated groups (Table 21Hausdorf and Hennig 2010 Corander et al2004 2008 Falush et al 2003 Pritchard et al2000) particularly for species delimitation prob-lems that exist at the interface of traditionalpopulation genetic and phylogenetic analyses(Carstens et al 2013 Weisrock et al 2010Knowles and Carstens 2007)
The programs STRUCTURE (Falush et al2003 Pritchard et al 2000) and STRUCTURA-MA (Huelsenbeck et al 2011) cluster samplesinto populations by minimizing HardyndashWeinbergdisequilibrium for a given number of populationclusters using unlinked genetic markers In gen-eral unlinked markers are not available for mostgroups of lichen-forming fungi and a numberof studies have used information from multilocussequence data for STRUCTURE analyses(Leavitt et al 2011a b c 2013e Fernaacutendez-Mendoza and Printzen 2013 Fernaacutendez-Mendoza et al 2011) Varying approaches havebeen implemented to convert DNA sequencedata to allelic data for Bayesian clustering (seeOrsquoNeill et al 2013) STRUCTURE is expectedto perform well when there is sufficient inde-pendence across regions such that linkage dis-equilibrium within regions does not dominate thedata (STRUCTURE manual) but can also beeffective using multilocus sequence data andtreating all SNPs as independent loci regardlessof physical linkage within each locus (OrsquoNeillet al 2013 Falush et al 2003) A recent study ofthe lichen-forming genus Letharia showed thatBayesian clustering implemented in the programSTRUCTURE was generally able to recover thesame putative Letharia lineages circumscribedemploying a gene genealogical approach inKroken and Taylorrsquos iconic species delimitation
28 SD Leavitt et al
study (Altermann et al 2014 Kroken and Taylor2001) Altermann et al (2014) show that popu-lation assignments were largely consistent acrossa range of scenarios including extensiveamounts of missing data the exclusion of SNPsfrom variable markers and inferences based onSNPs from as few as three gene regions Incontrast to STRUCTURE and STRUCTURA-MA the program BAPS (Corander et al 2008)can explicitly infer genetic structure using hap-loid sequence data or other linked geneticmarkers in the ldquoclustering with linked locirdquomodel Another advantage of BAPS is that theprogram is much more computationally efficientthan STRUCTURE or STRUCTURAMA Sim-ulation studies indicate that both BAPS andSTRUCTURE perform well at low levels ofpopulation differentiation and when clusters arenot well differentiated (Latch et al 2006)
In practice inferring the most appropriatelevel of population structure using Bayesianclustering algorithms remains challenging (Latchet al 2006 Evanno et al 2005) BAPS andSTRUCTURAMA can infer both individualassignments and the most likely number ofgenetic clusters (Corander et al 2008 Huelsen-beck et al 2011) and the ad hoc ΔK statisticproposed by Evanno et al (2005) provides anobjective approach for identifying the uppermosthierarchical level of structure However carefulconsideration of other levels of populationstructure may ultimately reveal more biologicallymeaning results and researchers should examineindividual assignments across a range of geneticgroupings Some markers generated fromindependent genomic regions such as SNPs andfast-evolving microsatellites can be used to dis-tinguish fine-scale population structuring on thebasis of allele frequencies and species delimita-tion analyses based on these markers may implythe risk of severe taxonomic oversplitting Inthese cases validation approaches such asBPampP and SpeDeSTEM and corroborationamong different species delimitation approachescan provide perspective between intraspecificpopulation structure and species-level clusters
Another limitation of clustering approaches isthat they do not assess or take into account
evolutionarily divergences and relationshipsamong population clusters Coalescent-basedspecies tree methods (discussed below) provide ameans to assess the diversification histories ofpopulations inferred from clustering analysesTherefore a potential working protocol for aninformed species delimitation study that takesinto account population structure could consist offirst applying a genetic clustering analysis under apopulation genetics criterion (eg BAPSSTRUCTURE STRUCTURAMA) to identifygenetically distinct population clusters that can beconsidered ldquocandidate speciesrdquo From these can-didate species a species tree can be inferred forfocal group using coalescent-based species treereconstruction methods (eg BEAST) Subse-quently a coalescent-based validation methodcan be applied to assess whether the distinctpopulation clusters represent independent evolu-tionary lineages (eg BPampP) This multistepapproach would provide a consistent and standardcriterion for distinguishing between population-and species-level lineages (Camargo and Sites2013) and has been applied to a number of casesincluding lichen-forming fungi (Leavitt et al2011a b c 2013e Leacheacute and Fujita 2010)
High-throughput sequencing methods providethe means to effectively sample hundreds tothousands of loci from across a species genome fora large number of species and continue to revolu-tionize studies that can be performed even in non-model organisms Targeted high-throughputsequencing approaches such as anchored phy-logenomics transcriptome sequencing reduced-representation genomic library sequencing(restriction-site-associated DNA sequencingRAD-Seq and genotype-by-sequencing GBS)and high-throughput amplicon sequencing pro-vide important insight into species boundaries fora number groups although none of these approa-ches has yet been applied to studies of lichenizedfungi
For example restriction-site-associated(RAD-Seq) DNA can simultaneously detect andgenotype thousands of genome-wide SNPs byfocusing the sequencing effort on a reducedrepresentation of the entire genome (Baird et al2008) and has been successfully applied to
2 The Dynamic Discipline of Species Delimitation hellip 29
intraspecies (Lewis et al 2007 Miller et al 2007Emerson et al 2010) and interspecies studies(Rubin et al 2012 Eaton and Ree 2013 Wagneret al 2013) This approach has provided strikinginsight into the recent adaption radiation of LakeVictoria cichlids (Wagner et al 2013) Analternative approach targeting the sequencing ofspecific loci using next-generation sequencingplatforms provides an efficient means of gener-ating data for loci of known genomic locationorthology size and expected level of variation(OrsquoNeill et al 2013) Markers can be targetedusing well-established PCR techniques or newerhybridization techniques and subsequentlypooled for high-throughput sequencing usingparallel-tagged sequencing of multiple individualsamples within a single sequencing run (OrsquoNeillet al 2013) A recent study of North Americantiger salamanders (Ambystoma tigrinum)demonstrated the potential for amplicon-basedparallel-tagged sequencing to rapidly generatelarge-scale genomic data for species delimitationand species tree research (OrsquoNeill et al 2013)
Although these methods provide an enormousamount of data and insight into diversification atan unprecedented scale computational limita-tions restrict the applicable analytical methodsfor large datasets although computational andanalytical advances are happening rapidly Mostrecent coalescent-based species delimitation andspecies tree models using gene trees have beenlimited to handle tens of loci for multiple indi-viduals and combining hundreds or thousands ofgene trees into a single species delimitationframework presents considerable computationalchallenges (Camargo and Sites 2013) Therecently developed method Bayes factor delim-itation (with genomic data BFD) combines adynamic programming algorithm for estimatingspecies trees that bypasses the computationallyintensive MCMC integration over gene trees toprovide a rigorous technique for species delimi-tation studies using genome-wide SNP data(Leacheacute et al 2014) Competing species delimi-tation models are compared using Bayes factorsand it appears that this approach is robust tosample sizes (ie few loci and limited samples
per species) and misspecification of the prior forpopulation size (θ) (Leacheacute et al 2014)
Fungi are an ideal model for assessing diver-gence in eukaryotes due to their simple mor-phologies small genomes broad ecological rolesand diverse lifestyles (Gladieux et al 2014)However the use of genomic data from high-throughput sequencing methods have not yet beenincluded in species delimitation studies of lichen-forming fungi This is due in part to challenges indealing with metagenomic data containing geno-mic information from a plethora of symbiontsassociated with a lichen thallus scant genomicresources and discipline-specific inertia A num-ber of lichen-forming fungal genomes are cur-rently available including Endocarponpusillum(Wang et al 2014) Cladonia spp (Park et al2013a 2014) Caloplaca flavorubescens (Parket al 2013b) and Cladonia greyi and Xanthoriaparientina through the Fungal Genomics Programat Joint Genome Institute of the United StatesDepartment of Energy (httpwwwjgidoegov)The foreseeable ongoing expansion of genomicresources for lichen-forming fungi will be centralto developing approaches for delimiting speciesusing high-throughput sequencing Specificallygenomic resources will be crucial in identifyingnovel markers (variable genes regions SNPsmicrosatellites etc) identifying conserved fungalmarkers for targeted high-throughput sequencingapproaches and references for entire genomecomparisons (Devkota et al 2014 Werth et al2013)
23 Can We Make SpeciesDelimitation in Lichen-FormingFungi Truly Integrative
The widespread availability of genetic data hascreated a biased viewpoint that only genetic datashould be used for statistical species delimitationHowever an ongoing appeal to researchers toassess species boundaries from multiple andcomplementary perspectives (phylogeneticspopulation genetics comparative morphologydevelopment ecology etc) has resulted in an
30 SD Leavitt et al
integrative taxonomic framework bringing theseconceptual and methodological perspectivestogether (Hamilton et al 2014 Fujita et al 2012Salicini et al 2011 Yeates et al 2011 Padialet al 2010 Dayrat 2005 Will et al 2005 Wiensand Penkrot 2002) In practice any study linkingdifferent kinds of data to support hypotheses ofspecies boundaries including mapping morpho-logical characters onto a molecular phylogenycan be considered integrative Integrative meth-ods for species delimitation fall across a broadspectrum ranging from verbal and qualitativeassessments of data classes to quantitative meth-ods that allow different data types to contribute tostatistical species delimitation (Yeates et al2011) In most taxonomic studies utilizing bothmolecular and morphological data expert opinionis eventually used to some degree to evaluate thefinal status of a candidate species (eg Bond andStockman 2008) In this sense many studiesusing evidence for independent data sources fordelimiting species boundaries use an iterativeapproach (Yeates et al 2011) where speciesboundaries can be tested within a hypothetico-deductive framework with diverse datasets
From a practical perspective we advocate aprocess of iterative taxonomy (sensu Yeates et al2011) to circumscribe and refine species limitsusing multiple lines of evidence This iterativeprocess involves comparisons of morphologicaldata with a phylogenetic hypothesis (ie single-locus gene tree or concatenated multilocusphylogeny) to identify the least inclusive mono-phyletic clade in the topology characterized by atleast one unambiguously diagnostic morphologi-cal character (Miralles and Vences 2013) Thisphylogenetic tree-informed approach to assessingspecies boundaries represents a common practicein studies of lichen-forming fungi Previouslyunrecognized species-level clades with corre-sponding subtle or overlooked phenotypiccharacters have been commonly observed in bothcrustose lichens [eg Acarospraceae (Wedin et al2009) Graphidaceae (Rivas Plata and Lumbsch2011 Papong et al 2009) Lecanoraceae (Leavittet al 2011a) Lecideaceae (Ruprecht et al 2010)Mycoblastaceae (Spribille et al 2011) andTeloschistaceae (Vondraacutek et al 2009 Muggia
et al 2008)] and foliose and fruticose lichens [egLobariaceae (Moncada et al 2014 McDonaldet al 2003) Parmeliaceae (Leavitt et al 2013aWirtz et al 2012 Divakar et al 2005 Molinaet al 2004 Kroken and Taylor 2001) Peltigera-ceae (OrsquoBrien et al 2009 Goffinet et al 2003)Physciaceae (Elix et al 2009 Divakar et al2007) and Sphaerophoraceae (Houmlgnabba andWedin 2003)] As a specific example Luumlckinget al (2008) used a combination of medullarychemistry and underside pigmentation in speci-mens from the Heterodermia obscurata group inCosta Rica to corroborate monophyletic clades inan ITS gene tree as species-level lineages
While this iterative approach to speciesdelimitation and taxonomy has proven valuablefor understanding species boundaries anddescribing new taxa in some groups of lichenizedfungi a posteriori examination of morphologicaland chemical features has failed to reveal diag-nostic phenotypic characters in a number ofstudies (Muggia et al 2014 Leavitt et al 2011aOtaacutelora et al 2010 OrsquoBrien et al 2009) Fur-thermore a study of widespread species in thegenus Melanelixia (Parmeliaceae) indicated thatphenotypically cryptic lichen-forming fungalspecies-level lineages may be relatively ancientand diagnosable phenotypic differences may beabsent even millions of years after the initialdivergence (Leavitt et al 2012b) The latter studyhighlights the fact that species-level lineages maycommonly exist without any observable diag-nostic phenotypic characters calling into ques-tion the impetus for a universal application ofintegrative taxonomy
In keeping with the principle that as manylines of evidence as available should be com-bined to delimit species (Dayrat 2005) the for-malized integrative taxonomic approach (ITAXMiralles and Vences 2013) uses a mtDNA guidetree and observations from different types of datathat might provide conclusive evidence for theindependence of lineages and thus their identityas different species Miralles and Vences (2013)provide a non-exhaustive list of species delimi-tation criteria to be integrated in the ITAXapproach including (a) sympatric occurrencewithout admixture as revealed by consistent
2 The Dynamic Discipline of Species Delimitation hellip 31
differences in morphological or molecular char-acters at the same geographic location (b) strongdifferences in a behavioral morphological orgenetic character known to mediate prematingisolation (c) unviability or infertility of hybrids(d) lack of gene flow across a geographicalhybrid zone (e) congruent diagnostic differencesbetween sister lineages in various unlinkedmorphological character (f) absence of haplo-type sharing in several unlinked nuclear loci and(g) a combination of criteria endashf Speciesboundaries are based on seeking the least inclu-sive monophyletic group in the mtDNA treewhich fulfills at least one of the criteria listedabove Clearly the ITAX approach is sensitive tosample size and in order to reliably support thedistinctiveness of a given species it has beenrecommended that the sampling strategy includesat least five individuals per species (Miralles andVences 2013) A similar approach could beadopted for lichenized fungi using an ITS genetopology as guide tree rather than mtDNA
A total evidence approach including concat-enation has been a common approach for inte-grating information from different sourcesincluding independent genetic markers in phy-logenetic reconstructions (Kluge 1989 Wiens1998 de Queiroz et al 1995) Proponents forconcatenation of independent data in phyloge-netic analyses argue that when combining alldata the underlying signal of speciation mayemerge even if weakly contradictory signalis contained in the individual data partitions(Gatesy et al 1999) For example establishing apreliminary perspective of species boundariesfrom multilocus sequence data using concatena-tion may provide a reasonable starting point forscreening for cryptic species and species treeinference (Šlapeta et al 2006 Leavitt et al2011a Le Gac et al 2007 Leacheacute 2009)However concatenation and consensus methodsimply a risk of obtaining inflated support forincorrect relationships and information aboutvariance in gene coalescence is lost (Degnan andRosenberg 2006 2009 Kubatko and Degnan2007 Edwards 2009) In spite of the impetus forintegrating evidence from independent data
sources into empirical species delimitation stud-ies and taxonomy (Fujita et al 2012 Padial et al2009) most currently available species delimi-tation methods are unable to accommodate non-genetic data sources in a statistical framework
231 Selecting the Appropriate Data
In the face of increasing availability of geneticdata and associated bioinformatical approachesfor delimiting species researchers should care-fully consider what information is being sacri-ficed by the failure to consider non-genetic datain species delimitation studies and whetheraccuracy could be improved by the addition ofmultiple data types Morphological data havehistorically served as a proxy to identify repro-ductively isolated groups (ie ldquospeciesrdquo) (Ray1686 Fujita et al 2012) Current methods fordelimiting species using non-genetic data (egchemistry morphology and ecology) remainwoefully understudied For example morphol-ogy-based species circumscriptions are generallybased on one or more qualitative (or quantitative)morphological characters that do not appear tooverlap with other species However ascertain-ing that a given trait is truly fixed within apopulation with statistical confidence requiresunrealistic sample sizes even when allowing forsome level of polymorphism in the diagnosticcharacter (Wiens and Servedio 2000) Now var-ious combinations of datamdashfrom morphologygenetics geography and ecologymdashare acceptedas standard information for species delimitationstudies (Ruiz-Sanchez and Sosa 2010 Ross et al2010 Edwards and Knowles 2014)
Below we briefly discuss appropriate datasources for species delimitation studies of lichen-forming fungi However homoplastic characters(similar traits that are not derived from a com-mon ancestor) are common among many traitscommonly used to circumscribe fungal taxa thebiological significance of secondary metabolitevariation remains largely unknown (Lawrey1986) and ecological niches may be difficultto adequately characterize and model due to
32 SD Leavitt et al
microhabitat requirements and data resolutionTherefore we advocate a cautious approach toselecting appropriate and relevant data forassessing species boundaries
In spite of the potential challenges and limi-tations of using phenotypic data these traits haveprovided a plethora of valuable information forunderstanding species boundaries For examplein the Melanelixia fuliginosa group (Parmelia-ceae) a morphometric analysis using color isi-dia and marginal zone free of isidia ascharacters revealed a general pattern of differ-entiation between material formerly recognizedas subspecies fuliginosa and glabratula in spiteof the fact that considerable overlap betweengroups occurs in some characters individually(Arup and Berlin 2011) Furthermore the dis-tinction of the two groups was supported by aphylogenetic analysis of the ITS marker andecological differences with M fuliginosaoccurring predominantly on rock and M glab-ratula on bark (Arup and Berlin 2011) Thisstudy of the M fuliginosa group provides a fit-ting example of using multiple independentsuites of data including ecology morphologyand genetic information to establish a robusthypothesis of species boundaries
In lichen systematics phenotypic dataincluding thallus organization secondary metab-olites mode of reproduction ascoma-type andontogeny ascus and ascospore characters havehistorically played a prominent role (Printzen2009) Ascomatal characters have traditionallyheld a major role in higher-level classification(Printzen 2009) in contrast to species-levelclassification which also tends to include a widearray of vegetative and chemical charactersCommonly assessed morphological charactersinclude thallus form and size cortical features(eg maculae and pseudocyphellae) presenceformcolor of attachment structure (eg rhizines)and reproductive mode (ascomata vs vegetativediaspores) (Printzen 2009) Different morpho-logical types of vegetative diasporesmdashcorticatedisidia and ecorticated sorediamdashand their locationare commonly used to distinguish speciesAscomatal characters including morphologylocation (laminal vs marginal) position (sessile
vs immersed) presence of thalline margins colorof an apothecial disc and presence or color ofpruina are also commonly used in speciesdelimitations of lichen-forming fungi (Printzen2009) Other important characters may includethalline characters form color size and septationof ascospores size and form and structure of ascithe hamathecium type of epihymenium andhypothecium and the type of excipulum orperidium conidiomatal characters etc (Printzen2009)
Assessments of secondary metabolites hasplayed an important role in lichen taxonomybeginning with the introduction of simple spottests by Nylander (1866a b) The use of chem-istry in lichen taxonomy has been discussed indetail in numerous reviews (Lumbsch 1998a bRogers 1989 Brodo 1986 Egan 1986 Leuckert1985 Brodo 1978 Hawksworth 1976 Culber-son 1969 1970) and we refer readers to thesevaluable sources for a more comprehensive per-spective on lichen chemistry In short extrolites(secondary metabolites) belong to various clas-ses the most common and diverse include dep-sides depsidones chlorinated xanthones andanthraquinones (Lumbsch 2002 Culberson1969) The presence or absence of specificextrolites or their replacements by another sub-stance is widely used to distinguish speciesparticularly when correlated with differences ingeographic distributions However if morpho-logical or geographical differences betweenpopulations containing different extrolites are notapparent the taxonomic significance has beendisputed with some authors distinguishing themas species and others preferring to regard them aschemical races within a species In addition tosimply using the presence or absence of extro-lites Culberson and Culberson (1976) proposedto arrange lichen substances into chemosyn-dromes of closely related substances The pres-ence or absence of these chemosyndromes maypotentially be used as characters to delimit spe-cies regarding differences involving the samechemosyndromes as intraspecific variation anddistinct chemosyndromes as evidence for inter-specific populations (Lumbsch 1994 Gowan1986)
2 The Dynamic Discipline of Species Delimitation hellip 33
Due to the fact that species delimitationstudies often incorporate a substantial biogeo-graphical or ecological component ecologicalniche modeling plays an increasing role in phy-logenetic and taxonomic research Niche mod-eling can provide evidence for ecologicalisolation between populations based on eitherconserved or divergent ecological niches andtherefore can provide additional evidence sup-porting lineage independence between putativespecies The application of ecological nichemodeling has been applied to species delimita-tion studies in a number of cases (Ruiz-Sanchezand Sosa 2010 Leacheacute et al 2009 Raxworthyet al 2007 Rissler and Apodaca 2007) althoughit has not been explicitly applied to assess speciesboundaries in lichen-forming fungi By mappingthe spatial distribution of environmental suit-ability of climatic variables for candidate speciesthe application of ecological niche modeling canbe particularly important in cases where specieshave allopatric distributions (Raxworthy et al2007)
Ecological niche modeling utilizes knownassociations between a speciesrsquo occurrencelocalities and environmental variables to defineabiotic conditions within which populations canbe maintained (Guisan and Thuiller 2005) Themethodological approach for modeling is basedon four general properties (i) The current knownspeciesrsquo localities is the dependent variable (ii)the distribution is modeled as a map composed ofgrid cells at a specified resolution (iii) a range ofenvironmental variables (eg temperature pre-cipitation and solar exposure) are collected todescribe the characteristics of each cell and (iv)classifying the degree to which each cell is eithersuitable or unsuitable for each species under arange of models (Guisan and Thuiller 2005)
Ecological data also have the potential to playan important role in understanding speciesboundaries in lichen-forming fungi For exampleNash and Zavada (1977) demonstrated that Xant-hoparmelia populations with distinct chemistriesoccurring in the northern portion of the SonoranDesert exhibit habitat selection among differentrock substrates within a region with relativelyuniform climate and topography In another case
the parmelioid species Parmelia mayi is mor-phologically indistinguishable from P saxatilisbut can be separated by bioclimatic features andgenetic and chemical characters (Molina et al2011) McCune and Printzen(2011) assess distri-butions and climatic niches of species in theLecanora varia group inwesternUSAandprovidea model that uses continental influence and annualtemperature as themajor factors predicting speciesdistributions The distribution of Usnea hirta alichen commonly used in air quality biomonitor-ing research was modeled for a section of theWhite River National Forest in central Coloradobased on the presence of U hirta at 72 biomoni-toring reference sites distributed in the inter-mountain western United States (Shrestha et al2012) The best model for predicting U hirtadistribution included four variablesmdashsolar radia-tion average monthly precipitation and averagemonthly minimum and maximum temperatures(Shrestha et al 2012) These studies supportthe potential use of ecological niche modelingmethods in species delimitation studies of lichen-forming fungi
24 Conclusions What AboutTaxonomy
In most cases species circumscription and tax-onomy requires some degree of qualitativejudgment and individual interpretation Integrat-ing multiple types of data into an empiricalframework for delimiting species boundarieswhere species boundaries can be tested within ahypothetico-deductive framework with diversedatasets can provide robust hypotheses of speciesboundaries and taxonomic stability (Yeates et al2011) However it has long been known toevolutionary biologists that distinct species donot need to have diagnosable morphologicaldifferences (Mayr 1963) and increasing avail-ability of genetic data has allowed researchers toidentify species and to rigorously test speciesboundaries with a level of precision that wasunimaginable a decade ago While analyticaladvances in statistical species delimitation havebeen largely based on genetic data the utility of
34 SD Leavitt et al
these approaches to formal taxonomy remainselusive Due to these challenges an eclecticapproach to delimiting species and cautionagainst the reliance on any single dataset ormethod is required when delimiting species
While results from many recent studies maycontradict traditional species boundaries acrossmany groups of lichen-forming fungi we areoptimistic that this research represents substantialprogress toward a more accurate perspective onspecies boundaries and diversity in fungi As aresult of ongoing research of species boundariesin lichen-forming fungi the taxonomic value ofmany phenotypes is now better understood ourunderstanding of ecological evolutionary andbiogeographic patterns has improved and we canbegin to better understand patterns of symbiontinteractions in lichens Integrating new data(including novel morphological characters andgenetic data) will be essential to accurately rep-resent species-level diversity across all groups oflichenized fungi Hopefully an improved per-spective on lichen diversity also increases ourappreciation of these incredible symbiotic sys-tems While we are strong advocates for theapplication of independent data types in devel-oping an integrative taxonomy there is anincreasing need to formally recognize the exis-tence of phenotypically cryptic species-levellineages in lichen-forming fungi (see Hibbettet al 2011) In some cases a molecular taxon-omy may provide the most practical approach toconsistent treatment of mycobiont species withinlichen groups where diagnostic morphologicalcharacters are unidentifiable or practically notfeasible (Leavitt et al 2013c d)
These are exciting times for taxonomists andphylogeneticists A closer look at lichen taxon-omy with the inclusion of new data will help usto better understand the diversity of these fasci-nating organisms accurately interpret distribu-tion patterns and play a more important role inmeaningful conservation practices Howeversome level of uncertainty will accompany pro-gress In many taxonomic groups our traditionalapproach for species identification will likelyneed to be substantially modified The search forcorroborating morphological support for cryptic
species identified using molecular data willrequire meticulous and creative approaches toassess phenotypic variation in potentially unor-thodox ways We are hopeful that lichenologistswho traditionally have been eager to include newmethods such as chromatography in their rou-tine identifications will be amenable to includemolecular techniques to their routine examina-tion of specimens for identification and classifi-cation Although this may prove difficult toachieve by single individuals especially citizenscientists that traditionally play an important rolein lichen taxonomy the increasing numberof collaborative projects in the discipline (egLumbsch et al 2011 Crespo et al 2010Gueidan et al 2009) make us optimistic thatbroad-scale collaborative approaches will facili-tate the inclusion of molecular data in lichenresearch at all levels
Acknowledgments We are indebted to various col-leagues for valuable thought-provoking discussionnotably Matthew Nelsen (University of Chicago) AnaCrespo (Universidad Complutense de Madrid) PradeepDivakar (Universidad Complutense de Madrid) BeckettSterner (The Field Museum) and Joyce Havstad (TheField Museum) We also thank anonymous reviewers whoprovided valuable comments that improved this chapterSupport by the US National Science Foundation isgratefully acknowledged (ldquoHidden diversity in parmelioidlichensrdquo DEB-0949147)
References
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Altermann S Leavitt SD Goward T Nelsen MPLumbsch HT (2014) How do you solve a problemlike Letharia A new look at cryptic species in lichen-forming fungi using Bayesian clustering and SNPSsfrom multilocus sequence data PLoS ONE 9(5)e97556 doi101371journalpone0097556
Amo de Paz G Cubas P Crespo A Elix JA Lumbsch HT(2012) Transoceanic dispersal and subsequent diver-sification on separate continents shaped diversity ofthe Xanthoparmelia pulla group (Ascomycota) PLoSONE 7(6)e39683 doi101371journalpone0039683
Arguumlello A Del Prado R Cubas P Crespo A (2007)Parmelina quercina (Parmeliaceae Lecanorales)
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includes four phylogenetically supported morphospe-cies Biol J Linn Soc 91(3)455ndash467 doi101111j1095-8312200700810x
Arup U Berlin ES (2011) A taxonomic study ofMelanelixia fuliginosa in Europe The Lichenologist43(02)89ndash97 doi101017S0024282910000678
Arup U Grube M (2000) Is Rhizoplaca (Lecanoraleslichenized Ascomycota) a monophyletic genus Can JBot 78(3)318ndash327 doi101139b00-006
Avise J Ball R (1990) Principles of genealogicalconcordance in species concepts and biological tax-onomy Oxf Surv Evol Biol 745ndash67
Baird NA Etter PD Atwood TS Currey MC Shiver ALLewis ZA Selker EU Cresko WA Johnson EA(2008) Rapid SNP discovery and genetic mappingusing sequenced RAD markers PLoS ONE 3(10)e3376 doi101371journalpone0003376
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Beaumont MA Nielsen R Robert C Hey J Gaggiotti OKnowles L Estoup A Panchal M Corander JHickerson M Sisson SA Fagundes N Chikhi LBeerli P Vitalis R Cornuet J-M Huelsenbeck J FollM Yang Z Rousset F Balding D Excoffier L (2010)In defence of model-based inference in phylogeogra-phy Mol Ecol 19(3)436ndash446 doi101111j1365-294X200904515x
Bickford D Lohman DJ Sodhi NS Ng PKL Meier RWinker K Ingram KK Das I (2007) Cryptic speciesas a window on diversity and conservation TrendsEcol Evol 22(3)148ndash155 doi101016jtree200611004
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Bond J Stockman A (2008) An integrative method fordelimiting cohesion species finding the population-species interface in a group of Californian trapdoorspiders with extreme genetic divergence and geo-graphic structuring Syst Biol 57(4)628ndash646 doi10108010635150802302443
Brodo IM (1978) Changing concepts regarding chemicaldiversity in lichens The Lichenologist 10(1)1ndash11doi101017S0024282978000031
Brodo IM (1986) Interpreting chemical variation inlichens for systematic purposes The Bryologist 89(2)132ndash138
Caley MJ Fisher R Mengersen K (2014) Global speciesrichness estimates have not converged Trends EcolEvol 29(4)187ndash188 doi101016jtree201402002
Camargo A Sites JW (2013) Species delimitation adecade after the Renaissance In Pavlinov I (ed) Thespecies problemmdashongoing issues InTech doi10577252664
Camargo A Morando M Avila LJ Sites JW (2012)Species delimitation with ABC and other coalescent-based methods a test of accuracy with simulations and
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Carstens BC Dewey TA (2010) Species delimitationusing a combined coalescent and information-theoreticapproach an example from North American Myotisbats Syst Biol 59(4)400ndash414 doi101093sysbiosyq024
Carstens BC Pelletier TA Reid NM Satler JD (2013)How to fail at species delimitation Mol Ecol 22(17)4369ndash4383 doi101111mec12413
Corander J Marttinen P (2006) Bayesian identification ofadmixture events using multi-locus molecular mark-ers Mol Ecol 15(10)2833ndash2843 doi101111j1365-294X200602994x
Corander J Waldmann P Marttinen P Sillanpaa M(2004) BAPS 2 enhanced possibilities for the analysisof genetic population structure Bioinformatics 20(15)2363ndash2369 doi101093bioinformaticsbth250
Corander J Marttinen P Mantyniemi S (2006) Bayesianidentification of stock mixtures from molecular markerdata Fish Bull 104550ndash558
Corander J Marttinen P Siren J Tang J (2008) EnhancedBayesian modelling in BAPS software for learninggenetic structures of populations BMC Bioinf 9(1)539 doi1011861471-2105-9-539
Coyne JA Orr HA (2004) Speciation Sinauer AssociatesSunderland
Cracraft J (1983) Species concepts and speciation anal-ysis Curr Ornithol 1159ndash187
Crespo A Lumbsch HT (2010) Cryptic species in lichen-forming fungi IMA Fungus 1167ndash170 doi105598imafungus2010010209
Crespo A Peacuterez-Ortega S (2009) Cryptic species andspecies pairs in lichens a discussion on the relation-ship between molecular phylogenies and morpholog-ical characters Anales del Jardin Botanico de Madrid66(S1)71ndash81 doi103989ajbm2225
Crespo A Kauff F Divakar PK del Prado R Perez-Ortega S Amo de Paz G Ferencova Z Blanco ORoca-Valiente B Nunez-Zapata J Cubas P ArguelloA Elix JA Esslinger TL Hawksworth DL MillanesA Molina MC Wedin M Ahti T Aptroot A et al(2010) Phylogenetic generic classification of parmeli-oid lichens (Parmeliaceae Ascomycota) based onmolecular morphological and chemical evidenceTaxon 59(6)1735ndash1753
Culberson WL (1969) The use of chemistry in thesystematics of the lichens Taxon 18498ndash505
Culberson WL (1970) Chemosystematics and ecology oflichen-forming fungi Annu Rev Ecol Syst 1153ndash170
Culberson CF Culberson WL (1976) Chemosyndromicvariation in lichens Syst Bot 1325ndash339
Darwin C (1859) On the origin of species by means ofnatural selection or the preservation of favoured racesin the struggle for life J Murray London
Dayrat B (2005) Towards integrative taxonomy Biol JLinn Soc 85(3)407ndash415 doi101111j1095-8312200500503x
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de Queiroz K (1998) The general lineage concept ofspecies species criteria and the process of speciationa conceptual unification and terminological recom-mendations In Howard DJ Berlocher SH (eds)Endless forms species and speciation Oxford Uni-versity Press Oxford pp 57ndash75
de Queiroz K (1999) The general lineage concept ofspecies and the defining properties of the speciescategory In Wilson RA (ed) Species new interdis-ciplinary essays MIT Press Cambridge pp 49ndash89
de Queiroz K (2007) Species concepts and speciesdelimitation Syst Biol 56(6)879ndash886 doi10108010635150701701083
de Queiroz A Donoghue MJ Kim J (1995) Separateversus combined analysis of phylogenetic evidenceAnnu Rev Ecol Syst 26(1)657ndash681 doi101146annureves26110195003301
Degnan J Rosenberg N (2006) Discordance of speciestrees with their most likely gene trees PLoS Genet 2(5)e68 doi101371journalpgen0020068
Degnan JH Rosenberg NA (2009) Gene tree discordancephylogenetic inference and the multispecies coales-cent Trends Ecol Evol 24(6)332ndash340 doihttpdxdoiorg101016jtree200901009
Del-Prado R Cubas P Lumbsch HT Divakar PK BlancoO de Paz GA Molina MC Crespo A (2010) Geneticdistances within and among species in monophyleticlineages of Parmeliaceae (Ascomycota) as a tool fortaxon delimitation Mol Phylogenet Evol 56(1)125ndash133 doi101016jympev201004014
Del-Prado R Divakar PK Crespo A (2011) Using geneticdistances in addition to ITS molecular phylogeny toidentify potential species in the Parmotrema reticul-atum complex a case study The Lichenologist 43(06)569ndash583 doi101017S0024282911000582
Del-Prado R Blanco O Lumbsch HT Divakar PK ElixJA Molina MC Crespo A (2013) Molecular phylog-eny and historical biogeography of the lichen-formingfungal genus Flavoparmelia (Ascomycota Parmelia-ceae) Taxon 62(5)928ndash939 doi101270562522
Dettman J Jacobson D Taylor J (2003a) A multilocusgenealogical approach to phylogenetic species recog-nition in the model eukaryote Neurospora Evolution57(12)2703ndash2720 doi101111j0014-38202003tb01514x
Dettman JR Jacobson DJ Turner E Pringle A TaylorJW (2003b) Reproductive isolation and phylogeneticdivergence in Neurospora comparing methods ofspecies recognition in a model eukaryote Evolution57(12)2721 doi10155403-074
Devkota S Cornejo C Werth S Chaudhary RP Sche-idegger C (2014) Characterization of microsatelliteloci in the Himalayan lichen fungus Lobaria pindar-ensis (Lobariaceae) Appl Plant Sci 2(5)1300101doi103732apps1300101
Divakar PK Molina MC Lumbsch HT Crespo A (2005)Parmelia barrenoae a new lichen species related toParmelia sulcata (Parmeliaceae) based on molecularand morphological data The Lichenologist 37(01)37ndash46 doi101017S0024282904014641
Divakar PK Amo De paz G del Prado R Esslinger TLCrespo A (2007) Upper cortex anatomy corroboratesphylogenetic hypothesis in species of Physconia(Ascomycota Lecanoromycetes) Mycol Res 111(11)1311ndash1320 doi101016jmycres200708009
Divakar PK Figueras G Hladun N Crespo A (2010)Molecular phylogenetic studies reveal an undescribedspecies within the North American concept of Mela-nelixia glabra (Parmeliaceae) Fungal Divers 42(1)47ndash55 doi101007s13225-010-0027-3
Donoghue MJ Gauthier A (2004) Implementing thephylocode Trends Ecol Evol 19(6)281ndash282 doi101016jtree200404004
Eaton DAR Ree RH (2013) Inferring phylogeny andintrogression using RADseq data an example fromflowering plants (Pedicularis Orobanchaceae) SystBiol 62(5)689ndash706 doi101093sysbiosyt032
Edwards SV (2009) Is a new and general theory ofmolecular systematics emerging Evolution 63(1)1ndash19 doi101111j1558-5646200800549x
Edwards DL Knowles LL (2014) Species detection andindividual assignment in species delimitation canintegrative data increase efficacy Proc R Soc B BiolSci 281(1777) doi101098rspb20132765
Egan RS (1986) Correlations and non-correlations ofchemical variation patterns with lichen morphologyand geography The Bryologist 8999ndash110
Elix JA Corush J Lumbsch HT (2009) Triterpenechemosyndromes and subtle morphological characterscharacterise lineages in the Physcia aipolia group inAustralia (Ascomycota) Syst Biodivers 7(04)479ndash487 doi101017S1477200009990223
Emerson KJ Merz CR Catchen JM Hohenlohe PACresko WA Bradshaw WE Holzapfel CM (2010)Resolving postglacial phylogeography using high-throughput sequencing Proc Natl Acad Sci 107(37)16196ndash16200 doi101073pnas1006538107
Ence DD Carstens BC (2011) SpedeSTEM a rapid andaccurate method for species delimitation Mol EcolResour 11(3)473ndash480 doi101111j1755-0998201002947x
EvannoGRegnaut SGoudet J (2005)Detecting thenumberof clusters of individuals using the software STRUC-TURE a simulation study Mol Ecol 14(8)2611ndash2620doi101111j1365-294X200502553x
Falush D Stephens M Pritchard JK (2003) Inference ofpopulation structure using multilocus genotype datalinked loci and correlated allele frequencies Genetics164(4)1567ndash1587 doi101111j1471-8286200701758x
Fan HH Kubatko LS (2011) Estimating species treesusing approximate Bayesian computation Mol Phylo-genet Evol 59(2)354ndash363 doi101016jympev201102019
Fernaacutendez-Mendoza F Printzen C (2013) Pleistoceneexpansion of the bipolar lichen Cetraria aculeata intothe Southern hemisphere Mol Ecol 22(7)1961ndash1983doi101111mec12210
Fernaacutendez-Mendoza F Domaschke S Garciacutea MA JordanP Martin MP Printzen C (2011) Population structure
2 The Dynamic Discipline of Species Delimitation hellip 37
of mycobionts and photobionts of the widespreadlichen Cetraria aculeata Mol Ecol 20(6)1208ndash1232doi101111j1365-294X201004993x
Fraley C Raftery A (2007) Model-based methods ofclassification Using the mclust software in chemo-metrics J Stat Softw 18i06 [Available online at httpwwwdoajorgdoajfunc=abstractampid=218544]
Fujisawa T Barraclough TG (2013) Delimiting speciesusing single-locus data and the generalized mixed yulecoalescent (GMYC) approach a revised method andevaluation on simulated datasets Syst Biol 62(5)707ndash724 doi101093sysbiosyt033
Fujita MK Leacheacute AD Burbrink FT McGuire JA MoritzC (2012) Coalescent-based species delimitation in anintegrative taxonomy Trends Ecol Evol 27(9)480ndash488 doi101016jtree201204012
Gatesy J OrsquoGrady P Baker RH (1999) Corroborationamong data sets in simultaneous analysis hiddensupport for phylogenetic relationships among higherlevel Artiodactyl taxa Cladistics 15(3)271ndash313doi101111j1096-00311999tb00268x
Gladieux P Ropars J Badouin H Branca A Aguileta Gde Vienne DM Rodriacuteguez de la Vega RC Branco SGiraud T (2014) Fungal evolutionary genomicsprovides insight into the mechanisms of adaptivedivergence in eukaryotes Mol Ecol 23(4)753ndash773doi101111mec12631
Goffinet B Miadlikowska J Goward T (2003) Phyloge-netic inferences based on nrDNA sequences supportfive morphospecies within the Peltigera didactylacomplex (Lichenized Ascomycota) The Bryologist106(3)349ndash364 doi10163901
Gowan SP (1986) Evolution of secondary natural productsin the genus Porpidia (Ascomycata Porpidiaceae) AmJ Bot 73606
Griffin PC Hoffmann AA (2014) Limited genetic diver-gence among Australian alpine Poa tussock grassescoupled with regional structuring points to ongoinggene flow and taxonomic challenges Ann Bot doi101093aobmcu017
Grube M Hawksworth DL (2007) Trouble with lichenthe re-evaluation and re-interpretation of thallus formand fruit body types in the molecular era Mycol Res111(9)1116ndash1132 doi101016jmycres200704008
Gueidan C Savi S Thues H Roux C Keller C Tibell LPrieto M Heimarsson S Breuss O Orange A FrobergL Wynns AA Navarro-Rosines P Krzewicka BPykaelae J Grube M Lutzoni F (2009) Genericclassification of the Verrucariaceae (Ascomycota)based on molecular and morphological evidencerecent progress and remaining challenges Taxon 58(1)184ndash208
Guillot G Mortier F Estoup A (2005) Geneland acomputer package for landscape genetics Mol EcolNotes 5(3)712ndash715 doi101111j1471-8286200501031x
Guillot G Renaud S Ledevin R Michaux J Claude J(2012) A unifying model for the analysis of pheno-typic genetic and geographic data Syst Biol 61(6)897ndash911 doi101093sysbiosys038
Guisan A Thuiller W (2005) Predicting species distribu-tion offering more than simple habitat models EcolLett 8(9)993ndash1009 doi101111j1461-0248200500792x
Hale ME (1990) A synopsis of the lichen genusXanthoparmelia (Vainio) Hale (AscomycotinaParmeliaceae) Smithsonian Institution Press Wash-ington DC
Hamilton CA Hendrixson BE Brewer MS Bond JE(2014) An evaluation of sampling effects on multipleDNA barcoding methods leads to an integrativeapproach for delimiting species a case study ofthe North American tarantula genus Aphonopelma(Araneae Mygalomorphae Theraphosidae) MolPhylogenet Evol 7179ndash93 doi101016jympev201311007
Hausdorf B (2011) Progress toward a general speciesconcept Evolution 65(4)923ndash931 doi101111j1558-5646201101231x
Hausdorf B Hennig C (2010) Species delimitation usingdominant and codominant multilocus markers SystBiol 59(5)491ndash503 doi101093sysbiosyq039
Hawksworth DL (1976) Lichen chemotaxonomy InBailey RH (ed) Lichenology progress and problemsAcademic London pp 139ndash184
Hebert PDN Cywinska A Ball SL deWaard JR (2003)Biological identifications through DNA barcodesProc R Soc Lond B Biol Sci 270(1512)313ndash321doi101098rspb20022218
Hebert PDN Stoeckle MY Zemlak TS Francis CM(2004) Identification of birds through DNA barcodesPLoS Biol 2(10)e312 doi101371journalpbio0020312
Heled J Drummond AJ (2010) Bayesian inference ofspecies trees from multilocus data Mol Biol Evol 27(3)570ndash580 doi101093molbevmsp274
Hey J (2006) On the failure of modern species conceptsTrends Ecol Evol 21(8)447ndash450 doi101016jtree200605011
Hibbett DS OhmanA Glotzer D NuhnMKirk P NilssonRH (2011) Progress in molecular and morphologicaltaxon discovery in Fungi and options for formalclassification on environmental sequences Fungal BiolRev 2538ndash47 doi101016jfbr201101001
Houmlgnabba F Wedin M (2003) Molecular phylogeny ofthe Sphaerophorus globosus species complex Cladis-tics 19(3)224ndash232 doi101111j1096-00312003tb00365x
Hudson RR Coyne JA (2002) Mathematical conse-quences of the genealogical species concept Evolu-tion 56(8)1557 doi101111j0014-38202002tb01467x
Huelsenbeck JP Andolfatto P Huelsenbeck ET (2011)Structurama Bayesian inference of population struc-ture Evol Bioinf Online 7(2011)55ndash59 doi104137EBOS6761
Jones G Oxelman B (2014) DISSECT an assignment-free Bayesian discovery method for species delimita-tion under the multispecies coalescent Bioinformaticsdoi101101003178
38 SD Leavitt et al
Kekkonen M Hebert PD (2014) DNA barcode-baseddelineation of putative species efficient start fortaxonomic workflows Mol Ecol Res doi1011111755-099812233
Kelly LJ Hollingsworth PM Coppins BJ Ellis CJHarrold P Tosh J Yahr R (2011) DNA barcoding oflichenized fungi demonstrates high identification suc-cess in a floristic context New Phytol 191(1)288ndash300doi101111j1469-8137201103677x
Kiss L (2012) Limits of nuclear ribosomal DNA internaltranscribed spacer (ITS) sequences as species barcodesfor fungi Proc Natl Acad Sci 109(27)E1811ndashE1811doi101073pnas1207143109
Kluge AG (1989) A concern for evidence and aphylogenetic hypothesis for relationships among Epi-crates (Boidae Serpentes) Syst Zool 38(1)7ndash25doi101093sysbio3817
Knowles LL Carstens BC (2007) Delimiting specieswithout monophyletic gene trees Syst Biol 56(6)887ndash895 doi10108010635150701701091
Kroken S Taylor JW (2001) A gene genealogical approachto recognize phylogenetic species boundaries in thelichenized fungus Letharia Mycologia 93(1)38ndash53
Kubatko LS Degnan JH (2007) Inconsistency of phylo-genetic estimates from concatenated data under coa-lescence Syst Biol 56(1)17ndash24 doi10108010635150601146041
Latch EK Dharmarajan G Glaubitz JC Rhodes OE Jr(2006) Relative performance of Bayesian clusteringsoftware for inferring population substructure andindividual assignments at low levels of populationdifferentiation Conserv Genet 7(2)295ndash302 doi101007s10592-005-9098-1
Lawrey JD (1986) Biological role of lichen substancesThe Bryologist 89(2)111ndash122
Le Gac M Hood ME Fournier E Giraud T (2007)Phylogenetic evidence of host-specific cryptic speciesin the anther smut fungus Evolution 61(1)15ndash26doi101111j1558-5646200700002x
Leacheacute AD (2009) Species tree discordance traces tophylogeographic clade boundaries in North Americanfence lizards (Sceloporus) Syst Biol 58(6)547ndash559doi101093sysbiosyp057
Leacheacute AD Fujita MK (2010) Bayesian species delim-itation in West African forest geckos (Hemidactylusfasciatus) Proc R Soc B Biol Sci 2773071ndash3077doi101098rspb20100662
Leacheacute AD Koo M Spencer C Papenfuss T Fisher RMcGuire J (2009) Quantifying ecological morpho-logical and genetic variation to delimit species in thecoast horned lizard species complex (Phrynosoma)Proc Natl Acad Sci USA 10612418ndash12423 doi101073pnas0906380106
Leacheacute AD Fujita MK Minin VN Bouckaert RR (2014)Species delimitation using genome-wide SNP dataSystematic Biology doi101093sysbiosyu018
Leavitt SD Fankhauser JD Leavitt DH Porter LDJohnson LA St Clair LL (2011a) Complex patterns ofspeciation in cosmopolitan ldquorock posyrdquo lichensmdashdiscovering and delimiting cryptic fungal species in
the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
Leavitt SD Esslinger TL Lumbsch HT (2012c) Neogene-dominated diversification in neotropical montanelichens dating divergence events in the lichen-form-ing fungal genus Oropogon (Parmeliaceae) Am J Bot99(11)1764ndash1777 doi103732ajb1200146
Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Lumbsch HT St Clair LL(2013c) DNA barcode identification of lichen-formingfungal species in the Rhizoplaca melanophthalmaspecies-complex (Lecanorales Lecanoraceae) includ-ing five new species MycoKeys 71ndash22 doi103897mycokeys74508
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Vondraacutek J Thorsten Lum-bsch H Clair LLS (2013d) Local representation ofglobal diversity in a cosmopolitan lichen-formingfungal species complex (Rhizoplaca Ascomycota)J Biogeogr 40(9)1792ndash1806 doi101111jbi12118
Leavitt SD Lumbsch HT Stenroos S St Clair LL(2013e) Pleistocene speciation in North American
2 The Dynamic Discipline of Species Delimitation hellip 39
lichenized fungi and the impact of alternative speciescircumscriptions and rates of molecular evolution ondivergence estimates PLoS ONE 8(12)e85240doi101371journalpone0085240
Leavitt SD Esslinger TL Hansen ES Divakar PK CrespoA Loomis BF Lumbsch HT (2014) DNA barcoding ofbrown Parmeliae (Parmeliaceae) species a molecularapproach for accurate specimen identification empha-sizing species in Greenland Organ Divers Evol 14(1)11ndash20 doi101007s13127-013-0147-1
Leliaert F Verbruggen H Vanormelingen P Steen FLopez-Bautista JM Zuccarello GC De Clerck O(2014) DNA-based species delimitation in algae Eur JPhycol 49(2) doi101080096702622014904524
Leuckert C (1985) Probleme der Flechten-Chemotaxon-omiemdashStoffkombinationen und ihre taxonomischeWertung Ber Deut Bot Ges 98401ndash408
Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
Lohtander K Kaumlllersjouml M Roland M Tehler A (2000)The family physciaceae in fennoscandia phylogenyinferred from its sequences Mycologia 92(4)728ndash735
Longton RE (1997) The role of bryophytes and lichens inpolar ecosystems In Woodin SJ Marquiss M (eds)Ecology of Arctic Environments Blackwell ScienceOxford pp 69ndash96 (Special publication No 13)
Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
Lumbsch HT (1994) Die Lecanora subfusca-Gruppe inAustralasien J Hattori Bot Lab 771ndash175
Lumbsch HT (1998a) Taxonomic use of metabolic data inlichen-forming fungi In Frisvad JC Bridge PDArora DK (eds) Chemical Fungal Taxonomy MarcelDekker New York pp 345ndash387
Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
Mayden RL (1997) A hierarchy of species concepts thedenouement in the saga of the species problem InClaridge MF Dawah HA Wilson MR (eds) Speciesthe units of biodiversity Chapman amp Hall Londonpp 381ndash424
Mayr E (1963) Animal species and evolution HarvardUniversity Press Cambridge
Mayr E (1970) Populations species and evolutionBelknap Press of Harvard University Press Cambridge
McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
McDonald T Miadlikowska J Lutzoni F (2003) Thelichen genus Sticta in the Great Smoky Mountains a
40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
McKay BD Mays HL Wu Y Li H Yao C-T Nishiumi IZou F (2013) An empirical comparison of character-based and coalescent-based approaches to speciesdelimitation in a young avian complex Mol Ecol 22(19)4943ndash4957 doi101111mec12446
Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
Mishler BD Brandon RN (1987) Individuality pluralismand the phylogenetic species concept Biol Philos 2397
Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
Nash TH Zavada M (1977) Population studies amongSonoran Desert species of Parmelia subg Xanthop-armelia (Parmeliaceae) Am J Bot 64(6)664ndash669
Nylander W (1866a) Circa novum in studio Lichenumcriterium chemicum Flora 49198ndash201
Nylander W (1866b) Hypochlorite of lime and hydrate ofpotash Two new criteria for the study of lichens(Tanslated and communicated by the Rev W ALeighton) Bot J Linn Soc 9358ndash365
OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
OrsquoNeill EM Schwartz R Bullock CT Williams JSShaffer HB Aguilar-Miguel X Parra-Olea G Weis-rock DW (2013) Parallel tagged amplicon sequencingreveals major lineages and phylogenetic structure inthe North American tiger salamander (Ambystomatigrinum) species complex Mol Ecol 22(1)111ndash129doi101111mec12049
Orock EA Leavitt SD Fonge BA St Clair LL LumbschHT (2012) DNA-based identification of lichen-form-ing fungi Can publicly available sequence databasesaid in lichen diversity inventories of Mount Cameroon(West Africa) The Lichenologist 44(6)833ndash839doi101017S0024282912000424
Otaacutelora MAG Martiacutenez I Aragoacuten G Molina MC (2010)Phylogeography and divergence date estimates of alichen species complex with a disjunct distributionpattern Am J Bot 97(2)216ndash223 doi103732ajb0900064
Padial J Castroviejo-Fisher S Kohler J Vila C ChaparroJ De la Riva I (2009) Deciphering the products ofevolution at the species level the need for anintegrative taxonomy Zoolog Scr 38(4)431ndash447doi101111j1463-6409200800381x
Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
Papong K Luumlcking R Thammathaworn A BoonpragobK (2009) Four new taxa of Chroodiscus (thelotremoidGraphidaceae) from Southeast Asia The Bryologist112(1)152ndash163 doi1016390007-2745-1121152
Park S-Y Choi J Kim JA Jeong M-H Kim S Lee Y-HHur J-S (2013a) Draft genome sequence of Cladoniamacilenta KoLRI003786 a lichen-forming fungusproducing biruloquinone Genome Announcements 1(5)e00695-13 doi101128genomeA00695-13
Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
Peacuterez-Ortega S Fernaacutendez-Mendoza F Raggio J VivasM Ascaso C Sancho LG Printzen C de los Riacuteos A(2012) Extreme phenotypic variation in Cetrariaaculeata (lichenized Ascomycota) adaptation or inci-dental modification Ann Bot 109(6)1133ndash1148doi101093aobmcs042
Pino-Bodas R Burgaz A Martiacuten M Lumbsch HT (2011)Phenotypical plasticity and homoplasy complicatespecies delimitation in the Cladonia gracilis group(Cladoniaceae Ascomycota) Organ Divers Evol 11(5)343ndash355 doi101007s13127-011-0062-2
Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
Pino-Bodas R Martiacuten M Burgaz A (2012b) Cladoniasubturgida and C iberica (Cladoniaceae) form asingle morphologically and chemically polymorphicspecies Mycol Prog 11(1)269ndash278 doi101007s11557-011-0746-1
Pino-Bodas R Ahti T Stenroos S Martiacuten MP BurgazAR (2013a) Multilocus approach to species recogni-tion in the Cladonia humilis complex (CladoniaceaeAscomycota) Am J Bot 100(4)664ndash678 doi103732ajb1200162
Pino-Bodas R Martiacuten AP Burgaz AR Lumbsch HT(2013b) Species delimitation in Cladonia (Ascomy-cota) a challenge to the DNA barcoding philosophyMol Ecol Resour 13(6)1058ndash1068 doi1011111755-099812086
Pons J Barraclough TG Gomez-Zurita J Cardoso ADuran DP Hazell S Kamoun S Sumlin WD VoglerAP (2006) Sequence-based species delimitation for theDNA taxonomy of undescribed insects Syst Biol 55(4)595ndash609 doi10108010635150600852011
Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
Pringle A Baker DM Platt JL Wares JP Latgeacute JPTaylor JW (2005) Cryptic speciation in the cosmo-politan and clonal human pathogenic fungus Asper-gillus fumigatus Evolution 59(9)1886ndash1899 doi10155404-2411
Printzen C (2009) Lichen systematics the role ofmorphological and molecular data to reconstructphylogenetic relationships Progress in botany vol71 Springer Berlin pp 233ndash275
Pritchard JK Stephens M Donnelly P (2000) Inference ofpopulation structure using multilocus genotype dataGenetics 155(2)945ndash959
Puillandre N Lambert A Brouillet S Achaz G (2012)ABGD automatic barcode gap discovery for primaryspecies delimitation Mol Ecol 21(8)1864ndash1877doi101111j1365-294X201105239x
Rannala B Yang Z (2003) Bayes estimation of speciesdivergence times and ancestral population sizes usingDNA sequences from multiple loci Genetics1641645ndash1656
Raxworthy CJ Ingram CM Rabibisoa N Pearson RG(2007) Applications of ecological niche modelingfor species delimitation a review and empiricalevaluation using day geckos (Phelsuma) fromMadagascar Syst Biol 56(6)907ndash923 doi10108010635150701775111
Ray J (1686) Historia planarum vol 1 Clark LondonReese Naeligsborg R Ekman S Tibell L (2007) Molecular
phylogeny of the genus Lecania (Ramalinaceaelichenized Ascomycota) Mycol Res 111(5)581ndash591doi101016jmycres200703001
Reid N Carstens B (2012) Phylogenetic estimation errorcan decrease the accuracy of species delimitation aBayesian implementation of the general mixed Yule-coalescent model BMC Evol Biol 12(1)196 doi1011861471-2148-12-196
Rissler L Apodaca J (2007) Adding more ecology intospecies delimitation ecological niche models andphylogeography help define cryptic species in theBlack Salamander (Aneides flavipunctatus) Syst Biol56924ndash942 doi10108010635150701703063
Rivas Plata E Luumlcking R (2013) High diversity ofGraphidaceae (lichenized Ascomycota Ostropales) inAmazonian Peruacute Fungal Divers 58(1)13ndash32 doi101007s13225-012-0172-y
Rivas Plata E Lumbsch HT (2011) Parallel evolution andphenotypic divergence in lichenized fungi a casestudy in the lichen-forming fungal family Graphida-ceae (Ascomycota Lecanoromycetes Ostropales)Mol Phylogenet Evol 61(1)45ndash63 doi101016jympev201104025
Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
Ross KG Gotzek D Ascunce MS Shoemaker DD (2010)Species delimitation a case study in a problematic anttaxon Syst Biol 59(2)162ndash184 doi101093sysbiosyp089
Rubin BER Ree RH Moreau CS (2012) Inferringphylogenies from RAD sequence data PLoS ONE 7(4)e33394 doi101371journalpone0033394
Rubinoff D (2006) Utility of mitochondrial DNA barcodesin species conservation Conserv Biol 20(4)1026ndash1033 doi101111j1523-1739200600372x
Ruiz-Sanchez E Sosa V (2010) Delimiting speciesboundaries within the Neotropical bamboo Otatea(Poaceae Bambusoideae) using molecular morpho-logical and ecological data Mol Phylogenet Evol 54(2)344ndash356 doi101016jympev200910035
Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
morphological characters Antarctic Sci 22 (SpecialIssue 06)727ndash741 doi101017S0954102010000477
Salicini I Ibaacutentildeez C Juste J (2011) Multilocus phylogenyand species delimitation within the Nattererrsquos batspecies complex in the Western Palearctic MolPhylogenet Evol 61(3)888ndash898 doi101016jympev201108010
Satler JD Carstens BC Hedin M (2013) Multilocusspecies delimitation in a complex of morphologicallyconserved trapdoor spiders (Mygalomorphae Antro-diaetidae Aliatypus) Syst Biol 62(6)805ndash823 doi101093sysbiosyt041
Schlick-Steiner BC Steiner FM Seifert B Stauffer CChristian E Crozier RH (2010) Integrative taxonomya multisource approach to exploring biodiversityAnnu Rev Entomol 55(1)421ndash438 doi101146annurev-ento-112408-085432
Schoch CL Seifert KA Huhndorf S Robert V SpougeJL Levesque CA Chen W Fungal Barcoding Con-sortium (2012) Nuclear ribosomal internal transcribedspacer (ITS) region as a universal DNA barcodemarker for Fungi Proc Natl Acad Sci 109(16)6241ndash6246 doi101073pnas1117018109
Seacuterusiaux E Villarreal AJC Wheeler T Goffinet B(2011) Recent origin active speciation and dispersalfor the lichen genus Nephroma (Peltigerales) inMacaronesia J Biogeogr 38(6)1138ndash1151 doi101111j1365-2699201002469x
Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
Simpson GG (1951) The species concept Evolution5285ndash298
Sites JW Marshall JC (2003) Delimiting species a renais-sance issue in systematic biology Trends Ecol Evol18462ndash470 doi101016S0169-5347(03)00184-8
Sites JW Marshall JC (2004) Operational criteria fordelimiting species Annu Rev Ecol Evol Syst 35(1)199ndash227 doi101146annurevecolsys35112202130128
Šlapeta J Loacutepez-Garciacutea P Moreira D (2006) Globaldispersal and ancient cryptic species in the smallestmarine eukaryotes Mol Biol Evol 23(1)23ndash29doi101093molbevmsj001
Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
Talavera G Dincă V Vila R (2013) Factors affectingspecies delimitations with the GMYC model insightsfrom a butterfly survey Methods Ecol Evol 4(12)1101ndash1110 doi1011112041-210X12107
Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
Tewksbury J Anderson JGT Bakker JD Billo TJDunwiddie PW Groom MJ Hampton SE HermanSG Levey DJ Machnicki NJ del Rio CM PowerME Rowell K Salomon AK Stacey L TrombulakSC Wheeler TA (2014) Natural historyrsquos place inscience and society BioScience doi101093bioscibiu032
Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
Werth S Cornejo C Scheidegger C (2013) Characteriza-tion of microsatellite loci in the lichen fungus Lobariapulmonaria (Lobariaceae) Appl Plant Sci 1(2)1200290 doi103732apps1200290
Westberg M Arup U Kaumlrnefelt I (2007) Phylogeneticstudies in the Candelariaceae (lichenized Ascomy-cota) based on nuclear ITS DNA sequence dataMycol Res 111(11)1277ndash1284 doi101017S0953756204002102
Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
2 The Dynamic Discipline of Species Delimitation hellip 43
(Lepidoptera Lycaenidae) Front Zoo 4(1)8 doi1011861742-9994-4-8
Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
21 Introduction Whatrsquosin a Name The Importanceof Accurate SpeciesDelimitations
Although there are over 15 million species for-mally named by scientists current estimates ofthe number of species alive on the planet todayrange from approximately two million to overone hundred million (Caley et al 2014) Docu-menting describing and naming this diversity isparamount for conservation human health foodsecurity and recreation (Tewksbury et al 2014)In a broad sense species delimitation is theprocess of identifying how individuals andpopulations fit into natural species-level clustersand not simply constructs of classification(Carstens et al 2013) Empirical species delimi-tation is a dynamic discipline with ongoingmethodological and bioinformatical develop-ments due to a growing interest in empiricalspecies delimitations Novel analytical methodsincreasing availability of geneticgenomic dataincreasing computation power reassessments ofmorphological and chemical characters andimproved availability of distributional and eco-logical records offer exciting avenues for empir-ically exploring species delimitation andevolutionary relationships in species all over theworld In this chapter we aim to contribute acontemporary perspective on delimiting speciesand offer practical direction for empirical speciesdelimitation studies
To illustrate the importance and difficulties indocumenting and describing biodiversity we willuse the lichens as an example Lichens describe amutualism between a fungus (mycobiont) and aphotosynthetic partner (photobiont) which can beeither a green algae andor cyanobacteriumLichens are ubiquitous components of most ter-restrial ecosystems playing important ecologicalroles and contributing to global biogeochemicalcycles (Porada et al 2014 Bonan and Shugart1989 Longton 1997) Due to the fact that manylichens live and grow continuously for decades oreven hundreds of years showing cumulativeresponses to changes in atmospheric pollutionlevels landmanagement practices and fluctuation
climate these are commonly used as bioindicatorsto monitor the impacts of air pollution forest agesoil quality and climate change (McCune 2000)As iconic examples of symbiosis lichens alsoprovide crucial insight into general patterns andprocesses in symbiotic systems Central tounderstanding the dynamic roles of lichens is ourability to accurately delimit and recognize speciesboundaries Increased accuracy in recognizingspecies boundaries in lichenized fungi has majorimplications for enhancing our perspective onbiological diversity evolution ecology symbi-otic interactions biomonitoring research andconservation policy
211 Species Concepts and Criteria
Species serve as a central unit for categorizingbiological diversity Humans including bird-watchers doctors fisherman gardeners politi-cians scientists and others rely to varyingdegrees on recognizing species for distinguishingdifferent kinds of organisms and effective com-munication In the biological sciences speciesrepresent one of the most fundamental units andprovide a valuable context for organizing eval-uating and communicating important biologicalconcepts and principles (Coyne and Orr 2004Mayr 1963 Darwin 1859) Due to the fact thatbiological information is commonly providedwith reference to a species unit accurate speciescircumscriptions are integral to interpreting bio-logical patterns and processes across a widerange of subdisciplines in biology (eg anatomybehavior ecology evolution physiology etc)
In spite of the underlying importance of spe-cies in biology the conceptualization of the termldquospeciesrdquo remains somewhat contentious (deQueiroz 2007 Hausdorf 2011 Coyne and Orr2004 Mayden 1997 Simpson 1951 Mayr1963) Most biologists agree that biological nat-ure is aggregated into discrete evolutionarilyindependent entities ie ldquospeciesrdquo (Coyne andOrr 2004) although theorists and empiricistsalike continue to debate over an all-encompass-ing species concept and appropriate operationalcriteria for delimiting species (Hausdorf 2011
12 SD Leavitt et al
de Queiroz 2007 Hey 2006 Donoghue andGauthier 2004 Cracraft 1983 Mishler andBrandon 1987 Mayr 1970) Over two-dozendifferent and at least partially incompatible spe-cies concepts have been proposed each based ondistinct biological properties eg differences ingenetic or morphological features adaptivezones or ecological niches mate recognitionsystems reproductive compatibility monophylyetc (de Queiroz 2007 Mayden 1997) Hausdorf(2011) argues that most species concepts areuseable but acceptance of a specific conceptrequires an appropriate adaptation of the termldquospeciesrdquo and of species delimitation In contrastde Queiroz (1998) and Mayden (1997) argue thatdistinct species concepts emphasize differentproperties of species rather than fundamentalconceptual differences and all modern speciesconcepts share an important commonalitymdashequating species with segments of metapopula-tion lineages This ldquogeneral lineage conceptrdquo(GLC de Queiroz 1999) highlights that no singleproperty should be regarded as defining for therecognition of species apart from forming lin-eages (Simpson 1951) and segments of meta-population lineages (ie ldquospeciesrdquo) may existregardless of our ability to empirically delimitthem (Camargo and Sites 2013)
We concur that the GLC provides a practicalsolution to the species concept impasse and ourdiscussion of species delimitation is based on theGLC Arguably the major implication of the GLCis that most of the earlier species concepts shouldbe regarded as secondary species ldquocriteriardquo ratherthan ldquoconceptsrdquo that can provide evidence oflineages separation (Sites and Marshall 2003Camargo and Sites 2013 Mayden 1997 de Que-iroz 2007) This pivotal distinction disentanglesthe conceptual issues of defining ldquospeciesrdquo frommethodological issues of delimiting speciesboundaries (Camargo and Sites 2013) The GLCallows researchers to delimit species using differ-ent empirical properties and facilitates the devel-opment of new methods to test hypotheses oflineage separation (de Queiroz 2007) Althoughdifferent datasets and operational criteria may giveconflicting or ambiguous results due to multipleevolutionary processes occurring within and
between populations (eg Miralles and Vences2013 Satler et al 2013) the use of several inde-pendent suites of characters such as genetic datamorphology geographic range host preferencechemistry and cross-validation using inferencesfrom multiple empirical operational criteria canprovide robust hypotheses of species boundaries(Carstens et al 2013 Fujita et al 2012)
212 Species in Lichenized FungiCases of Cryptic DiversityPolymorphic Lineagesand Striking BiogeographicPatterns
Similar to most major biological groups includ-ing birds (McKay et al 2013) fish (Wagner et al2013) plants (Griffin and Hoffmann 2014)arthropods (Schlick-Steiner et al 2010 Moreau2009) and many others finding and applying theappropriate character sets and analytical tools isone of the greatest challenges with empiricalspecies delimitation in lichen-forming fungi(Lumbsch and Leavitt 2011) Understanding thedifferences between morphological variationwithin a species and among closely related groupsis central to identifying diagnostic charactersrequired for establishing accurate phenotype-based taxonomic boundaries However in prac-tice a clear demarcation between intraspecificand interspecific variation is commonly subject toobservational bias and individual interpretation
Traditionally differences in morphologicalchemical and ecological features have been thepredominant source of diagnostic taxonomiccharacters for circumscribing lichen-formingfungal species (Printzen 2009) However lichensgenerally display few taxonomically useful char-acters relative to other groups (eg vascularplants vertebrates and arthropods) (Printzen2009) and varying levels of intraspecific variationamong different species groups may confoundaccurate taxonomic circumscriptions While somespecies may have little variation high levels ofintraspecific phenotypic variation are well docu-mented in some lichen-forming fungi (eg Xant-hoparmelia Hale 1990) Therefore molecular
2 The Dynamic Discipline of Species Delimitation hellip 13
genetic data now play a prominent role in delim-iting fungal species and understanding evolu-tionary relationships in lichens (Lumbsch andLeavitt 2011 Printzen 2009)
Arguably the use of molecular data has led toan improved perspective on the taxonomic valueof many phenotypic characters in lichenized fungiand species delimitation in general Cryptic spe-cies-level lineages are commonly identified usingmolecular data and in some cases these previ-ously unrecognized lineages are corroborated byformerly overlooked phenotypic characters (Pino-Bodas et al 2012a Spribille et al 2011 Divakaret al 2010 Arguumlello et al 2007) Other studieshave revealed the fact that some species-levellineages are likely comprised of chemically andmorphologically polymorphic individuals whichare conventionally considered as separate species(eg Leavitt et al 2011a Pino-Bodas et al 2011Velmala et al 2009) While these studies high-light the limitations of using traditional taxo-nomic characters for distinguishing naturalgroups in lichen-forming fungi they also providea valuable perspective on the importance ofongoing research in even the best-studied lichengroups Furthermore an improved perspective ofspecies boundaries has led to a strikingimprovement in understanding diversification anddistribution in many groups of lichenized fungi
Traditional phenotype-based approaches tospecies recognition appear to vastly underesti-mate diversity in lichen-forming fungi Whilemolecular research has corroborated traditionalphenotype-based hypotheses of species bound-aries in a number of cases studies repeatedlydemonstrate that our current interpretation ofmorphological and chemical characters oftenfails to accurately characterize species-leveldiversity (reviewed in Lumbsch and Leavitt2011) A growing body of evidence reveals that asignificant proportion of unknown diversityestimated for fungi including lichen-formingfungi is hidden under names of supposedlycommon and widespread species For exampleapproximately 80 unrecognized species-levellineages are estimated to occur in Parmeliaceae(Crespo and Lumbsch 2010) Even higher levels
of unrecognized species diversity are estimatedto occur in other families such as Graphidaceae(Rivas Plata and Luumlcking 2013 Rivas Plata andLumbsch 2011 Luumlcking 2012)
The topic of cryptic species cases wheretwo or more distinct species-level lineages areerroneously classified (and hidden) under onenominal taxon (Bickford et al 2007) has beenfrequently reviewed for lichen-forming fungi(Lumbsch and Leavitt 2011 Crespo and Lum-bsch 2010 Crespo and Peacuterez-Ortega 2009Printzen 2009) Although novel diagnostic phe-notypic characters may potentially be identifiedcorroborating ldquocrypticrdquo species these previouslyunrecognized lineages generally remain difficultto classify within a traditional phenotype-basedtaxonomy (Leavitt et al 2013c d) In somecases it appears that similar phenotypic charac-ters may arise in parallel at local or regionalscales but may not be correlated with naturalgroups or genetic isolation (Muggia et al 2014Rivas Plata and Lumbsch 2011 Grube andHawksworth 2007) For example in the cosmo-politan species Tephromela atra (Fig 21) up to15 independent lineages were identified usingphylogenetic analyses of molecular sequencedata However the continuum of morphologicaland chemical variability in the T atra complexcurrently prevents the description of new speciesusing traditional phenotype-based characters(Muggia et al 2014)
Recent research on the genus Cladonia(Cladoniaceae) highlights a fitting example of thecomplexities associated within using phenotypiccharacters for delimiting species in lichen-forming fungi (Fig 21 Pino-Bodas et al 20112012a b 2013a b) In the Cladonia gracilisgroup most currently accepted species were notrecovered as monophyletic clades and traditionaldiagnostic morphological characters were shownto be highly homoplasious (Pino-Bodas et al2011) Similarly C iberica and C subturgidahave been shown to constitute a single morpho-logically and chemically polymorphic species(Pino-Bodas et al 2012b) Similar patterns ofhigh degrees of morphological and chemicalpolymorphisms have also been observed in the
14 SD Leavitt et al
C cariosa group (Pino-Bodas et al 2012a) andC humilis species complex (Pino-Bodas et al2013a) High levels of intraspecific morphologi-cal and chemical polymorphisms are not restric-ted to Cladonia A clear demarcation betweenintraspecific and interspecific morphological andor chemical variation does not exist for a largeproportion of Xanthoparmelia species in westernNorth America and high intraspecific morpho-logical and chemical variation are common for anumber of species-level genetic groups (Fig 21
Leavitt et al 2011b c 2013e) In some cases asmany as eight traditionally circumscribed Xant-hoparmelia species were recovered in a singlespecies-level genetic group (Leavitt et al 2011c)High levels of intraspecific phenotypic variationhave been observed in a number of other generain Parmeliaceae including Bryoria (Velmalaet al 2009) Cetraria (Peacuterez-Ortega et al 2012)Vulpicida (Mark et al 2012) and others
The importance of biogeography in lichen-forming fungal evolution has remained somewhat
Fig 21 Examples of common lichens in which tradi-tional morphology-based species circumscriptions fail toreflect natural species-level fungal lineages a Cladoniagracilis photographed in the Clearwater Valley BritishColumbia Canada (see Pino-Bodas et al 2011) (photo-graph creditCurtis Bjoumlrk) bR shushanii a member of theRhizoplaca melanophthalma species group from the
Aquarius Plateau Utah USA (see Leavitt et al 2011a)(photograph credit S Leavitt) c Xanthoparmelia affwyomingica occurring in Coloradorsquos Front Range USA(see Leavitt et al 2011b c Leavitt et al 2013e) (photo-graph credit S Leavitt) d Tephromela atra sensu latofound in the Santa Monica Mountains California USA(see Muggia et al 2014) (photograph credit J Hollinger)
2 The Dynamic Discipline of Species Delimitation hellip 15
ambiguous due to the occurrence of phenotypi-cally similar lichens occurring across broadintercontinental distributions and uncertainty ofappropriate species circumscriptions While anumber of lichen-forming fungal species havebeen found to be truly widespread (eg Lindblomand Soslashchting 2008 Fernaacutendez-Mendoza et al2011 Ahti and Hawksworth 2005 Del-Pradoet al 2013) improved recognition of speciesboundaries has provided insight into importantbiogeographical patterns in lichens previouslyassumed to have cosmopolitan distribution pat-terns (Leavitt et al 2013d Del-Prado et al 2013Amo de Paz et al 2012 Seacuterusiaux et al 2011Otaacutelora et al 2010 Elix et al 2009) Crypticdiversity and complex biogeographic patterns arehighlighted in the Rhizoplaca melanophthalmaspecies group (Fig 21 Leavitt et al 2013d)Analyses of R melanophthalma sensu lato col-lected from five continents supported the presenceof cryptic species within this complex Two ofthese lineages were found to have broad inter-continental distributions while the other four werelimited to western North America (Leavitt et al2013d) Most strikingly of the six lineages fivewere found on a single mountain in the westernUSA and the sixth occurred no more than 200 kmaway from this mountain A recent study ofPseudocyphellaria (Lobariaceae) sensu lato inHawaii revealed a surprising number of previouslyunrecognized species hidden within nominal taxawith putative broad geographic distributionssuggesting a high degree of endemism in Hawaii(Moncada et al 2014) These studies providecrucial impetus to reevaluate species boundaries inorder to improve our understanding of diversitydistributions and evolution in lichenized fungi
22 A Practical Guideto Contemporary SpeciesDelimitation
As it has become clear that conventional phe-notype-based criteria for species circumscriptionsare often problematic (Bickford et al 2007)molecular data are thereby particularly valuablefor assessing traditional species boundaries and
for species delimitation in general (Lumbsch andLeavitt 2011 Fujita et al 2012) Below weprovide an overview of relevant operationalspecies delimitation methods the majority ofwhich are reliant on molecular data coupled withmolecular phylogenetic and population geneticanalyses Assuming that species do in fact rep-resent ldquosegments of metapopulation lineagesrdquo (deQueiroz 1998) direct genetic evidence of lineagestatus is particularly relevant to species delimi-tation studies when analyzed within a rigorousstatistical framework regardless of whether lin-eages differ in phenotypic characters that areapparent to human observers (Fujita et al 2012)This perspective should not be taken as supportfor disregarding phenotypic characters in speciesdelimitation studies (Edwards and Knowles2014 Fujita et al 2012 Yeates et al 2011)Rather hypotheses of species boundaries shouldbe considered more robust with increasing cor-roboration from independent data sources (iemolecular chemistry morphology ecologyetc) and the integration of independent data forempirical species delimitation studies should be amajor focus of species delimitation research(Fujita et al 2012)
Themajority of molecular phylogenetic studiesof lichenized fungi focus on generating sequencedata from a number of specimens representing thefocal group inferring a gene tree from the geneticdata matrix and assessing the monophyly of thesampled taxa This approach has provided valu-able acumen into evolutionary relationships thevalue of morphological characters for taxonomyand insight into diversity of lichenized fungi(Thell et al 2009 Reese Naeligsborg et al 2007Westberg et al 2007 Martiacuten et al 2003 Arup andGrube 2000 Lohtander et al 2000 Stenroos andDePriest 1998) However simply assessing themonophyly of traditional phenotype-based spe-cies often offers an incomplete perspective onspecies boundaries Studies explicitly designed forempirically delimiting species are pivotal toadvancing our understanding of speciation andspecies diversity in lichens For example althougha nominal species may be recovered as mono-phyletic in a gene tree intraspecific phylogeneticsubstructure may correspond to evolutionarily
16 SD Leavitt et al
independent lineages (eg Leavitt et al 2011a)As a construct of taxonomy recognizing amonophyletic clade comprised of multiple mor-phological indistinguishable species-level lin-eages as a single species may hold some appealhowever failing to formally recognize this diver-sity can have far-reaching implications in ourbiological (eg ecology evolution reproduction)interpretation of the group Alternatively well-supported intraspecific phylogenetic substructuremay be the result of stochastic evolutionary pro-cesses uniparental inheritance etc rather thanevolutionary independence Interpreting this typeof phylogenetic substructure as species-leveldiversity can introduce detrimental bias
Empirical species delimitation methods havebroadly focused on four main areas detectingputative species individual specimen assignmentto a species group or operational taxonomic unit(OTU) validation of candidate species or OTUsas evolutionarily distinct lineages and inferringspecies relationships (ie species tree inference)Ideally operational species delimitation criteriashould be based on explicit statistical protocolsin order to objectively assess species boundariesand minimize the need of subjective interpreta-tions or taxonomic expertise In this section weprovide a brief overview on a number of empir-ical species delimitation methods that to varyingdegrees fit these criteria For convenience wedivide these methods into three general catego-ries (i) species discovery methods for assigningsamples to populations without a priori infor-mation (ii) species validation approaches andcoestimating individual assignments and speciestrees and (iii) species delimitation using geno-mic data (Table 21)
Empirical species delimitation has receivedincreasing attention including ongoing develop-ment of bioinformatical approaches and themethods and programs provided in this chapterare by no means intended to be a comprehensivelist of all available analytical approaches Ratherthe methods provided here have been shown tobe useful in a number of previous studies orshow particular promise for future speciesdelimitation studies Our aim is that these can
serve as a starting point when designing studiesto assess species limits Not surprisingly variousapproaches to species delimitation may yielddifferent estimates of species boundaries and theresearcher may be required to make some degreeof subjective interpretation of the most biologi-cally appropriate species boundaries Comple-mentarily recently developed metrics forquantifying the congruence between two taxo-nomies (Ctax) and the potential for an approach tocapture a high number of species boundaries(Rtax) provide a means to objectively assess dis-crepancies among species delimitation methods(Miralles and Vences 2013) More sophisticatedapproaches including selection of speciesdelimitation models using approximate Bayesiancomputing (Camargo et al 2012 Fan andKubatko 2011 Beaumont et al 2010) anddesigning and conducting a simulation study thatmatches the characteristics of the empirical study(Carstens et al 2013 Camargo et al 2012) canbe used to more objectively evaluate competinghypotheses of species boundaries In most con-texts it is likely better to fail to delimit speciesthan it is to falsely circumscribe entities that donot represent actual species and therefore theinferences drawn from species delimitation stud-ies should generally be conservative (Carstenset al 2013 Miralles and Vences 2013)
221 Corroborating TraditionalTaxonomy and DiscoveringCryptic Species Using Single-Locus Data
Objectively defining a threshold separatingintraspecific population substructure from inter-specific divergence is the general aim of speciesdelimitation studies using single-marker datasetsMost species delimitation methods based onsingle-locus sequence data fall under two generalcategories either genetic distance or tree-basedapproaches (Sites and Marshall 2004) Distance-based approaches attempt to detect a differencebetween intra- and interspecific distances (ieldquobarcode gaprdquo) where the pairwise genetic
2 The Dynamic Discipline of Species Delimitation hellip 17
Table
21
Somemetho
dsused
forspeciesdelim
itatio
ninclud
ingspeciesdiscov
erymetho
dsforassign
ingsamples
topo
pulatio
nswith
outapriori
inform
ation(BAPS
Gaussianclustering
Guillo
trsquosUnified
Mod
elS
TRUCTURES
TRUCTURAMA)genetic
distance-based
metho
dforsortingsequ
encesinto
hypo
theticalspecies(A
BGD)tree-
basedspeciesdiscov
erymetho
ds(bGMYCG
MYCb
PTP
PTP
ldquoSpecies
Delim
itatio
nrdquoplug
-inforGeneiou
s)and
jointdiscov
eryandvalid
ationmetho
ds(BPamp
PBrownie
DISSE
CTSp
eDeStem)
Metho
dDescriptio
nInpu
tdata
BAPS
mdashpo
pulatio
nassignmentusing
Bayesianclustering
Aprogram
forBayesianinferenceof
thegenetic
structurein
apopulatio
nclustering
molecular
data
andperformingadmixture
analysesGenetic
mixture
analyses
canbe
performed
atbo
thgroupandindividu
allevelsusingeither
ano
n-spatialo
rspatialm
odel
BAPS
treatsboth
theallele
frequenciesof
themolecular
markers
(ornucleotid
efrequenciesforDNA
sequ
ence
data)andthenu
mberof
genetically
diverged
groups
inpopulatio
nas
random
variablesIn
theldquoclusteringwith
linkedlocirdquomod
elagenetic
mixture
analysiscanbe
done
usinghaploidsequ
ence
data
orotherlin
kedgenetic
markersA
nalysesandmodel
comparisons
canalso
beperformed
usingafixednu
mber
ofgenetically
diverged
grou
psor
prespecified
populatio
nstructures
Limita
tions
Tem
poraldivergence
andrelatio
nships
amongputativ
egroups
arenot
explicitlyestim
atedE
quivalence
togenetic
clusters
tospecies-levelg
roupsisuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
Availablefrom
httpwwwhelsinkifi
bsgsoftwareBAPS
describedin
Coran-
deret
al(200
420
0620
08)CoranderandMarttinen20
06)
Genotyp
icdatahaploidsequence
dataor
linkedmarkers
(AFL
Por
SNPs)
Gaussianclusteringmdashpo
pulatio
nassignment
usingGaussianclustering
Aprogram
forBayesianinferenceof
thegenetic
structurein
apopulatio
nModelgroups
sampleinto
popu
latio
nsusinggeno
typicdata
bysearchingforclusters
that
canbe
attributed
tobeingmixturesof
norm
alallelefrequencydistributio
nsG
aussianclustering
requires
adatasetwhere
thecasesaredefinedby
variable
ofmetricscaleandhasbeen
used
with
geneticenvironmentalandmorphologicaldatasetsindividuallyinadditio
nto
integrated
datasets
Limita
tions
Tem
poraldivergence
andrelatio
nships
amongputativ
egroups
arenot
explicitlyestim
atedE
quivalence
togenetic
clusters
tospecies-levelg
roupsisuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
Implem
entedin
Rusingtheprabclus
(HausdorfandHennig20
10)andmclust
packages
(FraleyandRaftery
2007)
Genotyp
icdata
(flexible)
(con
tinued)
18 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
Guillo
trsquosUnified
Mod
elmdashpo
pulatio
nassignmentusingBayesianclustering
Thisapproach
provides
astatistical
model
that
allowsoneto
analyzegenetic
and
phenotyp
ticdata
with
inaun
ified
mod
elandinferencefram
eworkandop
tionally
incorporateinform
ationaboutthespatialdistributio
nof
samplesA
Bayesianclustering
algorithm
assumes
that
each
clusterin
ageographical
domaincanbe
approxim
ated
bypo
lygons
that
arecentered
around
pointsgeneratedby
aPo
issonprocessGuillo
trsquos
Unified
Mod
elisflexiblein
term
sof
thegenetic
data
that
itcanutilize
andcapableof
accurately
delim
iting
species
Limita
tions
Genetic
andphenotypic
data
cantracedifferentevolutionary
historiesfor
instancephylogenetic
divergence
forneutralgenetic
markers
andadaptatio
nfora
morphological
structure
Source
Availableas
anextensionof
theRGENELAND
package(G
uillo
tet
al20
05
2012)(http
www2im
mdtudk
gigu
Geneland)
Genotyp
icandno
n-genetic
(eg
phenotypicalecolog
icalgeograph
ical
behavioral)data
STRUCTUREmdash
populatio
nassignment
usingBayesianclustering
Amodel-based
clustering
methodusingmultilocus
genotype
data
toinferpopulatio
nstructureandassign
individualsto
populatio
nsIndividualsin
thesampleareassigned
probabilistically
topopulatio
nso
rjointly
totwoor
morepopulatio
nsiftheirgenotypes
indicatethatthey
areadmixedT
hemodeldoes
notassum
eaparticular
mutationprocess
anditcanbe
appliedto
mosto
fthecommonly
used
genetic
markersp
rovidedthat
they
areno
tcloselylin
ked
The
methodcanproducehigh
lyaccurate
assignmentsusing
modestn
umbersof
loci(Pritchard
etal2
000)T
hemostappropriatelevelo
fpopulatio
nstructurecanbe
inferred
byassessinglik
elihoodscores
orthead
hocΔKstatistic
(Evann
oet
al20
05)
Limita
tions
Identifying
themostappropriatenumberof
genetic
clusters
ischallenging
clusters
produced
byST
RUCTUREcanbe
strongly
influenced
byvariationin
sample
sizeclusters
createdby
STRUCTUREmay
notbe
consistent
with
theevolutionary
historyof
thepopulatio
nswhentherearerelativ
elylong
divergence
times
with
inevolutionary
lineagesTem
poraldivergence
andrelatio
nships
amongputativ
egroups
isnotexplicitlyestim
atedEquivalence
togenetic
clusters
tospecies-levelgroups
isuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
httppritchardlabstanfordedustructurehtm
ldescribedin
Falush
etal(200
3)andPritchard
etal(200
0)
Genotyp
icdata
(con
tinued)
2 The Dynamic Discipline of Species Delimitation hellip 19
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
Structuram
amdashpo
pulatio
nassignmentusing
Bayesianclustering
Implem
entstheclustering
algorithm
used
inST
RUCTURE(see
above)
that
clusters
samples
into
populatio
nsby
minim
izingHardyndashWeinb
ergdisequ
ilibrium
foragiven
partitioninglevelHow
everStructuram
aalso
includes
theadditio
nof
reversible-jum
pMCMCto
identifytheop
timalpartition
inglevelNearlyanytype
ofgenetic
datacanbe
inpu
tinto
Structuram
aandtheprog
ram
canassign
individu
alsto
populatio
nwith
orwith
outtheadmixture
Limita
tions
Tem
poraldivergence
andrelatio
nships
amongputativ
egroups
isnot
explicitlyestim
atedE
quivalence
togenetic
clusters
tospecies-levelg
roupsisuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
httpctegberkeleyedu
structuram
aindexhtmldescribedin
Huelsenbeck
etal(201
1)
Genotyp
icdata
ABGDmdashbarcodegapusinggenetic
distances
ldquoAutom
aticBarcode
Gap
Discoveryrdquosortssequencesinto
hypotheticalspeciesbasedon
thebarcodegap
The
methoduses
arecursiveapproach
topartition
thedata
andtestfor
sign
ificant
gapsA
BGDisfastsim
plemethodto
split
asequence
alignm
entd
atasetinto
candidatespeciesthat
should
becomplem
entedwith
otherevidence
inan
integrative
taxono
mic
approach
Limita
tions
Single-locus
data
aloneshould
only
beused
toprovideaprelim
inary
perspectiveof
speciesboundaries
andnotas
thesole
evidence
inspecies
circum
scriptions
Source
httpwwwabisnv
jussieufrpublicabg
ddescribedin
Puillandreet
al(201
2)
Single-locus
sequence
alignm
ent
GMYCampbG
MYCmdashgene
tree
The
GMYC
approach
combinesacoalescent
model
ofintraspecificbranchingwith
aYulemod
elforinterspecificbranching
which
isthen
fitto
aninferred
sing
le-gene
topology
toestim
atespeciesboundaries
andastatistical
measure
ofconfi
denceforthe
inferred
boundariesA
san
inputtheGMYCapproach
requires
anultram
etricgene
tree
andrecent
refinementscanaccountforuncertaintyin
phylogenetic
relatio
nships
and
parametersof
theGMYCmod
elT
heGMYCisgenerally
stable
across
awiderangeof
circum
stancesincludingvariousmethods
ofphylogeneticreconstructio
nthepresence
ofahigh
numbersingletonshigh
numbers
ofsampled
speciesandgaps
inintraspecific
sampling
theaccuracy
oftheGMYCismostsign
ificantly
affected
bythemean
populatio
nsize
relativ
eto
divergence
times
betweenthem
Limita
tions
The
GMYCmay
delim
itwell-supportedclades
ofhaplotypes
asindependent
lineagesandas
such
may
beproneto
over-delim
itatio
nSingle-locus
data
aloneshould
only
beused
toprov
ideaprelim
inaryperspectiveof
speciesbo
undaries
andno
tas
the
sole
evidence
inspeciescircum
scriptions
Source
(http
r-forger-projecto
rgprojectssplitshttpssitesgooglecom
site
noahmreidhom
esoftware)describedin
Monaghanet
al(200
9)Fu
jisaw
aandBarrac-
loug
h(201
3)andPo
nset
al(200
6)
Sing
le-locus
ultram
etricgene
tree (con
tinued)
20 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
PTPamp
bPTPmdash
gene
tree
The
Poissontree
processes(PTP)
modelcanbe
used
toinferputativ
especiesboundaries
onagivenphylogenetic
inputtreePT
Pcaninferputativ
especiesboundaries
that
are
consistent
with
thePS
CAnim
portantadvantageof
thismethodisthat
itmod
els
speciatio
nin
term
sof
thenumberof
substitutionsthereby
circum
ventingthepotentially
error-proneandcompute-intensive
processof
generatin
gultram
etrictreeswhich
are
required
asan
inpu
tfor
GMYCmod
el(see
abov
e)F
urthermoreitappearsthatthePT
Pmodel
may
outperform
theGMYCandotherOTU-picking
methods
whenevolutionary
distancesaresm
all
Limita
tions
Single-locus
data
aloneshould
only
beused
toprovideaprelim
inary
perspectiveof
speciesboundaries
andnotas
thesole
evidence
inspecies
circum
scriptions
Source
httpscoh-itsorgexelix
iswebsoftwarePT
Pindexhtmldescribedin
Zhang
etal(201
3)
Sing
le-locus
gene
tree
GeneiousSp
eciesDelim
itatio
nplug
-inmdash
gene
tree
Aplug-into
theGeneioussoftwareprovides
anexploratorytool
allowingtheuser
toassess
phylog
enetic
supportanddiagno
sabilityof
speciesdefinedas
user-selected
collections
oftaxa
onuser-sup
pliedtreesThe
plug
-incompu
tesstatisticsrelatin
gto
the
probability
oftheob
served
mon
ophyly
orexclusivity
having
occurred
bychance
ina
coalescent
processandassesses
thewith
in-andbetween-speciesgenetic
distancesto
infertheprobability
with
which
mem
bers
ofaputativ
especiesmight
beidentifi
edsuccessfully
with
tree-based
methods
Limita
tions
The
plug-insummarizes
measuresof
phylogenetic
supportand
diagnosabilityof
speciesdefinedas
user-selectedcollections
oftaxabutitdoes
not
providedefinitiv
esupportforspeciesgroupsItassumes
speciesaremonophyletic
Source
Implem
entedas
aplug-into
Geneious(geneiouscom)describedin
Mastersetal
(201
1)
Sing
le-locus
gene
tree
BPamp
Pmdashmultispecies
coalescent
model
for
speciesvalid
ation
Thisapproach
tospeciesdelim
itatio
nuses
aBayesianmodelingapproach
togeneratethe
posteriorprobabilitiesof
speciesassignmentstaking
accountof
uncertaintiesdueto
unknow
ngene
treesandtheancestralcoalescent
processThe
methodrelieson
auser-
specified
guidetreeimplem
entin
gareversible-jum
pMarkovchainMonte
Carlo
search
ofparameter
spacethatincludes
θpopulatio
ndivergenceand
estim
ated
distributio
nsof
gene
treesfrom
multip
leloci
Limita
tions
Misspecificatio
nsof
priorsandthegu
idetree
canresultin
inflatedspeciatio
nprobabilitiesitassumes
norecombinatio
nandcomputatio
nallim
itatio
nsrestrict
itsutility
with
next-generationdatasetswith
100s
ofloci
Source
httpabacusgeneuclacuksoftwarehtmldescribedin
YangandRannala
(201
0)andRannala
andYang(200
3)
Multilocus
sequence
alignm
entsandgroup
mem
bership
(con
tinued)
2 The Dynamic Discipline of Species Delimitation hellip 21
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
DISSE
CTmdashmultispecies
coalescent
model
forspeciesdelim
itatio
nDISSE
CTexplores
thefullspaceof
possible
clusteringsof
individualsandspeciestree
topologies
inaBayesianfram
ework
Toavoidtheneed
forreversible-jum
pMCMCit
uses
anapproxim
ationin
theform
ofapriorthatisamodificatio
nof
thebirthndashdeathprior
forthespeciestreeItisim
plem
entedas
partof
BEAST
andrequires
only
afewchanges
from
astandard
BEAST
analysisA
nalysesof
simulated
andem
piricald
atasuggestthat
themethodisshow
nto
beinsensitive
tothedegree
ofapproxim
ation
butq
uitesensitive
tootherparameters
Limita
tions
Recently
describedmethodlackingathorough
theoretical
andem
pirical
evaluatio
nItappearsthat
alargenu
mberof
sequencesarerequ
ired
todraw
firm
conclusion
sSo
urce
httpcodegooglecompbeast-m
cmcamphttpwwwin
driid
com
dissectinbeast
htmldescribedin
JonesandOxelm
an(201
4)
Multilocus
sequence
data
SpeD
eSTEMmdashmultispecies
coalescent
mod
elfordiscov
ery
valid
ation
andjoint
estim
ation
Thismaxim
umlik
elihoo
dandorinform
ationtheory-based
methodwas
developedto
test
speciesboundaries
inasystem
with
existin
gsubspecies
taxonomy(CarstensandDew
ey20
10)andcomputestheprobability
ofthegene
treesgiventhespeciestree
forall
hierarchical
perm
utations
oflin
eage
grou
pingSp
eciesbo
undaries
arecomparedusing
Akaikeinform
ationcriteria
andphylogenetic
uncertaintyin
thespeciestree
topologies
does
notaffect
speciesdelim
itatio
nsLimita
tions
Accuracyisdependenton
quality
ofthegene
tree
estim
ates
Source
(http
carstenslaborgohio-stateedusoftwarehtml)criteria
describedin
Ence
andCarstens(201
1)
Multilocus
sequence
alignm
entsandgroup
mem
bership
Browniemdash
multispecies
coalescent
model
forspeciesdelim
itatio
nThe
nonp
aram
etricheuristic
speciesdelim
itatio
napproach
implem
entedin
theprog
ram
Brownie(O
rsquoMeara
2010)jointly
sortsanonym
oussamples
into
speciesandinfers
aspeciestree
from
inputg
enetreesfrom
differentlociassumingthatforaspeciatio
neven
thecorrespondingnodeson
gene
treeswill
bemoreconsistent
with
each
than
the
divergenceswith
inspecies
Limita
tions
Findingboth
theoptim
umspeciestree
andspeciesboundaries
remains
compu
tatio
nally
challenging
andBrowniehasbeen
show
nto
frequently
yieldincorrect
resultsT
heaccuracy
ofthemethodislik
elycorrelated
with
nodalsupportvalues
inthe
individu
algene
topo
logies
Source
httpwwwbrianom
earain
fobrownie
describedin
OrsquoM
eara
(201
0)
Individual
gene
trees
(con
tinued)
22 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
BFD
mdashmultispecies
coalescent
model
for
speciesdelim
itatio
nThe
recently
developedmethod
Bayes
factor
delim
itatio
n(with
genomicdataB
FD)
combinesady
namic
programmingalgorithm
forestim
atingspeciestreesthat
bypasses
thecompu
tatio
nally
intensiveMCMCintegrationov
ergene
treesto
prov
idearigorous
techniqueforspeciesdelim
itatio
nstudiesusinggenome-wideSN
PdataCom
petin
gspeciesdelim
itatio
nmodelsarecomparedusingBayes
factorsanditappearsthat
this
approach
isrobustto
samplesizes(iefew
loci
andlim
itedsamples
perspecies)
and
misspecificatio
nof
thepriorforpo
pulatio
nsize
(θ)
Limita
tions
Recently
describedmethodlackingathorough
theoretical
andem
pirical
evaluatio
nSo
urce
httpwwwbeast2orgwikiindexphpBFD
describedin
Leacheacuteet
al(201
4)
Genom
e-wideSN
Pdata
2 The Dynamic Discipline of Species Delimitation hellip 23
distances among organisms belonging to thesame species are smaller than distances amongorganisms from different species (Puillandreet al 2012 Hebert et al 2003) Genetic distanceapproaches hold particular promise as an identi-fication tool shortcutting the difficulties of mor-phology-based identification (Hebert et al 2004)although in practice a barcode gap may not existfor many groups (Wiemers and Fiedler 2007)Furthermore the role of distance-based approa-ches using a single genetic marker for speciesdiscovery remains more controversial (Rubinoff2006) and without other corroborating evidenceOTUs inferred from single-locus dataset shouldonly be considered ldquocandidaterdquo species Tree-based methods aim to detect monophyletic cladescorresponding to species-level diversity bydetecting discontinuities associated within inter-and intraspecific branching patterns (Fujisawaand Barraclough 2013 Zhang et al 2013Monaghan et al 2009 Pons et al 2006) Tree-based species delimitation methods can also beused on the basis of other properties related tophylogenetic tree topologies (monophyly con-cordance with geography etc (Sites and Marshall2003 2004) Both distance- and tree-basedmethods have been applied for assessing speciesboundaries in lichen-forming fungi (Leavitt et al2012c 2014 Parnmen et al 2012 Del-Pradoet al 2010 2011 Molina et al 2011)
A number of tree-based methods partiallyautomate the species delimitation process withspecific bioinformatical analyses (Table 21)including the general mixed Yule coalescent(GMYC) approach (Fujisawa and Barraclough2013 Monaghan et al 2009 Pons et al 2006)and the Poisson tree processes (PTP) model(Zhang et al 2013) These methods provide rel-atively straightforward and objective pipelinesfor delimiting putative species-level lineagesfrom inferred gene trees by fitting within- andbetween-species branching models to an inferredsingle-locus topology A number of other tree-based methods for species delimitation areeffectively summarized in Sites and Marshall(2004)
When only single-locus data are available theGMYC has been shown to be a relatively robust
tool for species delimitation The GMYCapproach combines a coalescent model of intra-specific branching with a Yule model for inter-specific branching which is then fit to an inferredsingle-gene topology to estimate species bound-aries and a statistical measure of confidence for theinferred boundaries As an input the GMYCapproach requires an ultrametric gene tree andrecent refinements can account for uncertainty inphylogenetic relationships and parameters of theGMYC model (Fujisawa and Barraclough 2013Reid and Carstens 2012) Regardless of theseimprovements it may be difficult to accuratelyinfer an adequate ultrametric tree for large data-sets Although it appears that the GMYC isgenerally stable across a wide range of circum-stances including various methods of phyloge-netic reconstruction the presence of a highnumber singletons high numbers of sampledspecies and gaps in intraspecific sampling theaccuracy of the GMYC is most significantlyaffected by the mean population size relative todivergence times between them (Fujisawa andBarraclough 2013 Talavera et al 2013) Fur-thermore research suggests that the so-calledsingle-threshold version of the GMYC methodlikely outperforms the multiple-thresholdapproach (Fujisawa and Barraclough 2013Monaghan et al 2009) However other studiessuggest that theGMYCmethodmay often providehigher estimates for the total number ofOTUs thanother molecular species delimitation methods(Hamilton et al 2014 Miralles and Vences 2013Talavera et al 2013) warranting a cautiousinterpretation of results from GMYC analyses
Compared to the GMYC approach therecently introduced PTP method for speciesdelimitation has been suggested to be moreaccurate for preliminary species delimitation(Zhang et al 2013) Relative to the GMYC PTPoffers a more straightforward implementationrequiring a simple phylogenetic tree rather thanan ultrametric chronogram However moreresearch is required to assess the general perfor-mance of PTP across wide range of empirical andsimulated species delimitation studies At thispoint data support the use of the GMYC andPTP methods as objective and reasonable starting
24 SD Leavitt et al
points for species delimitation using single-genetopologies
The ldquoSpecies Delimitationrdquo plug-in to theGeneious software provides statistical approachesfor exploring species boundaries in single-genetopologies (Masters et al 2011) Using a priorispecimen assignments to putative species theldquoSpecies Delimitationrdquo plug-in computes statis-tics relating to the probability of the observedmonophyly or exclusivity having occurred bychance in a coalescent process and assesses thewithin- and between-species genetic distances inorder to infer the probability with which membersof a putative species might be identified suc-cessfully with tree-based methods An importantcontribution of the ldquoSpecies Delimitationrdquo plug-in is that it provides an objective means for usersto assess putative species within an empiricalstatistic-based framework rather than qualitativeevaluations of what level of hierarchy constitutesa species in a phylogeny
In contrast to simple sequence similaritythresholds (OTU-picking) for delimiting putativeevolutionarily significant units the AutomaticBarcode Gap Discovery (ABGD) method is anautomated procedure that sorts sequences intohypothetical species based on the existence of abarcode gap observed whenever intraspecificgenetic distances are less than those amongorganisms from different species (Puillandreet al 2012) Ultimately ABGD is a fast simplemethod that can be used to group individualsrepresented in a single-locus sequence alignmentinto candidate species that should be comple-mented with other lines of evidence in an inte-grative taxonomic approach (Kekkonen andHebert 2014 Puillandre et al 2012)
In general the ABGD GMYC and PTP ana-lytical protocols using single-locus data arerepeatable and computationally relatively fastproviding a valuable starting point for a pre-liminary perspective into species boundaries inunderstudied groups that can be validated withsubsequent studies (Kekkonen and Hebert 2014)Similarly analyses of single-locus data can beused to corroborate traditional phenotype-basedspecies boundaries and identify candidate speciesthat have previously been hidden within nominal
species However single-locus species delimita-tionmethodsmaybeparticularly prone to failure inrecognizing significant proportions of species-level biodiversity due to the strict criterion forreciprocal monophyly or alternatively providespurious inflations of estimated species diversitybased on genetic differences that donot correspondto species-level lineages For example recentestimates suggest that the incidence of species-level gene tree paraphyly is approximately 20 (Ross 2014) suggesting that analyses of single-locus datasets would likely fail to accurately deli-mit species in one in every five cases In contrastrecent empirical studies suggest that in some casesthe GMYC provides a striking overestimate ofspecies diversity (Miralles and Vences 2013)
The internal transcribed spacer region (ITS)has played a central role in molecular phyloge-netic studies of lichenized fungi and has recentlybeen adopted as the official barcoding marker forfungi (Schoch et al 2012) In many cases the ITSis sufficiently variable to resolve species bound-aries for lichenized fungi (Schoch et al 2012Kelly et al 2011) although accurate specimenidentification using sequence-based identificationapproaches requires a thoroughly curated refer-ence database (Leavitt et al 2014 Orock et al2012 Kelly et al 2011) In spite of the generalutility of the ITS marker a number of issues maypotentially limit its effective use in speciesdelimitation studies including the potential forintragenomic variation of the nuclear ribosomaltandem repeat and difficulties in aligning ITSsequences from divergent taxa (Kiss 2012)Because the ITS has been formally adopted as theofficial barcoding marker for fungi we recom-mend that species delimitation studies attempt toinclude this region for consistency across studiesHowever we recognize that in some taxonomicgroups the inclusion of the ITS may be prob-lematic and therefore suggest a careful screeningof other markers to identify appropriate loci forresolving species-level relationships
Ultimately the success of any single-locusspecies delimitation method largely depends onthe evolutionary history of the species groupunder study and the variability of the selectedmarker (Fig 22) Species delimitation will be
2 The Dynamic Discipline of Species Delimitation hellip 25
more difficult in more recently diverged lineagesand in cases with some level of interspecific geneflow relative to older well-diverged lineages(eg Leavitt et al 2012c 2013e) In the end wereemphasize that single-locus data alone shouldonly be used to provide a preliminary perspectiveof species boundaries and not as the sole evi-dence in species circumscriptions
222 Sampling Across the GenomeMultilocus Sequence Dataand Genome-Wide Markers
Although single-locus methods can provide anefficient approach for preliminary large-scaleassessments of species diversity there are
significant limitations particularly in recentlydiverged species groups where retention ofancestral polymorphisms and incomplete lineagesorting will likely result in different neutral locihaving unique gene topologies that do not mirrorthe speciation process (Knowles and Carstens2007 Heled and Drummond 2010 Taylor et al2000) In contrast to single-locus and strictlyphenotype-based approaches for species delimi-tation analyses of genetic data collected fromindependent genomic regions can provide robusthypotheses of species boundaries with increasingconfidence (Satler et al 2013 Zhang et al 2011Yang and Rannala 2010) Sequences from mul-tiple independent loci provide an importantsource of data for species delimitation studiesincluding recently developed models that
Fig 22 A simplified diagram illustrating the process ofspeciation through time in a single gene history andresulting gene topologies sampled at two points in thespeciation history (modified from Leliaert et al 2014)aEach dot represents a distinct gene copy and each row onenon-overlapping generation with lines connecting genecopies to their ancestors in the previous generation (onerow below) dashed diagonal lines represent reproductivebarriers two hypothetical sampling intervals at differentpoints in the speciation history are shown Tx and Ty
species delimitation methods using single-locus data areeffective only when species are reciprocally monophyleticin the sampled gene tree genetic clustering and coalescent-based species delimitation methods can circumscribespecies when species may not be monophyletic in sampledgenetic loci b Gene topology representing sampledhaplotypes at time Tx (shown in panel a) hypotheticalspecies are reciprocally monophyletic c Gene topologyrepresenting sampled haplotypes at time Ty (shown in panela) hypothetical species are para- and polyphyletic
26 SD Leavitt et al
combine individual gene genealogies and speciesphylogenies via modeling the coalescent historyof markers (Yang and Rannala 2010 Edwards2009 Knowles and Carstens 2007) As aresponse to advances in sequencing technologiesbioinformatical approaches for multimarker spe-cies delimitation analyses continue to be devel-oped (Table 21 Camargo and Sites 2013)
Long-term reproductive isolation of candidatespecies can be assessed with multilocus sequencedata by evaluating genealogical concordance ofunlinked markers (Baum and Shaw 1995 Aviseand Ball 1990) Within species the mixingeffects of recombination cause unlinked loci tohave distinct genealogical histories but geneticdrift and long-term divergence leads to concor-dant genealogical histories at loci across thegenome Relationships of individuals belongingto distinct candidate species can be evaluatedusing gene genealogies (eg haplotype networksor single-gene topologies) to identify lineagesthat exhibit genealogical exclusivity acrossunlinked neutral loci (Hudson and Coyne 2002Dettman et al 2003b Avise and Ball 1990) Thepresence of the same clades in the majority ofsingle-locus genealogies is taken as evidence thatthe clades represent reproductively isolated lin-eages (Dettman et al 2003a Pringle et al 2005)In practice the criteria of reciprocal monophylyand genealogical concordance of unlinked lociprovide a conservative approach for assessingspecies boundaries due to the fact that a sub-stantial amount of time is required after the initialdivergence of species until ancestral polymor-phisms have fully sorted (Knowles and Carstens2007 Hudson and Coyne 2002) Consequentlygroups with recent divergence histories willlikely go undiscovered under a genealogicalconcordance criterion due to the fact that speciesboundaries likely are not reflected in genegenealogies For example it would take morethan 1 million years after speciation before spe-cies would be delimited if 15 loci were sampledin species with an effective population size (Ne)of 100000 assuming one generation a yearunder a strict reciprocal monophyly criterion(Knowles and Carstens 2007) The amount oftime required for a species to be recognized using
a criterion of reciprocal monophyly increasesproportionally with increasing population sizes(Hudson and Coyne 2002) In a number ofstudies of lichen-forming fungi a genealogicalconcordance criterion has been used to bothdelimit previously unrecognized species-levellineages and validate some conventional pheno-type-based species (Leavitt et al 2012a c2013b Molina et al 2011 Kroken and Taylor2001)
Although analyses of molecular sequence datarelying on reciprocal monophyly or fixed char-acter differences for species delimitation canserve as important criteria for identifying speciesthese criteria are not commonly met acrossmultiple loci particularly in recent speciationhistories (Fig 22) To accommodate theobserved conflict among genealogies from mul-tiple loci with the underlying speciation historythe recent merge of coalescent theory with phy-logenetics has driven a major paradigm shift inspecies delimitation and molecular systematics ingeneral (Fujita et al 2012 Edwards 2009) Themultispecies coalescent model can be applied togenealogical histories from multiple independentloci to assemble separate coalescent processesoccurring in populations into a species tree(Degnan and Rosenberg 2009 Rannala andYang 2003) Within this coalescent-basedframework multiple individuals can be assignedto a single speciespopulation and the speciationhistory of ancestral and descendant species-levellineages can be inferred as a ldquospecies treerdquo incontrast to estimating gene genealogies fromindividual samples (Degnan and Rosenberg2006 2009 Rannala and Yang 2003)
A number of multispecies coalescent-basedspecies delimitation methods have recently beenintroduced offering an exciting framework forempirically assessing species boundaries byselecting the best species tree model from a set ofalternative models representing differenthypotheses of species boundaries (Table 21)For example SpeDeSTEM (Ence and Carstens2011) finds the maximum likelihood values for aspecies tree assuming all putative species areseparate lineages and for alternative species treeswhere two or more species are collapsed into a
2 The Dynamic Discipline of Species Delimitation hellip 27
single lineage The best fitting model is thenselected using the Akaike information criterionunder the assumption of constant populationsizes and fixed gene topologies across the speciestree The species delimitation program BayesianPhylogenetic and Phylogeography (BPampPYangand Rannala 2010) accommodates gene treeuncertainty and variable population sizes andsamples from the Bayesian posterior distributionof species delimitation models using reversible-jump Markov chain Monte Carlo (rjMCMC)sampling BPampP requires a user-provided guidetree with resolution finer than the species leveland evaluates alternative modes derived from allpossible subtrees that are generated by collapsingor splitting nodes on the guide tree (Yang andRannala 2010 Rannala and Yang 2003) Theproportion of time spent on each model is pro-portional to the posterior probability of themodel ie ldquospeciation probabilitiesrdquo WhileBPampP ranks among the most popular coalescent-based species delimitation programs (Fujita et al2012) misspecifications of the guide tree andorthe prior distribution for population size (θ) canresult in strong support for models containing aninflated number of species (Leacheacute and Fujita2010 Zhang et al 2011) The nonparametricheuristic species delimitation approach imple-mented in the program Brownie (OrsquoMeara 2010)jointly sorts anonymous samples into species andinfers a species tree from input gene trees fromdifferent loci assuming that for a speciationevent the corresponding nodes on gene trees willbe more consistent with each than the diver-gences within species However finding both theoptimum species tree and species boundariesremains computationally challenging andBrownie has been shown to frequently yieldincorrect results (OrsquoMeara 2010)
Most recently available coalescent-basedspecies delimitation methods require individualassignments to a species or population a priori Ina number of scenarios correct assignments ofsamples to species may be difficult includingpresence of cryptic species incongruencebetween conventional species and moleculardata or simply the fact that accurate specimenidentification in complex groups with subtle or
difficult to discern diagnostic characters is anontrivial task (Leavitt et al 2013e) Statisticalmethods for assessing individual assignment andspecies detection prior to coalescent-based spe-cies delimitation and species tree reconstructionand species provide a more objective approachfor understanding species boundaries
Population assignment tests using a variety ofclustering algorithms using genetic data offer auseful approach for grouping individuals intoputative reproductively isolated groups (Table 21Hausdorf and Hennig 2010 Corander et al2004 2008 Falush et al 2003 Pritchard et al2000) particularly for species delimitation prob-lems that exist at the interface of traditionalpopulation genetic and phylogenetic analyses(Carstens et al 2013 Weisrock et al 2010Knowles and Carstens 2007)
The programs STRUCTURE (Falush et al2003 Pritchard et al 2000) and STRUCTURA-MA (Huelsenbeck et al 2011) cluster samplesinto populations by minimizing HardyndashWeinbergdisequilibrium for a given number of populationclusters using unlinked genetic markers In gen-eral unlinked markers are not available for mostgroups of lichen-forming fungi and a numberof studies have used information from multilocussequence data for STRUCTURE analyses(Leavitt et al 2011a b c 2013e Fernaacutendez-Mendoza and Printzen 2013 Fernaacutendez-Mendoza et al 2011) Varying approaches havebeen implemented to convert DNA sequencedata to allelic data for Bayesian clustering (seeOrsquoNeill et al 2013) STRUCTURE is expectedto perform well when there is sufficient inde-pendence across regions such that linkage dis-equilibrium within regions does not dominate thedata (STRUCTURE manual) but can also beeffective using multilocus sequence data andtreating all SNPs as independent loci regardlessof physical linkage within each locus (OrsquoNeillet al 2013 Falush et al 2003) A recent study ofthe lichen-forming genus Letharia showed thatBayesian clustering implemented in the programSTRUCTURE was generally able to recover thesame putative Letharia lineages circumscribedemploying a gene genealogical approach inKroken and Taylorrsquos iconic species delimitation
28 SD Leavitt et al
study (Altermann et al 2014 Kroken and Taylor2001) Altermann et al (2014) show that popu-lation assignments were largely consistent acrossa range of scenarios including extensiveamounts of missing data the exclusion of SNPsfrom variable markers and inferences based onSNPs from as few as three gene regions Incontrast to STRUCTURE and STRUCTURA-MA the program BAPS (Corander et al 2008)can explicitly infer genetic structure using hap-loid sequence data or other linked geneticmarkers in the ldquoclustering with linked locirdquomodel Another advantage of BAPS is that theprogram is much more computationally efficientthan STRUCTURE or STRUCTURAMA Sim-ulation studies indicate that both BAPS andSTRUCTURE perform well at low levels ofpopulation differentiation and when clusters arenot well differentiated (Latch et al 2006)
In practice inferring the most appropriatelevel of population structure using Bayesianclustering algorithms remains challenging (Latchet al 2006 Evanno et al 2005) BAPS andSTRUCTURAMA can infer both individualassignments and the most likely number ofgenetic clusters (Corander et al 2008 Huelsen-beck et al 2011) and the ad hoc ΔK statisticproposed by Evanno et al (2005) provides anobjective approach for identifying the uppermosthierarchical level of structure However carefulconsideration of other levels of populationstructure may ultimately reveal more biologicallymeaning results and researchers should examineindividual assignments across a range of geneticgroupings Some markers generated fromindependent genomic regions such as SNPs andfast-evolving microsatellites can be used to dis-tinguish fine-scale population structuring on thebasis of allele frequencies and species delimita-tion analyses based on these markers may implythe risk of severe taxonomic oversplitting Inthese cases validation approaches such asBPampP and SpeDeSTEM and corroborationamong different species delimitation approachescan provide perspective between intraspecificpopulation structure and species-level clusters
Another limitation of clustering approaches isthat they do not assess or take into account
evolutionarily divergences and relationshipsamong population clusters Coalescent-basedspecies tree methods (discussed below) provide ameans to assess the diversification histories ofpopulations inferred from clustering analysesTherefore a potential working protocol for aninformed species delimitation study that takesinto account population structure could consist offirst applying a genetic clustering analysis under apopulation genetics criterion (eg BAPSSTRUCTURE STRUCTURAMA) to identifygenetically distinct population clusters that can beconsidered ldquocandidate speciesrdquo From these can-didate species a species tree can be inferred forfocal group using coalescent-based species treereconstruction methods (eg BEAST) Subse-quently a coalescent-based validation methodcan be applied to assess whether the distinctpopulation clusters represent independent evolu-tionary lineages (eg BPampP) This multistepapproach would provide a consistent and standardcriterion for distinguishing between population-and species-level lineages (Camargo and Sites2013) and has been applied to a number of casesincluding lichen-forming fungi (Leavitt et al2011a b c 2013e Leacheacute and Fujita 2010)
High-throughput sequencing methods providethe means to effectively sample hundreds tothousands of loci from across a species genome fora large number of species and continue to revolu-tionize studies that can be performed even in non-model organisms Targeted high-throughputsequencing approaches such as anchored phy-logenomics transcriptome sequencing reduced-representation genomic library sequencing(restriction-site-associated DNA sequencingRAD-Seq and genotype-by-sequencing GBS)and high-throughput amplicon sequencing pro-vide important insight into species boundaries fora number groups although none of these approa-ches has yet been applied to studies of lichenizedfungi
For example restriction-site-associated(RAD-Seq) DNA can simultaneously detect andgenotype thousands of genome-wide SNPs byfocusing the sequencing effort on a reducedrepresentation of the entire genome (Baird et al2008) and has been successfully applied to
2 The Dynamic Discipline of Species Delimitation hellip 29
intraspecies (Lewis et al 2007 Miller et al 2007Emerson et al 2010) and interspecies studies(Rubin et al 2012 Eaton and Ree 2013 Wagneret al 2013) This approach has provided strikinginsight into the recent adaption radiation of LakeVictoria cichlids (Wagner et al 2013) Analternative approach targeting the sequencing ofspecific loci using next-generation sequencingplatforms provides an efficient means of gener-ating data for loci of known genomic locationorthology size and expected level of variation(OrsquoNeill et al 2013) Markers can be targetedusing well-established PCR techniques or newerhybridization techniques and subsequentlypooled for high-throughput sequencing usingparallel-tagged sequencing of multiple individualsamples within a single sequencing run (OrsquoNeillet al 2013) A recent study of North Americantiger salamanders (Ambystoma tigrinum)demonstrated the potential for amplicon-basedparallel-tagged sequencing to rapidly generatelarge-scale genomic data for species delimitationand species tree research (OrsquoNeill et al 2013)
Although these methods provide an enormousamount of data and insight into diversification atan unprecedented scale computational limita-tions restrict the applicable analytical methodsfor large datasets although computational andanalytical advances are happening rapidly Mostrecent coalescent-based species delimitation andspecies tree models using gene trees have beenlimited to handle tens of loci for multiple indi-viduals and combining hundreds or thousands ofgene trees into a single species delimitationframework presents considerable computationalchallenges (Camargo and Sites 2013) Therecently developed method Bayes factor delim-itation (with genomic data BFD) combines adynamic programming algorithm for estimatingspecies trees that bypasses the computationallyintensive MCMC integration over gene trees toprovide a rigorous technique for species delimi-tation studies using genome-wide SNP data(Leacheacute et al 2014) Competing species delimi-tation models are compared using Bayes factorsand it appears that this approach is robust tosample sizes (ie few loci and limited samples
per species) and misspecification of the prior forpopulation size (θ) (Leacheacute et al 2014)
Fungi are an ideal model for assessing diver-gence in eukaryotes due to their simple mor-phologies small genomes broad ecological rolesand diverse lifestyles (Gladieux et al 2014)However the use of genomic data from high-throughput sequencing methods have not yet beenincluded in species delimitation studies of lichen-forming fungi This is due in part to challenges indealing with metagenomic data containing geno-mic information from a plethora of symbiontsassociated with a lichen thallus scant genomicresources and discipline-specific inertia A num-ber of lichen-forming fungal genomes are cur-rently available including Endocarponpusillum(Wang et al 2014) Cladonia spp (Park et al2013a 2014) Caloplaca flavorubescens (Parket al 2013b) and Cladonia greyi and Xanthoriaparientina through the Fungal Genomics Programat Joint Genome Institute of the United StatesDepartment of Energy (httpwwwjgidoegov)The foreseeable ongoing expansion of genomicresources for lichen-forming fungi will be centralto developing approaches for delimiting speciesusing high-throughput sequencing Specificallygenomic resources will be crucial in identifyingnovel markers (variable genes regions SNPsmicrosatellites etc) identifying conserved fungalmarkers for targeted high-throughput sequencingapproaches and references for entire genomecomparisons (Devkota et al 2014 Werth et al2013)
23 Can We Make SpeciesDelimitation in Lichen-FormingFungi Truly Integrative
The widespread availability of genetic data hascreated a biased viewpoint that only genetic datashould be used for statistical species delimitationHowever an ongoing appeal to researchers toassess species boundaries from multiple andcomplementary perspectives (phylogeneticspopulation genetics comparative morphologydevelopment ecology etc) has resulted in an
30 SD Leavitt et al
integrative taxonomic framework bringing theseconceptual and methodological perspectivestogether (Hamilton et al 2014 Fujita et al 2012Salicini et al 2011 Yeates et al 2011 Padialet al 2010 Dayrat 2005 Will et al 2005 Wiensand Penkrot 2002) In practice any study linkingdifferent kinds of data to support hypotheses ofspecies boundaries including mapping morpho-logical characters onto a molecular phylogenycan be considered integrative Integrative meth-ods for species delimitation fall across a broadspectrum ranging from verbal and qualitativeassessments of data classes to quantitative meth-ods that allow different data types to contribute tostatistical species delimitation (Yeates et al2011) In most taxonomic studies utilizing bothmolecular and morphological data expert opinionis eventually used to some degree to evaluate thefinal status of a candidate species (eg Bond andStockman 2008) In this sense many studiesusing evidence for independent data sources fordelimiting species boundaries use an iterativeapproach (Yeates et al 2011) where speciesboundaries can be tested within a hypothetico-deductive framework with diverse datasets
From a practical perspective we advocate aprocess of iterative taxonomy (sensu Yeates et al2011) to circumscribe and refine species limitsusing multiple lines of evidence This iterativeprocess involves comparisons of morphologicaldata with a phylogenetic hypothesis (ie single-locus gene tree or concatenated multilocusphylogeny) to identify the least inclusive mono-phyletic clade in the topology characterized by atleast one unambiguously diagnostic morphologi-cal character (Miralles and Vences 2013) Thisphylogenetic tree-informed approach to assessingspecies boundaries represents a common practicein studies of lichen-forming fungi Previouslyunrecognized species-level clades with corre-sponding subtle or overlooked phenotypiccharacters have been commonly observed in bothcrustose lichens [eg Acarospraceae (Wedin et al2009) Graphidaceae (Rivas Plata and Lumbsch2011 Papong et al 2009) Lecanoraceae (Leavittet al 2011a) Lecideaceae (Ruprecht et al 2010)Mycoblastaceae (Spribille et al 2011) andTeloschistaceae (Vondraacutek et al 2009 Muggia
et al 2008)] and foliose and fruticose lichens [egLobariaceae (Moncada et al 2014 McDonaldet al 2003) Parmeliaceae (Leavitt et al 2013aWirtz et al 2012 Divakar et al 2005 Molinaet al 2004 Kroken and Taylor 2001) Peltigera-ceae (OrsquoBrien et al 2009 Goffinet et al 2003)Physciaceae (Elix et al 2009 Divakar et al2007) and Sphaerophoraceae (Houmlgnabba andWedin 2003)] As a specific example Luumlckinget al (2008) used a combination of medullarychemistry and underside pigmentation in speci-mens from the Heterodermia obscurata group inCosta Rica to corroborate monophyletic clades inan ITS gene tree as species-level lineages
While this iterative approach to speciesdelimitation and taxonomy has proven valuablefor understanding species boundaries anddescribing new taxa in some groups of lichenizedfungi a posteriori examination of morphologicaland chemical features has failed to reveal diag-nostic phenotypic characters in a number ofstudies (Muggia et al 2014 Leavitt et al 2011aOtaacutelora et al 2010 OrsquoBrien et al 2009) Fur-thermore a study of widespread species in thegenus Melanelixia (Parmeliaceae) indicated thatphenotypically cryptic lichen-forming fungalspecies-level lineages may be relatively ancientand diagnosable phenotypic differences may beabsent even millions of years after the initialdivergence (Leavitt et al 2012b) The latter studyhighlights the fact that species-level lineages maycommonly exist without any observable diag-nostic phenotypic characters calling into ques-tion the impetus for a universal application ofintegrative taxonomy
In keeping with the principle that as manylines of evidence as available should be com-bined to delimit species (Dayrat 2005) the for-malized integrative taxonomic approach (ITAXMiralles and Vences 2013) uses a mtDNA guidetree and observations from different types of datathat might provide conclusive evidence for theindependence of lineages and thus their identityas different species Miralles and Vences (2013)provide a non-exhaustive list of species delimi-tation criteria to be integrated in the ITAXapproach including (a) sympatric occurrencewithout admixture as revealed by consistent
2 The Dynamic Discipline of Species Delimitation hellip 31
differences in morphological or molecular char-acters at the same geographic location (b) strongdifferences in a behavioral morphological orgenetic character known to mediate prematingisolation (c) unviability or infertility of hybrids(d) lack of gene flow across a geographicalhybrid zone (e) congruent diagnostic differencesbetween sister lineages in various unlinkedmorphological character (f) absence of haplo-type sharing in several unlinked nuclear loci and(g) a combination of criteria endashf Speciesboundaries are based on seeking the least inclu-sive monophyletic group in the mtDNA treewhich fulfills at least one of the criteria listedabove Clearly the ITAX approach is sensitive tosample size and in order to reliably support thedistinctiveness of a given species it has beenrecommended that the sampling strategy includesat least five individuals per species (Miralles andVences 2013) A similar approach could beadopted for lichenized fungi using an ITS genetopology as guide tree rather than mtDNA
A total evidence approach including concat-enation has been a common approach for inte-grating information from different sourcesincluding independent genetic markers in phy-logenetic reconstructions (Kluge 1989 Wiens1998 de Queiroz et al 1995) Proponents forconcatenation of independent data in phyloge-netic analyses argue that when combining alldata the underlying signal of speciation mayemerge even if weakly contradictory signalis contained in the individual data partitions(Gatesy et al 1999) For example establishing apreliminary perspective of species boundariesfrom multilocus sequence data using concatena-tion may provide a reasonable starting point forscreening for cryptic species and species treeinference (Šlapeta et al 2006 Leavitt et al2011a Le Gac et al 2007 Leacheacute 2009)However concatenation and consensus methodsimply a risk of obtaining inflated support forincorrect relationships and information aboutvariance in gene coalescence is lost (Degnan andRosenberg 2006 2009 Kubatko and Degnan2007 Edwards 2009) In spite of the impetus forintegrating evidence from independent data
sources into empirical species delimitation stud-ies and taxonomy (Fujita et al 2012 Padial et al2009) most currently available species delimi-tation methods are unable to accommodate non-genetic data sources in a statistical framework
231 Selecting the Appropriate Data
In the face of increasing availability of geneticdata and associated bioinformatical approachesfor delimiting species researchers should care-fully consider what information is being sacri-ficed by the failure to consider non-genetic datain species delimitation studies and whetheraccuracy could be improved by the addition ofmultiple data types Morphological data havehistorically served as a proxy to identify repro-ductively isolated groups (ie ldquospeciesrdquo) (Ray1686 Fujita et al 2012) Current methods fordelimiting species using non-genetic data (egchemistry morphology and ecology) remainwoefully understudied For example morphol-ogy-based species circumscriptions are generallybased on one or more qualitative (or quantitative)morphological characters that do not appear tooverlap with other species However ascertain-ing that a given trait is truly fixed within apopulation with statistical confidence requiresunrealistic sample sizes even when allowing forsome level of polymorphism in the diagnosticcharacter (Wiens and Servedio 2000) Now var-ious combinations of datamdashfrom morphologygenetics geography and ecologymdashare acceptedas standard information for species delimitationstudies (Ruiz-Sanchez and Sosa 2010 Ross et al2010 Edwards and Knowles 2014)
Below we briefly discuss appropriate datasources for species delimitation studies of lichen-forming fungi However homoplastic characters(similar traits that are not derived from a com-mon ancestor) are common among many traitscommonly used to circumscribe fungal taxa thebiological significance of secondary metabolitevariation remains largely unknown (Lawrey1986) and ecological niches may be difficultto adequately characterize and model due to
32 SD Leavitt et al
microhabitat requirements and data resolutionTherefore we advocate a cautious approach toselecting appropriate and relevant data forassessing species boundaries
In spite of the potential challenges and limi-tations of using phenotypic data these traits haveprovided a plethora of valuable information forunderstanding species boundaries For examplein the Melanelixia fuliginosa group (Parmelia-ceae) a morphometric analysis using color isi-dia and marginal zone free of isidia ascharacters revealed a general pattern of differ-entiation between material formerly recognizedas subspecies fuliginosa and glabratula in spiteof the fact that considerable overlap betweengroups occurs in some characters individually(Arup and Berlin 2011) Furthermore the dis-tinction of the two groups was supported by aphylogenetic analysis of the ITS marker andecological differences with M fuliginosaoccurring predominantly on rock and M glab-ratula on bark (Arup and Berlin 2011) Thisstudy of the M fuliginosa group provides a fit-ting example of using multiple independentsuites of data including ecology morphologyand genetic information to establish a robusthypothesis of species boundaries
In lichen systematics phenotypic dataincluding thallus organization secondary metab-olites mode of reproduction ascoma-type andontogeny ascus and ascospore characters havehistorically played a prominent role (Printzen2009) Ascomatal characters have traditionallyheld a major role in higher-level classification(Printzen 2009) in contrast to species-levelclassification which also tends to include a widearray of vegetative and chemical charactersCommonly assessed morphological charactersinclude thallus form and size cortical features(eg maculae and pseudocyphellae) presenceformcolor of attachment structure (eg rhizines)and reproductive mode (ascomata vs vegetativediaspores) (Printzen 2009) Different morpho-logical types of vegetative diasporesmdashcorticatedisidia and ecorticated sorediamdashand their locationare commonly used to distinguish speciesAscomatal characters including morphologylocation (laminal vs marginal) position (sessile
vs immersed) presence of thalline margins colorof an apothecial disc and presence or color ofpruina are also commonly used in speciesdelimitations of lichen-forming fungi (Printzen2009) Other important characters may includethalline characters form color size and septationof ascospores size and form and structure of ascithe hamathecium type of epihymenium andhypothecium and the type of excipulum orperidium conidiomatal characters etc (Printzen2009)
Assessments of secondary metabolites hasplayed an important role in lichen taxonomybeginning with the introduction of simple spottests by Nylander (1866a b) The use of chem-istry in lichen taxonomy has been discussed indetail in numerous reviews (Lumbsch 1998a bRogers 1989 Brodo 1986 Egan 1986 Leuckert1985 Brodo 1978 Hawksworth 1976 Culber-son 1969 1970) and we refer readers to thesevaluable sources for a more comprehensive per-spective on lichen chemistry In short extrolites(secondary metabolites) belong to various clas-ses the most common and diverse include dep-sides depsidones chlorinated xanthones andanthraquinones (Lumbsch 2002 Culberson1969) The presence or absence of specificextrolites or their replacements by another sub-stance is widely used to distinguish speciesparticularly when correlated with differences ingeographic distributions However if morpho-logical or geographical differences betweenpopulations containing different extrolites are notapparent the taxonomic significance has beendisputed with some authors distinguishing themas species and others preferring to regard them aschemical races within a species In addition tosimply using the presence or absence of extro-lites Culberson and Culberson (1976) proposedto arrange lichen substances into chemosyn-dromes of closely related substances The pres-ence or absence of these chemosyndromes maypotentially be used as characters to delimit spe-cies regarding differences involving the samechemosyndromes as intraspecific variation anddistinct chemosyndromes as evidence for inter-specific populations (Lumbsch 1994 Gowan1986)
2 The Dynamic Discipline of Species Delimitation hellip 33
Due to the fact that species delimitationstudies often incorporate a substantial biogeo-graphical or ecological component ecologicalniche modeling plays an increasing role in phy-logenetic and taxonomic research Niche mod-eling can provide evidence for ecologicalisolation between populations based on eitherconserved or divergent ecological niches andtherefore can provide additional evidence sup-porting lineage independence between putativespecies The application of ecological nichemodeling has been applied to species delimita-tion studies in a number of cases (Ruiz-Sanchezand Sosa 2010 Leacheacute et al 2009 Raxworthyet al 2007 Rissler and Apodaca 2007) althoughit has not been explicitly applied to assess speciesboundaries in lichen-forming fungi By mappingthe spatial distribution of environmental suit-ability of climatic variables for candidate speciesthe application of ecological niche modeling canbe particularly important in cases where specieshave allopatric distributions (Raxworthy et al2007)
Ecological niche modeling utilizes knownassociations between a speciesrsquo occurrencelocalities and environmental variables to defineabiotic conditions within which populations canbe maintained (Guisan and Thuiller 2005) Themethodological approach for modeling is basedon four general properties (i) The current knownspeciesrsquo localities is the dependent variable (ii)the distribution is modeled as a map composed ofgrid cells at a specified resolution (iii) a range ofenvironmental variables (eg temperature pre-cipitation and solar exposure) are collected todescribe the characteristics of each cell and (iv)classifying the degree to which each cell is eithersuitable or unsuitable for each species under arange of models (Guisan and Thuiller 2005)
Ecological data also have the potential to playan important role in understanding speciesboundaries in lichen-forming fungi For exampleNash and Zavada (1977) demonstrated that Xant-hoparmelia populations with distinct chemistriesoccurring in the northern portion of the SonoranDesert exhibit habitat selection among differentrock substrates within a region with relativelyuniform climate and topography In another case
the parmelioid species Parmelia mayi is mor-phologically indistinguishable from P saxatilisbut can be separated by bioclimatic features andgenetic and chemical characters (Molina et al2011) McCune and Printzen(2011) assess distri-butions and climatic niches of species in theLecanora varia group inwesternUSAandprovidea model that uses continental influence and annualtemperature as themajor factors predicting speciesdistributions The distribution of Usnea hirta alichen commonly used in air quality biomonitor-ing research was modeled for a section of theWhite River National Forest in central Coloradobased on the presence of U hirta at 72 biomoni-toring reference sites distributed in the inter-mountain western United States (Shrestha et al2012) The best model for predicting U hirtadistribution included four variablesmdashsolar radia-tion average monthly precipitation and averagemonthly minimum and maximum temperatures(Shrestha et al 2012) These studies supportthe potential use of ecological niche modelingmethods in species delimitation studies of lichen-forming fungi
24 Conclusions What AboutTaxonomy
In most cases species circumscription and tax-onomy requires some degree of qualitativejudgment and individual interpretation Integrat-ing multiple types of data into an empiricalframework for delimiting species boundarieswhere species boundaries can be tested within ahypothetico-deductive framework with diversedatasets can provide robust hypotheses of speciesboundaries and taxonomic stability (Yeates et al2011) However it has long been known toevolutionary biologists that distinct species donot need to have diagnosable morphologicaldifferences (Mayr 1963) and increasing avail-ability of genetic data has allowed researchers toidentify species and to rigorously test speciesboundaries with a level of precision that wasunimaginable a decade ago While analyticaladvances in statistical species delimitation havebeen largely based on genetic data the utility of
34 SD Leavitt et al
these approaches to formal taxonomy remainselusive Due to these challenges an eclecticapproach to delimiting species and cautionagainst the reliance on any single dataset ormethod is required when delimiting species
While results from many recent studies maycontradict traditional species boundaries acrossmany groups of lichen-forming fungi we areoptimistic that this research represents substantialprogress toward a more accurate perspective onspecies boundaries and diversity in fungi As aresult of ongoing research of species boundariesin lichen-forming fungi the taxonomic value ofmany phenotypes is now better understood ourunderstanding of ecological evolutionary andbiogeographic patterns has improved and we canbegin to better understand patterns of symbiontinteractions in lichens Integrating new data(including novel morphological characters andgenetic data) will be essential to accurately rep-resent species-level diversity across all groups oflichenized fungi Hopefully an improved per-spective on lichen diversity also increases ourappreciation of these incredible symbiotic sys-tems While we are strong advocates for theapplication of independent data types in devel-oping an integrative taxonomy there is anincreasing need to formally recognize the exis-tence of phenotypically cryptic species-levellineages in lichen-forming fungi (see Hibbettet al 2011) In some cases a molecular taxon-omy may provide the most practical approach toconsistent treatment of mycobiont species withinlichen groups where diagnostic morphologicalcharacters are unidentifiable or practically notfeasible (Leavitt et al 2013c d)
These are exciting times for taxonomists andphylogeneticists A closer look at lichen taxon-omy with the inclusion of new data will help usto better understand the diversity of these fasci-nating organisms accurately interpret distribu-tion patterns and play a more important role inmeaningful conservation practices Howeversome level of uncertainty will accompany pro-gress In many taxonomic groups our traditionalapproach for species identification will likelyneed to be substantially modified The search forcorroborating morphological support for cryptic
species identified using molecular data willrequire meticulous and creative approaches toassess phenotypic variation in potentially unor-thodox ways We are hopeful that lichenologistswho traditionally have been eager to include newmethods such as chromatography in their rou-tine identifications will be amenable to includemolecular techniques to their routine examina-tion of specimens for identification and classifi-cation Although this may prove difficult toachieve by single individuals especially citizenscientists that traditionally play an important rolein lichen taxonomy the increasing numberof collaborative projects in the discipline (egLumbsch et al 2011 Crespo et al 2010Gueidan et al 2009) make us optimistic thatbroad-scale collaborative approaches will facili-tate the inclusion of molecular data in lichenresearch at all levels
Acknowledgments We are indebted to various col-leagues for valuable thought-provoking discussionnotably Matthew Nelsen (University of Chicago) AnaCrespo (Universidad Complutense de Madrid) PradeepDivakar (Universidad Complutense de Madrid) BeckettSterner (The Field Museum) and Joyce Havstad (TheField Museum) We also thank anonymous reviewers whoprovided valuable comments that improved this chapterSupport by the US National Science Foundation isgratefully acknowledged (ldquoHidden diversity in parmelioidlichensrdquo DEB-0949147)
References
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Altermann S Leavitt SD Goward T Nelsen MPLumbsch HT (2014) How do you solve a problemlike Letharia A new look at cryptic species in lichen-forming fungi using Bayesian clustering and SNPSsfrom multilocus sequence data PLoS ONE 9(5)e97556 doi101371journalpone0097556
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2 The Dynamic Discipline of Species Delimitation hellip 35
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Corander J Marttinen P Mantyniemi S (2006) Bayesianidentification of stock mixtures from molecular markerdata Fish Bull 104550ndash558
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Culberson WL (1969) The use of chemistry in thesystematics of the lichens Taxon 18498ndash505
Culberson WL (1970) Chemosystematics and ecology oflichen-forming fungi Annu Rev Ecol Syst 1153ndash170
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Darwin C (1859) On the origin of species by means ofnatural selection or the preservation of favoured racesin the struggle for life J Murray London
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de Queiroz K (1998) The general lineage concept ofspecies species criteria and the process of speciationa conceptual unification and terminological recom-mendations In Howard DJ Berlocher SH (eds)Endless forms species and speciation Oxford Uni-versity Press Oxford pp 57ndash75
de Queiroz K (1999) The general lineage concept ofspecies and the defining properties of the speciescategory In Wilson RA (ed) Species new interdis-ciplinary essays MIT Press Cambridge pp 49ndash89
de Queiroz K (2007) Species concepts and speciesdelimitation Syst Biol 56(6)879ndash886 doi10108010635150701701083
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Degnan JH Rosenberg NA (2009) Gene tree discordancephylogenetic inference and the multispecies coales-cent Trends Ecol Evol 24(6)332ndash340 doihttpdxdoiorg101016jtree200901009
Del-Prado R Cubas P Lumbsch HT Divakar PK BlancoO de Paz GA Molina MC Crespo A (2010) Geneticdistances within and among species in monophyleticlineages of Parmeliaceae (Ascomycota) as a tool fortaxon delimitation Mol Phylogenet Evol 56(1)125ndash133 doi101016jympev201004014
Del-Prado R Divakar PK Crespo A (2011) Using geneticdistances in addition to ITS molecular phylogeny toidentify potential species in the Parmotrema reticul-atum complex a case study The Lichenologist 43(06)569ndash583 doi101017S0024282911000582
Del-Prado R Blanco O Lumbsch HT Divakar PK ElixJA Molina MC Crespo A (2013) Molecular phylog-eny and historical biogeography of the lichen-formingfungal genus Flavoparmelia (Ascomycota Parmelia-ceae) Taxon 62(5)928ndash939 doi101270562522
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Dettman JR Jacobson DJ Turner E Pringle A TaylorJW (2003b) Reproductive isolation and phylogeneticdivergence in Neurospora comparing methods ofspecies recognition in a model eukaryote Evolution57(12)2721 doi10155403-074
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Divakar PK Molina MC Lumbsch HT Crespo A (2005)Parmelia barrenoae a new lichen species related toParmelia sulcata (Parmeliaceae) based on molecularand morphological data The Lichenologist 37(01)37ndash46 doi101017S0024282904014641
Divakar PK Amo De paz G del Prado R Esslinger TLCrespo A (2007) Upper cortex anatomy corroboratesphylogenetic hypothesis in species of Physconia(Ascomycota Lecanoromycetes) Mycol Res 111(11)1311ndash1320 doi101016jmycres200708009
Divakar PK Figueras G Hladun N Crespo A (2010)Molecular phylogenetic studies reveal an undescribedspecies within the North American concept of Mela-nelixia glabra (Parmeliaceae) Fungal Divers 42(1)47ndash55 doi101007s13225-010-0027-3
Donoghue MJ Gauthier A (2004) Implementing thephylocode Trends Ecol Evol 19(6)281ndash282 doi101016jtree200404004
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Edwards SV (2009) Is a new and general theory ofmolecular systematics emerging Evolution 63(1)1ndash19 doi101111j1558-5646200800549x
Edwards DL Knowles LL (2014) Species detection andindividual assignment in species delimitation canintegrative data increase efficacy Proc R Soc B BiolSci 281(1777) doi101098rspb20132765
Egan RS (1986) Correlations and non-correlations ofchemical variation patterns with lichen morphologyand geography The Bryologist 8999ndash110
Elix JA Corush J Lumbsch HT (2009) Triterpenechemosyndromes and subtle morphological characterscharacterise lineages in the Physcia aipolia group inAustralia (Ascomycota) Syst Biodivers 7(04)479ndash487 doi101017S1477200009990223
Emerson KJ Merz CR Catchen JM Hohenlohe PACresko WA Bradshaw WE Holzapfel CM (2010)Resolving postglacial phylogeography using high-throughput sequencing Proc Natl Acad Sci 107(37)16196ndash16200 doi101073pnas1006538107
Ence DD Carstens BC (2011) SpedeSTEM a rapid andaccurate method for species delimitation Mol EcolResour 11(3)473ndash480 doi101111j1755-0998201002947x
EvannoGRegnaut SGoudet J (2005)Detecting thenumberof clusters of individuals using the software STRUC-TURE a simulation study Mol Ecol 14(8)2611ndash2620doi101111j1365-294X200502553x
Falush D Stephens M Pritchard JK (2003) Inference ofpopulation structure using multilocus genotype datalinked loci and correlated allele frequencies Genetics164(4)1567ndash1587 doi101111j1471-8286200701758x
Fan HH Kubatko LS (2011) Estimating species treesusing approximate Bayesian computation Mol Phylo-genet Evol 59(2)354ndash363 doi101016jympev201102019
Fernaacutendez-Mendoza F Printzen C (2013) Pleistoceneexpansion of the bipolar lichen Cetraria aculeata intothe Southern hemisphere Mol Ecol 22(7)1961ndash1983doi101111mec12210
Fernaacutendez-Mendoza F Domaschke S Garciacutea MA JordanP Martin MP Printzen C (2011) Population structure
2 The Dynamic Discipline of Species Delimitation hellip 37
of mycobionts and photobionts of the widespreadlichen Cetraria aculeata Mol Ecol 20(6)1208ndash1232doi101111j1365-294X201004993x
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Fujita MK Leacheacute AD Burbrink FT McGuire JA MoritzC (2012) Coalescent-based species delimitation in anintegrative taxonomy Trends Ecol Evol 27(9)480ndash488 doi101016jtree201204012
Gatesy J OrsquoGrady P Baker RH (1999) Corroborationamong data sets in simultaneous analysis hiddensupport for phylogenetic relationships among higherlevel Artiodactyl taxa Cladistics 15(3)271ndash313doi101111j1096-00311999tb00268x
Gladieux P Ropars J Badouin H Branca A Aguileta Gde Vienne DM Rodriacuteguez de la Vega RC Branco SGiraud T (2014) Fungal evolutionary genomicsprovides insight into the mechanisms of adaptivedivergence in eukaryotes Mol Ecol 23(4)753ndash773doi101111mec12631
Goffinet B Miadlikowska J Goward T (2003) Phyloge-netic inferences based on nrDNA sequences supportfive morphospecies within the Peltigera didactylacomplex (Lichenized Ascomycota) The Bryologist106(3)349ndash364 doi10163901
Gowan SP (1986) Evolution of secondary natural productsin the genus Porpidia (Ascomycata Porpidiaceae) AmJ Bot 73606
Griffin PC Hoffmann AA (2014) Limited genetic diver-gence among Australian alpine Poa tussock grassescoupled with regional structuring points to ongoinggene flow and taxonomic challenges Ann Bot doi101093aobmcu017
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Gueidan C Savi S Thues H Roux C Keller C Tibell LPrieto M Heimarsson S Breuss O Orange A FrobergL Wynns AA Navarro-Rosines P Krzewicka BPykaelae J Grube M Lutzoni F (2009) Genericclassification of the Verrucariaceae (Ascomycota)based on molecular and morphological evidencerecent progress and remaining challenges Taxon 58(1)184ndash208
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Guillot G Renaud S Ledevin R Michaux J Claude J(2012) A unifying model for the analysis of pheno-typic genetic and geographic data Syst Biol 61(6)897ndash911 doi101093sysbiosys038
Guisan A Thuiller W (2005) Predicting species distribu-tion offering more than simple habitat models EcolLett 8(9)993ndash1009 doi101111j1461-0248200500792x
Hale ME (1990) A synopsis of the lichen genusXanthoparmelia (Vainio) Hale (AscomycotinaParmeliaceae) Smithsonian Institution Press Wash-ington DC
Hamilton CA Hendrixson BE Brewer MS Bond JE(2014) An evaluation of sampling effects on multipleDNA barcoding methods leads to an integrativeapproach for delimiting species a case study ofthe North American tarantula genus Aphonopelma(Araneae Mygalomorphae Theraphosidae) MolPhylogenet Evol 7179ndash93 doi101016jympev201311007
Hausdorf B (2011) Progress toward a general speciesconcept Evolution 65(4)923ndash931 doi101111j1558-5646201101231x
Hausdorf B Hennig C (2010) Species delimitation usingdominant and codominant multilocus markers SystBiol 59(5)491ndash503 doi101093sysbiosyq039
Hawksworth DL (1976) Lichen chemotaxonomy InBailey RH (ed) Lichenology progress and problemsAcademic London pp 139ndash184
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Hebert PDN Stoeckle MY Zemlak TS Francis CM(2004) Identification of birds through DNA barcodesPLoS Biol 2(10)e312 doi101371journalpbio0020312
Heled J Drummond AJ (2010) Bayesian inference ofspecies trees from multilocus data Mol Biol Evol 27(3)570ndash580 doi101093molbevmsp274
Hey J (2006) On the failure of modern species conceptsTrends Ecol Evol 21(8)447ndash450 doi101016jtree200605011
Hibbett DS OhmanA Glotzer D NuhnMKirk P NilssonRH (2011) Progress in molecular and morphologicaltaxon discovery in Fungi and options for formalclassification on environmental sequences Fungal BiolRev 2538ndash47 doi101016jfbr201101001
Houmlgnabba F Wedin M (2003) Molecular phylogeny ofthe Sphaerophorus globosus species complex Cladis-tics 19(3)224ndash232 doi101111j1096-00312003tb00365x
Hudson RR Coyne JA (2002) Mathematical conse-quences of the genealogical species concept Evolu-tion 56(8)1557 doi101111j0014-38202002tb01467x
Huelsenbeck JP Andolfatto P Huelsenbeck ET (2011)Structurama Bayesian inference of population struc-ture Evol Bioinf Online 7(2011)55ndash59 doi104137EBOS6761
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Leavitt SD Fankhauser JD Leavitt DH Porter LDJohnson LA St Clair LL (2011a) Complex patterns ofspeciation in cosmopolitan ldquorock posyrdquo lichensmdashdiscovering and delimiting cryptic fungal species in
the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
Leavitt SD Esslinger TL Lumbsch HT (2012c) Neogene-dominated diversification in neotropical montanelichens dating divergence events in the lichen-form-ing fungal genus Oropogon (Parmeliaceae) Am J Bot99(11)1764ndash1777 doi103732ajb1200146
Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Lumbsch HT St Clair LL(2013c) DNA barcode identification of lichen-formingfungal species in the Rhizoplaca melanophthalmaspecies-complex (Lecanorales Lecanoraceae) includ-ing five new species MycoKeys 71ndash22 doi103897mycokeys74508
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Vondraacutek J Thorsten Lum-bsch H Clair LLS (2013d) Local representation ofglobal diversity in a cosmopolitan lichen-formingfungal species complex (Rhizoplaca Ascomycota)J Biogeogr 40(9)1792ndash1806 doi101111jbi12118
Leavitt SD Lumbsch HT Stenroos S St Clair LL(2013e) Pleistocene speciation in North American
2 The Dynamic Discipline of Species Delimitation hellip 39
lichenized fungi and the impact of alternative speciescircumscriptions and rates of molecular evolution ondivergence estimates PLoS ONE 8(12)e85240doi101371journalpone0085240
Leavitt SD Esslinger TL Hansen ES Divakar PK CrespoA Loomis BF Lumbsch HT (2014) DNA barcoding ofbrown Parmeliae (Parmeliaceae) species a molecularapproach for accurate specimen identification empha-sizing species in Greenland Organ Divers Evol 14(1)11ndash20 doi101007s13127-013-0147-1
Leliaert F Verbruggen H Vanormelingen P Steen FLopez-Bautista JM Zuccarello GC De Clerck O(2014) DNA-based species delimitation in algae Eur JPhycol 49(2) doi101080096702622014904524
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Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
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Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
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Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
Mayden RL (1997) A hierarchy of species concepts thedenouement in the saga of the species problem InClaridge MF Dawah HA Wilson MR (eds) Speciesthe units of biodiversity Chapman amp Hall Londonpp 381ndash424
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McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
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40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
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Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
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Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
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OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
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Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
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Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
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Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
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Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
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Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
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Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
morphological characters Antarctic Sci 22 (SpecialIssue 06)727ndash741 doi101017S0954102010000477
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Seacuterusiaux E Villarreal AJC Wheeler T Goffinet B(2011) Recent origin active speciation and dispersalfor the lichen genus Nephroma (Peltigerales) inMacaronesia J Biogeogr 38(6)1138ndash1151 doi101111j1365-2699201002469x
Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
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Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
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Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
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Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
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Westberg M Arup U Kaumlrnefelt I (2007) Phylogeneticstudies in the Candelariaceae (lichenized Ascomy-cota) based on nuclear ITS DNA sequence dataMycol Res 111(11)1277ndash1284 doi101017S0953756204002102
Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
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(Lepidoptera Lycaenidae) Front Zoo 4(1)8 doi1011861742-9994-4-8
Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
de Queiroz 2007 Hey 2006 Donoghue andGauthier 2004 Cracraft 1983 Mishler andBrandon 1987 Mayr 1970) Over two-dozendifferent and at least partially incompatible spe-cies concepts have been proposed each based ondistinct biological properties eg differences ingenetic or morphological features adaptivezones or ecological niches mate recognitionsystems reproductive compatibility monophylyetc (de Queiroz 2007 Mayden 1997) Hausdorf(2011) argues that most species concepts areuseable but acceptance of a specific conceptrequires an appropriate adaptation of the termldquospeciesrdquo and of species delimitation In contrastde Queiroz (1998) and Mayden (1997) argue thatdistinct species concepts emphasize differentproperties of species rather than fundamentalconceptual differences and all modern speciesconcepts share an important commonalitymdashequating species with segments of metapopula-tion lineages This ldquogeneral lineage conceptrdquo(GLC de Queiroz 1999) highlights that no singleproperty should be regarded as defining for therecognition of species apart from forming lin-eages (Simpson 1951) and segments of meta-population lineages (ie ldquospeciesrdquo) may existregardless of our ability to empirically delimitthem (Camargo and Sites 2013)
We concur that the GLC provides a practicalsolution to the species concept impasse and ourdiscussion of species delimitation is based on theGLC Arguably the major implication of the GLCis that most of the earlier species concepts shouldbe regarded as secondary species ldquocriteriardquo ratherthan ldquoconceptsrdquo that can provide evidence oflineages separation (Sites and Marshall 2003Camargo and Sites 2013 Mayden 1997 de Que-iroz 2007) This pivotal distinction disentanglesthe conceptual issues of defining ldquospeciesrdquo frommethodological issues of delimiting speciesboundaries (Camargo and Sites 2013) The GLCallows researchers to delimit species using differ-ent empirical properties and facilitates the devel-opment of new methods to test hypotheses oflineage separation (de Queiroz 2007) Althoughdifferent datasets and operational criteria may giveconflicting or ambiguous results due to multipleevolutionary processes occurring within and
between populations (eg Miralles and Vences2013 Satler et al 2013) the use of several inde-pendent suites of characters such as genetic datamorphology geographic range host preferencechemistry and cross-validation using inferencesfrom multiple empirical operational criteria canprovide robust hypotheses of species boundaries(Carstens et al 2013 Fujita et al 2012)
212 Species in Lichenized FungiCases of Cryptic DiversityPolymorphic Lineagesand Striking BiogeographicPatterns
Similar to most major biological groups includ-ing birds (McKay et al 2013) fish (Wagner et al2013) plants (Griffin and Hoffmann 2014)arthropods (Schlick-Steiner et al 2010 Moreau2009) and many others finding and applying theappropriate character sets and analytical tools isone of the greatest challenges with empiricalspecies delimitation in lichen-forming fungi(Lumbsch and Leavitt 2011) Understanding thedifferences between morphological variationwithin a species and among closely related groupsis central to identifying diagnostic charactersrequired for establishing accurate phenotype-based taxonomic boundaries However in prac-tice a clear demarcation between intraspecificand interspecific variation is commonly subject toobservational bias and individual interpretation
Traditionally differences in morphologicalchemical and ecological features have been thepredominant source of diagnostic taxonomiccharacters for circumscribing lichen-formingfungal species (Printzen 2009) However lichensgenerally display few taxonomically useful char-acters relative to other groups (eg vascularplants vertebrates and arthropods) (Printzen2009) and varying levels of intraspecific variationamong different species groups may confoundaccurate taxonomic circumscriptions While somespecies may have little variation high levels ofintraspecific phenotypic variation are well docu-mented in some lichen-forming fungi (eg Xant-hoparmelia Hale 1990) Therefore molecular
2 The Dynamic Discipline of Species Delimitation hellip 13
genetic data now play a prominent role in delim-iting fungal species and understanding evolu-tionary relationships in lichens (Lumbsch andLeavitt 2011 Printzen 2009)
Arguably the use of molecular data has led toan improved perspective on the taxonomic valueof many phenotypic characters in lichenized fungiand species delimitation in general Cryptic spe-cies-level lineages are commonly identified usingmolecular data and in some cases these previ-ously unrecognized lineages are corroborated byformerly overlooked phenotypic characters (Pino-Bodas et al 2012a Spribille et al 2011 Divakaret al 2010 Arguumlello et al 2007) Other studieshave revealed the fact that some species-levellineages are likely comprised of chemically andmorphologically polymorphic individuals whichare conventionally considered as separate species(eg Leavitt et al 2011a Pino-Bodas et al 2011Velmala et al 2009) While these studies high-light the limitations of using traditional taxo-nomic characters for distinguishing naturalgroups in lichen-forming fungi they also providea valuable perspective on the importance ofongoing research in even the best-studied lichengroups Furthermore an improved perspective ofspecies boundaries has led to a strikingimprovement in understanding diversification anddistribution in many groups of lichenized fungi
Traditional phenotype-based approaches tospecies recognition appear to vastly underesti-mate diversity in lichen-forming fungi Whilemolecular research has corroborated traditionalphenotype-based hypotheses of species bound-aries in a number of cases studies repeatedlydemonstrate that our current interpretation ofmorphological and chemical characters oftenfails to accurately characterize species-leveldiversity (reviewed in Lumbsch and Leavitt2011) A growing body of evidence reveals that asignificant proportion of unknown diversityestimated for fungi including lichen-formingfungi is hidden under names of supposedlycommon and widespread species For exampleapproximately 80 unrecognized species-levellineages are estimated to occur in Parmeliaceae(Crespo and Lumbsch 2010) Even higher levels
of unrecognized species diversity are estimatedto occur in other families such as Graphidaceae(Rivas Plata and Luumlcking 2013 Rivas Plata andLumbsch 2011 Luumlcking 2012)
The topic of cryptic species cases wheretwo or more distinct species-level lineages areerroneously classified (and hidden) under onenominal taxon (Bickford et al 2007) has beenfrequently reviewed for lichen-forming fungi(Lumbsch and Leavitt 2011 Crespo and Lum-bsch 2010 Crespo and Peacuterez-Ortega 2009Printzen 2009) Although novel diagnostic phe-notypic characters may potentially be identifiedcorroborating ldquocrypticrdquo species these previouslyunrecognized lineages generally remain difficultto classify within a traditional phenotype-basedtaxonomy (Leavitt et al 2013c d) In somecases it appears that similar phenotypic charac-ters may arise in parallel at local or regionalscales but may not be correlated with naturalgroups or genetic isolation (Muggia et al 2014Rivas Plata and Lumbsch 2011 Grube andHawksworth 2007) For example in the cosmo-politan species Tephromela atra (Fig 21) up to15 independent lineages were identified usingphylogenetic analyses of molecular sequencedata However the continuum of morphologicaland chemical variability in the T atra complexcurrently prevents the description of new speciesusing traditional phenotype-based characters(Muggia et al 2014)
Recent research on the genus Cladonia(Cladoniaceae) highlights a fitting example of thecomplexities associated within using phenotypiccharacters for delimiting species in lichen-forming fungi (Fig 21 Pino-Bodas et al 20112012a b 2013a b) In the Cladonia gracilisgroup most currently accepted species were notrecovered as monophyletic clades and traditionaldiagnostic morphological characters were shownto be highly homoplasious (Pino-Bodas et al2011) Similarly C iberica and C subturgidahave been shown to constitute a single morpho-logically and chemically polymorphic species(Pino-Bodas et al 2012b) Similar patterns ofhigh degrees of morphological and chemicalpolymorphisms have also been observed in the
14 SD Leavitt et al
C cariosa group (Pino-Bodas et al 2012a) andC humilis species complex (Pino-Bodas et al2013a) High levels of intraspecific morphologi-cal and chemical polymorphisms are not restric-ted to Cladonia A clear demarcation betweenintraspecific and interspecific morphological andor chemical variation does not exist for a largeproportion of Xanthoparmelia species in westernNorth America and high intraspecific morpho-logical and chemical variation are common for anumber of species-level genetic groups (Fig 21
Leavitt et al 2011b c 2013e) In some cases asmany as eight traditionally circumscribed Xant-hoparmelia species were recovered in a singlespecies-level genetic group (Leavitt et al 2011c)High levels of intraspecific phenotypic variationhave been observed in a number of other generain Parmeliaceae including Bryoria (Velmalaet al 2009) Cetraria (Peacuterez-Ortega et al 2012)Vulpicida (Mark et al 2012) and others
The importance of biogeography in lichen-forming fungal evolution has remained somewhat
Fig 21 Examples of common lichens in which tradi-tional morphology-based species circumscriptions fail toreflect natural species-level fungal lineages a Cladoniagracilis photographed in the Clearwater Valley BritishColumbia Canada (see Pino-Bodas et al 2011) (photo-graph creditCurtis Bjoumlrk) bR shushanii a member of theRhizoplaca melanophthalma species group from the
Aquarius Plateau Utah USA (see Leavitt et al 2011a)(photograph credit S Leavitt) c Xanthoparmelia affwyomingica occurring in Coloradorsquos Front Range USA(see Leavitt et al 2011b c Leavitt et al 2013e) (photo-graph credit S Leavitt) d Tephromela atra sensu latofound in the Santa Monica Mountains California USA(see Muggia et al 2014) (photograph credit J Hollinger)
2 The Dynamic Discipline of Species Delimitation hellip 15
ambiguous due to the occurrence of phenotypi-cally similar lichens occurring across broadintercontinental distributions and uncertainty ofappropriate species circumscriptions While anumber of lichen-forming fungal species havebeen found to be truly widespread (eg Lindblomand Soslashchting 2008 Fernaacutendez-Mendoza et al2011 Ahti and Hawksworth 2005 Del-Pradoet al 2013) improved recognition of speciesboundaries has provided insight into importantbiogeographical patterns in lichens previouslyassumed to have cosmopolitan distribution pat-terns (Leavitt et al 2013d Del-Prado et al 2013Amo de Paz et al 2012 Seacuterusiaux et al 2011Otaacutelora et al 2010 Elix et al 2009) Crypticdiversity and complex biogeographic patterns arehighlighted in the Rhizoplaca melanophthalmaspecies group (Fig 21 Leavitt et al 2013d)Analyses of R melanophthalma sensu lato col-lected from five continents supported the presenceof cryptic species within this complex Two ofthese lineages were found to have broad inter-continental distributions while the other four werelimited to western North America (Leavitt et al2013d) Most strikingly of the six lineages fivewere found on a single mountain in the westernUSA and the sixth occurred no more than 200 kmaway from this mountain A recent study ofPseudocyphellaria (Lobariaceae) sensu lato inHawaii revealed a surprising number of previouslyunrecognized species hidden within nominal taxawith putative broad geographic distributionssuggesting a high degree of endemism in Hawaii(Moncada et al 2014) These studies providecrucial impetus to reevaluate species boundaries inorder to improve our understanding of diversitydistributions and evolution in lichenized fungi
22 A Practical Guideto Contemporary SpeciesDelimitation
As it has become clear that conventional phe-notype-based criteria for species circumscriptionsare often problematic (Bickford et al 2007)molecular data are thereby particularly valuablefor assessing traditional species boundaries and
for species delimitation in general (Lumbsch andLeavitt 2011 Fujita et al 2012) Below weprovide an overview of relevant operationalspecies delimitation methods the majority ofwhich are reliant on molecular data coupled withmolecular phylogenetic and population geneticanalyses Assuming that species do in fact rep-resent ldquosegments of metapopulation lineagesrdquo (deQueiroz 1998) direct genetic evidence of lineagestatus is particularly relevant to species delimi-tation studies when analyzed within a rigorousstatistical framework regardless of whether lin-eages differ in phenotypic characters that areapparent to human observers (Fujita et al 2012)This perspective should not be taken as supportfor disregarding phenotypic characters in speciesdelimitation studies (Edwards and Knowles2014 Fujita et al 2012 Yeates et al 2011)Rather hypotheses of species boundaries shouldbe considered more robust with increasing cor-roboration from independent data sources (iemolecular chemistry morphology ecologyetc) and the integration of independent data forempirical species delimitation studies should be amajor focus of species delimitation research(Fujita et al 2012)
Themajority of molecular phylogenetic studiesof lichenized fungi focus on generating sequencedata from a number of specimens representing thefocal group inferring a gene tree from the geneticdata matrix and assessing the monophyly of thesampled taxa This approach has provided valu-able acumen into evolutionary relationships thevalue of morphological characters for taxonomyand insight into diversity of lichenized fungi(Thell et al 2009 Reese Naeligsborg et al 2007Westberg et al 2007 Martiacuten et al 2003 Arup andGrube 2000 Lohtander et al 2000 Stenroos andDePriest 1998) However simply assessing themonophyly of traditional phenotype-based spe-cies often offers an incomplete perspective onspecies boundaries Studies explicitly designed forempirically delimiting species are pivotal toadvancing our understanding of speciation andspecies diversity in lichens For example althougha nominal species may be recovered as mono-phyletic in a gene tree intraspecific phylogeneticsubstructure may correspond to evolutionarily
16 SD Leavitt et al
independent lineages (eg Leavitt et al 2011a)As a construct of taxonomy recognizing amonophyletic clade comprised of multiple mor-phological indistinguishable species-level lin-eages as a single species may hold some appealhowever failing to formally recognize this diver-sity can have far-reaching implications in ourbiological (eg ecology evolution reproduction)interpretation of the group Alternatively well-supported intraspecific phylogenetic substructuremay be the result of stochastic evolutionary pro-cesses uniparental inheritance etc rather thanevolutionary independence Interpreting this typeof phylogenetic substructure as species-leveldiversity can introduce detrimental bias
Empirical species delimitation methods havebroadly focused on four main areas detectingputative species individual specimen assignmentto a species group or operational taxonomic unit(OTU) validation of candidate species or OTUsas evolutionarily distinct lineages and inferringspecies relationships (ie species tree inference)Ideally operational species delimitation criteriashould be based on explicit statistical protocolsin order to objectively assess species boundariesand minimize the need of subjective interpreta-tions or taxonomic expertise In this section weprovide a brief overview on a number of empir-ical species delimitation methods that to varyingdegrees fit these criteria For convenience wedivide these methods into three general catego-ries (i) species discovery methods for assigningsamples to populations without a priori infor-mation (ii) species validation approaches andcoestimating individual assignments and speciestrees and (iii) species delimitation using geno-mic data (Table 21)
Empirical species delimitation has receivedincreasing attention including ongoing develop-ment of bioinformatical approaches and themethods and programs provided in this chapterare by no means intended to be a comprehensivelist of all available analytical approaches Ratherthe methods provided here have been shown tobe useful in a number of previous studies orshow particular promise for future speciesdelimitation studies Our aim is that these can
serve as a starting point when designing studiesto assess species limits Not surprisingly variousapproaches to species delimitation may yielddifferent estimates of species boundaries and theresearcher may be required to make some degreeof subjective interpretation of the most biologi-cally appropriate species boundaries Comple-mentarily recently developed metrics forquantifying the congruence between two taxo-nomies (Ctax) and the potential for an approach tocapture a high number of species boundaries(Rtax) provide a means to objectively assess dis-crepancies among species delimitation methods(Miralles and Vences 2013) More sophisticatedapproaches including selection of speciesdelimitation models using approximate Bayesiancomputing (Camargo et al 2012 Fan andKubatko 2011 Beaumont et al 2010) anddesigning and conducting a simulation study thatmatches the characteristics of the empirical study(Carstens et al 2013 Camargo et al 2012) canbe used to more objectively evaluate competinghypotheses of species boundaries In most con-texts it is likely better to fail to delimit speciesthan it is to falsely circumscribe entities that donot represent actual species and therefore theinferences drawn from species delimitation stud-ies should generally be conservative (Carstenset al 2013 Miralles and Vences 2013)
221 Corroborating TraditionalTaxonomy and DiscoveringCryptic Species Using Single-Locus Data
Objectively defining a threshold separatingintraspecific population substructure from inter-specific divergence is the general aim of speciesdelimitation studies using single-marker datasetsMost species delimitation methods based onsingle-locus sequence data fall under two generalcategories either genetic distance or tree-basedapproaches (Sites and Marshall 2004) Distance-based approaches attempt to detect a differencebetween intra- and interspecific distances (ieldquobarcode gaprdquo) where the pairwise genetic
2 The Dynamic Discipline of Species Delimitation hellip 17
Table
21
Somemetho
dsused
forspeciesdelim
itatio
ninclud
ingspeciesdiscov
erymetho
dsforassign
ingsamples
topo
pulatio
nswith
outapriori
inform
ation(BAPS
Gaussianclustering
Guillo
trsquosUnified
Mod
elS
TRUCTURES
TRUCTURAMA)genetic
distance-based
metho
dforsortingsequ
encesinto
hypo
theticalspecies(A
BGD)tree-
basedspeciesdiscov
erymetho
ds(bGMYCG
MYCb
PTP
PTP
ldquoSpecies
Delim
itatio
nrdquoplug
-inforGeneiou
s)and
jointdiscov
eryandvalid
ationmetho
ds(BPamp
PBrownie
DISSE
CTSp
eDeStem)
Metho
dDescriptio
nInpu
tdata
BAPS
mdashpo
pulatio
nassignmentusing
Bayesianclustering
Aprogram
forBayesianinferenceof
thegenetic
structurein
apopulatio
nclustering
molecular
data
andperformingadmixture
analysesGenetic
mixture
analyses
canbe
performed
atbo
thgroupandindividu
allevelsusingeither
ano
n-spatialo
rspatialm
odel
BAPS
treatsboth
theallele
frequenciesof
themolecular
markers
(ornucleotid
efrequenciesforDNA
sequ
ence
data)andthenu
mberof
genetically
diverged
groups
inpopulatio
nas
random
variablesIn
theldquoclusteringwith
linkedlocirdquomod
elagenetic
mixture
analysiscanbe
done
usinghaploidsequ
ence
data
orotherlin
kedgenetic
markersA
nalysesandmodel
comparisons
canalso
beperformed
usingafixednu
mber
ofgenetically
diverged
grou
psor
prespecified
populatio
nstructures
Limita
tions
Tem
poraldivergence
andrelatio
nships
amongputativ
egroups
arenot
explicitlyestim
atedE
quivalence
togenetic
clusters
tospecies-levelg
roupsisuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
Availablefrom
httpwwwhelsinkifi
bsgsoftwareBAPS
describedin
Coran-
deret
al(200
420
0620
08)CoranderandMarttinen20
06)
Genotyp
icdatahaploidsequence
dataor
linkedmarkers
(AFL
Por
SNPs)
Gaussianclusteringmdashpo
pulatio
nassignment
usingGaussianclustering
Aprogram
forBayesianinferenceof
thegenetic
structurein
apopulatio
nModelgroups
sampleinto
popu
latio
nsusinggeno
typicdata
bysearchingforclusters
that
canbe
attributed
tobeingmixturesof
norm
alallelefrequencydistributio
nsG
aussianclustering
requires
adatasetwhere
thecasesaredefinedby
variable
ofmetricscaleandhasbeen
used
with
geneticenvironmentalandmorphologicaldatasetsindividuallyinadditio
nto
integrated
datasets
Limita
tions
Tem
poraldivergence
andrelatio
nships
amongputativ
egroups
arenot
explicitlyestim
atedE
quivalence
togenetic
clusters
tospecies-levelg
roupsisuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
Implem
entedin
Rusingtheprabclus
(HausdorfandHennig20
10)andmclust
packages
(FraleyandRaftery
2007)
Genotyp
icdata
(flexible)
(con
tinued)
18 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
Guillo
trsquosUnified
Mod
elmdashpo
pulatio
nassignmentusingBayesianclustering
Thisapproach
provides
astatistical
model
that
allowsoneto
analyzegenetic
and
phenotyp
ticdata
with
inaun
ified
mod
elandinferencefram
eworkandop
tionally
incorporateinform
ationaboutthespatialdistributio
nof
samplesA
Bayesianclustering
algorithm
assumes
that
each
clusterin
ageographical
domaincanbe
approxim
ated
bypo
lygons
that
arecentered
around
pointsgeneratedby
aPo
issonprocessGuillo
trsquos
Unified
Mod
elisflexiblein
term
sof
thegenetic
data
that
itcanutilize
andcapableof
accurately
delim
iting
species
Limita
tions
Genetic
andphenotypic
data
cantracedifferentevolutionary
historiesfor
instancephylogenetic
divergence
forneutralgenetic
markers
andadaptatio
nfora
morphological
structure
Source
Availableas
anextensionof
theRGENELAND
package(G
uillo
tet
al20
05
2012)(http
www2im
mdtudk
gigu
Geneland)
Genotyp
icandno
n-genetic
(eg
phenotypicalecolog
icalgeograph
ical
behavioral)data
STRUCTUREmdash
populatio
nassignment
usingBayesianclustering
Amodel-based
clustering
methodusingmultilocus
genotype
data
toinferpopulatio
nstructureandassign
individualsto
populatio
nsIndividualsin
thesampleareassigned
probabilistically
topopulatio
nso
rjointly
totwoor
morepopulatio
nsiftheirgenotypes
indicatethatthey
areadmixedT
hemodeldoes
notassum
eaparticular
mutationprocess
anditcanbe
appliedto
mosto
fthecommonly
used
genetic
markersp
rovidedthat
they
areno
tcloselylin
ked
The
methodcanproducehigh
lyaccurate
assignmentsusing
modestn
umbersof
loci(Pritchard
etal2
000)T
hemostappropriatelevelo
fpopulatio
nstructurecanbe
inferred
byassessinglik
elihoodscores
orthead
hocΔKstatistic
(Evann
oet
al20
05)
Limita
tions
Identifying
themostappropriatenumberof
genetic
clusters
ischallenging
clusters
produced
byST
RUCTUREcanbe
strongly
influenced
byvariationin
sample
sizeclusters
createdby
STRUCTUREmay
notbe
consistent
with
theevolutionary
historyof
thepopulatio
nswhentherearerelativ
elylong
divergence
times
with
inevolutionary
lineagesTem
poraldivergence
andrelatio
nships
amongputativ
egroups
isnotexplicitlyestim
atedEquivalence
togenetic
clusters
tospecies-levelgroups
isuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
httppritchardlabstanfordedustructurehtm
ldescribedin
Falush
etal(200
3)andPritchard
etal(200
0)
Genotyp
icdata
(con
tinued)
2 The Dynamic Discipline of Species Delimitation hellip 19
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
Structuram
amdashpo
pulatio
nassignmentusing
Bayesianclustering
Implem
entstheclustering
algorithm
used
inST
RUCTURE(see
above)
that
clusters
samples
into
populatio
nsby
minim
izingHardyndashWeinb
ergdisequ
ilibrium
foragiven
partitioninglevelHow
everStructuram
aalso
includes
theadditio
nof
reversible-jum
pMCMCto
identifytheop
timalpartition
inglevelNearlyanytype
ofgenetic
datacanbe
inpu
tinto
Structuram
aandtheprog
ram
canassign
individu
alsto
populatio
nwith
orwith
outtheadmixture
Limita
tions
Tem
poraldivergence
andrelatio
nships
amongputativ
egroups
isnot
explicitlyestim
atedE
quivalence
togenetic
clusters
tospecies-levelg
roupsisuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
httpctegberkeleyedu
structuram
aindexhtmldescribedin
Huelsenbeck
etal(201
1)
Genotyp
icdata
ABGDmdashbarcodegapusinggenetic
distances
ldquoAutom
aticBarcode
Gap
Discoveryrdquosortssequencesinto
hypotheticalspeciesbasedon
thebarcodegap
The
methoduses
arecursiveapproach
topartition
thedata
andtestfor
sign
ificant
gapsA
BGDisfastsim
plemethodto
split
asequence
alignm
entd
atasetinto
candidatespeciesthat
should
becomplem
entedwith
otherevidence
inan
integrative
taxono
mic
approach
Limita
tions
Single-locus
data
aloneshould
only
beused
toprovideaprelim
inary
perspectiveof
speciesboundaries
andnotas
thesole
evidence
inspecies
circum
scriptions
Source
httpwwwabisnv
jussieufrpublicabg
ddescribedin
Puillandreet
al(201
2)
Single-locus
sequence
alignm
ent
GMYCampbG
MYCmdashgene
tree
The
GMYC
approach
combinesacoalescent
model
ofintraspecificbranchingwith
aYulemod
elforinterspecificbranching
which
isthen
fitto
aninferred
sing
le-gene
topology
toestim
atespeciesboundaries
andastatistical
measure
ofconfi
denceforthe
inferred
boundariesA
san
inputtheGMYCapproach
requires
anultram
etricgene
tree
andrecent
refinementscanaccountforuncertaintyin
phylogenetic
relatio
nships
and
parametersof
theGMYCmod
elT
heGMYCisgenerally
stable
across
awiderangeof
circum
stancesincludingvariousmethods
ofphylogeneticreconstructio
nthepresence
ofahigh
numbersingletonshigh
numbers
ofsampled
speciesandgaps
inintraspecific
sampling
theaccuracy
oftheGMYCismostsign
ificantly
affected
bythemean
populatio
nsize
relativ
eto
divergence
times
betweenthem
Limita
tions
The
GMYCmay
delim
itwell-supportedclades
ofhaplotypes
asindependent
lineagesandas
such
may
beproneto
over-delim
itatio
nSingle-locus
data
aloneshould
only
beused
toprov
ideaprelim
inaryperspectiveof
speciesbo
undaries
andno
tas
the
sole
evidence
inspeciescircum
scriptions
Source
(http
r-forger-projecto
rgprojectssplitshttpssitesgooglecom
site
noahmreidhom
esoftware)describedin
Monaghanet
al(200
9)Fu
jisaw
aandBarrac-
loug
h(201
3)andPo
nset
al(200
6)
Sing
le-locus
ultram
etricgene
tree (con
tinued)
20 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
PTPamp
bPTPmdash
gene
tree
The
Poissontree
processes(PTP)
modelcanbe
used
toinferputativ
especiesboundaries
onagivenphylogenetic
inputtreePT
Pcaninferputativ
especiesboundaries
that
are
consistent
with
thePS
CAnim
portantadvantageof
thismethodisthat
itmod
els
speciatio
nin
term
sof
thenumberof
substitutionsthereby
circum
ventingthepotentially
error-proneandcompute-intensive
processof
generatin
gultram
etrictreeswhich
are
required
asan
inpu
tfor
GMYCmod
el(see
abov
e)F
urthermoreitappearsthatthePT
Pmodel
may
outperform
theGMYCandotherOTU-picking
methods
whenevolutionary
distancesaresm
all
Limita
tions
Single-locus
data
aloneshould
only
beused
toprovideaprelim
inary
perspectiveof
speciesboundaries
andnotas
thesole
evidence
inspecies
circum
scriptions
Source
httpscoh-itsorgexelix
iswebsoftwarePT
Pindexhtmldescribedin
Zhang
etal(201
3)
Sing
le-locus
gene
tree
GeneiousSp
eciesDelim
itatio
nplug
-inmdash
gene
tree
Aplug-into
theGeneioussoftwareprovides
anexploratorytool
allowingtheuser
toassess
phylog
enetic
supportanddiagno
sabilityof
speciesdefinedas
user-selected
collections
oftaxa
onuser-sup
pliedtreesThe
plug
-incompu
tesstatisticsrelatin
gto
the
probability
oftheob
served
mon
ophyly
orexclusivity
having
occurred
bychance
ina
coalescent
processandassesses
thewith
in-andbetween-speciesgenetic
distancesto
infertheprobability
with
which
mem
bers
ofaputativ
especiesmight
beidentifi
edsuccessfully
with
tree-based
methods
Limita
tions
The
plug-insummarizes
measuresof
phylogenetic
supportand
diagnosabilityof
speciesdefinedas
user-selectedcollections
oftaxabutitdoes
not
providedefinitiv
esupportforspeciesgroupsItassumes
speciesaremonophyletic
Source
Implem
entedas
aplug-into
Geneious(geneiouscom)describedin
Mastersetal
(201
1)
Sing
le-locus
gene
tree
BPamp
Pmdashmultispecies
coalescent
model
for
speciesvalid
ation
Thisapproach
tospeciesdelim
itatio
nuses
aBayesianmodelingapproach
togeneratethe
posteriorprobabilitiesof
speciesassignmentstaking
accountof
uncertaintiesdueto
unknow
ngene
treesandtheancestralcoalescent
processThe
methodrelieson
auser-
specified
guidetreeimplem
entin
gareversible-jum
pMarkovchainMonte
Carlo
search
ofparameter
spacethatincludes
θpopulatio
ndivergenceand
estim
ated
distributio
nsof
gene
treesfrom
multip
leloci
Limita
tions
Misspecificatio
nsof
priorsandthegu
idetree
canresultin
inflatedspeciatio
nprobabilitiesitassumes
norecombinatio
nandcomputatio
nallim
itatio
nsrestrict
itsutility
with
next-generationdatasetswith
100s
ofloci
Source
httpabacusgeneuclacuksoftwarehtmldescribedin
YangandRannala
(201
0)andRannala
andYang(200
3)
Multilocus
sequence
alignm
entsandgroup
mem
bership
(con
tinued)
2 The Dynamic Discipline of Species Delimitation hellip 21
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
DISSE
CTmdashmultispecies
coalescent
model
forspeciesdelim
itatio
nDISSE
CTexplores
thefullspaceof
possible
clusteringsof
individualsandspeciestree
topologies
inaBayesianfram
ework
Toavoidtheneed
forreversible-jum
pMCMCit
uses
anapproxim
ationin
theform
ofapriorthatisamodificatio
nof
thebirthndashdeathprior
forthespeciestreeItisim
plem
entedas
partof
BEAST
andrequires
only
afewchanges
from
astandard
BEAST
analysisA
nalysesof
simulated
andem
piricald
atasuggestthat
themethodisshow
nto
beinsensitive
tothedegree
ofapproxim
ation
butq
uitesensitive
tootherparameters
Limita
tions
Recently
describedmethodlackingathorough
theoretical
andem
pirical
evaluatio
nItappearsthat
alargenu
mberof
sequencesarerequ
ired
todraw
firm
conclusion
sSo
urce
httpcodegooglecompbeast-m
cmcamphttpwwwin
driid
com
dissectinbeast
htmldescribedin
JonesandOxelm
an(201
4)
Multilocus
sequence
data
SpeD
eSTEMmdashmultispecies
coalescent
mod
elfordiscov
ery
valid
ation
andjoint
estim
ation
Thismaxim
umlik
elihoo
dandorinform
ationtheory-based
methodwas
developedto
test
speciesboundaries
inasystem
with
existin
gsubspecies
taxonomy(CarstensandDew
ey20
10)andcomputestheprobability
ofthegene
treesgiventhespeciestree
forall
hierarchical
perm
utations
oflin
eage
grou
pingSp
eciesbo
undaries
arecomparedusing
Akaikeinform
ationcriteria
andphylogenetic
uncertaintyin
thespeciestree
topologies
does
notaffect
speciesdelim
itatio
nsLimita
tions
Accuracyisdependenton
quality
ofthegene
tree
estim
ates
Source
(http
carstenslaborgohio-stateedusoftwarehtml)criteria
describedin
Ence
andCarstens(201
1)
Multilocus
sequence
alignm
entsandgroup
mem
bership
Browniemdash
multispecies
coalescent
model
forspeciesdelim
itatio
nThe
nonp
aram
etricheuristic
speciesdelim
itatio
napproach
implem
entedin
theprog
ram
Brownie(O
rsquoMeara
2010)jointly
sortsanonym
oussamples
into
speciesandinfers
aspeciestree
from
inputg
enetreesfrom
differentlociassumingthatforaspeciatio
neven
thecorrespondingnodeson
gene
treeswill
bemoreconsistent
with
each
than
the
divergenceswith
inspecies
Limita
tions
Findingboth
theoptim
umspeciestree
andspeciesboundaries
remains
compu
tatio
nally
challenging
andBrowniehasbeen
show
nto
frequently
yieldincorrect
resultsT
heaccuracy
ofthemethodislik
elycorrelated
with
nodalsupportvalues
inthe
individu
algene
topo
logies
Source
httpwwwbrianom
earain
fobrownie
describedin
OrsquoM
eara
(201
0)
Individual
gene
trees
(con
tinued)
22 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
BFD
mdashmultispecies
coalescent
model
for
speciesdelim
itatio
nThe
recently
developedmethod
Bayes
factor
delim
itatio
n(with
genomicdataB
FD)
combinesady
namic
programmingalgorithm
forestim
atingspeciestreesthat
bypasses
thecompu
tatio
nally
intensiveMCMCintegrationov
ergene
treesto
prov
idearigorous
techniqueforspeciesdelim
itatio
nstudiesusinggenome-wideSN
PdataCom
petin
gspeciesdelim
itatio
nmodelsarecomparedusingBayes
factorsanditappearsthat
this
approach
isrobustto
samplesizes(iefew
loci
andlim
itedsamples
perspecies)
and
misspecificatio
nof
thepriorforpo
pulatio
nsize
(θ)
Limita
tions
Recently
describedmethodlackingathorough
theoretical
andem
pirical
evaluatio
nSo
urce
httpwwwbeast2orgwikiindexphpBFD
describedin
Leacheacuteet
al(201
4)
Genom
e-wideSN
Pdata
2 The Dynamic Discipline of Species Delimitation hellip 23
distances among organisms belonging to thesame species are smaller than distances amongorganisms from different species (Puillandreet al 2012 Hebert et al 2003) Genetic distanceapproaches hold particular promise as an identi-fication tool shortcutting the difficulties of mor-phology-based identification (Hebert et al 2004)although in practice a barcode gap may not existfor many groups (Wiemers and Fiedler 2007)Furthermore the role of distance-based approa-ches using a single genetic marker for speciesdiscovery remains more controversial (Rubinoff2006) and without other corroborating evidenceOTUs inferred from single-locus dataset shouldonly be considered ldquocandidaterdquo species Tree-based methods aim to detect monophyletic cladescorresponding to species-level diversity bydetecting discontinuities associated within inter-and intraspecific branching patterns (Fujisawaand Barraclough 2013 Zhang et al 2013Monaghan et al 2009 Pons et al 2006) Tree-based species delimitation methods can also beused on the basis of other properties related tophylogenetic tree topologies (monophyly con-cordance with geography etc (Sites and Marshall2003 2004) Both distance- and tree-basedmethods have been applied for assessing speciesboundaries in lichen-forming fungi (Leavitt et al2012c 2014 Parnmen et al 2012 Del-Pradoet al 2010 2011 Molina et al 2011)
A number of tree-based methods partiallyautomate the species delimitation process withspecific bioinformatical analyses (Table 21)including the general mixed Yule coalescent(GMYC) approach (Fujisawa and Barraclough2013 Monaghan et al 2009 Pons et al 2006)and the Poisson tree processes (PTP) model(Zhang et al 2013) These methods provide rel-atively straightforward and objective pipelinesfor delimiting putative species-level lineagesfrom inferred gene trees by fitting within- andbetween-species branching models to an inferredsingle-locus topology A number of other tree-based methods for species delimitation areeffectively summarized in Sites and Marshall(2004)
When only single-locus data are available theGMYC has been shown to be a relatively robust
tool for species delimitation The GMYCapproach combines a coalescent model of intra-specific branching with a Yule model for inter-specific branching which is then fit to an inferredsingle-gene topology to estimate species bound-aries and a statistical measure of confidence for theinferred boundaries As an input the GMYCapproach requires an ultrametric gene tree andrecent refinements can account for uncertainty inphylogenetic relationships and parameters of theGMYC model (Fujisawa and Barraclough 2013Reid and Carstens 2012) Regardless of theseimprovements it may be difficult to accuratelyinfer an adequate ultrametric tree for large data-sets Although it appears that the GMYC isgenerally stable across a wide range of circum-stances including various methods of phyloge-netic reconstruction the presence of a highnumber singletons high numbers of sampledspecies and gaps in intraspecific sampling theaccuracy of the GMYC is most significantlyaffected by the mean population size relative todivergence times between them (Fujisawa andBarraclough 2013 Talavera et al 2013) Fur-thermore research suggests that the so-calledsingle-threshold version of the GMYC methodlikely outperforms the multiple-thresholdapproach (Fujisawa and Barraclough 2013Monaghan et al 2009) However other studiessuggest that theGMYCmethodmay often providehigher estimates for the total number ofOTUs thanother molecular species delimitation methods(Hamilton et al 2014 Miralles and Vences 2013Talavera et al 2013) warranting a cautiousinterpretation of results from GMYC analyses
Compared to the GMYC approach therecently introduced PTP method for speciesdelimitation has been suggested to be moreaccurate for preliminary species delimitation(Zhang et al 2013) Relative to the GMYC PTPoffers a more straightforward implementationrequiring a simple phylogenetic tree rather thanan ultrametric chronogram However moreresearch is required to assess the general perfor-mance of PTP across wide range of empirical andsimulated species delimitation studies At thispoint data support the use of the GMYC andPTP methods as objective and reasonable starting
24 SD Leavitt et al
points for species delimitation using single-genetopologies
The ldquoSpecies Delimitationrdquo plug-in to theGeneious software provides statistical approachesfor exploring species boundaries in single-genetopologies (Masters et al 2011) Using a priorispecimen assignments to putative species theldquoSpecies Delimitationrdquo plug-in computes statis-tics relating to the probability of the observedmonophyly or exclusivity having occurred bychance in a coalescent process and assesses thewithin- and between-species genetic distances inorder to infer the probability with which membersof a putative species might be identified suc-cessfully with tree-based methods An importantcontribution of the ldquoSpecies Delimitationrdquo plug-in is that it provides an objective means for usersto assess putative species within an empiricalstatistic-based framework rather than qualitativeevaluations of what level of hierarchy constitutesa species in a phylogeny
In contrast to simple sequence similaritythresholds (OTU-picking) for delimiting putativeevolutionarily significant units the AutomaticBarcode Gap Discovery (ABGD) method is anautomated procedure that sorts sequences intohypothetical species based on the existence of abarcode gap observed whenever intraspecificgenetic distances are less than those amongorganisms from different species (Puillandreet al 2012) Ultimately ABGD is a fast simplemethod that can be used to group individualsrepresented in a single-locus sequence alignmentinto candidate species that should be comple-mented with other lines of evidence in an inte-grative taxonomic approach (Kekkonen andHebert 2014 Puillandre et al 2012)
In general the ABGD GMYC and PTP ana-lytical protocols using single-locus data arerepeatable and computationally relatively fastproviding a valuable starting point for a pre-liminary perspective into species boundaries inunderstudied groups that can be validated withsubsequent studies (Kekkonen and Hebert 2014)Similarly analyses of single-locus data can beused to corroborate traditional phenotype-basedspecies boundaries and identify candidate speciesthat have previously been hidden within nominal
species However single-locus species delimita-tionmethodsmaybeparticularly prone to failure inrecognizing significant proportions of species-level biodiversity due to the strict criterion forreciprocal monophyly or alternatively providespurious inflations of estimated species diversitybased on genetic differences that donot correspondto species-level lineages For example recentestimates suggest that the incidence of species-level gene tree paraphyly is approximately 20 (Ross 2014) suggesting that analyses of single-locus datasets would likely fail to accurately deli-mit species in one in every five cases In contrastrecent empirical studies suggest that in some casesthe GMYC provides a striking overestimate ofspecies diversity (Miralles and Vences 2013)
The internal transcribed spacer region (ITS)has played a central role in molecular phyloge-netic studies of lichenized fungi and has recentlybeen adopted as the official barcoding marker forfungi (Schoch et al 2012) In many cases the ITSis sufficiently variable to resolve species bound-aries for lichenized fungi (Schoch et al 2012Kelly et al 2011) although accurate specimenidentification using sequence-based identificationapproaches requires a thoroughly curated refer-ence database (Leavitt et al 2014 Orock et al2012 Kelly et al 2011) In spite of the generalutility of the ITS marker a number of issues maypotentially limit its effective use in speciesdelimitation studies including the potential forintragenomic variation of the nuclear ribosomaltandem repeat and difficulties in aligning ITSsequences from divergent taxa (Kiss 2012)Because the ITS has been formally adopted as theofficial barcoding marker for fungi we recom-mend that species delimitation studies attempt toinclude this region for consistency across studiesHowever we recognize that in some taxonomicgroups the inclusion of the ITS may be prob-lematic and therefore suggest a careful screeningof other markers to identify appropriate loci forresolving species-level relationships
Ultimately the success of any single-locusspecies delimitation method largely depends onthe evolutionary history of the species groupunder study and the variability of the selectedmarker (Fig 22) Species delimitation will be
2 The Dynamic Discipline of Species Delimitation hellip 25
more difficult in more recently diverged lineagesand in cases with some level of interspecific geneflow relative to older well-diverged lineages(eg Leavitt et al 2012c 2013e) In the end wereemphasize that single-locus data alone shouldonly be used to provide a preliminary perspectiveof species boundaries and not as the sole evi-dence in species circumscriptions
222 Sampling Across the GenomeMultilocus Sequence Dataand Genome-Wide Markers
Although single-locus methods can provide anefficient approach for preliminary large-scaleassessments of species diversity there are
significant limitations particularly in recentlydiverged species groups where retention ofancestral polymorphisms and incomplete lineagesorting will likely result in different neutral locihaving unique gene topologies that do not mirrorthe speciation process (Knowles and Carstens2007 Heled and Drummond 2010 Taylor et al2000) In contrast to single-locus and strictlyphenotype-based approaches for species delimi-tation analyses of genetic data collected fromindependent genomic regions can provide robusthypotheses of species boundaries with increasingconfidence (Satler et al 2013 Zhang et al 2011Yang and Rannala 2010) Sequences from mul-tiple independent loci provide an importantsource of data for species delimitation studiesincluding recently developed models that
Fig 22 A simplified diagram illustrating the process ofspeciation through time in a single gene history andresulting gene topologies sampled at two points in thespeciation history (modified from Leliaert et al 2014)aEach dot represents a distinct gene copy and each row onenon-overlapping generation with lines connecting genecopies to their ancestors in the previous generation (onerow below) dashed diagonal lines represent reproductivebarriers two hypothetical sampling intervals at differentpoints in the speciation history are shown Tx and Ty
species delimitation methods using single-locus data areeffective only when species are reciprocally monophyleticin the sampled gene tree genetic clustering and coalescent-based species delimitation methods can circumscribespecies when species may not be monophyletic in sampledgenetic loci b Gene topology representing sampledhaplotypes at time Tx (shown in panel a) hypotheticalspecies are reciprocally monophyletic c Gene topologyrepresenting sampled haplotypes at time Ty (shown in panela) hypothetical species are para- and polyphyletic
26 SD Leavitt et al
combine individual gene genealogies and speciesphylogenies via modeling the coalescent historyof markers (Yang and Rannala 2010 Edwards2009 Knowles and Carstens 2007) As aresponse to advances in sequencing technologiesbioinformatical approaches for multimarker spe-cies delimitation analyses continue to be devel-oped (Table 21 Camargo and Sites 2013)
Long-term reproductive isolation of candidatespecies can be assessed with multilocus sequencedata by evaluating genealogical concordance ofunlinked markers (Baum and Shaw 1995 Aviseand Ball 1990) Within species the mixingeffects of recombination cause unlinked loci tohave distinct genealogical histories but geneticdrift and long-term divergence leads to concor-dant genealogical histories at loci across thegenome Relationships of individuals belongingto distinct candidate species can be evaluatedusing gene genealogies (eg haplotype networksor single-gene topologies) to identify lineagesthat exhibit genealogical exclusivity acrossunlinked neutral loci (Hudson and Coyne 2002Dettman et al 2003b Avise and Ball 1990) Thepresence of the same clades in the majority ofsingle-locus genealogies is taken as evidence thatthe clades represent reproductively isolated lin-eages (Dettman et al 2003a Pringle et al 2005)In practice the criteria of reciprocal monophylyand genealogical concordance of unlinked lociprovide a conservative approach for assessingspecies boundaries due to the fact that a sub-stantial amount of time is required after the initialdivergence of species until ancestral polymor-phisms have fully sorted (Knowles and Carstens2007 Hudson and Coyne 2002) Consequentlygroups with recent divergence histories willlikely go undiscovered under a genealogicalconcordance criterion due to the fact that speciesboundaries likely are not reflected in genegenealogies For example it would take morethan 1 million years after speciation before spe-cies would be delimited if 15 loci were sampledin species with an effective population size (Ne)of 100000 assuming one generation a yearunder a strict reciprocal monophyly criterion(Knowles and Carstens 2007) The amount oftime required for a species to be recognized using
a criterion of reciprocal monophyly increasesproportionally with increasing population sizes(Hudson and Coyne 2002) In a number ofstudies of lichen-forming fungi a genealogicalconcordance criterion has been used to bothdelimit previously unrecognized species-levellineages and validate some conventional pheno-type-based species (Leavitt et al 2012a c2013b Molina et al 2011 Kroken and Taylor2001)
Although analyses of molecular sequence datarelying on reciprocal monophyly or fixed char-acter differences for species delimitation canserve as important criteria for identifying speciesthese criteria are not commonly met acrossmultiple loci particularly in recent speciationhistories (Fig 22) To accommodate theobserved conflict among genealogies from mul-tiple loci with the underlying speciation historythe recent merge of coalescent theory with phy-logenetics has driven a major paradigm shift inspecies delimitation and molecular systematics ingeneral (Fujita et al 2012 Edwards 2009) Themultispecies coalescent model can be applied togenealogical histories from multiple independentloci to assemble separate coalescent processesoccurring in populations into a species tree(Degnan and Rosenberg 2009 Rannala andYang 2003) Within this coalescent-basedframework multiple individuals can be assignedto a single speciespopulation and the speciationhistory of ancestral and descendant species-levellineages can be inferred as a ldquospecies treerdquo incontrast to estimating gene genealogies fromindividual samples (Degnan and Rosenberg2006 2009 Rannala and Yang 2003)
A number of multispecies coalescent-basedspecies delimitation methods have recently beenintroduced offering an exciting framework forempirically assessing species boundaries byselecting the best species tree model from a set ofalternative models representing differenthypotheses of species boundaries (Table 21)For example SpeDeSTEM (Ence and Carstens2011) finds the maximum likelihood values for aspecies tree assuming all putative species areseparate lineages and for alternative species treeswhere two or more species are collapsed into a
2 The Dynamic Discipline of Species Delimitation hellip 27
single lineage The best fitting model is thenselected using the Akaike information criterionunder the assumption of constant populationsizes and fixed gene topologies across the speciestree The species delimitation program BayesianPhylogenetic and Phylogeography (BPampPYangand Rannala 2010) accommodates gene treeuncertainty and variable population sizes andsamples from the Bayesian posterior distributionof species delimitation models using reversible-jump Markov chain Monte Carlo (rjMCMC)sampling BPampP requires a user-provided guidetree with resolution finer than the species leveland evaluates alternative modes derived from allpossible subtrees that are generated by collapsingor splitting nodes on the guide tree (Yang andRannala 2010 Rannala and Yang 2003) Theproportion of time spent on each model is pro-portional to the posterior probability of themodel ie ldquospeciation probabilitiesrdquo WhileBPampP ranks among the most popular coalescent-based species delimitation programs (Fujita et al2012) misspecifications of the guide tree andorthe prior distribution for population size (θ) canresult in strong support for models containing aninflated number of species (Leacheacute and Fujita2010 Zhang et al 2011) The nonparametricheuristic species delimitation approach imple-mented in the program Brownie (OrsquoMeara 2010)jointly sorts anonymous samples into species andinfers a species tree from input gene trees fromdifferent loci assuming that for a speciationevent the corresponding nodes on gene trees willbe more consistent with each than the diver-gences within species However finding both theoptimum species tree and species boundariesremains computationally challenging andBrownie has been shown to frequently yieldincorrect results (OrsquoMeara 2010)
Most recently available coalescent-basedspecies delimitation methods require individualassignments to a species or population a priori Ina number of scenarios correct assignments ofsamples to species may be difficult includingpresence of cryptic species incongruencebetween conventional species and moleculardata or simply the fact that accurate specimenidentification in complex groups with subtle or
difficult to discern diagnostic characters is anontrivial task (Leavitt et al 2013e) Statisticalmethods for assessing individual assignment andspecies detection prior to coalescent-based spe-cies delimitation and species tree reconstructionand species provide a more objective approachfor understanding species boundaries
Population assignment tests using a variety ofclustering algorithms using genetic data offer auseful approach for grouping individuals intoputative reproductively isolated groups (Table 21Hausdorf and Hennig 2010 Corander et al2004 2008 Falush et al 2003 Pritchard et al2000) particularly for species delimitation prob-lems that exist at the interface of traditionalpopulation genetic and phylogenetic analyses(Carstens et al 2013 Weisrock et al 2010Knowles and Carstens 2007)
The programs STRUCTURE (Falush et al2003 Pritchard et al 2000) and STRUCTURA-MA (Huelsenbeck et al 2011) cluster samplesinto populations by minimizing HardyndashWeinbergdisequilibrium for a given number of populationclusters using unlinked genetic markers In gen-eral unlinked markers are not available for mostgroups of lichen-forming fungi and a numberof studies have used information from multilocussequence data for STRUCTURE analyses(Leavitt et al 2011a b c 2013e Fernaacutendez-Mendoza and Printzen 2013 Fernaacutendez-Mendoza et al 2011) Varying approaches havebeen implemented to convert DNA sequencedata to allelic data for Bayesian clustering (seeOrsquoNeill et al 2013) STRUCTURE is expectedto perform well when there is sufficient inde-pendence across regions such that linkage dis-equilibrium within regions does not dominate thedata (STRUCTURE manual) but can also beeffective using multilocus sequence data andtreating all SNPs as independent loci regardlessof physical linkage within each locus (OrsquoNeillet al 2013 Falush et al 2003) A recent study ofthe lichen-forming genus Letharia showed thatBayesian clustering implemented in the programSTRUCTURE was generally able to recover thesame putative Letharia lineages circumscribedemploying a gene genealogical approach inKroken and Taylorrsquos iconic species delimitation
28 SD Leavitt et al
study (Altermann et al 2014 Kroken and Taylor2001) Altermann et al (2014) show that popu-lation assignments were largely consistent acrossa range of scenarios including extensiveamounts of missing data the exclusion of SNPsfrom variable markers and inferences based onSNPs from as few as three gene regions Incontrast to STRUCTURE and STRUCTURA-MA the program BAPS (Corander et al 2008)can explicitly infer genetic structure using hap-loid sequence data or other linked geneticmarkers in the ldquoclustering with linked locirdquomodel Another advantage of BAPS is that theprogram is much more computationally efficientthan STRUCTURE or STRUCTURAMA Sim-ulation studies indicate that both BAPS andSTRUCTURE perform well at low levels ofpopulation differentiation and when clusters arenot well differentiated (Latch et al 2006)
In practice inferring the most appropriatelevel of population structure using Bayesianclustering algorithms remains challenging (Latchet al 2006 Evanno et al 2005) BAPS andSTRUCTURAMA can infer both individualassignments and the most likely number ofgenetic clusters (Corander et al 2008 Huelsen-beck et al 2011) and the ad hoc ΔK statisticproposed by Evanno et al (2005) provides anobjective approach for identifying the uppermosthierarchical level of structure However carefulconsideration of other levels of populationstructure may ultimately reveal more biologicallymeaning results and researchers should examineindividual assignments across a range of geneticgroupings Some markers generated fromindependent genomic regions such as SNPs andfast-evolving microsatellites can be used to dis-tinguish fine-scale population structuring on thebasis of allele frequencies and species delimita-tion analyses based on these markers may implythe risk of severe taxonomic oversplitting Inthese cases validation approaches such asBPampP and SpeDeSTEM and corroborationamong different species delimitation approachescan provide perspective between intraspecificpopulation structure and species-level clusters
Another limitation of clustering approaches isthat they do not assess or take into account
evolutionarily divergences and relationshipsamong population clusters Coalescent-basedspecies tree methods (discussed below) provide ameans to assess the diversification histories ofpopulations inferred from clustering analysesTherefore a potential working protocol for aninformed species delimitation study that takesinto account population structure could consist offirst applying a genetic clustering analysis under apopulation genetics criterion (eg BAPSSTRUCTURE STRUCTURAMA) to identifygenetically distinct population clusters that can beconsidered ldquocandidate speciesrdquo From these can-didate species a species tree can be inferred forfocal group using coalescent-based species treereconstruction methods (eg BEAST) Subse-quently a coalescent-based validation methodcan be applied to assess whether the distinctpopulation clusters represent independent evolu-tionary lineages (eg BPampP) This multistepapproach would provide a consistent and standardcriterion for distinguishing between population-and species-level lineages (Camargo and Sites2013) and has been applied to a number of casesincluding lichen-forming fungi (Leavitt et al2011a b c 2013e Leacheacute and Fujita 2010)
High-throughput sequencing methods providethe means to effectively sample hundreds tothousands of loci from across a species genome fora large number of species and continue to revolu-tionize studies that can be performed even in non-model organisms Targeted high-throughputsequencing approaches such as anchored phy-logenomics transcriptome sequencing reduced-representation genomic library sequencing(restriction-site-associated DNA sequencingRAD-Seq and genotype-by-sequencing GBS)and high-throughput amplicon sequencing pro-vide important insight into species boundaries fora number groups although none of these approa-ches has yet been applied to studies of lichenizedfungi
For example restriction-site-associated(RAD-Seq) DNA can simultaneously detect andgenotype thousands of genome-wide SNPs byfocusing the sequencing effort on a reducedrepresentation of the entire genome (Baird et al2008) and has been successfully applied to
2 The Dynamic Discipline of Species Delimitation hellip 29
intraspecies (Lewis et al 2007 Miller et al 2007Emerson et al 2010) and interspecies studies(Rubin et al 2012 Eaton and Ree 2013 Wagneret al 2013) This approach has provided strikinginsight into the recent adaption radiation of LakeVictoria cichlids (Wagner et al 2013) Analternative approach targeting the sequencing ofspecific loci using next-generation sequencingplatforms provides an efficient means of gener-ating data for loci of known genomic locationorthology size and expected level of variation(OrsquoNeill et al 2013) Markers can be targetedusing well-established PCR techniques or newerhybridization techniques and subsequentlypooled for high-throughput sequencing usingparallel-tagged sequencing of multiple individualsamples within a single sequencing run (OrsquoNeillet al 2013) A recent study of North Americantiger salamanders (Ambystoma tigrinum)demonstrated the potential for amplicon-basedparallel-tagged sequencing to rapidly generatelarge-scale genomic data for species delimitationand species tree research (OrsquoNeill et al 2013)
Although these methods provide an enormousamount of data and insight into diversification atan unprecedented scale computational limita-tions restrict the applicable analytical methodsfor large datasets although computational andanalytical advances are happening rapidly Mostrecent coalescent-based species delimitation andspecies tree models using gene trees have beenlimited to handle tens of loci for multiple indi-viduals and combining hundreds or thousands ofgene trees into a single species delimitationframework presents considerable computationalchallenges (Camargo and Sites 2013) Therecently developed method Bayes factor delim-itation (with genomic data BFD) combines adynamic programming algorithm for estimatingspecies trees that bypasses the computationallyintensive MCMC integration over gene trees toprovide a rigorous technique for species delimi-tation studies using genome-wide SNP data(Leacheacute et al 2014) Competing species delimi-tation models are compared using Bayes factorsand it appears that this approach is robust tosample sizes (ie few loci and limited samples
per species) and misspecification of the prior forpopulation size (θ) (Leacheacute et al 2014)
Fungi are an ideal model for assessing diver-gence in eukaryotes due to their simple mor-phologies small genomes broad ecological rolesand diverse lifestyles (Gladieux et al 2014)However the use of genomic data from high-throughput sequencing methods have not yet beenincluded in species delimitation studies of lichen-forming fungi This is due in part to challenges indealing with metagenomic data containing geno-mic information from a plethora of symbiontsassociated with a lichen thallus scant genomicresources and discipline-specific inertia A num-ber of lichen-forming fungal genomes are cur-rently available including Endocarponpusillum(Wang et al 2014) Cladonia spp (Park et al2013a 2014) Caloplaca flavorubescens (Parket al 2013b) and Cladonia greyi and Xanthoriaparientina through the Fungal Genomics Programat Joint Genome Institute of the United StatesDepartment of Energy (httpwwwjgidoegov)The foreseeable ongoing expansion of genomicresources for lichen-forming fungi will be centralto developing approaches for delimiting speciesusing high-throughput sequencing Specificallygenomic resources will be crucial in identifyingnovel markers (variable genes regions SNPsmicrosatellites etc) identifying conserved fungalmarkers for targeted high-throughput sequencingapproaches and references for entire genomecomparisons (Devkota et al 2014 Werth et al2013)
23 Can We Make SpeciesDelimitation in Lichen-FormingFungi Truly Integrative
The widespread availability of genetic data hascreated a biased viewpoint that only genetic datashould be used for statistical species delimitationHowever an ongoing appeal to researchers toassess species boundaries from multiple andcomplementary perspectives (phylogeneticspopulation genetics comparative morphologydevelopment ecology etc) has resulted in an
30 SD Leavitt et al
integrative taxonomic framework bringing theseconceptual and methodological perspectivestogether (Hamilton et al 2014 Fujita et al 2012Salicini et al 2011 Yeates et al 2011 Padialet al 2010 Dayrat 2005 Will et al 2005 Wiensand Penkrot 2002) In practice any study linkingdifferent kinds of data to support hypotheses ofspecies boundaries including mapping morpho-logical characters onto a molecular phylogenycan be considered integrative Integrative meth-ods for species delimitation fall across a broadspectrum ranging from verbal and qualitativeassessments of data classes to quantitative meth-ods that allow different data types to contribute tostatistical species delimitation (Yeates et al2011) In most taxonomic studies utilizing bothmolecular and morphological data expert opinionis eventually used to some degree to evaluate thefinal status of a candidate species (eg Bond andStockman 2008) In this sense many studiesusing evidence for independent data sources fordelimiting species boundaries use an iterativeapproach (Yeates et al 2011) where speciesboundaries can be tested within a hypothetico-deductive framework with diverse datasets
From a practical perspective we advocate aprocess of iterative taxonomy (sensu Yeates et al2011) to circumscribe and refine species limitsusing multiple lines of evidence This iterativeprocess involves comparisons of morphologicaldata with a phylogenetic hypothesis (ie single-locus gene tree or concatenated multilocusphylogeny) to identify the least inclusive mono-phyletic clade in the topology characterized by atleast one unambiguously diagnostic morphologi-cal character (Miralles and Vences 2013) Thisphylogenetic tree-informed approach to assessingspecies boundaries represents a common practicein studies of lichen-forming fungi Previouslyunrecognized species-level clades with corre-sponding subtle or overlooked phenotypiccharacters have been commonly observed in bothcrustose lichens [eg Acarospraceae (Wedin et al2009) Graphidaceae (Rivas Plata and Lumbsch2011 Papong et al 2009) Lecanoraceae (Leavittet al 2011a) Lecideaceae (Ruprecht et al 2010)Mycoblastaceae (Spribille et al 2011) andTeloschistaceae (Vondraacutek et al 2009 Muggia
et al 2008)] and foliose and fruticose lichens [egLobariaceae (Moncada et al 2014 McDonaldet al 2003) Parmeliaceae (Leavitt et al 2013aWirtz et al 2012 Divakar et al 2005 Molinaet al 2004 Kroken and Taylor 2001) Peltigera-ceae (OrsquoBrien et al 2009 Goffinet et al 2003)Physciaceae (Elix et al 2009 Divakar et al2007) and Sphaerophoraceae (Houmlgnabba andWedin 2003)] As a specific example Luumlckinget al (2008) used a combination of medullarychemistry and underside pigmentation in speci-mens from the Heterodermia obscurata group inCosta Rica to corroborate monophyletic clades inan ITS gene tree as species-level lineages
While this iterative approach to speciesdelimitation and taxonomy has proven valuablefor understanding species boundaries anddescribing new taxa in some groups of lichenizedfungi a posteriori examination of morphologicaland chemical features has failed to reveal diag-nostic phenotypic characters in a number ofstudies (Muggia et al 2014 Leavitt et al 2011aOtaacutelora et al 2010 OrsquoBrien et al 2009) Fur-thermore a study of widespread species in thegenus Melanelixia (Parmeliaceae) indicated thatphenotypically cryptic lichen-forming fungalspecies-level lineages may be relatively ancientand diagnosable phenotypic differences may beabsent even millions of years after the initialdivergence (Leavitt et al 2012b) The latter studyhighlights the fact that species-level lineages maycommonly exist without any observable diag-nostic phenotypic characters calling into ques-tion the impetus for a universal application ofintegrative taxonomy
In keeping with the principle that as manylines of evidence as available should be com-bined to delimit species (Dayrat 2005) the for-malized integrative taxonomic approach (ITAXMiralles and Vences 2013) uses a mtDNA guidetree and observations from different types of datathat might provide conclusive evidence for theindependence of lineages and thus their identityas different species Miralles and Vences (2013)provide a non-exhaustive list of species delimi-tation criteria to be integrated in the ITAXapproach including (a) sympatric occurrencewithout admixture as revealed by consistent
2 The Dynamic Discipline of Species Delimitation hellip 31
differences in morphological or molecular char-acters at the same geographic location (b) strongdifferences in a behavioral morphological orgenetic character known to mediate prematingisolation (c) unviability or infertility of hybrids(d) lack of gene flow across a geographicalhybrid zone (e) congruent diagnostic differencesbetween sister lineages in various unlinkedmorphological character (f) absence of haplo-type sharing in several unlinked nuclear loci and(g) a combination of criteria endashf Speciesboundaries are based on seeking the least inclu-sive monophyletic group in the mtDNA treewhich fulfills at least one of the criteria listedabove Clearly the ITAX approach is sensitive tosample size and in order to reliably support thedistinctiveness of a given species it has beenrecommended that the sampling strategy includesat least five individuals per species (Miralles andVences 2013) A similar approach could beadopted for lichenized fungi using an ITS genetopology as guide tree rather than mtDNA
A total evidence approach including concat-enation has been a common approach for inte-grating information from different sourcesincluding independent genetic markers in phy-logenetic reconstructions (Kluge 1989 Wiens1998 de Queiroz et al 1995) Proponents forconcatenation of independent data in phyloge-netic analyses argue that when combining alldata the underlying signal of speciation mayemerge even if weakly contradictory signalis contained in the individual data partitions(Gatesy et al 1999) For example establishing apreliminary perspective of species boundariesfrom multilocus sequence data using concatena-tion may provide a reasonable starting point forscreening for cryptic species and species treeinference (Šlapeta et al 2006 Leavitt et al2011a Le Gac et al 2007 Leacheacute 2009)However concatenation and consensus methodsimply a risk of obtaining inflated support forincorrect relationships and information aboutvariance in gene coalescence is lost (Degnan andRosenberg 2006 2009 Kubatko and Degnan2007 Edwards 2009) In spite of the impetus forintegrating evidence from independent data
sources into empirical species delimitation stud-ies and taxonomy (Fujita et al 2012 Padial et al2009) most currently available species delimi-tation methods are unable to accommodate non-genetic data sources in a statistical framework
231 Selecting the Appropriate Data
In the face of increasing availability of geneticdata and associated bioinformatical approachesfor delimiting species researchers should care-fully consider what information is being sacri-ficed by the failure to consider non-genetic datain species delimitation studies and whetheraccuracy could be improved by the addition ofmultiple data types Morphological data havehistorically served as a proxy to identify repro-ductively isolated groups (ie ldquospeciesrdquo) (Ray1686 Fujita et al 2012) Current methods fordelimiting species using non-genetic data (egchemistry morphology and ecology) remainwoefully understudied For example morphol-ogy-based species circumscriptions are generallybased on one or more qualitative (or quantitative)morphological characters that do not appear tooverlap with other species However ascertain-ing that a given trait is truly fixed within apopulation with statistical confidence requiresunrealistic sample sizes even when allowing forsome level of polymorphism in the diagnosticcharacter (Wiens and Servedio 2000) Now var-ious combinations of datamdashfrom morphologygenetics geography and ecologymdashare acceptedas standard information for species delimitationstudies (Ruiz-Sanchez and Sosa 2010 Ross et al2010 Edwards and Knowles 2014)
Below we briefly discuss appropriate datasources for species delimitation studies of lichen-forming fungi However homoplastic characters(similar traits that are not derived from a com-mon ancestor) are common among many traitscommonly used to circumscribe fungal taxa thebiological significance of secondary metabolitevariation remains largely unknown (Lawrey1986) and ecological niches may be difficultto adequately characterize and model due to
32 SD Leavitt et al
microhabitat requirements and data resolutionTherefore we advocate a cautious approach toselecting appropriate and relevant data forassessing species boundaries
In spite of the potential challenges and limi-tations of using phenotypic data these traits haveprovided a plethora of valuable information forunderstanding species boundaries For examplein the Melanelixia fuliginosa group (Parmelia-ceae) a morphometric analysis using color isi-dia and marginal zone free of isidia ascharacters revealed a general pattern of differ-entiation between material formerly recognizedas subspecies fuliginosa and glabratula in spiteof the fact that considerable overlap betweengroups occurs in some characters individually(Arup and Berlin 2011) Furthermore the dis-tinction of the two groups was supported by aphylogenetic analysis of the ITS marker andecological differences with M fuliginosaoccurring predominantly on rock and M glab-ratula on bark (Arup and Berlin 2011) Thisstudy of the M fuliginosa group provides a fit-ting example of using multiple independentsuites of data including ecology morphologyand genetic information to establish a robusthypothesis of species boundaries
In lichen systematics phenotypic dataincluding thallus organization secondary metab-olites mode of reproduction ascoma-type andontogeny ascus and ascospore characters havehistorically played a prominent role (Printzen2009) Ascomatal characters have traditionallyheld a major role in higher-level classification(Printzen 2009) in contrast to species-levelclassification which also tends to include a widearray of vegetative and chemical charactersCommonly assessed morphological charactersinclude thallus form and size cortical features(eg maculae and pseudocyphellae) presenceformcolor of attachment structure (eg rhizines)and reproductive mode (ascomata vs vegetativediaspores) (Printzen 2009) Different morpho-logical types of vegetative diasporesmdashcorticatedisidia and ecorticated sorediamdashand their locationare commonly used to distinguish speciesAscomatal characters including morphologylocation (laminal vs marginal) position (sessile
vs immersed) presence of thalline margins colorof an apothecial disc and presence or color ofpruina are also commonly used in speciesdelimitations of lichen-forming fungi (Printzen2009) Other important characters may includethalline characters form color size and septationof ascospores size and form and structure of ascithe hamathecium type of epihymenium andhypothecium and the type of excipulum orperidium conidiomatal characters etc (Printzen2009)
Assessments of secondary metabolites hasplayed an important role in lichen taxonomybeginning with the introduction of simple spottests by Nylander (1866a b) The use of chem-istry in lichen taxonomy has been discussed indetail in numerous reviews (Lumbsch 1998a bRogers 1989 Brodo 1986 Egan 1986 Leuckert1985 Brodo 1978 Hawksworth 1976 Culber-son 1969 1970) and we refer readers to thesevaluable sources for a more comprehensive per-spective on lichen chemistry In short extrolites(secondary metabolites) belong to various clas-ses the most common and diverse include dep-sides depsidones chlorinated xanthones andanthraquinones (Lumbsch 2002 Culberson1969) The presence or absence of specificextrolites or their replacements by another sub-stance is widely used to distinguish speciesparticularly when correlated with differences ingeographic distributions However if morpho-logical or geographical differences betweenpopulations containing different extrolites are notapparent the taxonomic significance has beendisputed with some authors distinguishing themas species and others preferring to regard them aschemical races within a species In addition tosimply using the presence or absence of extro-lites Culberson and Culberson (1976) proposedto arrange lichen substances into chemosyn-dromes of closely related substances The pres-ence or absence of these chemosyndromes maypotentially be used as characters to delimit spe-cies regarding differences involving the samechemosyndromes as intraspecific variation anddistinct chemosyndromes as evidence for inter-specific populations (Lumbsch 1994 Gowan1986)
2 The Dynamic Discipline of Species Delimitation hellip 33
Due to the fact that species delimitationstudies often incorporate a substantial biogeo-graphical or ecological component ecologicalniche modeling plays an increasing role in phy-logenetic and taxonomic research Niche mod-eling can provide evidence for ecologicalisolation between populations based on eitherconserved or divergent ecological niches andtherefore can provide additional evidence sup-porting lineage independence between putativespecies The application of ecological nichemodeling has been applied to species delimita-tion studies in a number of cases (Ruiz-Sanchezand Sosa 2010 Leacheacute et al 2009 Raxworthyet al 2007 Rissler and Apodaca 2007) althoughit has not been explicitly applied to assess speciesboundaries in lichen-forming fungi By mappingthe spatial distribution of environmental suit-ability of climatic variables for candidate speciesthe application of ecological niche modeling canbe particularly important in cases where specieshave allopatric distributions (Raxworthy et al2007)
Ecological niche modeling utilizes knownassociations between a speciesrsquo occurrencelocalities and environmental variables to defineabiotic conditions within which populations canbe maintained (Guisan and Thuiller 2005) Themethodological approach for modeling is basedon four general properties (i) The current knownspeciesrsquo localities is the dependent variable (ii)the distribution is modeled as a map composed ofgrid cells at a specified resolution (iii) a range ofenvironmental variables (eg temperature pre-cipitation and solar exposure) are collected todescribe the characteristics of each cell and (iv)classifying the degree to which each cell is eithersuitable or unsuitable for each species under arange of models (Guisan and Thuiller 2005)
Ecological data also have the potential to playan important role in understanding speciesboundaries in lichen-forming fungi For exampleNash and Zavada (1977) demonstrated that Xant-hoparmelia populations with distinct chemistriesoccurring in the northern portion of the SonoranDesert exhibit habitat selection among differentrock substrates within a region with relativelyuniform climate and topography In another case
the parmelioid species Parmelia mayi is mor-phologically indistinguishable from P saxatilisbut can be separated by bioclimatic features andgenetic and chemical characters (Molina et al2011) McCune and Printzen(2011) assess distri-butions and climatic niches of species in theLecanora varia group inwesternUSAandprovidea model that uses continental influence and annualtemperature as themajor factors predicting speciesdistributions The distribution of Usnea hirta alichen commonly used in air quality biomonitor-ing research was modeled for a section of theWhite River National Forest in central Coloradobased on the presence of U hirta at 72 biomoni-toring reference sites distributed in the inter-mountain western United States (Shrestha et al2012) The best model for predicting U hirtadistribution included four variablesmdashsolar radia-tion average monthly precipitation and averagemonthly minimum and maximum temperatures(Shrestha et al 2012) These studies supportthe potential use of ecological niche modelingmethods in species delimitation studies of lichen-forming fungi
24 Conclusions What AboutTaxonomy
In most cases species circumscription and tax-onomy requires some degree of qualitativejudgment and individual interpretation Integrat-ing multiple types of data into an empiricalframework for delimiting species boundarieswhere species boundaries can be tested within ahypothetico-deductive framework with diversedatasets can provide robust hypotheses of speciesboundaries and taxonomic stability (Yeates et al2011) However it has long been known toevolutionary biologists that distinct species donot need to have diagnosable morphologicaldifferences (Mayr 1963) and increasing avail-ability of genetic data has allowed researchers toidentify species and to rigorously test speciesboundaries with a level of precision that wasunimaginable a decade ago While analyticaladvances in statistical species delimitation havebeen largely based on genetic data the utility of
34 SD Leavitt et al
these approaches to formal taxonomy remainselusive Due to these challenges an eclecticapproach to delimiting species and cautionagainst the reliance on any single dataset ormethod is required when delimiting species
While results from many recent studies maycontradict traditional species boundaries acrossmany groups of lichen-forming fungi we areoptimistic that this research represents substantialprogress toward a more accurate perspective onspecies boundaries and diversity in fungi As aresult of ongoing research of species boundariesin lichen-forming fungi the taxonomic value ofmany phenotypes is now better understood ourunderstanding of ecological evolutionary andbiogeographic patterns has improved and we canbegin to better understand patterns of symbiontinteractions in lichens Integrating new data(including novel morphological characters andgenetic data) will be essential to accurately rep-resent species-level diversity across all groups oflichenized fungi Hopefully an improved per-spective on lichen diversity also increases ourappreciation of these incredible symbiotic sys-tems While we are strong advocates for theapplication of independent data types in devel-oping an integrative taxonomy there is anincreasing need to formally recognize the exis-tence of phenotypically cryptic species-levellineages in lichen-forming fungi (see Hibbettet al 2011) In some cases a molecular taxon-omy may provide the most practical approach toconsistent treatment of mycobiont species withinlichen groups where diagnostic morphologicalcharacters are unidentifiable or practically notfeasible (Leavitt et al 2013c d)
These are exciting times for taxonomists andphylogeneticists A closer look at lichen taxon-omy with the inclusion of new data will help usto better understand the diversity of these fasci-nating organisms accurately interpret distribu-tion patterns and play a more important role inmeaningful conservation practices Howeversome level of uncertainty will accompany pro-gress In many taxonomic groups our traditionalapproach for species identification will likelyneed to be substantially modified The search forcorroborating morphological support for cryptic
species identified using molecular data willrequire meticulous and creative approaches toassess phenotypic variation in potentially unor-thodox ways We are hopeful that lichenologistswho traditionally have been eager to include newmethods such as chromatography in their rou-tine identifications will be amenable to includemolecular techniques to their routine examina-tion of specimens for identification and classifi-cation Although this may prove difficult toachieve by single individuals especially citizenscientists that traditionally play an important rolein lichen taxonomy the increasing numberof collaborative projects in the discipline (egLumbsch et al 2011 Crespo et al 2010Gueidan et al 2009) make us optimistic thatbroad-scale collaborative approaches will facili-tate the inclusion of molecular data in lichenresearch at all levels
Acknowledgments We are indebted to various col-leagues for valuable thought-provoking discussionnotably Matthew Nelsen (University of Chicago) AnaCrespo (Universidad Complutense de Madrid) PradeepDivakar (Universidad Complutense de Madrid) BeckettSterner (The Field Museum) and Joyce Havstad (TheField Museum) We also thank anonymous reviewers whoprovided valuable comments that improved this chapterSupport by the US National Science Foundation isgratefully acknowledged (ldquoHidden diversity in parmelioidlichensrdquo DEB-0949147)
References
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Altermann S Leavitt SD Goward T Nelsen MPLumbsch HT (2014) How do you solve a problemlike Letharia A new look at cryptic species in lichen-forming fungi using Bayesian clustering and SNPSsfrom multilocus sequence data PLoS ONE 9(5)e97556 doi101371journalpone0097556
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Culberson WL (1970) Chemosystematics and ecology oflichen-forming fungi Annu Rev Ecol Syst 1153ndash170
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de Queiroz K (1999) The general lineage concept ofspecies and the defining properties of the speciescategory In Wilson RA (ed) Species new interdis-ciplinary essays MIT Press Cambridge pp 49ndash89
de Queiroz K (2007) Species concepts and speciesdelimitation Syst Biol 56(6)879ndash886 doi10108010635150701701083
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Degnan JH Rosenberg NA (2009) Gene tree discordancephylogenetic inference and the multispecies coales-cent Trends Ecol Evol 24(6)332ndash340 doihttpdxdoiorg101016jtree200901009
Del-Prado R Cubas P Lumbsch HT Divakar PK BlancoO de Paz GA Molina MC Crespo A (2010) Geneticdistances within and among species in monophyleticlineages of Parmeliaceae (Ascomycota) as a tool fortaxon delimitation Mol Phylogenet Evol 56(1)125ndash133 doi101016jympev201004014
Del-Prado R Divakar PK Crespo A (2011) Using geneticdistances in addition to ITS molecular phylogeny toidentify potential species in the Parmotrema reticul-atum complex a case study The Lichenologist 43(06)569ndash583 doi101017S0024282911000582
Del-Prado R Blanco O Lumbsch HT Divakar PK ElixJA Molina MC Crespo A (2013) Molecular phylog-eny and historical biogeography of the lichen-formingfungal genus Flavoparmelia (Ascomycota Parmelia-ceae) Taxon 62(5)928ndash939 doi101270562522
Dettman J Jacobson D Taylor J (2003a) A multilocusgenealogical approach to phylogenetic species recog-nition in the model eukaryote Neurospora Evolution57(12)2703ndash2720 doi101111j0014-38202003tb01514x
Dettman JR Jacobson DJ Turner E Pringle A TaylorJW (2003b) Reproductive isolation and phylogeneticdivergence in Neurospora comparing methods ofspecies recognition in a model eukaryote Evolution57(12)2721 doi10155403-074
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Divakar PK Molina MC Lumbsch HT Crespo A (2005)Parmelia barrenoae a new lichen species related toParmelia sulcata (Parmeliaceae) based on molecularand morphological data The Lichenologist 37(01)37ndash46 doi101017S0024282904014641
Divakar PK Amo De paz G del Prado R Esslinger TLCrespo A (2007) Upper cortex anatomy corroboratesphylogenetic hypothesis in species of Physconia(Ascomycota Lecanoromycetes) Mycol Res 111(11)1311ndash1320 doi101016jmycres200708009
Divakar PK Figueras G Hladun N Crespo A (2010)Molecular phylogenetic studies reveal an undescribedspecies within the North American concept of Mela-nelixia glabra (Parmeliaceae) Fungal Divers 42(1)47ndash55 doi101007s13225-010-0027-3
Donoghue MJ Gauthier A (2004) Implementing thephylocode Trends Ecol Evol 19(6)281ndash282 doi101016jtree200404004
Eaton DAR Ree RH (2013) Inferring phylogeny andintrogression using RADseq data an example fromflowering plants (Pedicularis Orobanchaceae) SystBiol 62(5)689ndash706 doi101093sysbiosyt032
Edwards SV (2009) Is a new and general theory ofmolecular systematics emerging Evolution 63(1)1ndash19 doi101111j1558-5646200800549x
Edwards DL Knowles LL (2014) Species detection andindividual assignment in species delimitation canintegrative data increase efficacy Proc R Soc B BiolSci 281(1777) doi101098rspb20132765
Egan RS (1986) Correlations and non-correlations ofchemical variation patterns with lichen morphologyand geography The Bryologist 8999ndash110
Elix JA Corush J Lumbsch HT (2009) Triterpenechemosyndromes and subtle morphological characterscharacterise lineages in the Physcia aipolia group inAustralia (Ascomycota) Syst Biodivers 7(04)479ndash487 doi101017S1477200009990223
Emerson KJ Merz CR Catchen JM Hohenlohe PACresko WA Bradshaw WE Holzapfel CM (2010)Resolving postglacial phylogeography using high-throughput sequencing Proc Natl Acad Sci 107(37)16196ndash16200 doi101073pnas1006538107
Ence DD Carstens BC (2011) SpedeSTEM a rapid andaccurate method for species delimitation Mol EcolResour 11(3)473ndash480 doi101111j1755-0998201002947x
EvannoGRegnaut SGoudet J (2005)Detecting thenumberof clusters of individuals using the software STRUC-TURE a simulation study Mol Ecol 14(8)2611ndash2620doi101111j1365-294X200502553x
Falush D Stephens M Pritchard JK (2003) Inference ofpopulation structure using multilocus genotype datalinked loci and correlated allele frequencies Genetics164(4)1567ndash1587 doi101111j1471-8286200701758x
Fan HH Kubatko LS (2011) Estimating species treesusing approximate Bayesian computation Mol Phylo-genet Evol 59(2)354ndash363 doi101016jympev201102019
Fernaacutendez-Mendoza F Printzen C (2013) Pleistoceneexpansion of the bipolar lichen Cetraria aculeata intothe Southern hemisphere Mol Ecol 22(7)1961ndash1983doi101111mec12210
Fernaacutendez-Mendoza F Domaschke S Garciacutea MA JordanP Martin MP Printzen C (2011) Population structure
2 The Dynamic Discipline of Species Delimitation hellip 37
of mycobionts and photobionts of the widespreadlichen Cetraria aculeata Mol Ecol 20(6)1208ndash1232doi101111j1365-294X201004993x
Fraley C Raftery A (2007) Model-based methods ofclassification Using the mclust software in chemo-metrics J Stat Softw 18i06 [Available online at httpwwwdoajorgdoajfunc=abstractampid=218544]
Fujisawa T Barraclough TG (2013) Delimiting speciesusing single-locus data and the generalized mixed yulecoalescent (GMYC) approach a revised method andevaluation on simulated datasets Syst Biol 62(5)707ndash724 doi101093sysbiosyt033
Fujita MK Leacheacute AD Burbrink FT McGuire JA MoritzC (2012) Coalescent-based species delimitation in anintegrative taxonomy Trends Ecol Evol 27(9)480ndash488 doi101016jtree201204012
Gatesy J OrsquoGrady P Baker RH (1999) Corroborationamong data sets in simultaneous analysis hiddensupport for phylogenetic relationships among higherlevel Artiodactyl taxa Cladistics 15(3)271ndash313doi101111j1096-00311999tb00268x
Gladieux P Ropars J Badouin H Branca A Aguileta Gde Vienne DM Rodriacuteguez de la Vega RC Branco SGiraud T (2014) Fungal evolutionary genomicsprovides insight into the mechanisms of adaptivedivergence in eukaryotes Mol Ecol 23(4)753ndash773doi101111mec12631
Goffinet B Miadlikowska J Goward T (2003) Phyloge-netic inferences based on nrDNA sequences supportfive morphospecies within the Peltigera didactylacomplex (Lichenized Ascomycota) The Bryologist106(3)349ndash364 doi10163901
Gowan SP (1986) Evolution of secondary natural productsin the genus Porpidia (Ascomycata Porpidiaceae) AmJ Bot 73606
Griffin PC Hoffmann AA (2014) Limited genetic diver-gence among Australian alpine Poa tussock grassescoupled with regional structuring points to ongoinggene flow and taxonomic challenges Ann Bot doi101093aobmcu017
Grube M Hawksworth DL (2007) Trouble with lichenthe re-evaluation and re-interpretation of thallus formand fruit body types in the molecular era Mycol Res111(9)1116ndash1132 doi101016jmycres200704008
Gueidan C Savi S Thues H Roux C Keller C Tibell LPrieto M Heimarsson S Breuss O Orange A FrobergL Wynns AA Navarro-Rosines P Krzewicka BPykaelae J Grube M Lutzoni F (2009) Genericclassification of the Verrucariaceae (Ascomycota)based on molecular and morphological evidencerecent progress and remaining challenges Taxon 58(1)184ndash208
Guillot G Mortier F Estoup A (2005) Geneland acomputer package for landscape genetics Mol EcolNotes 5(3)712ndash715 doi101111j1471-8286200501031x
Guillot G Renaud S Ledevin R Michaux J Claude J(2012) A unifying model for the analysis of pheno-typic genetic and geographic data Syst Biol 61(6)897ndash911 doi101093sysbiosys038
Guisan A Thuiller W (2005) Predicting species distribu-tion offering more than simple habitat models EcolLett 8(9)993ndash1009 doi101111j1461-0248200500792x
Hale ME (1990) A synopsis of the lichen genusXanthoparmelia (Vainio) Hale (AscomycotinaParmeliaceae) Smithsonian Institution Press Wash-ington DC
Hamilton CA Hendrixson BE Brewer MS Bond JE(2014) An evaluation of sampling effects on multipleDNA barcoding methods leads to an integrativeapproach for delimiting species a case study ofthe North American tarantula genus Aphonopelma(Araneae Mygalomorphae Theraphosidae) MolPhylogenet Evol 7179ndash93 doi101016jympev201311007
Hausdorf B (2011) Progress toward a general speciesconcept Evolution 65(4)923ndash931 doi101111j1558-5646201101231x
Hausdorf B Hennig C (2010) Species delimitation usingdominant and codominant multilocus markers SystBiol 59(5)491ndash503 doi101093sysbiosyq039
Hawksworth DL (1976) Lichen chemotaxonomy InBailey RH (ed) Lichenology progress and problemsAcademic London pp 139ndash184
Hebert PDN Cywinska A Ball SL deWaard JR (2003)Biological identifications through DNA barcodesProc R Soc Lond B Biol Sci 270(1512)313ndash321doi101098rspb20022218
Hebert PDN Stoeckle MY Zemlak TS Francis CM(2004) Identification of birds through DNA barcodesPLoS Biol 2(10)e312 doi101371journalpbio0020312
Heled J Drummond AJ (2010) Bayesian inference ofspecies trees from multilocus data Mol Biol Evol 27(3)570ndash580 doi101093molbevmsp274
Hey J (2006) On the failure of modern species conceptsTrends Ecol Evol 21(8)447ndash450 doi101016jtree200605011
Hibbett DS OhmanA Glotzer D NuhnMKirk P NilssonRH (2011) Progress in molecular and morphologicaltaxon discovery in Fungi and options for formalclassification on environmental sequences Fungal BiolRev 2538ndash47 doi101016jfbr201101001
Houmlgnabba F Wedin M (2003) Molecular phylogeny ofthe Sphaerophorus globosus species complex Cladis-tics 19(3)224ndash232 doi101111j1096-00312003tb00365x
Hudson RR Coyne JA (2002) Mathematical conse-quences of the genealogical species concept Evolu-tion 56(8)1557 doi101111j0014-38202002tb01467x
Huelsenbeck JP Andolfatto P Huelsenbeck ET (2011)Structurama Bayesian inference of population struc-ture Evol Bioinf Online 7(2011)55ndash59 doi104137EBOS6761
Jones G Oxelman B (2014) DISSECT an assignment-free Bayesian discovery method for species delimita-tion under the multispecies coalescent Bioinformaticsdoi101101003178
38 SD Leavitt et al
Kekkonen M Hebert PD (2014) DNA barcode-baseddelineation of putative species efficient start fortaxonomic workflows Mol Ecol Res doi1011111755-099812233
Kelly LJ Hollingsworth PM Coppins BJ Ellis CJHarrold P Tosh J Yahr R (2011) DNA barcoding oflichenized fungi demonstrates high identification suc-cess in a floristic context New Phytol 191(1)288ndash300doi101111j1469-8137201103677x
Kiss L (2012) Limits of nuclear ribosomal DNA internaltranscribed spacer (ITS) sequences as species barcodesfor fungi Proc Natl Acad Sci 109(27)E1811ndashE1811doi101073pnas1207143109
Kluge AG (1989) A concern for evidence and aphylogenetic hypothesis for relationships among Epi-crates (Boidae Serpentes) Syst Zool 38(1)7ndash25doi101093sysbio3817
Knowles LL Carstens BC (2007) Delimiting specieswithout monophyletic gene trees Syst Biol 56(6)887ndash895 doi10108010635150701701091
Kroken S Taylor JW (2001) A gene genealogical approachto recognize phylogenetic species boundaries in thelichenized fungus Letharia Mycologia 93(1)38ndash53
Kubatko LS Degnan JH (2007) Inconsistency of phylo-genetic estimates from concatenated data under coa-lescence Syst Biol 56(1)17ndash24 doi10108010635150601146041
Latch EK Dharmarajan G Glaubitz JC Rhodes OE Jr(2006) Relative performance of Bayesian clusteringsoftware for inferring population substructure andindividual assignments at low levels of populationdifferentiation Conserv Genet 7(2)295ndash302 doi101007s10592-005-9098-1
Lawrey JD (1986) Biological role of lichen substancesThe Bryologist 89(2)111ndash122
Le Gac M Hood ME Fournier E Giraud T (2007)Phylogenetic evidence of host-specific cryptic speciesin the anther smut fungus Evolution 61(1)15ndash26doi101111j1558-5646200700002x
Leacheacute AD (2009) Species tree discordance traces tophylogeographic clade boundaries in North Americanfence lizards (Sceloporus) Syst Biol 58(6)547ndash559doi101093sysbiosyp057
Leacheacute AD Fujita MK (2010) Bayesian species delim-itation in West African forest geckos (Hemidactylusfasciatus) Proc R Soc B Biol Sci 2773071ndash3077doi101098rspb20100662
Leacheacute AD Koo M Spencer C Papenfuss T Fisher RMcGuire J (2009) Quantifying ecological morpho-logical and genetic variation to delimit species in thecoast horned lizard species complex (Phrynosoma)Proc Natl Acad Sci USA 10612418ndash12423 doi101073pnas0906380106
Leacheacute AD Fujita MK Minin VN Bouckaert RR (2014)Species delimitation using genome-wide SNP dataSystematic Biology doi101093sysbiosyu018
Leavitt SD Fankhauser JD Leavitt DH Porter LDJohnson LA St Clair LL (2011a) Complex patterns ofspeciation in cosmopolitan ldquorock posyrdquo lichensmdashdiscovering and delimiting cryptic fungal species in
the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
Leavitt SD Esslinger TL Lumbsch HT (2012c) Neogene-dominated diversification in neotropical montanelichens dating divergence events in the lichen-form-ing fungal genus Oropogon (Parmeliaceae) Am J Bot99(11)1764ndash1777 doi103732ajb1200146
Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Lumbsch HT St Clair LL(2013c) DNA barcode identification of lichen-formingfungal species in the Rhizoplaca melanophthalmaspecies-complex (Lecanorales Lecanoraceae) includ-ing five new species MycoKeys 71ndash22 doi103897mycokeys74508
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Vondraacutek J Thorsten Lum-bsch H Clair LLS (2013d) Local representation ofglobal diversity in a cosmopolitan lichen-formingfungal species complex (Rhizoplaca Ascomycota)J Biogeogr 40(9)1792ndash1806 doi101111jbi12118
Leavitt SD Lumbsch HT Stenroos S St Clair LL(2013e) Pleistocene speciation in North American
2 The Dynamic Discipline of Species Delimitation hellip 39
lichenized fungi and the impact of alternative speciescircumscriptions and rates of molecular evolution ondivergence estimates PLoS ONE 8(12)e85240doi101371journalpone0085240
Leavitt SD Esslinger TL Hansen ES Divakar PK CrespoA Loomis BF Lumbsch HT (2014) DNA barcoding ofbrown Parmeliae (Parmeliaceae) species a molecularapproach for accurate specimen identification empha-sizing species in Greenland Organ Divers Evol 14(1)11ndash20 doi101007s13127-013-0147-1
Leliaert F Verbruggen H Vanormelingen P Steen FLopez-Bautista JM Zuccarello GC De Clerck O(2014) DNA-based species delimitation in algae Eur JPhycol 49(2) doi101080096702622014904524
Leuckert C (1985) Probleme der Flechten-Chemotaxon-omiemdashStoffkombinationen und ihre taxonomischeWertung Ber Deut Bot Ges 98401ndash408
Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
Lohtander K Kaumlllersjouml M Roland M Tehler A (2000)The family physciaceae in fennoscandia phylogenyinferred from its sequences Mycologia 92(4)728ndash735
Longton RE (1997) The role of bryophytes and lichens inpolar ecosystems In Woodin SJ Marquiss M (eds)Ecology of Arctic Environments Blackwell ScienceOxford pp 69ndash96 (Special publication No 13)
Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
Lumbsch HT (1994) Die Lecanora subfusca-Gruppe inAustralasien J Hattori Bot Lab 771ndash175
Lumbsch HT (1998a) Taxonomic use of metabolic data inlichen-forming fungi In Frisvad JC Bridge PDArora DK (eds) Chemical Fungal Taxonomy MarcelDekker New York pp 345ndash387
Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
Mayden RL (1997) A hierarchy of species concepts thedenouement in the saga of the species problem InClaridge MF Dawah HA Wilson MR (eds) Speciesthe units of biodiversity Chapman amp Hall Londonpp 381ndash424
Mayr E (1963) Animal species and evolution HarvardUniversity Press Cambridge
Mayr E (1970) Populations species and evolutionBelknap Press of Harvard University Press Cambridge
McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
McDonald T Miadlikowska J Lutzoni F (2003) Thelichen genus Sticta in the Great Smoky Mountains a
40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
McKay BD Mays HL Wu Y Li H Yao C-T Nishiumi IZou F (2013) An empirical comparison of character-based and coalescent-based approaches to speciesdelimitation in a young avian complex Mol Ecol 22(19)4943ndash4957 doi101111mec12446
Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
Mishler BD Brandon RN (1987) Individuality pluralismand the phylogenetic species concept Biol Philos 2397
Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
Nash TH Zavada M (1977) Population studies amongSonoran Desert species of Parmelia subg Xanthop-armelia (Parmeliaceae) Am J Bot 64(6)664ndash669
Nylander W (1866a) Circa novum in studio Lichenumcriterium chemicum Flora 49198ndash201
Nylander W (1866b) Hypochlorite of lime and hydrate ofpotash Two new criteria for the study of lichens(Tanslated and communicated by the Rev W ALeighton) Bot J Linn Soc 9358ndash365
OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
OrsquoNeill EM Schwartz R Bullock CT Williams JSShaffer HB Aguilar-Miguel X Parra-Olea G Weis-rock DW (2013) Parallel tagged amplicon sequencingreveals major lineages and phylogenetic structure inthe North American tiger salamander (Ambystomatigrinum) species complex Mol Ecol 22(1)111ndash129doi101111mec12049
Orock EA Leavitt SD Fonge BA St Clair LL LumbschHT (2012) DNA-based identification of lichen-form-ing fungi Can publicly available sequence databasesaid in lichen diversity inventories of Mount Cameroon(West Africa) The Lichenologist 44(6)833ndash839doi101017S0024282912000424
Otaacutelora MAG Martiacutenez I Aragoacuten G Molina MC (2010)Phylogeography and divergence date estimates of alichen species complex with a disjunct distributionpattern Am J Bot 97(2)216ndash223 doi103732ajb0900064
Padial J Castroviejo-Fisher S Kohler J Vila C ChaparroJ De la Riva I (2009) Deciphering the products ofevolution at the species level the need for anintegrative taxonomy Zoolog Scr 38(4)431ndash447doi101111j1463-6409200800381x
Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
Papong K Luumlcking R Thammathaworn A BoonpragobK (2009) Four new taxa of Chroodiscus (thelotremoidGraphidaceae) from Southeast Asia The Bryologist112(1)152ndash163 doi1016390007-2745-1121152
Park S-Y Choi J Kim JA Jeong M-H Kim S Lee Y-HHur J-S (2013a) Draft genome sequence of Cladoniamacilenta KoLRI003786 a lichen-forming fungusproducing biruloquinone Genome Announcements 1(5)e00695-13 doi101128genomeA00695-13
Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
Peacuterez-Ortega S Fernaacutendez-Mendoza F Raggio J VivasM Ascaso C Sancho LG Printzen C de los Riacuteos A(2012) Extreme phenotypic variation in Cetrariaaculeata (lichenized Ascomycota) adaptation or inci-dental modification Ann Bot 109(6)1133ndash1148doi101093aobmcs042
Pino-Bodas R Burgaz A Martiacuten M Lumbsch HT (2011)Phenotypical plasticity and homoplasy complicatespecies delimitation in the Cladonia gracilis group(Cladoniaceae Ascomycota) Organ Divers Evol 11(5)343ndash355 doi101007s13127-011-0062-2
Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
Pino-Bodas R Martiacuten M Burgaz A (2012b) Cladoniasubturgida and C iberica (Cladoniaceae) form asingle morphologically and chemically polymorphicspecies Mycol Prog 11(1)269ndash278 doi101007s11557-011-0746-1
Pino-Bodas R Ahti T Stenroos S Martiacuten MP BurgazAR (2013a) Multilocus approach to species recogni-tion in the Cladonia humilis complex (CladoniaceaeAscomycota) Am J Bot 100(4)664ndash678 doi103732ajb1200162
Pino-Bodas R Martiacuten AP Burgaz AR Lumbsch HT(2013b) Species delimitation in Cladonia (Ascomy-cota) a challenge to the DNA barcoding philosophyMol Ecol Resour 13(6)1058ndash1068 doi1011111755-099812086
Pons J Barraclough TG Gomez-Zurita J Cardoso ADuran DP Hazell S Kamoun S Sumlin WD VoglerAP (2006) Sequence-based species delimitation for theDNA taxonomy of undescribed insects Syst Biol 55(4)595ndash609 doi10108010635150600852011
Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
Pringle A Baker DM Platt JL Wares JP Latgeacute JPTaylor JW (2005) Cryptic speciation in the cosmo-politan and clonal human pathogenic fungus Asper-gillus fumigatus Evolution 59(9)1886ndash1899 doi10155404-2411
Printzen C (2009) Lichen systematics the role ofmorphological and molecular data to reconstructphylogenetic relationships Progress in botany vol71 Springer Berlin pp 233ndash275
Pritchard JK Stephens M Donnelly P (2000) Inference ofpopulation structure using multilocus genotype dataGenetics 155(2)945ndash959
Puillandre N Lambert A Brouillet S Achaz G (2012)ABGD automatic barcode gap discovery for primaryspecies delimitation Mol Ecol 21(8)1864ndash1877doi101111j1365-294X201105239x
Rannala B Yang Z (2003) Bayes estimation of speciesdivergence times and ancestral population sizes usingDNA sequences from multiple loci Genetics1641645ndash1656
Raxworthy CJ Ingram CM Rabibisoa N Pearson RG(2007) Applications of ecological niche modelingfor species delimitation a review and empiricalevaluation using day geckos (Phelsuma) fromMadagascar Syst Biol 56(6)907ndash923 doi10108010635150701775111
Ray J (1686) Historia planarum vol 1 Clark LondonReese Naeligsborg R Ekman S Tibell L (2007) Molecular
phylogeny of the genus Lecania (Ramalinaceaelichenized Ascomycota) Mycol Res 111(5)581ndash591doi101016jmycres200703001
Reid N Carstens B (2012) Phylogenetic estimation errorcan decrease the accuracy of species delimitation aBayesian implementation of the general mixed Yule-coalescent model BMC Evol Biol 12(1)196 doi1011861471-2148-12-196
Rissler L Apodaca J (2007) Adding more ecology intospecies delimitation ecological niche models andphylogeography help define cryptic species in theBlack Salamander (Aneides flavipunctatus) Syst Biol56924ndash942 doi10108010635150701703063
Rivas Plata E Luumlcking R (2013) High diversity ofGraphidaceae (lichenized Ascomycota Ostropales) inAmazonian Peruacute Fungal Divers 58(1)13ndash32 doi101007s13225-012-0172-y
Rivas Plata E Lumbsch HT (2011) Parallel evolution andphenotypic divergence in lichenized fungi a casestudy in the lichen-forming fungal family Graphida-ceae (Ascomycota Lecanoromycetes Ostropales)Mol Phylogenet Evol 61(1)45ndash63 doi101016jympev201104025
Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
Ross KG Gotzek D Ascunce MS Shoemaker DD (2010)Species delimitation a case study in a problematic anttaxon Syst Biol 59(2)162ndash184 doi101093sysbiosyp089
Rubin BER Ree RH Moreau CS (2012) Inferringphylogenies from RAD sequence data PLoS ONE 7(4)e33394 doi101371journalpone0033394
Rubinoff D (2006) Utility of mitochondrial DNA barcodesin species conservation Conserv Biol 20(4)1026ndash1033 doi101111j1523-1739200600372x
Ruiz-Sanchez E Sosa V (2010) Delimiting speciesboundaries within the Neotropical bamboo Otatea(Poaceae Bambusoideae) using molecular morpho-logical and ecological data Mol Phylogenet Evol 54(2)344ndash356 doi101016jympev200910035
Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
morphological characters Antarctic Sci 22 (SpecialIssue 06)727ndash741 doi101017S0954102010000477
Salicini I Ibaacutentildeez C Juste J (2011) Multilocus phylogenyand species delimitation within the Nattererrsquos batspecies complex in the Western Palearctic MolPhylogenet Evol 61(3)888ndash898 doi101016jympev201108010
Satler JD Carstens BC Hedin M (2013) Multilocusspecies delimitation in a complex of morphologicallyconserved trapdoor spiders (Mygalomorphae Antro-diaetidae Aliatypus) Syst Biol 62(6)805ndash823 doi101093sysbiosyt041
Schlick-Steiner BC Steiner FM Seifert B Stauffer CChristian E Crozier RH (2010) Integrative taxonomya multisource approach to exploring biodiversityAnnu Rev Entomol 55(1)421ndash438 doi101146annurev-ento-112408-085432
Schoch CL Seifert KA Huhndorf S Robert V SpougeJL Levesque CA Chen W Fungal Barcoding Con-sortium (2012) Nuclear ribosomal internal transcribedspacer (ITS) region as a universal DNA barcodemarker for Fungi Proc Natl Acad Sci 109(16)6241ndash6246 doi101073pnas1117018109
Seacuterusiaux E Villarreal AJC Wheeler T Goffinet B(2011) Recent origin active speciation and dispersalfor the lichen genus Nephroma (Peltigerales) inMacaronesia J Biogeogr 38(6)1138ndash1151 doi101111j1365-2699201002469x
Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
Simpson GG (1951) The species concept Evolution5285ndash298
Sites JW Marshall JC (2003) Delimiting species a renais-sance issue in systematic biology Trends Ecol Evol18462ndash470 doi101016S0169-5347(03)00184-8
Sites JW Marshall JC (2004) Operational criteria fordelimiting species Annu Rev Ecol Evol Syst 35(1)199ndash227 doi101146annurevecolsys35112202130128
Šlapeta J Loacutepez-Garciacutea P Moreira D (2006) Globaldispersal and ancient cryptic species in the smallestmarine eukaryotes Mol Biol Evol 23(1)23ndash29doi101093molbevmsj001
Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
Talavera G Dincă V Vila R (2013) Factors affectingspecies delimitations with the GMYC model insightsfrom a butterfly survey Methods Ecol Evol 4(12)1101ndash1110 doi1011112041-210X12107
Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
Tewksbury J Anderson JGT Bakker JD Billo TJDunwiddie PW Groom MJ Hampton SE HermanSG Levey DJ Machnicki NJ del Rio CM PowerME Rowell K Salomon AK Stacey L TrombulakSC Wheeler TA (2014) Natural historyrsquos place inscience and society BioScience doi101093bioscibiu032
Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
Werth S Cornejo C Scheidegger C (2013) Characteriza-tion of microsatellite loci in the lichen fungus Lobariapulmonaria (Lobariaceae) Appl Plant Sci 1(2)1200290 doi103732apps1200290
Westberg M Arup U Kaumlrnefelt I (2007) Phylogeneticstudies in the Candelariaceae (lichenized Ascomy-cota) based on nuclear ITS DNA sequence dataMycol Res 111(11)1277ndash1284 doi101017S0953756204002102
Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
2 The Dynamic Discipline of Species Delimitation hellip 43
(Lepidoptera Lycaenidae) Front Zoo 4(1)8 doi1011861742-9994-4-8
Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
genetic data now play a prominent role in delim-iting fungal species and understanding evolu-tionary relationships in lichens (Lumbsch andLeavitt 2011 Printzen 2009)
Arguably the use of molecular data has led toan improved perspective on the taxonomic valueof many phenotypic characters in lichenized fungiand species delimitation in general Cryptic spe-cies-level lineages are commonly identified usingmolecular data and in some cases these previ-ously unrecognized lineages are corroborated byformerly overlooked phenotypic characters (Pino-Bodas et al 2012a Spribille et al 2011 Divakaret al 2010 Arguumlello et al 2007) Other studieshave revealed the fact that some species-levellineages are likely comprised of chemically andmorphologically polymorphic individuals whichare conventionally considered as separate species(eg Leavitt et al 2011a Pino-Bodas et al 2011Velmala et al 2009) While these studies high-light the limitations of using traditional taxo-nomic characters for distinguishing naturalgroups in lichen-forming fungi they also providea valuable perspective on the importance ofongoing research in even the best-studied lichengroups Furthermore an improved perspective ofspecies boundaries has led to a strikingimprovement in understanding diversification anddistribution in many groups of lichenized fungi
Traditional phenotype-based approaches tospecies recognition appear to vastly underesti-mate diversity in lichen-forming fungi Whilemolecular research has corroborated traditionalphenotype-based hypotheses of species bound-aries in a number of cases studies repeatedlydemonstrate that our current interpretation ofmorphological and chemical characters oftenfails to accurately characterize species-leveldiversity (reviewed in Lumbsch and Leavitt2011) A growing body of evidence reveals that asignificant proportion of unknown diversityestimated for fungi including lichen-formingfungi is hidden under names of supposedlycommon and widespread species For exampleapproximately 80 unrecognized species-levellineages are estimated to occur in Parmeliaceae(Crespo and Lumbsch 2010) Even higher levels
of unrecognized species diversity are estimatedto occur in other families such as Graphidaceae(Rivas Plata and Luumlcking 2013 Rivas Plata andLumbsch 2011 Luumlcking 2012)
The topic of cryptic species cases wheretwo or more distinct species-level lineages areerroneously classified (and hidden) under onenominal taxon (Bickford et al 2007) has beenfrequently reviewed for lichen-forming fungi(Lumbsch and Leavitt 2011 Crespo and Lum-bsch 2010 Crespo and Peacuterez-Ortega 2009Printzen 2009) Although novel diagnostic phe-notypic characters may potentially be identifiedcorroborating ldquocrypticrdquo species these previouslyunrecognized lineages generally remain difficultto classify within a traditional phenotype-basedtaxonomy (Leavitt et al 2013c d) In somecases it appears that similar phenotypic charac-ters may arise in parallel at local or regionalscales but may not be correlated with naturalgroups or genetic isolation (Muggia et al 2014Rivas Plata and Lumbsch 2011 Grube andHawksworth 2007) For example in the cosmo-politan species Tephromela atra (Fig 21) up to15 independent lineages were identified usingphylogenetic analyses of molecular sequencedata However the continuum of morphologicaland chemical variability in the T atra complexcurrently prevents the description of new speciesusing traditional phenotype-based characters(Muggia et al 2014)
Recent research on the genus Cladonia(Cladoniaceae) highlights a fitting example of thecomplexities associated within using phenotypiccharacters for delimiting species in lichen-forming fungi (Fig 21 Pino-Bodas et al 20112012a b 2013a b) In the Cladonia gracilisgroup most currently accepted species were notrecovered as monophyletic clades and traditionaldiagnostic morphological characters were shownto be highly homoplasious (Pino-Bodas et al2011) Similarly C iberica and C subturgidahave been shown to constitute a single morpho-logically and chemically polymorphic species(Pino-Bodas et al 2012b) Similar patterns ofhigh degrees of morphological and chemicalpolymorphisms have also been observed in the
14 SD Leavitt et al
C cariosa group (Pino-Bodas et al 2012a) andC humilis species complex (Pino-Bodas et al2013a) High levels of intraspecific morphologi-cal and chemical polymorphisms are not restric-ted to Cladonia A clear demarcation betweenintraspecific and interspecific morphological andor chemical variation does not exist for a largeproportion of Xanthoparmelia species in westernNorth America and high intraspecific morpho-logical and chemical variation are common for anumber of species-level genetic groups (Fig 21
Leavitt et al 2011b c 2013e) In some cases asmany as eight traditionally circumscribed Xant-hoparmelia species were recovered in a singlespecies-level genetic group (Leavitt et al 2011c)High levels of intraspecific phenotypic variationhave been observed in a number of other generain Parmeliaceae including Bryoria (Velmalaet al 2009) Cetraria (Peacuterez-Ortega et al 2012)Vulpicida (Mark et al 2012) and others
The importance of biogeography in lichen-forming fungal evolution has remained somewhat
Fig 21 Examples of common lichens in which tradi-tional morphology-based species circumscriptions fail toreflect natural species-level fungal lineages a Cladoniagracilis photographed in the Clearwater Valley BritishColumbia Canada (see Pino-Bodas et al 2011) (photo-graph creditCurtis Bjoumlrk) bR shushanii a member of theRhizoplaca melanophthalma species group from the
Aquarius Plateau Utah USA (see Leavitt et al 2011a)(photograph credit S Leavitt) c Xanthoparmelia affwyomingica occurring in Coloradorsquos Front Range USA(see Leavitt et al 2011b c Leavitt et al 2013e) (photo-graph credit S Leavitt) d Tephromela atra sensu latofound in the Santa Monica Mountains California USA(see Muggia et al 2014) (photograph credit J Hollinger)
2 The Dynamic Discipline of Species Delimitation hellip 15
ambiguous due to the occurrence of phenotypi-cally similar lichens occurring across broadintercontinental distributions and uncertainty ofappropriate species circumscriptions While anumber of lichen-forming fungal species havebeen found to be truly widespread (eg Lindblomand Soslashchting 2008 Fernaacutendez-Mendoza et al2011 Ahti and Hawksworth 2005 Del-Pradoet al 2013) improved recognition of speciesboundaries has provided insight into importantbiogeographical patterns in lichens previouslyassumed to have cosmopolitan distribution pat-terns (Leavitt et al 2013d Del-Prado et al 2013Amo de Paz et al 2012 Seacuterusiaux et al 2011Otaacutelora et al 2010 Elix et al 2009) Crypticdiversity and complex biogeographic patterns arehighlighted in the Rhizoplaca melanophthalmaspecies group (Fig 21 Leavitt et al 2013d)Analyses of R melanophthalma sensu lato col-lected from five continents supported the presenceof cryptic species within this complex Two ofthese lineages were found to have broad inter-continental distributions while the other four werelimited to western North America (Leavitt et al2013d) Most strikingly of the six lineages fivewere found on a single mountain in the westernUSA and the sixth occurred no more than 200 kmaway from this mountain A recent study ofPseudocyphellaria (Lobariaceae) sensu lato inHawaii revealed a surprising number of previouslyunrecognized species hidden within nominal taxawith putative broad geographic distributionssuggesting a high degree of endemism in Hawaii(Moncada et al 2014) These studies providecrucial impetus to reevaluate species boundaries inorder to improve our understanding of diversitydistributions and evolution in lichenized fungi
22 A Practical Guideto Contemporary SpeciesDelimitation
As it has become clear that conventional phe-notype-based criteria for species circumscriptionsare often problematic (Bickford et al 2007)molecular data are thereby particularly valuablefor assessing traditional species boundaries and
for species delimitation in general (Lumbsch andLeavitt 2011 Fujita et al 2012) Below weprovide an overview of relevant operationalspecies delimitation methods the majority ofwhich are reliant on molecular data coupled withmolecular phylogenetic and population geneticanalyses Assuming that species do in fact rep-resent ldquosegments of metapopulation lineagesrdquo (deQueiroz 1998) direct genetic evidence of lineagestatus is particularly relevant to species delimi-tation studies when analyzed within a rigorousstatistical framework regardless of whether lin-eages differ in phenotypic characters that areapparent to human observers (Fujita et al 2012)This perspective should not be taken as supportfor disregarding phenotypic characters in speciesdelimitation studies (Edwards and Knowles2014 Fujita et al 2012 Yeates et al 2011)Rather hypotheses of species boundaries shouldbe considered more robust with increasing cor-roboration from independent data sources (iemolecular chemistry morphology ecologyetc) and the integration of independent data forempirical species delimitation studies should be amajor focus of species delimitation research(Fujita et al 2012)
Themajority of molecular phylogenetic studiesof lichenized fungi focus on generating sequencedata from a number of specimens representing thefocal group inferring a gene tree from the geneticdata matrix and assessing the monophyly of thesampled taxa This approach has provided valu-able acumen into evolutionary relationships thevalue of morphological characters for taxonomyand insight into diversity of lichenized fungi(Thell et al 2009 Reese Naeligsborg et al 2007Westberg et al 2007 Martiacuten et al 2003 Arup andGrube 2000 Lohtander et al 2000 Stenroos andDePriest 1998) However simply assessing themonophyly of traditional phenotype-based spe-cies often offers an incomplete perspective onspecies boundaries Studies explicitly designed forempirically delimiting species are pivotal toadvancing our understanding of speciation andspecies diversity in lichens For example althougha nominal species may be recovered as mono-phyletic in a gene tree intraspecific phylogeneticsubstructure may correspond to evolutionarily
16 SD Leavitt et al
independent lineages (eg Leavitt et al 2011a)As a construct of taxonomy recognizing amonophyletic clade comprised of multiple mor-phological indistinguishable species-level lin-eages as a single species may hold some appealhowever failing to formally recognize this diver-sity can have far-reaching implications in ourbiological (eg ecology evolution reproduction)interpretation of the group Alternatively well-supported intraspecific phylogenetic substructuremay be the result of stochastic evolutionary pro-cesses uniparental inheritance etc rather thanevolutionary independence Interpreting this typeof phylogenetic substructure as species-leveldiversity can introduce detrimental bias
Empirical species delimitation methods havebroadly focused on four main areas detectingputative species individual specimen assignmentto a species group or operational taxonomic unit(OTU) validation of candidate species or OTUsas evolutionarily distinct lineages and inferringspecies relationships (ie species tree inference)Ideally operational species delimitation criteriashould be based on explicit statistical protocolsin order to objectively assess species boundariesand minimize the need of subjective interpreta-tions or taxonomic expertise In this section weprovide a brief overview on a number of empir-ical species delimitation methods that to varyingdegrees fit these criteria For convenience wedivide these methods into three general catego-ries (i) species discovery methods for assigningsamples to populations without a priori infor-mation (ii) species validation approaches andcoestimating individual assignments and speciestrees and (iii) species delimitation using geno-mic data (Table 21)
Empirical species delimitation has receivedincreasing attention including ongoing develop-ment of bioinformatical approaches and themethods and programs provided in this chapterare by no means intended to be a comprehensivelist of all available analytical approaches Ratherthe methods provided here have been shown tobe useful in a number of previous studies orshow particular promise for future speciesdelimitation studies Our aim is that these can
serve as a starting point when designing studiesto assess species limits Not surprisingly variousapproaches to species delimitation may yielddifferent estimates of species boundaries and theresearcher may be required to make some degreeof subjective interpretation of the most biologi-cally appropriate species boundaries Comple-mentarily recently developed metrics forquantifying the congruence between two taxo-nomies (Ctax) and the potential for an approach tocapture a high number of species boundaries(Rtax) provide a means to objectively assess dis-crepancies among species delimitation methods(Miralles and Vences 2013) More sophisticatedapproaches including selection of speciesdelimitation models using approximate Bayesiancomputing (Camargo et al 2012 Fan andKubatko 2011 Beaumont et al 2010) anddesigning and conducting a simulation study thatmatches the characteristics of the empirical study(Carstens et al 2013 Camargo et al 2012) canbe used to more objectively evaluate competinghypotheses of species boundaries In most con-texts it is likely better to fail to delimit speciesthan it is to falsely circumscribe entities that donot represent actual species and therefore theinferences drawn from species delimitation stud-ies should generally be conservative (Carstenset al 2013 Miralles and Vences 2013)
221 Corroborating TraditionalTaxonomy and DiscoveringCryptic Species Using Single-Locus Data
Objectively defining a threshold separatingintraspecific population substructure from inter-specific divergence is the general aim of speciesdelimitation studies using single-marker datasetsMost species delimitation methods based onsingle-locus sequence data fall under two generalcategories either genetic distance or tree-basedapproaches (Sites and Marshall 2004) Distance-based approaches attempt to detect a differencebetween intra- and interspecific distances (ieldquobarcode gaprdquo) where the pairwise genetic
2 The Dynamic Discipline of Species Delimitation hellip 17
Table
21
Somemetho
dsused
forspeciesdelim
itatio
ninclud
ingspeciesdiscov
erymetho
dsforassign
ingsamples
topo
pulatio
nswith
outapriori
inform
ation(BAPS
Gaussianclustering
Guillo
trsquosUnified
Mod
elS
TRUCTURES
TRUCTURAMA)genetic
distance-based
metho
dforsortingsequ
encesinto
hypo
theticalspecies(A
BGD)tree-
basedspeciesdiscov
erymetho
ds(bGMYCG
MYCb
PTP
PTP
ldquoSpecies
Delim
itatio
nrdquoplug
-inforGeneiou
s)and
jointdiscov
eryandvalid
ationmetho
ds(BPamp
PBrownie
DISSE
CTSp
eDeStem)
Metho
dDescriptio
nInpu
tdata
BAPS
mdashpo
pulatio
nassignmentusing
Bayesianclustering
Aprogram
forBayesianinferenceof
thegenetic
structurein
apopulatio
nclustering
molecular
data
andperformingadmixture
analysesGenetic
mixture
analyses
canbe
performed
atbo
thgroupandindividu
allevelsusingeither
ano
n-spatialo
rspatialm
odel
BAPS
treatsboth
theallele
frequenciesof
themolecular
markers
(ornucleotid
efrequenciesforDNA
sequ
ence
data)andthenu
mberof
genetically
diverged
groups
inpopulatio
nas
random
variablesIn
theldquoclusteringwith
linkedlocirdquomod
elagenetic
mixture
analysiscanbe
done
usinghaploidsequ
ence
data
orotherlin
kedgenetic
markersA
nalysesandmodel
comparisons
canalso
beperformed
usingafixednu
mber
ofgenetically
diverged
grou
psor
prespecified
populatio
nstructures
Limita
tions
Tem
poraldivergence
andrelatio
nships
amongputativ
egroups
arenot
explicitlyestim
atedE
quivalence
togenetic
clusters
tospecies-levelg
roupsisuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
Availablefrom
httpwwwhelsinkifi
bsgsoftwareBAPS
describedin
Coran-
deret
al(200
420
0620
08)CoranderandMarttinen20
06)
Genotyp
icdatahaploidsequence
dataor
linkedmarkers
(AFL
Por
SNPs)
Gaussianclusteringmdashpo
pulatio
nassignment
usingGaussianclustering
Aprogram
forBayesianinferenceof
thegenetic
structurein
apopulatio
nModelgroups
sampleinto
popu
latio
nsusinggeno
typicdata
bysearchingforclusters
that
canbe
attributed
tobeingmixturesof
norm
alallelefrequencydistributio
nsG
aussianclustering
requires
adatasetwhere
thecasesaredefinedby
variable
ofmetricscaleandhasbeen
used
with
geneticenvironmentalandmorphologicaldatasetsindividuallyinadditio
nto
integrated
datasets
Limita
tions
Tem
poraldivergence
andrelatio
nships
amongputativ
egroups
arenot
explicitlyestim
atedE
quivalence
togenetic
clusters
tospecies-levelg
roupsisuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
Implem
entedin
Rusingtheprabclus
(HausdorfandHennig20
10)andmclust
packages
(FraleyandRaftery
2007)
Genotyp
icdata
(flexible)
(con
tinued)
18 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
Guillo
trsquosUnified
Mod
elmdashpo
pulatio
nassignmentusingBayesianclustering
Thisapproach
provides
astatistical
model
that
allowsoneto
analyzegenetic
and
phenotyp
ticdata
with
inaun
ified
mod
elandinferencefram
eworkandop
tionally
incorporateinform
ationaboutthespatialdistributio
nof
samplesA
Bayesianclustering
algorithm
assumes
that
each
clusterin
ageographical
domaincanbe
approxim
ated
bypo
lygons
that
arecentered
around
pointsgeneratedby
aPo
issonprocessGuillo
trsquos
Unified
Mod
elisflexiblein
term
sof
thegenetic
data
that
itcanutilize
andcapableof
accurately
delim
iting
species
Limita
tions
Genetic
andphenotypic
data
cantracedifferentevolutionary
historiesfor
instancephylogenetic
divergence
forneutralgenetic
markers
andadaptatio
nfora
morphological
structure
Source
Availableas
anextensionof
theRGENELAND
package(G
uillo
tet
al20
05
2012)(http
www2im
mdtudk
gigu
Geneland)
Genotyp
icandno
n-genetic
(eg
phenotypicalecolog
icalgeograph
ical
behavioral)data
STRUCTUREmdash
populatio
nassignment
usingBayesianclustering
Amodel-based
clustering
methodusingmultilocus
genotype
data
toinferpopulatio
nstructureandassign
individualsto
populatio
nsIndividualsin
thesampleareassigned
probabilistically
topopulatio
nso
rjointly
totwoor
morepopulatio
nsiftheirgenotypes
indicatethatthey
areadmixedT
hemodeldoes
notassum
eaparticular
mutationprocess
anditcanbe
appliedto
mosto
fthecommonly
used
genetic
markersp
rovidedthat
they
areno
tcloselylin
ked
The
methodcanproducehigh
lyaccurate
assignmentsusing
modestn
umbersof
loci(Pritchard
etal2
000)T
hemostappropriatelevelo
fpopulatio
nstructurecanbe
inferred
byassessinglik
elihoodscores
orthead
hocΔKstatistic
(Evann
oet
al20
05)
Limita
tions
Identifying
themostappropriatenumberof
genetic
clusters
ischallenging
clusters
produced
byST
RUCTUREcanbe
strongly
influenced
byvariationin
sample
sizeclusters
createdby
STRUCTUREmay
notbe
consistent
with
theevolutionary
historyof
thepopulatio
nswhentherearerelativ
elylong
divergence
times
with
inevolutionary
lineagesTem
poraldivergence
andrelatio
nships
amongputativ
egroups
isnotexplicitlyestim
atedEquivalence
togenetic
clusters
tospecies-levelgroups
isuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
httppritchardlabstanfordedustructurehtm
ldescribedin
Falush
etal(200
3)andPritchard
etal(200
0)
Genotyp
icdata
(con
tinued)
2 The Dynamic Discipline of Species Delimitation hellip 19
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
Structuram
amdashpo
pulatio
nassignmentusing
Bayesianclustering
Implem
entstheclustering
algorithm
used
inST
RUCTURE(see
above)
that
clusters
samples
into
populatio
nsby
minim
izingHardyndashWeinb
ergdisequ
ilibrium
foragiven
partitioninglevelHow
everStructuram
aalso
includes
theadditio
nof
reversible-jum
pMCMCto
identifytheop
timalpartition
inglevelNearlyanytype
ofgenetic
datacanbe
inpu
tinto
Structuram
aandtheprog
ram
canassign
individu
alsto
populatio
nwith
orwith
outtheadmixture
Limita
tions
Tem
poraldivergence
andrelatio
nships
amongputativ
egroups
isnot
explicitlyestim
atedE
quivalence
togenetic
clusters
tospecies-levelg
roupsisuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
httpctegberkeleyedu
structuram
aindexhtmldescribedin
Huelsenbeck
etal(201
1)
Genotyp
icdata
ABGDmdashbarcodegapusinggenetic
distances
ldquoAutom
aticBarcode
Gap
Discoveryrdquosortssequencesinto
hypotheticalspeciesbasedon
thebarcodegap
The
methoduses
arecursiveapproach
topartition
thedata
andtestfor
sign
ificant
gapsA
BGDisfastsim
plemethodto
split
asequence
alignm
entd
atasetinto
candidatespeciesthat
should
becomplem
entedwith
otherevidence
inan
integrative
taxono
mic
approach
Limita
tions
Single-locus
data
aloneshould
only
beused
toprovideaprelim
inary
perspectiveof
speciesboundaries
andnotas
thesole
evidence
inspecies
circum
scriptions
Source
httpwwwabisnv
jussieufrpublicabg
ddescribedin
Puillandreet
al(201
2)
Single-locus
sequence
alignm
ent
GMYCampbG
MYCmdashgene
tree
The
GMYC
approach
combinesacoalescent
model
ofintraspecificbranchingwith
aYulemod
elforinterspecificbranching
which
isthen
fitto
aninferred
sing
le-gene
topology
toestim
atespeciesboundaries
andastatistical
measure
ofconfi
denceforthe
inferred
boundariesA
san
inputtheGMYCapproach
requires
anultram
etricgene
tree
andrecent
refinementscanaccountforuncertaintyin
phylogenetic
relatio
nships
and
parametersof
theGMYCmod
elT
heGMYCisgenerally
stable
across
awiderangeof
circum
stancesincludingvariousmethods
ofphylogeneticreconstructio
nthepresence
ofahigh
numbersingletonshigh
numbers
ofsampled
speciesandgaps
inintraspecific
sampling
theaccuracy
oftheGMYCismostsign
ificantly
affected
bythemean
populatio
nsize
relativ
eto
divergence
times
betweenthem
Limita
tions
The
GMYCmay
delim
itwell-supportedclades
ofhaplotypes
asindependent
lineagesandas
such
may
beproneto
over-delim
itatio
nSingle-locus
data
aloneshould
only
beused
toprov
ideaprelim
inaryperspectiveof
speciesbo
undaries
andno
tas
the
sole
evidence
inspeciescircum
scriptions
Source
(http
r-forger-projecto
rgprojectssplitshttpssitesgooglecom
site
noahmreidhom
esoftware)describedin
Monaghanet
al(200
9)Fu
jisaw
aandBarrac-
loug
h(201
3)andPo
nset
al(200
6)
Sing
le-locus
ultram
etricgene
tree (con
tinued)
20 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
PTPamp
bPTPmdash
gene
tree
The
Poissontree
processes(PTP)
modelcanbe
used
toinferputativ
especiesboundaries
onagivenphylogenetic
inputtreePT
Pcaninferputativ
especiesboundaries
that
are
consistent
with
thePS
CAnim
portantadvantageof
thismethodisthat
itmod
els
speciatio
nin
term
sof
thenumberof
substitutionsthereby
circum
ventingthepotentially
error-proneandcompute-intensive
processof
generatin
gultram
etrictreeswhich
are
required
asan
inpu
tfor
GMYCmod
el(see
abov
e)F
urthermoreitappearsthatthePT
Pmodel
may
outperform
theGMYCandotherOTU-picking
methods
whenevolutionary
distancesaresm
all
Limita
tions
Single-locus
data
aloneshould
only
beused
toprovideaprelim
inary
perspectiveof
speciesboundaries
andnotas
thesole
evidence
inspecies
circum
scriptions
Source
httpscoh-itsorgexelix
iswebsoftwarePT
Pindexhtmldescribedin
Zhang
etal(201
3)
Sing
le-locus
gene
tree
GeneiousSp
eciesDelim
itatio
nplug
-inmdash
gene
tree
Aplug-into
theGeneioussoftwareprovides
anexploratorytool
allowingtheuser
toassess
phylog
enetic
supportanddiagno
sabilityof
speciesdefinedas
user-selected
collections
oftaxa
onuser-sup
pliedtreesThe
plug
-incompu
tesstatisticsrelatin
gto
the
probability
oftheob
served
mon
ophyly
orexclusivity
having
occurred
bychance
ina
coalescent
processandassesses
thewith
in-andbetween-speciesgenetic
distancesto
infertheprobability
with
which
mem
bers
ofaputativ
especiesmight
beidentifi
edsuccessfully
with
tree-based
methods
Limita
tions
The
plug-insummarizes
measuresof
phylogenetic
supportand
diagnosabilityof
speciesdefinedas
user-selectedcollections
oftaxabutitdoes
not
providedefinitiv
esupportforspeciesgroupsItassumes
speciesaremonophyletic
Source
Implem
entedas
aplug-into
Geneious(geneiouscom)describedin
Mastersetal
(201
1)
Sing
le-locus
gene
tree
BPamp
Pmdashmultispecies
coalescent
model
for
speciesvalid
ation
Thisapproach
tospeciesdelim
itatio
nuses
aBayesianmodelingapproach
togeneratethe
posteriorprobabilitiesof
speciesassignmentstaking
accountof
uncertaintiesdueto
unknow
ngene
treesandtheancestralcoalescent
processThe
methodrelieson
auser-
specified
guidetreeimplem
entin
gareversible-jum
pMarkovchainMonte
Carlo
search
ofparameter
spacethatincludes
θpopulatio
ndivergenceand
estim
ated
distributio
nsof
gene
treesfrom
multip
leloci
Limita
tions
Misspecificatio
nsof
priorsandthegu
idetree
canresultin
inflatedspeciatio
nprobabilitiesitassumes
norecombinatio
nandcomputatio
nallim
itatio
nsrestrict
itsutility
with
next-generationdatasetswith
100s
ofloci
Source
httpabacusgeneuclacuksoftwarehtmldescribedin
YangandRannala
(201
0)andRannala
andYang(200
3)
Multilocus
sequence
alignm
entsandgroup
mem
bership
(con
tinued)
2 The Dynamic Discipline of Species Delimitation hellip 21
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
DISSE
CTmdashmultispecies
coalescent
model
forspeciesdelim
itatio
nDISSE
CTexplores
thefullspaceof
possible
clusteringsof
individualsandspeciestree
topologies
inaBayesianfram
ework
Toavoidtheneed
forreversible-jum
pMCMCit
uses
anapproxim
ationin
theform
ofapriorthatisamodificatio
nof
thebirthndashdeathprior
forthespeciestreeItisim
plem
entedas
partof
BEAST
andrequires
only
afewchanges
from
astandard
BEAST
analysisA
nalysesof
simulated
andem
piricald
atasuggestthat
themethodisshow
nto
beinsensitive
tothedegree
ofapproxim
ation
butq
uitesensitive
tootherparameters
Limita
tions
Recently
describedmethodlackingathorough
theoretical
andem
pirical
evaluatio
nItappearsthat
alargenu
mberof
sequencesarerequ
ired
todraw
firm
conclusion
sSo
urce
httpcodegooglecompbeast-m
cmcamphttpwwwin
driid
com
dissectinbeast
htmldescribedin
JonesandOxelm
an(201
4)
Multilocus
sequence
data
SpeD
eSTEMmdashmultispecies
coalescent
mod
elfordiscov
ery
valid
ation
andjoint
estim
ation
Thismaxim
umlik
elihoo
dandorinform
ationtheory-based
methodwas
developedto
test
speciesboundaries
inasystem
with
existin
gsubspecies
taxonomy(CarstensandDew
ey20
10)andcomputestheprobability
ofthegene
treesgiventhespeciestree
forall
hierarchical
perm
utations
oflin
eage
grou
pingSp
eciesbo
undaries
arecomparedusing
Akaikeinform
ationcriteria
andphylogenetic
uncertaintyin
thespeciestree
topologies
does
notaffect
speciesdelim
itatio
nsLimita
tions
Accuracyisdependenton
quality
ofthegene
tree
estim
ates
Source
(http
carstenslaborgohio-stateedusoftwarehtml)criteria
describedin
Ence
andCarstens(201
1)
Multilocus
sequence
alignm
entsandgroup
mem
bership
Browniemdash
multispecies
coalescent
model
forspeciesdelim
itatio
nThe
nonp
aram
etricheuristic
speciesdelim
itatio
napproach
implem
entedin
theprog
ram
Brownie(O
rsquoMeara
2010)jointly
sortsanonym
oussamples
into
speciesandinfers
aspeciestree
from
inputg
enetreesfrom
differentlociassumingthatforaspeciatio
neven
thecorrespondingnodeson
gene
treeswill
bemoreconsistent
with
each
than
the
divergenceswith
inspecies
Limita
tions
Findingboth
theoptim
umspeciestree
andspeciesboundaries
remains
compu
tatio
nally
challenging
andBrowniehasbeen
show
nto
frequently
yieldincorrect
resultsT
heaccuracy
ofthemethodislik
elycorrelated
with
nodalsupportvalues
inthe
individu
algene
topo
logies
Source
httpwwwbrianom
earain
fobrownie
describedin
OrsquoM
eara
(201
0)
Individual
gene
trees
(con
tinued)
22 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
BFD
mdashmultispecies
coalescent
model
for
speciesdelim
itatio
nThe
recently
developedmethod
Bayes
factor
delim
itatio
n(with
genomicdataB
FD)
combinesady
namic
programmingalgorithm
forestim
atingspeciestreesthat
bypasses
thecompu
tatio
nally
intensiveMCMCintegrationov
ergene
treesto
prov
idearigorous
techniqueforspeciesdelim
itatio
nstudiesusinggenome-wideSN
PdataCom
petin
gspeciesdelim
itatio
nmodelsarecomparedusingBayes
factorsanditappearsthat
this
approach
isrobustto
samplesizes(iefew
loci
andlim
itedsamples
perspecies)
and
misspecificatio
nof
thepriorforpo
pulatio
nsize
(θ)
Limita
tions
Recently
describedmethodlackingathorough
theoretical
andem
pirical
evaluatio
nSo
urce
httpwwwbeast2orgwikiindexphpBFD
describedin
Leacheacuteet
al(201
4)
Genom
e-wideSN
Pdata
2 The Dynamic Discipline of Species Delimitation hellip 23
distances among organisms belonging to thesame species are smaller than distances amongorganisms from different species (Puillandreet al 2012 Hebert et al 2003) Genetic distanceapproaches hold particular promise as an identi-fication tool shortcutting the difficulties of mor-phology-based identification (Hebert et al 2004)although in practice a barcode gap may not existfor many groups (Wiemers and Fiedler 2007)Furthermore the role of distance-based approa-ches using a single genetic marker for speciesdiscovery remains more controversial (Rubinoff2006) and without other corroborating evidenceOTUs inferred from single-locus dataset shouldonly be considered ldquocandidaterdquo species Tree-based methods aim to detect monophyletic cladescorresponding to species-level diversity bydetecting discontinuities associated within inter-and intraspecific branching patterns (Fujisawaand Barraclough 2013 Zhang et al 2013Monaghan et al 2009 Pons et al 2006) Tree-based species delimitation methods can also beused on the basis of other properties related tophylogenetic tree topologies (monophyly con-cordance with geography etc (Sites and Marshall2003 2004) Both distance- and tree-basedmethods have been applied for assessing speciesboundaries in lichen-forming fungi (Leavitt et al2012c 2014 Parnmen et al 2012 Del-Pradoet al 2010 2011 Molina et al 2011)
A number of tree-based methods partiallyautomate the species delimitation process withspecific bioinformatical analyses (Table 21)including the general mixed Yule coalescent(GMYC) approach (Fujisawa and Barraclough2013 Monaghan et al 2009 Pons et al 2006)and the Poisson tree processes (PTP) model(Zhang et al 2013) These methods provide rel-atively straightforward and objective pipelinesfor delimiting putative species-level lineagesfrom inferred gene trees by fitting within- andbetween-species branching models to an inferredsingle-locus topology A number of other tree-based methods for species delimitation areeffectively summarized in Sites and Marshall(2004)
When only single-locus data are available theGMYC has been shown to be a relatively robust
tool for species delimitation The GMYCapproach combines a coalescent model of intra-specific branching with a Yule model for inter-specific branching which is then fit to an inferredsingle-gene topology to estimate species bound-aries and a statistical measure of confidence for theinferred boundaries As an input the GMYCapproach requires an ultrametric gene tree andrecent refinements can account for uncertainty inphylogenetic relationships and parameters of theGMYC model (Fujisawa and Barraclough 2013Reid and Carstens 2012) Regardless of theseimprovements it may be difficult to accuratelyinfer an adequate ultrametric tree for large data-sets Although it appears that the GMYC isgenerally stable across a wide range of circum-stances including various methods of phyloge-netic reconstruction the presence of a highnumber singletons high numbers of sampledspecies and gaps in intraspecific sampling theaccuracy of the GMYC is most significantlyaffected by the mean population size relative todivergence times between them (Fujisawa andBarraclough 2013 Talavera et al 2013) Fur-thermore research suggests that the so-calledsingle-threshold version of the GMYC methodlikely outperforms the multiple-thresholdapproach (Fujisawa and Barraclough 2013Monaghan et al 2009) However other studiessuggest that theGMYCmethodmay often providehigher estimates for the total number ofOTUs thanother molecular species delimitation methods(Hamilton et al 2014 Miralles and Vences 2013Talavera et al 2013) warranting a cautiousinterpretation of results from GMYC analyses
Compared to the GMYC approach therecently introduced PTP method for speciesdelimitation has been suggested to be moreaccurate for preliminary species delimitation(Zhang et al 2013) Relative to the GMYC PTPoffers a more straightforward implementationrequiring a simple phylogenetic tree rather thanan ultrametric chronogram However moreresearch is required to assess the general perfor-mance of PTP across wide range of empirical andsimulated species delimitation studies At thispoint data support the use of the GMYC andPTP methods as objective and reasonable starting
24 SD Leavitt et al
points for species delimitation using single-genetopologies
The ldquoSpecies Delimitationrdquo plug-in to theGeneious software provides statistical approachesfor exploring species boundaries in single-genetopologies (Masters et al 2011) Using a priorispecimen assignments to putative species theldquoSpecies Delimitationrdquo plug-in computes statis-tics relating to the probability of the observedmonophyly or exclusivity having occurred bychance in a coalescent process and assesses thewithin- and between-species genetic distances inorder to infer the probability with which membersof a putative species might be identified suc-cessfully with tree-based methods An importantcontribution of the ldquoSpecies Delimitationrdquo plug-in is that it provides an objective means for usersto assess putative species within an empiricalstatistic-based framework rather than qualitativeevaluations of what level of hierarchy constitutesa species in a phylogeny
In contrast to simple sequence similaritythresholds (OTU-picking) for delimiting putativeevolutionarily significant units the AutomaticBarcode Gap Discovery (ABGD) method is anautomated procedure that sorts sequences intohypothetical species based on the existence of abarcode gap observed whenever intraspecificgenetic distances are less than those amongorganisms from different species (Puillandreet al 2012) Ultimately ABGD is a fast simplemethod that can be used to group individualsrepresented in a single-locus sequence alignmentinto candidate species that should be comple-mented with other lines of evidence in an inte-grative taxonomic approach (Kekkonen andHebert 2014 Puillandre et al 2012)
In general the ABGD GMYC and PTP ana-lytical protocols using single-locus data arerepeatable and computationally relatively fastproviding a valuable starting point for a pre-liminary perspective into species boundaries inunderstudied groups that can be validated withsubsequent studies (Kekkonen and Hebert 2014)Similarly analyses of single-locus data can beused to corroborate traditional phenotype-basedspecies boundaries and identify candidate speciesthat have previously been hidden within nominal
species However single-locus species delimita-tionmethodsmaybeparticularly prone to failure inrecognizing significant proportions of species-level biodiversity due to the strict criterion forreciprocal monophyly or alternatively providespurious inflations of estimated species diversitybased on genetic differences that donot correspondto species-level lineages For example recentestimates suggest that the incidence of species-level gene tree paraphyly is approximately 20 (Ross 2014) suggesting that analyses of single-locus datasets would likely fail to accurately deli-mit species in one in every five cases In contrastrecent empirical studies suggest that in some casesthe GMYC provides a striking overestimate ofspecies diversity (Miralles and Vences 2013)
The internal transcribed spacer region (ITS)has played a central role in molecular phyloge-netic studies of lichenized fungi and has recentlybeen adopted as the official barcoding marker forfungi (Schoch et al 2012) In many cases the ITSis sufficiently variable to resolve species bound-aries for lichenized fungi (Schoch et al 2012Kelly et al 2011) although accurate specimenidentification using sequence-based identificationapproaches requires a thoroughly curated refer-ence database (Leavitt et al 2014 Orock et al2012 Kelly et al 2011) In spite of the generalutility of the ITS marker a number of issues maypotentially limit its effective use in speciesdelimitation studies including the potential forintragenomic variation of the nuclear ribosomaltandem repeat and difficulties in aligning ITSsequences from divergent taxa (Kiss 2012)Because the ITS has been formally adopted as theofficial barcoding marker for fungi we recom-mend that species delimitation studies attempt toinclude this region for consistency across studiesHowever we recognize that in some taxonomicgroups the inclusion of the ITS may be prob-lematic and therefore suggest a careful screeningof other markers to identify appropriate loci forresolving species-level relationships
Ultimately the success of any single-locusspecies delimitation method largely depends onthe evolutionary history of the species groupunder study and the variability of the selectedmarker (Fig 22) Species delimitation will be
2 The Dynamic Discipline of Species Delimitation hellip 25
more difficult in more recently diverged lineagesand in cases with some level of interspecific geneflow relative to older well-diverged lineages(eg Leavitt et al 2012c 2013e) In the end wereemphasize that single-locus data alone shouldonly be used to provide a preliminary perspectiveof species boundaries and not as the sole evi-dence in species circumscriptions
222 Sampling Across the GenomeMultilocus Sequence Dataand Genome-Wide Markers
Although single-locus methods can provide anefficient approach for preliminary large-scaleassessments of species diversity there are
significant limitations particularly in recentlydiverged species groups where retention ofancestral polymorphisms and incomplete lineagesorting will likely result in different neutral locihaving unique gene topologies that do not mirrorthe speciation process (Knowles and Carstens2007 Heled and Drummond 2010 Taylor et al2000) In contrast to single-locus and strictlyphenotype-based approaches for species delimi-tation analyses of genetic data collected fromindependent genomic regions can provide robusthypotheses of species boundaries with increasingconfidence (Satler et al 2013 Zhang et al 2011Yang and Rannala 2010) Sequences from mul-tiple independent loci provide an importantsource of data for species delimitation studiesincluding recently developed models that
Fig 22 A simplified diagram illustrating the process ofspeciation through time in a single gene history andresulting gene topologies sampled at two points in thespeciation history (modified from Leliaert et al 2014)aEach dot represents a distinct gene copy and each row onenon-overlapping generation with lines connecting genecopies to their ancestors in the previous generation (onerow below) dashed diagonal lines represent reproductivebarriers two hypothetical sampling intervals at differentpoints in the speciation history are shown Tx and Ty
species delimitation methods using single-locus data areeffective only when species are reciprocally monophyleticin the sampled gene tree genetic clustering and coalescent-based species delimitation methods can circumscribespecies when species may not be monophyletic in sampledgenetic loci b Gene topology representing sampledhaplotypes at time Tx (shown in panel a) hypotheticalspecies are reciprocally monophyletic c Gene topologyrepresenting sampled haplotypes at time Ty (shown in panela) hypothetical species are para- and polyphyletic
26 SD Leavitt et al
combine individual gene genealogies and speciesphylogenies via modeling the coalescent historyof markers (Yang and Rannala 2010 Edwards2009 Knowles and Carstens 2007) As aresponse to advances in sequencing technologiesbioinformatical approaches for multimarker spe-cies delimitation analyses continue to be devel-oped (Table 21 Camargo and Sites 2013)
Long-term reproductive isolation of candidatespecies can be assessed with multilocus sequencedata by evaluating genealogical concordance ofunlinked markers (Baum and Shaw 1995 Aviseand Ball 1990) Within species the mixingeffects of recombination cause unlinked loci tohave distinct genealogical histories but geneticdrift and long-term divergence leads to concor-dant genealogical histories at loci across thegenome Relationships of individuals belongingto distinct candidate species can be evaluatedusing gene genealogies (eg haplotype networksor single-gene topologies) to identify lineagesthat exhibit genealogical exclusivity acrossunlinked neutral loci (Hudson and Coyne 2002Dettman et al 2003b Avise and Ball 1990) Thepresence of the same clades in the majority ofsingle-locus genealogies is taken as evidence thatthe clades represent reproductively isolated lin-eages (Dettman et al 2003a Pringle et al 2005)In practice the criteria of reciprocal monophylyand genealogical concordance of unlinked lociprovide a conservative approach for assessingspecies boundaries due to the fact that a sub-stantial amount of time is required after the initialdivergence of species until ancestral polymor-phisms have fully sorted (Knowles and Carstens2007 Hudson and Coyne 2002) Consequentlygroups with recent divergence histories willlikely go undiscovered under a genealogicalconcordance criterion due to the fact that speciesboundaries likely are not reflected in genegenealogies For example it would take morethan 1 million years after speciation before spe-cies would be delimited if 15 loci were sampledin species with an effective population size (Ne)of 100000 assuming one generation a yearunder a strict reciprocal monophyly criterion(Knowles and Carstens 2007) The amount oftime required for a species to be recognized using
a criterion of reciprocal monophyly increasesproportionally with increasing population sizes(Hudson and Coyne 2002) In a number ofstudies of lichen-forming fungi a genealogicalconcordance criterion has been used to bothdelimit previously unrecognized species-levellineages and validate some conventional pheno-type-based species (Leavitt et al 2012a c2013b Molina et al 2011 Kroken and Taylor2001)
Although analyses of molecular sequence datarelying on reciprocal monophyly or fixed char-acter differences for species delimitation canserve as important criteria for identifying speciesthese criteria are not commonly met acrossmultiple loci particularly in recent speciationhistories (Fig 22) To accommodate theobserved conflict among genealogies from mul-tiple loci with the underlying speciation historythe recent merge of coalescent theory with phy-logenetics has driven a major paradigm shift inspecies delimitation and molecular systematics ingeneral (Fujita et al 2012 Edwards 2009) Themultispecies coalescent model can be applied togenealogical histories from multiple independentloci to assemble separate coalescent processesoccurring in populations into a species tree(Degnan and Rosenberg 2009 Rannala andYang 2003) Within this coalescent-basedframework multiple individuals can be assignedto a single speciespopulation and the speciationhistory of ancestral and descendant species-levellineages can be inferred as a ldquospecies treerdquo incontrast to estimating gene genealogies fromindividual samples (Degnan and Rosenberg2006 2009 Rannala and Yang 2003)
A number of multispecies coalescent-basedspecies delimitation methods have recently beenintroduced offering an exciting framework forempirically assessing species boundaries byselecting the best species tree model from a set ofalternative models representing differenthypotheses of species boundaries (Table 21)For example SpeDeSTEM (Ence and Carstens2011) finds the maximum likelihood values for aspecies tree assuming all putative species areseparate lineages and for alternative species treeswhere two or more species are collapsed into a
2 The Dynamic Discipline of Species Delimitation hellip 27
single lineage The best fitting model is thenselected using the Akaike information criterionunder the assumption of constant populationsizes and fixed gene topologies across the speciestree The species delimitation program BayesianPhylogenetic and Phylogeography (BPampPYangand Rannala 2010) accommodates gene treeuncertainty and variable population sizes andsamples from the Bayesian posterior distributionof species delimitation models using reversible-jump Markov chain Monte Carlo (rjMCMC)sampling BPampP requires a user-provided guidetree with resolution finer than the species leveland evaluates alternative modes derived from allpossible subtrees that are generated by collapsingor splitting nodes on the guide tree (Yang andRannala 2010 Rannala and Yang 2003) Theproportion of time spent on each model is pro-portional to the posterior probability of themodel ie ldquospeciation probabilitiesrdquo WhileBPampP ranks among the most popular coalescent-based species delimitation programs (Fujita et al2012) misspecifications of the guide tree andorthe prior distribution for population size (θ) canresult in strong support for models containing aninflated number of species (Leacheacute and Fujita2010 Zhang et al 2011) The nonparametricheuristic species delimitation approach imple-mented in the program Brownie (OrsquoMeara 2010)jointly sorts anonymous samples into species andinfers a species tree from input gene trees fromdifferent loci assuming that for a speciationevent the corresponding nodes on gene trees willbe more consistent with each than the diver-gences within species However finding both theoptimum species tree and species boundariesremains computationally challenging andBrownie has been shown to frequently yieldincorrect results (OrsquoMeara 2010)
Most recently available coalescent-basedspecies delimitation methods require individualassignments to a species or population a priori Ina number of scenarios correct assignments ofsamples to species may be difficult includingpresence of cryptic species incongruencebetween conventional species and moleculardata or simply the fact that accurate specimenidentification in complex groups with subtle or
difficult to discern diagnostic characters is anontrivial task (Leavitt et al 2013e) Statisticalmethods for assessing individual assignment andspecies detection prior to coalescent-based spe-cies delimitation and species tree reconstructionand species provide a more objective approachfor understanding species boundaries
Population assignment tests using a variety ofclustering algorithms using genetic data offer auseful approach for grouping individuals intoputative reproductively isolated groups (Table 21Hausdorf and Hennig 2010 Corander et al2004 2008 Falush et al 2003 Pritchard et al2000) particularly for species delimitation prob-lems that exist at the interface of traditionalpopulation genetic and phylogenetic analyses(Carstens et al 2013 Weisrock et al 2010Knowles and Carstens 2007)
The programs STRUCTURE (Falush et al2003 Pritchard et al 2000) and STRUCTURA-MA (Huelsenbeck et al 2011) cluster samplesinto populations by minimizing HardyndashWeinbergdisequilibrium for a given number of populationclusters using unlinked genetic markers In gen-eral unlinked markers are not available for mostgroups of lichen-forming fungi and a numberof studies have used information from multilocussequence data for STRUCTURE analyses(Leavitt et al 2011a b c 2013e Fernaacutendez-Mendoza and Printzen 2013 Fernaacutendez-Mendoza et al 2011) Varying approaches havebeen implemented to convert DNA sequencedata to allelic data for Bayesian clustering (seeOrsquoNeill et al 2013) STRUCTURE is expectedto perform well when there is sufficient inde-pendence across regions such that linkage dis-equilibrium within regions does not dominate thedata (STRUCTURE manual) but can also beeffective using multilocus sequence data andtreating all SNPs as independent loci regardlessof physical linkage within each locus (OrsquoNeillet al 2013 Falush et al 2003) A recent study ofthe lichen-forming genus Letharia showed thatBayesian clustering implemented in the programSTRUCTURE was generally able to recover thesame putative Letharia lineages circumscribedemploying a gene genealogical approach inKroken and Taylorrsquos iconic species delimitation
28 SD Leavitt et al
study (Altermann et al 2014 Kroken and Taylor2001) Altermann et al (2014) show that popu-lation assignments were largely consistent acrossa range of scenarios including extensiveamounts of missing data the exclusion of SNPsfrom variable markers and inferences based onSNPs from as few as three gene regions Incontrast to STRUCTURE and STRUCTURA-MA the program BAPS (Corander et al 2008)can explicitly infer genetic structure using hap-loid sequence data or other linked geneticmarkers in the ldquoclustering with linked locirdquomodel Another advantage of BAPS is that theprogram is much more computationally efficientthan STRUCTURE or STRUCTURAMA Sim-ulation studies indicate that both BAPS andSTRUCTURE perform well at low levels ofpopulation differentiation and when clusters arenot well differentiated (Latch et al 2006)
In practice inferring the most appropriatelevel of population structure using Bayesianclustering algorithms remains challenging (Latchet al 2006 Evanno et al 2005) BAPS andSTRUCTURAMA can infer both individualassignments and the most likely number ofgenetic clusters (Corander et al 2008 Huelsen-beck et al 2011) and the ad hoc ΔK statisticproposed by Evanno et al (2005) provides anobjective approach for identifying the uppermosthierarchical level of structure However carefulconsideration of other levels of populationstructure may ultimately reveal more biologicallymeaning results and researchers should examineindividual assignments across a range of geneticgroupings Some markers generated fromindependent genomic regions such as SNPs andfast-evolving microsatellites can be used to dis-tinguish fine-scale population structuring on thebasis of allele frequencies and species delimita-tion analyses based on these markers may implythe risk of severe taxonomic oversplitting Inthese cases validation approaches such asBPampP and SpeDeSTEM and corroborationamong different species delimitation approachescan provide perspective between intraspecificpopulation structure and species-level clusters
Another limitation of clustering approaches isthat they do not assess or take into account
evolutionarily divergences and relationshipsamong population clusters Coalescent-basedspecies tree methods (discussed below) provide ameans to assess the diversification histories ofpopulations inferred from clustering analysesTherefore a potential working protocol for aninformed species delimitation study that takesinto account population structure could consist offirst applying a genetic clustering analysis under apopulation genetics criterion (eg BAPSSTRUCTURE STRUCTURAMA) to identifygenetically distinct population clusters that can beconsidered ldquocandidate speciesrdquo From these can-didate species a species tree can be inferred forfocal group using coalescent-based species treereconstruction methods (eg BEAST) Subse-quently a coalescent-based validation methodcan be applied to assess whether the distinctpopulation clusters represent independent evolu-tionary lineages (eg BPampP) This multistepapproach would provide a consistent and standardcriterion for distinguishing between population-and species-level lineages (Camargo and Sites2013) and has been applied to a number of casesincluding lichen-forming fungi (Leavitt et al2011a b c 2013e Leacheacute and Fujita 2010)
High-throughput sequencing methods providethe means to effectively sample hundreds tothousands of loci from across a species genome fora large number of species and continue to revolu-tionize studies that can be performed even in non-model organisms Targeted high-throughputsequencing approaches such as anchored phy-logenomics transcriptome sequencing reduced-representation genomic library sequencing(restriction-site-associated DNA sequencingRAD-Seq and genotype-by-sequencing GBS)and high-throughput amplicon sequencing pro-vide important insight into species boundaries fora number groups although none of these approa-ches has yet been applied to studies of lichenizedfungi
For example restriction-site-associated(RAD-Seq) DNA can simultaneously detect andgenotype thousands of genome-wide SNPs byfocusing the sequencing effort on a reducedrepresentation of the entire genome (Baird et al2008) and has been successfully applied to
2 The Dynamic Discipline of Species Delimitation hellip 29
intraspecies (Lewis et al 2007 Miller et al 2007Emerson et al 2010) and interspecies studies(Rubin et al 2012 Eaton and Ree 2013 Wagneret al 2013) This approach has provided strikinginsight into the recent adaption radiation of LakeVictoria cichlids (Wagner et al 2013) Analternative approach targeting the sequencing ofspecific loci using next-generation sequencingplatforms provides an efficient means of gener-ating data for loci of known genomic locationorthology size and expected level of variation(OrsquoNeill et al 2013) Markers can be targetedusing well-established PCR techniques or newerhybridization techniques and subsequentlypooled for high-throughput sequencing usingparallel-tagged sequencing of multiple individualsamples within a single sequencing run (OrsquoNeillet al 2013) A recent study of North Americantiger salamanders (Ambystoma tigrinum)demonstrated the potential for amplicon-basedparallel-tagged sequencing to rapidly generatelarge-scale genomic data for species delimitationand species tree research (OrsquoNeill et al 2013)
Although these methods provide an enormousamount of data and insight into diversification atan unprecedented scale computational limita-tions restrict the applicable analytical methodsfor large datasets although computational andanalytical advances are happening rapidly Mostrecent coalescent-based species delimitation andspecies tree models using gene trees have beenlimited to handle tens of loci for multiple indi-viduals and combining hundreds or thousands ofgene trees into a single species delimitationframework presents considerable computationalchallenges (Camargo and Sites 2013) Therecently developed method Bayes factor delim-itation (with genomic data BFD) combines adynamic programming algorithm for estimatingspecies trees that bypasses the computationallyintensive MCMC integration over gene trees toprovide a rigorous technique for species delimi-tation studies using genome-wide SNP data(Leacheacute et al 2014) Competing species delimi-tation models are compared using Bayes factorsand it appears that this approach is robust tosample sizes (ie few loci and limited samples
per species) and misspecification of the prior forpopulation size (θ) (Leacheacute et al 2014)
Fungi are an ideal model for assessing diver-gence in eukaryotes due to their simple mor-phologies small genomes broad ecological rolesand diverse lifestyles (Gladieux et al 2014)However the use of genomic data from high-throughput sequencing methods have not yet beenincluded in species delimitation studies of lichen-forming fungi This is due in part to challenges indealing with metagenomic data containing geno-mic information from a plethora of symbiontsassociated with a lichen thallus scant genomicresources and discipline-specific inertia A num-ber of lichen-forming fungal genomes are cur-rently available including Endocarponpusillum(Wang et al 2014) Cladonia spp (Park et al2013a 2014) Caloplaca flavorubescens (Parket al 2013b) and Cladonia greyi and Xanthoriaparientina through the Fungal Genomics Programat Joint Genome Institute of the United StatesDepartment of Energy (httpwwwjgidoegov)The foreseeable ongoing expansion of genomicresources for lichen-forming fungi will be centralto developing approaches for delimiting speciesusing high-throughput sequencing Specificallygenomic resources will be crucial in identifyingnovel markers (variable genes regions SNPsmicrosatellites etc) identifying conserved fungalmarkers for targeted high-throughput sequencingapproaches and references for entire genomecomparisons (Devkota et al 2014 Werth et al2013)
23 Can We Make SpeciesDelimitation in Lichen-FormingFungi Truly Integrative
The widespread availability of genetic data hascreated a biased viewpoint that only genetic datashould be used for statistical species delimitationHowever an ongoing appeal to researchers toassess species boundaries from multiple andcomplementary perspectives (phylogeneticspopulation genetics comparative morphologydevelopment ecology etc) has resulted in an
30 SD Leavitt et al
integrative taxonomic framework bringing theseconceptual and methodological perspectivestogether (Hamilton et al 2014 Fujita et al 2012Salicini et al 2011 Yeates et al 2011 Padialet al 2010 Dayrat 2005 Will et al 2005 Wiensand Penkrot 2002) In practice any study linkingdifferent kinds of data to support hypotheses ofspecies boundaries including mapping morpho-logical characters onto a molecular phylogenycan be considered integrative Integrative meth-ods for species delimitation fall across a broadspectrum ranging from verbal and qualitativeassessments of data classes to quantitative meth-ods that allow different data types to contribute tostatistical species delimitation (Yeates et al2011) In most taxonomic studies utilizing bothmolecular and morphological data expert opinionis eventually used to some degree to evaluate thefinal status of a candidate species (eg Bond andStockman 2008) In this sense many studiesusing evidence for independent data sources fordelimiting species boundaries use an iterativeapproach (Yeates et al 2011) where speciesboundaries can be tested within a hypothetico-deductive framework with diverse datasets
From a practical perspective we advocate aprocess of iterative taxonomy (sensu Yeates et al2011) to circumscribe and refine species limitsusing multiple lines of evidence This iterativeprocess involves comparisons of morphologicaldata with a phylogenetic hypothesis (ie single-locus gene tree or concatenated multilocusphylogeny) to identify the least inclusive mono-phyletic clade in the topology characterized by atleast one unambiguously diagnostic morphologi-cal character (Miralles and Vences 2013) Thisphylogenetic tree-informed approach to assessingspecies boundaries represents a common practicein studies of lichen-forming fungi Previouslyunrecognized species-level clades with corre-sponding subtle or overlooked phenotypiccharacters have been commonly observed in bothcrustose lichens [eg Acarospraceae (Wedin et al2009) Graphidaceae (Rivas Plata and Lumbsch2011 Papong et al 2009) Lecanoraceae (Leavittet al 2011a) Lecideaceae (Ruprecht et al 2010)Mycoblastaceae (Spribille et al 2011) andTeloschistaceae (Vondraacutek et al 2009 Muggia
et al 2008)] and foliose and fruticose lichens [egLobariaceae (Moncada et al 2014 McDonaldet al 2003) Parmeliaceae (Leavitt et al 2013aWirtz et al 2012 Divakar et al 2005 Molinaet al 2004 Kroken and Taylor 2001) Peltigera-ceae (OrsquoBrien et al 2009 Goffinet et al 2003)Physciaceae (Elix et al 2009 Divakar et al2007) and Sphaerophoraceae (Houmlgnabba andWedin 2003)] As a specific example Luumlckinget al (2008) used a combination of medullarychemistry and underside pigmentation in speci-mens from the Heterodermia obscurata group inCosta Rica to corroborate monophyletic clades inan ITS gene tree as species-level lineages
While this iterative approach to speciesdelimitation and taxonomy has proven valuablefor understanding species boundaries anddescribing new taxa in some groups of lichenizedfungi a posteriori examination of morphologicaland chemical features has failed to reveal diag-nostic phenotypic characters in a number ofstudies (Muggia et al 2014 Leavitt et al 2011aOtaacutelora et al 2010 OrsquoBrien et al 2009) Fur-thermore a study of widespread species in thegenus Melanelixia (Parmeliaceae) indicated thatphenotypically cryptic lichen-forming fungalspecies-level lineages may be relatively ancientand diagnosable phenotypic differences may beabsent even millions of years after the initialdivergence (Leavitt et al 2012b) The latter studyhighlights the fact that species-level lineages maycommonly exist without any observable diag-nostic phenotypic characters calling into ques-tion the impetus for a universal application ofintegrative taxonomy
In keeping with the principle that as manylines of evidence as available should be com-bined to delimit species (Dayrat 2005) the for-malized integrative taxonomic approach (ITAXMiralles and Vences 2013) uses a mtDNA guidetree and observations from different types of datathat might provide conclusive evidence for theindependence of lineages and thus their identityas different species Miralles and Vences (2013)provide a non-exhaustive list of species delimi-tation criteria to be integrated in the ITAXapproach including (a) sympatric occurrencewithout admixture as revealed by consistent
2 The Dynamic Discipline of Species Delimitation hellip 31
differences in morphological or molecular char-acters at the same geographic location (b) strongdifferences in a behavioral morphological orgenetic character known to mediate prematingisolation (c) unviability or infertility of hybrids(d) lack of gene flow across a geographicalhybrid zone (e) congruent diagnostic differencesbetween sister lineages in various unlinkedmorphological character (f) absence of haplo-type sharing in several unlinked nuclear loci and(g) a combination of criteria endashf Speciesboundaries are based on seeking the least inclu-sive monophyletic group in the mtDNA treewhich fulfills at least one of the criteria listedabove Clearly the ITAX approach is sensitive tosample size and in order to reliably support thedistinctiveness of a given species it has beenrecommended that the sampling strategy includesat least five individuals per species (Miralles andVences 2013) A similar approach could beadopted for lichenized fungi using an ITS genetopology as guide tree rather than mtDNA
A total evidence approach including concat-enation has been a common approach for inte-grating information from different sourcesincluding independent genetic markers in phy-logenetic reconstructions (Kluge 1989 Wiens1998 de Queiroz et al 1995) Proponents forconcatenation of independent data in phyloge-netic analyses argue that when combining alldata the underlying signal of speciation mayemerge even if weakly contradictory signalis contained in the individual data partitions(Gatesy et al 1999) For example establishing apreliminary perspective of species boundariesfrom multilocus sequence data using concatena-tion may provide a reasonable starting point forscreening for cryptic species and species treeinference (Šlapeta et al 2006 Leavitt et al2011a Le Gac et al 2007 Leacheacute 2009)However concatenation and consensus methodsimply a risk of obtaining inflated support forincorrect relationships and information aboutvariance in gene coalescence is lost (Degnan andRosenberg 2006 2009 Kubatko and Degnan2007 Edwards 2009) In spite of the impetus forintegrating evidence from independent data
sources into empirical species delimitation stud-ies and taxonomy (Fujita et al 2012 Padial et al2009) most currently available species delimi-tation methods are unable to accommodate non-genetic data sources in a statistical framework
231 Selecting the Appropriate Data
In the face of increasing availability of geneticdata and associated bioinformatical approachesfor delimiting species researchers should care-fully consider what information is being sacri-ficed by the failure to consider non-genetic datain species delimitation studies and whetheraccuracy could be improved by the addition ofmultiple data types Morphological data havehistorically served as a proxy to identify repro-ductively isolated groups (ie ldquospeciesrdquo) (Ray1686 Fujita et al 2012) Current methods fordelimiting species using non-genetic data (egchemistry morphology and ecology) remainwoefully understudied For example morphol-ogy-based species circumscriptions are generallybased on one or more qualitative (or quantitative)morphological characters that do not appear tooverlap with other species However ascertain-ing that a given trait is truly fixed within apopulation with statistical confidence requiresunrealistic sample sizes even when allowing forsome level of polymorphism in the diagnosticcharacter (Wiens and Servedio 2000) Now var-ious combinations of datamdashfrom morphologygenetics geography and ecologymdashare acceptedas standard information for species delimitationstudies (Ruiz-Sanchez and Sosa 2010 Ross et al2010 Edwards and Knowles 2014)
Below we briefly discuss appropriate datasources for species delimitation studies of lichen-forming fungi However homoplastic characters(similar traits that are not derived from a com-mon ancestor) are common among many traitscommonly used to circumscribe fungal taxa thebiological significance of secondary metabolitevariation remains largely unknown (Lawrey1986) and ecological niches may be difficultto adequately characterize and model due to
32 SD Leavitt et al
microhabitat requirements and data resolutionTherefore we advocate a cautious approach toselecting appropriate and relevant data forassessing species boundaries
In spite of the potential challenges and limi-tations of using phenotypic data these traits haveprovided a plethora of valuable information forunderstanding species boundaries For examplein the Melanelixia fuliginosa group (Parmelia-ceae) a morphometric analysis using color isi-dia and marginal zone free of isidia ascharacters revealed a general pattern of differ-entiation between material formerly recognizedas subspecies fuliginosa and glabratula in spiteof the fact that considerable overlap betweengroups occurs in some characters individually(Arup and Berlin 2011) Furthermore the dis-tinction of the two groups was supported by aphylogenetic analysis of the ITS marker andecological differences with M fuliginosaoccurring predominantly on rock and M glab-ratula on bark (Arup and Berlin 2011) Thisstudy of the M fuliginosa group provides a fit-ting example of using multiple independentsuites of data including ecology morphologyand genetic information to establish a robusthypothesis of species boundaries
In lichen systematics phenotypic dataincluding thallus organization secondary metab-olites mode of reproduction ascoma-type andontogeny ascus and ascospore characters havehistorically played a prominent role (Printzen2009) Ascomatal characters have traditionallyheld a major role in higher-level classification(Printzen 2009) in contrast to species-levelclassification which also tends to include a widearray of vegetative and chemical charactersCommonly assessed morphological charactersinclude thallus form and size cortical features(eg maculae and pseudocyphellae) presenceformcolor of attachment structure (eg rhizines)and reproductive mode (ascomata vs vegetativediaspores) (Printzen 2009) Different morpho-logical types of vegetative diasporesmdashcorticatedisidia and ecorticated sorediamdashand their locationare commonly used to distinguish speciesAscomatal characters including morphologylocation (laminal vs marginal) position (sessile
vs immersed) presence of thalline margins colorof an apothecial disc and presence or color ofpruina are also commonly used in speciesdelimitations of lichen-forming fungi (Printzen2009) Other important characters may includethalline characters form color size and septationof ascospores size and form and structure of ascithe hamathecium type of epihymenium andhypothecium and the type of excipulum orperidium conidiomatal characters etc (Printzen2009)
Assessments of secondary metabolites hasplayed an important role in lichen taxonomybeginning with the introduction of simple spottests by Nylander (1866a b) The use of chem-istry in lichen taxonomy has been discussed indetail in numerous reviews (Lumbsch 1998a bRogers 1989 Brodo 1986 Egan 1986 Leuckert1985 Brodo 1978 Hawksworth 1976 Culber-son 1969 1970) and we refer readers to thesevaluable sources for a more comprehensive per-spective on lichen chemistry In short extrolites(secondary metabolites) belong to various clas-ses the most common and diverse include dep-sides depsidones chlorinated xanthones andanthraquinones (Lumbsch 2002 Culberson1969) The presence or absence of specificextrolites or their replacements by another sub-stance is widely used to distinguish speciesparticularly when correlated with differences ingeographic distributions However if morpho-logical or geographical differences betweenpopulations containing different extrolites are notapparent the taxonomic significance has beendisputed with some authors distinguishing themas species and others preferring to regard them aschemical races within a species In addition tosimply using the presence or absence of extro-lites Culberson and Culberson (1976) proposedto arrange lichen substances into chemosyn-dromes of closely related substances The pres-ence or absence of these chemosyndromes maypotentially be used as characters to delimit spe-cies regarding differences involving the samechemosyndromes as intraspecific variation anddistinct chemosyndromes as evidence for inter-specific populations (Lumbsch 1994 Gowan1986)
2 The Dynamic Discipline of Species Delimitation hellip 33
Due to the fact that species delimitationstudies often incorporate a substantial biogeo-graphical or ecological component ecologicalniche modeling plays an increasing role in phy-logenetic and taxonomic research Niche mod-eling can provide evidence for ecologicalisolation between populations based on eitherconserved or divergent ecological niches andtherefore can provide additional evidence sup-porting lineage independence between putativespecies The application of ecological nichemodeling has been applied to species delimita-tion studies in a number of cases (Ruiz-Sanchezand Sosa 2010 Leacheacute et al 2009 Raxworthyet al 2007 Rissler and Apodaca 2007) althoughit has not been explicitly applied to assess speciesboundaries in lichen-forming fungi By mappingthe spatial distribution of environmental suit-ability of climatic variables for candidate speciesthe application of ecological niche modeling canbe particularly important in cases where specieshave allopatric distributions (Raxworthy et al2007)
Ecological niche modeling utilizes knownassociations between a speciesrsquo occurrencelocalities and environmental variables to defineabiotic conditions within which populations canbe maintained (Guisan and Thuiller 2005) Themethodological approach for modeling is basedon four general properties (i) The current knownspeciesrsquo localities is the dependent variable (ii)the distribution is modeled as a map composed ofgrid cells at a specified resolution (iii) a range ofenvironmental variables (eg temperature pre-cipitation and solar exposure) are collected todescribe the characteristics of each cell and (iv)classifying the degree to which each cell is eithersuitable or unsuitable for each species under arange of models (Guisan and Thuiller 2005)
Ecological data also have the potential to playan important role in understanding speciesboundaries in lichen-forming fungi For exampleNash and Zavada (1977) demonstrated that Xant-hoparmelia populations with distinct chemistriesoccurring in the northern portion of the SonoranDesert exhibit habitat selection among differentrock substrates within a region with relativelyuniform climate and topography In another case
the parmelioid species Parmelia mayi is mor-phologically indistinguishable from P saxatilisbut can be separated by bioclimatic features andgenetic and chemical characters (Molina et al2011) McCune and Printzen(2011) assess distri-butions and climatic niches of species in theLecanora varia group inwesternUSAandprovidea model that uses continental influence and annualtemperature as themajor factors predicting speciesdistributions The distribution of Usnea hirta alichen commonly used in air quality biomonitor-ing research was modeled for a section of theWhite River National Forest in central Coloradobased on the presence of U hirta at 72 biomoni-toring reference sites distributed in the inter-mountain western United States (Shrestha et al2012) The best model for predicting U hirtadistribution included four variablesmdashsolar radia-tion average monthly precipitation and averagemonthly minimum and maximum temperatures(Shrestha et al 2012) These studies supportthe potential use of ecological niche modelingmethods in species delimitation studies of lichen-forming fungi
24 Conclusions What AboutTaxonomy
In most cases species circumscription and tax-onomy requires some degree of qualitativejudgment and individual interpretation Integrat-ing multiple types of data into an empiricalframework for delimiting species boundarieswhere species boundaries can be tested within ahypothetico-deductive framework with diversedatasets can provide robust hypotheses of speciesboundaries and taxonomic stability (Yeates et al2011) However it has long been known toevolutionary biologists that distinct species donot need to have diagnosable morphologicaldifferences (Mayr 1963) and increasing avail-ability of genetic data has allowed researchers toidentify species and to rigorously test speciesboundaries with a level of precision that wasunimaginable a decade ago While analyticaladvances in statistical species delimitation havebeen largely based on genetic data the utility of
34 SD Leavitt et al
these approaches to formal taxonomy remainselusive Due to these challenges an eclecticapproach to delimiting species and cautionagainst the reliance on any single dataset ormethod is required when delimiting species
While results from many recent studies maycontradict traditional species boundaries acrossmany groups of lichen-forming fungi we areoptimistic that this research represents substantialprogress toward a more accurate perspective onspecies boundaries and diversity in fungi As aresult of ongoing research of species boundariesin lichen-forming fungi the taxonomic value ofmany phenotypes is now better understood ourunderstanding of ecological evolutionary andbiogeographic patterns has improved and we canbegin to better understand patterns of symbiontinteractions in lichens Integrating new data(including novel morphological characters andgenetic data) will be essential to accurately rep-resent species-level diversity across all groups oflichenized fungi Hopefully an improved per-spective on lichen diversity also increases ourappreciation of these incredible symbiotic sys-tems While we are strong advocates for theapplication of independent data types in devel-oping an integrative taxonomy there is anincreasing need to formally recognize the exis-tence of phenotypically cryptic species-levellineages in lichen-forming fungi (see Hibbettet al 2011) In some cases a molecular taxon-omy may provide the most practical approach toconsistent treatment of mycobiont species withinlichen groups where diagnostic morphologicalcharacters are unidentifiable or practically notfeasible (Leavitt et al 2013c d)
These are exciting times for taxonomists andphylogeneticists A closer look at lichen taxon-omy with the inclusion of new data will help usto better understand the diversity of these fasci-nating organisms accurately interpret distribu-tion patterns and play a more important role inmeaningful conservation practices Howeversome level of uncertainty will accompany pro-gress In many taxonomic groups our traditionalapproach for species identification will likelyneed to be substantially modified The search forcorroborating morphological support for cryptic
species identified using molecular data willrequire meticulous and creative approaches toassess phenotypic variation in potentially unor-thodox ways We are hopeful that lichenologistswho traditionally have been eager to include newmethods such as chromatography in their rou-tine identifications will be amenable to includemolecular techniques to their routine examina-tion of specimens for identification and classifi-cation Although this may prove difficult toachieve by single individuals especially citizenscientists that traditionally play an important rolein lichen taxonomy the increasing numberof collaborative projects in the discipline (egLumbsch et al 2011 Crespo et al 2010Gueidan et al 2009) make us optimistic thatbroad-scale collaborative approaches will facili-tate the inclusion of molecular data in lichenresearch at all levels
Acknowledgments We are indebted to various col-leagues for valuable thought-provoking discussionnotably Matthew Nelsen (University of Chicago) AnaCrespo (Universidad Complutense de Madrid) PradeepDivakar (Universidad Complutense de Madrid) BeckettSterner (The Field Museum) and Joyce Havstad (TheField Museum) We also thank anonymous reviewers whoprovided valuable comments that improved this chapterSupport by the US National Science Foundation isgratefully acknowledged (ldquoHidden diversity in parmelioidlichensrdquo DEB-0949147)
References
Ahti T Hawksworth DL (2005) Xanthoparmelia steno-phylla the correct name for X somloeumlnsis one of themost widespread usnic acid containing species of thegenus The Lichenologist 37(4)363ndash366 doi101017S0024282905015197
Altermann S Leavitt SD Goward T Nelsen MPLumbsch HT (2014) How do you solve a problemlike Letharia A new look at cryptic species in lichen-forming fungi using Bayesian clustering and SNPSsfrom multilocus sequence data PLoS ONE 9(5)e97556 doi101371journalpone0097556
Amo de Paz G Cubas P Crespo A Elix JA Lumbsch HT(2012) Transoceanic dispersal and subsequent diver-sification on separate continents shaped diversity ofthe Xanthoparmelia pulla group (Ascomycota) PLoSONE 7(6)e39683 doi101371journalpone0039683
Arguumlello A Del Prado R Cubas P Crespo A (2007)Parmelina quercina (Parmeliaceae Lecanorales)
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Arup U Berlin ES (2011) A taxonomic study ofMelanelixia fuliginosa in Europe The Lichenologist43(02)89ndash97 doi101017S0024282910000678
Arup U Grube M (2000) Is Rhizoplaca (Lecanoraleslichenized Ascomycota) a monophyletic genus Can JBot 78(3)318ndash327 doi101139b00-006
Avise J Ball R (1990) Principles of genealogicalconcordance in species concepts and biological tax-onomy Oxf Surv Evol Biol 745ndash67
Baird NA Etter PD Atwood TS Currey MC Shiver ALLewis ZA Selker EU Cresko WA Johnson EA(2008) Rapid SNP discovery and genetic mappingusing sequenced RAD markers PLoS ONE 3(10)e3376 doi101371journalpone0003376
Baum DA Shaw KL (1995) Genealogical perspectives onthe species problem In Hoch PC Stephenson AG(eds) Experimental and molecular approaches to plantbiosystematics Missouri Botanical Garden St Louispp 289ndash303
Beaumont MA Nielsen R Robert C Hey J Gaggiotti OKnowles L Estoup A Panchal M Corander JHickerson M Sisson SA Fagundes N Chikhi LBeerli P Vitalis R Cornuet J-M Huelsenbeck J FollM Yang Z Rousset F Balding D Excoffier L (2010)In defence of model-based inference in phylogeogra-phy Mol Ecol 19(3)436ndash446 doi101111j1365-294X200904515x
Bickford D Lohman DJ Sodhi NS Ng PKL Meier RWinker K Ingram KK Das I (2007) Cryptic speciesas a window on diversity and conservation TrendsEcol Evol 22(3)148ndash155 doi101016jtree200611004
Bonan GB Shugart HH (1989) Environmental factors andecological processes in boreal forests Annu Rev EcolSyst 20(1989)1ndash28
Bond J Stockman A (2008) An integrative method fordelimiting cohesion species finding the population-species interface in a group of Californian trapdoorspiders with extreme genetic divergence and geo-graphic structuring Syst Biol 57(4)628ndash646 doi10108010635150802302443
Brodo IM (1978) Changing concepts regarding chemicaldiversity in lichens The Lichenologist 10(1)1ndash11doi101017S0024282978000031
Brodo IM (1986) Interpreting chemical variation inlichens for systematic purposes The Bryologist 89(2)132ndash138
Caley MJ Fisher R Mengersen K (2014) Global speciesrichness estimates have not converged Trends EcolEvol 29(4)187ndash188 doi101016jtree201402002
Camargo A Sites JW (2013) Species delimitation adecade after the Renaissance In Pavlinov I (ed) Thespecies problemmdashongoing issues InTech doi10577252664
Camargo A Morando M Avila LJ Sites JW (2012)Species delimitation with ABC and other coalescent-based methods a test of accuracy with simulations and
an empirical example with lizards of the Liolaemusdarwinii complex (Squamata Liolaemidae) Evolution66(9)2834ndash2849 doi101111j1558-5646201201640x
Carstens BC Dewey TA (2010) Species delimitationusing a combined coalescent and information-theoreticapproach an example from North American Myotisbats Syst Biol 59(4)400ndash414 doi101093sysbiosyq024
Carstens BC Pelletier TA Reid NM Satler JD (2013)How to fail at species delimitation Mol Ecol 22(17)4369ndash4383 doi101111mec12413
Corander J Marttinen P (2006) Bayesian identification ofadmixture events using multi-locus molecular mark-ers Mol Ecol 15(10)2833ndash2843 doi101111j1365-294X200602994x
Corander J Waldmann P Marttinen P Sillanpaa M(2004) BAPS 2 enhanced possibilities for the analysisof genetic population structure Bioinformatics 20(15)2363ndash2369 doi101093bioinformaticsbth250
Corander J Marttinen P Mantyniemi S (2006) Bayesianidentification of stock mixtures from molecular markerdata Fish Bull 104550ndash558
Corander J Marttinen P Siren J Tang J (2008) EnhancedBayesian modelling in BAPS software for learninggenetic structures of populations BMC Bioinf 9(1)539 doi1011861471-2105-9-539
Coyne JA Orr HA (2004) Speciation Sinauer AssociatesSunderland
Cracraft J (1983) Species concepts and speciation anal-ysis Curr Ornithol 1159ndash187
Crespo A Lumbsch HT (2010) Cryptic species in lichen-forming fungi IMA Fungus 1167ndash170 doi105598imafungus2010010209
Crespo A Peacuterez-Ortega S (2009) Cryptic species andspecies pairs in lichens a discussion on the relation-ship between molecular phylogenies and morpholog-ical characters Anales del Jardin Botanico de Madrid66(S1)71ndash81 doi103989ajbm2225
Crespo A Kauff F Divakar PK del Prado R Perez-Ortega S Amo de Paz G Ferencova Z Blanco ORoca-Valiente B Nunez-Zapata J Cubas P ArguelloA Elix JA Esslinger TL Hawksworth DL MillanesA Molina MC Wedin M Ahti T Aptroot A et al(2010) Phylogenetic generic classification of parmeli-oid lichens (Parmeliaceae Ascomycota) based onmolecular morphological and chemical evidenceTaxon 59(6)1735ndash1753
Culberson WL (1969) The use of chemistry in thesystematics of the lichens Taxon 18498ndash505
Culberson WL (1970) Chemosystematics and ecology oflichen-forming fungi Annu Rev Ecol Syst 1153ndash170
Culberson CF Culberson WL (1976) Chemosyndromicvariation in lichens Syst Bot 1325ndash339
Darwin C (1859) On the origin of species by means ofnatural selection or the preservation of favoured racesin the struggle for life J Murray London
Dayrat B (2005) Towards integrative taxonomy Biol JLinn Soc 85(3)407ndash415 doi101111j1095-8312200500503x
36 SD Leavitt et al
de Queiroz K (1998) The general lineage concept ofspecies species criteria and the process of speciationa conceptual unification and terminological recom-mendations In Howard DJ Berlocher SH (eds)Endless forms species and speciation Oxford Uni-versity Press Oxford pp 57ndash75
de Queiroz K (1999) The general lineage concept ofspecies and the defining properties of the speciescategory In Wilson RA (ed) Species new interdis-ciplinary essays MIT Press Cambridge pp 49ndash89
de Queiroz K (2007) Species concepts and speciesdelimitation Syst Biol 56(6)879ndash886 doi10108010635150701701083
de Queiroz A Donoghue MJ Kim J (1995) Separateversus combined analysis of phylogenetic evidenceAnnu Rev Ecol Syst 26(1)657ndash681 doi101146annureves26110195003301
Degnan J Rosenberg N (2006) Discordance of speciestrees with their most likely gene trees PLoS Genet 2(5)e68 doi101371journalpgen0020068
Degnan JH Rosenberg NA (2009) Gene tree discordancephylogenetic inference and the multispecies coales-cent Trends Ecol Evol 24(6)332ndash340 doihttpdxdoiorg101016jtree200901009
Del-Prado R Cubas P Lumbsch HT Divakar PK BlancoO de Paz GA Molina MC Crespo A (2010) Geneticdistances within and among species in monophyleticlineages of Parmeliaceae (Ascomycota) as a tool fortaxon delimitation Mol Phylogenet Evol 56(1)125ndash133 doi101016jympev201004014
Del-Prado R Divakar PK Crespo A (2011) Using geneticdistances in addition to ITS molecular phylogeny toidentify potential species in the Parmotrema reticul-atum complex a case study The Lichenologist 43(06)569ndash583 doi101017S0024282911000582
Del-Prado R Blanco O Lumbsch HT Divakar PK ElixJA Molina MC Crespo A (2013) Molecular phylog-eny and historical biogeography of the lichen-formingfungal genus Flavoparmelia (Ascomycota Parmelia-ceae) Taxon 62(5)928ndash939 doi101270562522
Dettman J Jacobson D Taylor J (2003a) A multilocusgenealogical approach to phylogenetic species recog-nition in the model eukaryote Neurospora Evolution57(12)2703ndash2720 doi101111j0014-38202003tb01514x
Dettman JR Jacobson DJ Turner E Pringle A TaylorJW (2003b) Reproductive isolation and phylogeneticdivergence in Neurospora comparing methods ofspecies recognition in a model eukaryote Evolution57(12)2721 doi10155403-074
Devkota S Cornejo C Werth S Chaudhary RP Sche-idegger C (2014) Characterization of microsatelliteloci in the Himalayan lichen fungus Lobaria pindar-ensis (Lobariaceae) Appl Plant Sci 2(5)1300101doi103732apps1300101
Divakar PK Molina MC Lumbsch HT Crespo A (2005)Parmelia barrenoae a new lichen species related toParmelia sulcata (Parmeliaceae) based on molecularand morphological data The Lichenologist 37(01)37ndash46 doi101017S0024282904014641
Divakar PK Amo De paz G del Prado R Esslinger TLCrespo A (2007) Upper cortex anatomy corroboratesphylogenetic hypothesis in species of Physconia(Ascomycota Lecanoromycetes) Mycol Res 111(11)1311ndash1320 doi101016jmycres200708009
Divakar PK Figueras G Hladun N Crespo A (2010)Molecular phylogenetic studies reveal an undescribedspecies within the North American concept of Mela-nelixia glabra (Parmeliaceae) Fungal Divers 42(1)47ndash55 doi101007s13225-010-0027-3
Donoghue MJ Gauthier A (2004) Implementing thephylocode Trends Ecol Evol 19(6)281ndash282 doi101016jtree200404004
Eaton DAR Ree RH (2013) Inferring phylogeny andintrogression using RADseq data an example fromflowering plants (Pedicularis Orobanchaceae) SystBiol 62(5)689ndash706 doi101093sysbiosyt032
Edwards SV (2009) Is a new and general theory ofmolecular systematics emerging Evolution 63(1)1ndash19 doi101111j1558-5646200800549x
Edwards DL Knowles LL (2014) Species detection andindividual assignment in species delimitation canintegrative data increase efficacy Proc R Soc B BiolSci 281(1777) doi101098rspb20132765
Egan RS (1986) Correlations and non-correlations ofchemical variation patterns with lichen morphologyand geography The Bryologist 8999ndash110
Elix JA Corush J Lumbsch HT (2009) Triterpenechemosyndromes and subtle morphological characterscharacterise lineages in the Physcia aipolia group inAustralia (Ascomycota) Syst Biodivers 7(04)479ndash487 doi101017S1477200009990223
Emerson KJ Merz CR Catchen JM Hohenlohe PACresko WA Bradshaw WE Holzapfel CM (2010)Resolving postglacial phylogeography using high-throughput sequencing Proc Natl Acad Sci 107(37)16196ndash16200 doi101073pnas1006538107
Ence DD Carstens BC (2011) SpedeSTEM a rapid andaccurate method for species delimitation Mol EcolResour 11(3)473ndash480 doi101111j1755-0998201002947x
EvannoGRegnaut SGoudet J (2005)Detecting thenumberof clusters of individuals using the software STRUC-TURE a simulation study Mol Ecol 14(8)2611ndash2620doi101111j1365-294X200502553x
Falush D Stephens M Pritchard JK (2003) Inference ofpopulation structure using multilocus genotype datalinked loci and correlated allele frequencies Genetics164(4)1567ndash1587 doi101111j1471-8286200701758x
Fan HH Kubatko LS (2011) Estimating species treesusing approximate Bayesian computation Mol Phylo-genet Evol 59(2)354ndash363 doi101016jympev201102019
Fernaacutendez-Mendoza F Printzen C (2013) Pleistoceneexpansion of the bipolar lichen Cetraria aculeata intothe Southern hemisphere Mol Ecol 22(7)1961ndash1983doi101111mec12210
Fernaacutendez-Mendoza F Domaschke S Garciacutea MA JordanP Martin MP Printzen C (2011) Population structure
2 The Dynamic Discipline of Species Delimitation hellip 37
of mycobionts and photobionts of the widespreadlichen Cetraria aculeata Mol Ecol 20(6)1208ndash1232doi101111j1365-294X201004993x
Fraley C Raftery A (2007) Model-based methods ofclassification Using the mclust software in chemo-metrics J Stat Softw 18i06 [Available online at httpwwwdoajorgdoajfunc=abstractampid=218544]
Fujisawa T Barraclough TG (2013) Delimiting speciesusing single-locus data and the generalized mixed yulecoalescent (GMYC) approach a revised method andevaluation on simulated datasets Syst Biol 62(5)707ndash724 doi101093sysbiosyt033
Fujita MK Leacheacute AD Burbrink FT McGuire JA MoritzC (2012) Coalescent-based species delimitation in anintegrative taxonomy Trends Ecol Evol 27(9)480ndash488 doi101016jtree201204012
Gatesy J OrsquoGrady P Baker RH (1999) Corroborationamong data sets in simultaneous analysis hiddensupport for phylogenetic relationships among higherlevel Artiodactyl taxa Cladistics 15(3)271ndash313doi101111j1096-00311999tb00268x
Gladieux P Ropars J Badouin H Branca A Aguileta Gde Vienne DM Rodriacuteguez de la Vega RC Branco SGiraud T (2014) Fungal evolutionary genomicsprovides insight into the mechanisms of adaptivedivergence in eukaryotes Mol Ecol 23(4)753ndash773doi101111mec12631
Goffinet B Miadlikowska J Goward T (2003) Phyloge-netic inferences based on nrDNA sequences supportfive morphospecies within the Peltigera didactylacomplex (Lichenized Ascomycota) The Bryologist106(3)349ndash364 doi10163901
Gowan SP (1986) Evolution of secondary natural productsin the genus Porpidia (Ascomycata Porpidiaceae) AmJ Bot 73606
Griffin PC Hoffmann AA (2014) Limited genetic diver-gence among Australian alpine Poa tussock grassescoupled with regional structuring points to ongoinggene flow and taxonomic challenges Ann Bot doi101093aobmcu017
Grube M Hawksworth DL (2007) Trouble with lichenthe re-evaluation and re-interpretation of thallus formand fruit body types in the molecular era Mycol Res111(9)1116ndash1132 doi101016jmycres200704008
Gueidan C Savi S Thues H Roux C Keller C Tibell LPrieto M Heimarsson S Breuss O Orange A FrobergL Wynns AA Navarro-Rosines P Krzewicka BPykaelae J Grube M Lutzoni F (2009) Genericclassification of the Verrucariaceae (Ascomycota)based on molecular and morphological evidencerecent progress and remaining challenges Taxon 58(1)184ndash208
Guillot G Mortier F Estoup A (2005) Geneland acomputer package for landscape genetics Mol EcolNotes 5(3)712ndash715 doi101111j1471-8286200501031x
Guillot G Renaud S Ledevin R Michaux J Claude J(2012) A unifying model for the analysis of pheno-typic genetic and geographic data Syst Biol 61(6)897ndash911 doi101093sysbiosys038
Guisan A Thuiller W (2005) Predicting species distribu-tion offering more than simple habitat models EcolLett 8(9)993ndash1009 doi101111j1461-0248200500792x
Hale ME (1990) A synopsis of the lichen genusXanthoparmelia (Vainio) Hale (AscomycotinaParmeliaceae) Smithsonian Institution Press Wash-ington DC
Hamilton CA Hendrixson BE Brewer MS Bond JE(2014) An evaluation of sampling effects on multipleDNA barcoding methods leads to an integrativeapproach for delimiting species a case study ofthe North American tarantula genus Aphonopelma(Araneae Mygalomorphae Theraphosidae) MolPhylogenet Evol 7179ndash93 doi101016jympev201311007
Hausdorf B (2011) Progress toward a general speciesconcept Evolution 65(4)923ndash931 doi101111j1558-5646201101231x
Hausdorf B Hennig C (2010) Species delimitation usingdominant and codominant multilocus markers SystBiol 59(5)491ndash503 doi101093sysbiosyq039
Hawksworth DL (1976) Lichen chemotaxonomy InBailey RH (ed) Lichenology progress and problemsAcademic London pp 139ndash184
Hebert PDN Cywinska A Ball SL deWaard JR (2003)Biological identifications through DNA barcodesProc R Soc Lond B Biol Sci 270(1512)313ndash321doi101098rspb20022218
Hebert PDN Stoeckle MY Zemlak TS Francis CM(2004) Identification of birds through DNA barcodesPLoS Biol 2(10)e312 doi101371journalpbio0020312
Heled J Drummond AJ (2010) Bayesian inference ofspecies trees from multilocus data Mol Biol Evol 27(3)570ndash580 doi101093molbevmsp274
Hey J (2006) On the failure of modern species conceptsTrends Ecol Evol 21(8)447ndash450 doi101016jtree200605011
Hibbett DS OhmanA Glotzer D NuhnMKirk P NilssonRH (2011) Progress in molecular and morphologicaltaxon discovery in Fungi and options for formalclassification on environmental sequences Fungal BiolRev 2538ndash47 doi101016jfbr201101001
Houmlgnabba F Wedin M (2003) Molecular phylogeny ofthe Sphaerophorus globosus species complex Cladis-tics 19(3)224ndash232 doi101111j1096-00312003tb00365x
Hudson RR Coyne JA (2002) Mathematical conse-quences of the genealogical species concept Evolu-tion 56(8)1557 doi101111j0014-38202002tb01467x
Huelsenbeck JP Andolfatto P Huelsenbeck ET (2011)Structurama Bayesian inference of population struc-ture Evol Bioinf Online 7(2011)55ndash59 doi104137EBOS6761
Jones G Oxelman B (2014) DISSECT an assignment-free Bayesian discovery method for species delimita-tion under the multispecies coalescent Bioinformaticsdoi101101003178
38 SD Leavitt et al
Kekkonen M Hebert PD (2014) DNA barcode-baseddelineation of putative species efficient start fortaxonomic workflows Mol Ecol Res doi1011111755-099812233
Kelly LJ Hollingsworth PM Coppins BJ Ellis CJHarrold P Tosh J Yahr R (2011) DNA barcoding oflichenized fungi demonstrates high identification suc-cess in a floristic context New Phytol 191(1)288ndash300doi101111j1469-8137201103677x
Kiss L (2012) Limits of nuclear ribosomal DNA internaltranscribed spacer (ITS) sequences as species barcodesfor fungi Proc Natl Acad Sci 109(27)E1811ndashE1811doi101073pnas1207143109
Kluge AG (1989) A concern for evidence and aphylogenetic hypothesis for relationships among Epi-crates (Boidae Serpentes) Syst Zool 38(1)7ndash25doi101093sysbio3817
Knowles LL Carstens BC (2007) Delimiting specieswithout monophyletic gene trees Syst Biol 56(6)887ndash895 doi10108010635150701701091
Kroken S Taylor JW (2001) A gene genealogical approachto recognize phylogenetic species boundaries in thelichenized fungus Letharia Mycologia 93(1)38ndash53
Kubatko LS Degnan JH (2007) Inconsistency of phylo-genetic estimates from concatenated data under coa-lescence Syst Biol 56(1)17ndash24 doi10108010635150601146041
Latch EK Dharmarajan G Glaubitz JC Rhodes OE Jr(2006) Relative performance of Bayesian clusteringsoftware for inferring population substructure andindividual assignments at low levels of populationdifferentiation Conserv Genet 7(2)295ndash302 doi101007s10592-005-9098-1
Lawrey JD (1986) Biological role of lichen substancesThe Bryologist 89(2)111ndash122
Le Gac M Hood ME Fournier E Giraud T (2007)Phylogenetic evidence of host-specific cryptic speciesin the anther smut fungus Evolution 61(1)15ndash26doi101111j1558-5646200700002x
Leacheacute AD (2009) Species tree discordance traces tophylogeographic clade boundaries in North Americanfence lizards (Sceloporus) Syst Biol 58(6)547ndash559doi101093sysbiosyp057
Leacheacute AD Fujita MK (2010) Bayesian species delim-itation in West African forest geckos (Hemidactylusfasciatus) Proc R Soc B Biol Sci 2773071ndash3077doi101098rspb20100662
Leacheacute AD Koo M Spencer C Papenfuss T Fisher RMcGuire J (2009) Quantifying ecological morpho-logical and genetic variation to delimit species in thecoast horned lizard species complex (Phrynosoma)Proc Natl Acad Sci USA 10612418ndash12423 doi101073pnas0906380106
Leacheacute AD Fujita MK Minin VN Bouckaert RR (2014)Species delimitation using genome-wide SNP dataSystematic Biology doi101093sysbiosyu018
Leavitt SD Fankhauser JD Leavitt DH Porter LDJohnson LA St Clair LL (2011a) Complex patterns ofspeciation in cosmopolitan ldquorock posyrdquo lichensmdashdiscovering and delimiting cryptic fungal species in
the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
Leavitt SD Esslinger TL Lumbsch HT (2012c) Neogene-dominated diversification in neotropical montanelichens dating divergence events in the lichen-form-ing fungal genus Oropogon (Parmeliaceae) Am J Bot99(11)1764ndash1777 doi103732ajb1200146
Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Lumbsch HT St Clair LL(2013c) DNA barcode identification of lichen-formingfungal species in the Rhizoplaca melanophthalmaspecies-complex (Lecanorales Lecanoraceae) includ-ing five new species MycoKeys 71ndash22 doi103897mycokeys74508
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Vondraacutek J Thorsten Lum-bsch H Clair LLS (2013d) Local representation ofglobal diversity in a cosmopolitan lichen-formingfungal species complex (Rhizoplaca Ascomycota)J Biogeogr 40(9)1792ndash1806 doi101111jbi12118
Leavitt SD Lumbsch HT Stenroos S St Clair LL(2013e) Pleistocene speciation in North American
2 The Dynamic Discipline of Species Delimitation hellip 39
lichenized fungi and the impact of alternative speciescircumscriptions and rates of molecular evolution ondivergence estimates PLoS ONE 8(12)e85240doi101371journalpone0085240
Leavitt SD Esslinger TL Hansen ES Divakar PK CrespoA Loomis BF Lumbsch HT (2014) DNA barcoding ofbrown Parmeliae (Parmeliaceae) species a molecularapproach for accurate specimen identification empha-sizing species in Greenland Organ Divers Evol 14(1)11ndash20 doi101007s13127-013-0147-1
Leliaert F Verbruggen H Vanormelingen P Steen FLopez-Bautista JM Zuccarello GC De Clerck O(2014) DNA-based species delimitation in algae Eur JPhycol 49(2) doi101080096702622014904524
Leuckert C (1985) Probleme der Flechten-Chemotaxon-omiemdashStoffkombinationen und ihre taxonomischeWertung Ber Deut Bot Ges 98401ndash408
Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
Lohtander K Kaumlllersjouml M Roland M Tehler A (2000)The family physciaceae in fennoscandia phylogenyinferred from its sequences Mycologia 92(4)728ndash735
Longton RE (1997) The role of bryophytes and lichens inpolar ecosystems In Woodin SJ Marquiss M (eds)Ecology of Arctic Environments Blackwell ScienceOxford pp 69ndash96 (Special publication No 13)
Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
Lumbsch HT (1994) Die Lecanora subfusca-Gruppe inAustralasien J Hattori Bot Lab 771ndash175
Lumbsch HT (1998a) Taxonomic use of metabolic data inlichen-forming fungi In Frisvad JC Bridge PDArora DK (eds) Chemical Fungal Taxonomy MarcelDekker New York pp 345ndash387
Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
Mayden RL (1997) A hierarchy of species concepts thedenouement in the saga of the species problem InClaridge MF Dawah HA Wilson MR (eds) Speciesthe units of biodiversity Chapman amp Hall Londonpp 381ndash424
Mayr E (1963) Animal species and evolution HarvardUniversity Press Cambridge
Mayr E (1970) Populations species and evolutionBelknap Press of Harvard University Press Cambridge
McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
McDonald T Miadlikowska J Lutzoni F (2003) Thelichen genus Sticta in the Great Smoky Mountains a
40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
McKay BD Mays HL Wu Y Li H Yao C-T Nishiumi IZou F (2013) An empirical comparison of character-based and coalescent-based approaches to speciesdelimitation in a young avian complex Mol Ecol 22(19)4943ndash4957 doi101111mec12446
Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
Mishler BD Brandon RN (1987) Individuality pluralismand the phylogenetic species concept Biol Philos 2397
Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
Nash TH Zavada M (1977) Population studies amongSonoran Desert species of Parmelia subg Xanthop-armelia (Parmeliaceae) Am J Bot 64(6)664ndash669
Nylander W (1866a) Circa novum in studio Lichenumcriterium chemicum Flora 49198ndash201
Nylander W (1866b) Hypochlorite of lime and hydrate ofpotash Two new criteria for the study of lichens(Tanslated and communicated by the Rev W ALeighton) Bot J Linn Soc 9358ndash365
OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
OrsquoNeill EM Schwartz R Bullock CT Williams JSShaffer HB Aguilar-Miguel X Parra-Olea G Weis-rock DW (2013) Parallel tagged amplicon sequencingreveals major lineages and phylogenetic structure inthe North American tiger salamander (Ambystomatigrinum) species complex Mol Ecol 22(1)111ndash129doi101111mec12049
Orock EA Leavitt SD Fonge BA St Clair LL LumbschHT (2012) DNA-based identification of lichen-form-ing fungi Can publicly available sequence databasesaid in lichen diversity inventories of Mount Cameroon(West Africa) The Lichenologist 44(6)833ndash839doi101017S0024282912000424
Otaacutelora MAG Martiacutenez I Aragoacuten G Molina MC (2010)Phylogeography and divergence date estimates of alichen species complex with a disjunct distributionpattern Am J Bot 97(2)216ndash223 doi103732ajb0900064
Padial J Castroviejo-Fisher S Kohler J Vila C ChaparroJ De la Riva I (2009) Deciphering the products ofevolution at the species level the need for anintegrative taxonomy Zoolog Scr 38(4)431ndash447doi101111j1463-6409200800381x
Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
Papong K Luumlcking R Thammathaworn A BoonpragobK (2009) Four new taxa of Chroodiscus (thelotremoidGraphidaceae) from Southeast Asia The Bryologist112(1)152ndash163 doi1016390007-2745-1121152
Park S-Y Choi J Kim JA Jeong M-H Kim S Lee Y-HHur J-S (2013a) Draft genome sequence of Cladoniamacilenta KoLRI003786 a lichen-forming fungusproducing biruloquinone Genome Announcements 1(5)e00695-13 doi101128genomeA00695-13
Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
Peacuterez-Ortega S Fernaacutendez-Mendoza F Raggio J VivasM Ascaso C Sancho LG Printzen C de los Riacuteos A(2012) Extreme phenotypic variation in Cetrariaaculeata (lichenized Ascomycota) adaptation or inci-dental modification Ann Bot 109(6)1133ndash1148doi101093aobmcs042
Pino-Bodas R Burgaz A Martiacuten M Lumbsch HT (2011)Phenotypical plasticity and homoplasy complicatespecies delimitation in the Cladonia gracilis group(Cladoniaceae Ascomycota) Organ Divers Evol 11(5)343ndash355 doi101007s13127-011-0062-2
Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
Pino-Bodas R Martiacuten M Burgaz A (2012b) Cladoniasubturgida and C iberica (Cladoniaceae) form asingle morphologically and chemically polymorphicspecies Mycol Prog 11(1)269ndash278 doi101007s11557-011-0746-1
Pino-Bodas R Ahti T Stenroos S Martiacuten MP BurgazAR (2013a) Multilocus approach to species recogni-tion in the Cladonia humilis complex (CladoniaceaeAscomycota) Am J Bot 100(4)664ndash678 doi103732ajb1200162
Pino-Bodas R Martiacuten AP Burgaz AR Lumbsch HT(2013b) Species delimitation in Cladonia (Ascomy-cota) a challenge to the DNA barcoding philosophyMol Ecol Resour 13(6)1058ndash1068 doi1011111755-099812086
Pons J Barraclough TG Gomez-Zurita J Cardoso ADuran DP Hazell S Kamoun S Sumlin WD VoglerAP (2006) Sequence-based species delimitation for theDNA taxonomy of undescribed insects Syst Biol 55(4)595ndash609 doi10108010635150600852011
Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
Pringle A Baker DM Platt JL Wares JP Latgeacute JPTaylor JW (2005) Cryptic speciation in the cosmo-politan and clonal human pathogenic fungus Asper-gillus fumigatus Evolution 59(9)1886ndash1899 doi10155404-2411
Printzen C (2009) Lichen systematics the role ofmorphological and molecular data to reconstructphylogenetic relationships Progress in botany vol71 Springer Berlin pp 233ndash275
Pritchard JK Stephens M Donnelly P (2000) Inference ofpopulation structure using multilocus genotype dataGenetics 155(2)945ndash959
Puillandre N Lambert A Brouillet S Achaz G (2012)ABGD automatic barcode gap discovery for primaryspecies delimitation Mol Ecol 21(8)1864ndash1877doi101111j1365-294X201105239x
Rannala B Yang Z (2003) Bayes estimation of speciesdivergence times and ancestral population sizes usingDNA sequences from multiple loci Genetics1641645ndash1656
Raxworthy CJ Ingram CM Rabibisoa N Pearson RG(2007) Applications of ecological niche modelingfor species delimitation a review and empiricalevaluation using day geckos (Phelsuma) fromMadagascar Syst Biol 56(6)907ndash923 doi10108010635150701775111
Ray J (1686) Historia planarum vol 1 Clark LondonReese Naeligsborg R Ekman S Tibell L (2007) Molecular
phylogeny of the genus Lecania (Ramalinaceaelichenized Ascomycota) Mycol Res 111(5)581ndash591doi101016jmycres200703001
Reid N Carstens B (2012) Phylogenetic estimation errorcan decrease the accuracy of species delimitation aBayesian implementation of the general mixed Yule-coalescent model BMC Evol Biol 12(1)196 doi1011861471-2148-12-196
Rissler L Apodaca J (2007) Adding more ecology intospecies delimitation ecological niche models andphylogeography help define cryptic species in theBlack Salamander (Aneides flavipunctatus) Syst Biol56924ndash942 doi10108010635150701703063
Rivas Plata E Luumlcking R (2013) High diversity ofGraphidaceae (lichenized Ascomycota Ostropales) inAmazonian Peruacute Fungal Divers 58(1)13ndash32 doi101007s13225-012-0172-y
Rivas Plata E Lumbsch HT (2011) Parallel evolution andphenotypic divergence in lichenized fungi a casestudy in the lichen-forming fungal family Graphida-ceae (Ascomycota Lecanoromycetes Ostropales)Mol Phylogenet Evol 61(1)45ndash63 doi101016jympev201104025
Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
Ross KG Gotzek D Ascunce MS Shoemaker DD (2010)Species delimitation a case study in a problematic anttaxon Syst Biol 59(2)162ndash184 doi101093sysbiosyp089
Rubin BER Ree RH Moreau CS (2012) Inferringphylogenies from RAD sequence data PLoS ONE 7(4)e33394 doi101371journalpone0033394
Rubinoff D (2006) Utility of mitochondrial DNA barcodesin species conservation Conserv Biol 20(4)1026ndash1033 doi101111j1523-1739200600372x
Ruiz-Sanchez E Sosa V (2010) Delimiting speciesboundaries within the Neotropical bamboo Otatea(Poaceae Bambusoideae) using molecular morpho-logical and ecological data Mol Phylogenet Evol 54(2)344ndash356 doi101016jympev200910035
Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
morphological characters Antarctic Sci 22 (SpecialIssue 06)727ndash741 doi101017S0954102010000477
Salicini I Ibaacutentildeez C Juste J (2011) Multilocus phylogenyand species delimitation within the Nattererrsquos batspecies complex in the Western Palearctic MolPhylogenet Evol 61(3)888ndash898 doi101016jympev201108010
Satler JD Carstens BC Hedin M (2013) Multilocusspecies delimitation in a complex of morphologicallyconserved trapdoor spiders (Mygalomorphae Antro-diaetidae Aliatypus) Syst Biol 62(6)805ndash823 doi101093sysbiosyt041
Schlick-Steiner BC Steiner FM Seifert B Stauffer CChristian E Crozier RH (2010) Integrative taxonomya multisource approach to exploring biodiversityAnnu Rev Entomol 55(1)421ndash438 doi101146annurev-ento-112408-085432
Schoch CL Seifert KA Huhndorf S Robert V SpougeJL Levesque CA Chen W Fungal Barcoding Con-sortium (2012) Nuclear ribosomal internal transcribedspacer (ITS) region as a universal DNA barcodemarker for Fungi Proc Natl Acad Sci 109(16)6241ndash6246 doi101073pnas1117018109
Seacuterusiaux E Villarreal AJC Wheeler T Goffinet B(2011) Recent origin active speciation and dispersalfor the lichen genus Nephroma (Peltigerales) inMacaronesia J Biogeogr 38(6)1138ndash1151 doi101111j1365-2699201002469x
Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
Simpson GG (1951) The species concept Evolution5285ndash298
Sites JW Marshall JC (2003) Delimiting species a renais-sance issue in systematic biology Trends Ecol Evol18462ndash470 doi101016S0169-5347(03)00184-8
Sites JW Marshall JC (2004) Operational criteria fordelimiting species Annu Rev Ecol Evol Syst 35(1)199ndash227 doi101146annurevecolsys35112202130128
Šlapeta J Loacutepez-Garciacutea P Moreira D (2006) Globaldispersal and ancient cryptic species in the smallestmarine eukaryotes Mol Biol Evol 23(1)23ndash29doi101093molbevmsj001
Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
Talavera G Dincă V Vila R (2013) Factors affectingspecies delimitations with the GMYC model insightsfrom a butterfly survey Methods Ecol Evol 4(12)1101ndash1110 doi1011112041-210X12107
Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
Tewksbury J Anderson JGT Bakker JD Billo TJDunwiddie PW Groom MJ Hampton SE HermanSG Levey DJ Machnicki NJ del Rio CM PowerME Rowell K Salomon AK Stacey L TrombulakSC Wheeler TA (2014) Natural historyrsquos place inscience and society BioScience doi101093bioscibiu032
Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
Werth S Cornejo C Scheidegger C (2013) Characteriza-tion of microsatellite loci in the lichen fungus Lobariapulmonaria (Lobariaceae) Appl Plant Sci 1(2)1200290 doi103732apps1200290
Westberg M Arup U Kaumlrnefelt I (2007) Phylogeneticstudies in the Candelariaceae (lichenized Ascomy-cota) based on nuclear ITS DNA sequence dataMycol Res 111(11)1277ndash1284 doi101017S0953756204002102
Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
2 The Dynamic Discipline of Species Delimitation hellip 43
(Lepidoptera Lycaenidae) Front Zoo 4(1)8 doi1011861742-9994-4-8
Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
C cariosa group (Pino-Bodas et al 2012a) andC humilis species complex (Pino-Bodas et al2013a) High levels of intraspecific morphologi-cal and chemical polymorphisms are not restric-ted to Cladonia A clear demarcation betweenintraspecific and interspecific morphological andor chemical variation does not exist for a largeproportion of Xanthoparmelia species in westernNorth America and high intraspecific morpho-logical and chemical variation are common for anumber of species-level genetic groups (Fig 21
Leavitt et al 2011b c 2013e) In some cases asmany as eight traditionally circumscribed Xant-hoparmelia species were recovered in a singlespecies-level genetic group (Leavitt et al 2011c)High levels of intraspecific phenotypic variationhave been observed in a number of other generain Parmeliaceae including Bryoria (Velmalaet al 2009) Cetraria (Peacuterez-Ortega et al 2012)Vulpicida (Mark et al 2012) and others
The importance of biogeography in lichen-forming fungal evolution has remained somewhat
Fig 21 Examples of common lichens in which tradi-tional morphology-based species circumscriptions fail toreflect natural species-level fungal lineages a Cladoniagracilis photographed in the Clearwater Valley BritishColumbia Canada (see Pino-Bodas et al 2011) (photo-graph creditCurtis Bjoumlrk) bR shushanii a member of theRhizoplaca melanophthalma species group from the
Aquarius Plateau Utah USA (see Leavitt et al 2011a)(photograph credit S Leavitt) c Xanthoparmelia affwyomingica occurring in Coloradorsquos Front Range USA(see Leavitt et al 2011b c Leavitt et al 2013e) (photo-graph credit S Leavitt) d Tephromela atra sensu latofound in the Santa Monica Mountains California USA(see Muggia et al 2014) (photograph credit J Hollinger)
2 The Dynamic Discipline of Species Delimitation hellip 15
ambiguous due to the occurrence of phenotypi-cally similar lichens occurring across broadintercontinental distributions and uncertainty ofappropriate species circumscriptions While anumber of lichen-forming fungal species havebeen found to be truly widespread (eg Lindblomand Soslashchting 2008 Fernaacutendez-Mendoza et al2011 Ahti and Hawksworth 2005 Del-Pradoet al 2013) improved recognition of speciesboundaries has provided insight into importantbiogeographical patterns in lichens previouslyassumed to have cosmopolitan distribution pat-terns (Leavitt et al 2013d Del-Prado et al 2013Amo de Paz et al 2012 Seacuterusiaux et al 2011Otaacutelora et al 2010 Elix et al 2009) Crypticdiversity and complex biogeographic patterns arehighlighted in the Rhizoplaca melanophthalmaspecies group (Fig 21 Leavitt et al 2013d)Analyses of R melanophthalma sensu lato col-lected from five continents supported the presenceof cryptic species within this complex Two ofthese lineages were found to have broad inter-continental distributions while the other four werelimited to western North America (Leavitt et al2013d) Most strikingly of the six lineages fivewere found on a single mountain in the westernUSA and the sixth occurred no more than 200 kmaway from this mountain A recent study ofPseudocyphellaria (Lobariaceae) sensu lato inHawaii revealed a surprising number of previouslyunrecognized species hidden within nominal taxawith putative broad geographic distributionssuggesting a high degree of endemism in Hawaii(Moncada et al 2014) These studies providecrucial impetus to reevaluate species boundaries inorder to improve our understanding of diversitydistributions and evolution in lichenized fungi
22 A Practical Guideto Contemporary SpeciesDelimitation
As it has become clear that conventional phe-notype-based criteria for species circumscriptionsare often problematic (Bickford et al 2007)molecular data are thereby particularly valuablefor assessing traditional species boundaries and
for species delimitation in general (Lumbsch andLeavitt 2011 Fujita et al 2012) Below weprovide an overview of relevant operationalspecies delimitation methods the majority ofwhich are reliant on molecular data coupled withmolecular phylogenetic and population geneticanalyses Assuming that species do in fact rep-resent ldquosegments of metapopulation lineagesrdquo (deQueiroz 1998) direct genetic evidence of lineagestatus is particularly relevant to species delimi-tation studies when analyzed within a rigorousstatistical framework regardless of whether lin-eages differ in phenotypic characters that areapparent to human observers (Fujita et al 2012)This perspective should not be taken as supportfor disregarding phenotypic characters in speciesdelimitation studies (Edwards and Knowles2014 Fujita et al 2012 Yeates et al 2011)Rather hypotheses of species boundaries shouldbe considered more robust with increasing cor-roboration from independent data sources (iemolecular chemistry morphology ecologyetc) and the integration of independent data forempirical species delimitation studies should be amajor focus of species delimitation research(Fujita et al 2012)
Themajority of molecular phylogenetic studiesof lichenized fungi focus on generating sequencedata from a number of specimens representing thefocal group inferring a gene tree from the geneticdata matrix and assessing the monophyly of thesampled taxa This approach has provided valu-able acumen into evolutionary relationships thevalue of morphological characters for taxonomyand insight into diversity of lichenized fungi(Thell et al 2009 Reese Naeligsborg et al 2007Westberg et al 2007 Martiacuten et al 2003 Arup andGrube 2000 Lohtander et al 2000 Stenroos andDePriest 1998) However simply assessing themonophyly of traditional phenotype-based spe-cies often offers an incomplete perspective onspecies boundaries Studies explicitly designed forempirically delimiting species are pivotal toadvancing our understanding of speciation andspecies diversity in lichens For example althougha nominal species may be recovered as mono-phyletic in a gene tree intraspecific phylogeneticsubstructure may correspond to evolutionarily
16 SD Leavitt et al
independent lineages (eg Leavitt et al 2011a)As a construct of taxonomy recognizing amonophyletic clade comprised of multiple mor-phological indistinguishable species-level lin-eages as a single species may hold some appealhowever failing to formally recognize this diver-sity can have far-reaching implications in ourbiological (eg ecology evolution reproduction)interpretation of the group Alternatively well-supported intraspecific phylogenetic substructuremay be the result of stochastic evolutionary pro-cesses uniparental inheritance etc rather thanevolutionary independence Interpreting this typeof phylogenetic substructure as species-leveldiversity can introduce detrimental bias
Empirical species delimitation methods havebroadly focused on four main areas detectingputative species individual specimen assignmentto a species group or operational taxonomic unit(OTU) validation of candidate species or OTUsas evolutionarily distinct lineages and inferringspecies relationships (ie species tree inference)Ideally operational species delimitation criteriashould be based on explicit statistical protocolsin order to objectively assess species boundariesand minimize the need of subjective interpreta-tions or taxonomic expertise In this section weprovide a brief overview on a number of empir-ical species delimitation methods that to varyingdegrees fit these criteria For convenience wedivide these methods into three general catego-ries (i) species discovery methods for assigningsamples to populations without a priori infor-mation (ii) species validation approaches andcoestimating individual assignments and speciestrees and (iii) species delimitation using geno-mic data (Table 21)
Empirical species delimitation has receivedincreasing attention including ongoing develop-ment of bioinformatical approaches and themethods and programs provided in this chapterare by no means intended to be a comprehensivelist of all available analytical approaches Ratherthe methods provided here have been shown tobe useful in a number of previous studies orshow particular promise for future speciesdelimitation studies Our aim is that these can
serve as a starting point when designing studiesto assess species limits Not surprisingly variousapproaches to species delimitation may yielddifferent estimates of species boundaries and theresearcher may be required to make some degreeof subjective interpretation of the most biologi-cally appropriate species boundaries Comple-mentarily recently developed metrics forquantifying the congruence between two taxo-nomies (Ctax) and the potential for an approach tocapture a high number of species boundaries(Rtax) provide a means to objectively assess dis-crepancies among species delimitation methods(Miralles and Vences 2013) More sophisticatedapproaches including selection of speciesdelimitation models using approximate Bayesiancomputing (Camargo et al 2012 Fan andKubatko 2011 Beaumont et al 2010) anddesigning and conducting a simulation study thatmatches the characteristics of the empirical study(Carstens et al 2013 Camargo et al 2012) canbe used to more objectively evaluate competinghypotheses of species boundaries In most con-texts it is likely better to fail to delimit speciesthan it is to falsely circumscribe entities that donot represent actual species and therefore theinferences drawn from species delimitation stud-ies should generally be conservative (Carstenset al 2013 Miralles and Vences 2013)
221 Corroborating TraditionalTaxonomy and DiscoveringCryptic Species Using Single-Locus Data
Objectively defining a threshold separatingintraspecific population substructure from inter-specific divergence is the general aim of speciesdelimitation studies using single-marker datasetsMost species delimitation methods based onsingle-locus sequence data fall under two generalcategories either genetic distance or tree-basedapproaches (Sites and Marshall 2004) Distance-based approaches attempt to detect a differencebetween intra- and interspecific distances (ieldquobarcode gaprdquo) where the pairwise genetic
2 The Dynamic Discipline of Species Delimitation hellip 17
Table
21
Somemetho
dsused
forspeciesdelim
itatio
ninclud
ingspeciesdiscov
erymetho
dsforassign
ingsamples
topo
pulatio
nswith
outapriori
inform
ation(BAPS
Gaussianclustering
Guillo
trsquosUnified
Mod
elS
TRUCTURES
TRUCTURAMA)genetic
distance-based
metho
dforsortingsequ
encesinto
hypo
theticalspecies(A
BGD)tree-
basedspeciesdiscov
erymetho
ds(bGMYCG
MYCb
PTP
PTP
ldquoSpecies
Delim
itatio
nrdquoplug
-inforGeneiou
s)and
jointdiscov
eryandvalid
ationmetho
ds(BPamp
PBrownie
DISSE
CTSp
eDeStem)
Metho
dDescriptio
nInpu
tdata
BAPS
mdashpo
pulatio
nassignmentusing
Bayesianclustering
Aprogram
forBayesianinferenceof
thegenetic
structurein
apopulatio
nclustering
molecular
data
andperformingadmixture
analysesGenetic
mixture
analyses
canbe
performed
atbo
thgroupandindividu
allevelsusingeither
ano
n-spatialo
rspatialm
odel
BAPS
treatsboth
theallele
frequenciesof
themolecular
markers
(ornucleotid
efrequenciesforDNA
sequ
ence
data)andthenu
mberof
genetically
diverged
groups
inpopulatio
nas
random
variablesIn
theldquoclusteringwith
linkedlocirdquomod
elagenetic
mixture
analysiscanbe
done
usinghaploidsequ
ence
data
orotherlin
kedgenetic
markersA
nalysesandmodel
comparisons
canalso
beperformed
usingafixednu
mber
ofgenetically
diverged
grou
psor
prespecified
populatio
nstructures
Limita
tions
Tem
poraldivergence
andrelatio
nships
amongputativ
egroups
arenot
explicitlyestim
atedE
quivalence
togenetic
clusters
tospecies-levelg
roupsisuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
Availablefrom
httpwwwhelsinkifi
bsgsoftwareBAPS
describedin
Coran-
deret
al(200
420
0620
08)CoranderandMarttinen20
06)
Genotyp
icdatahaploidsequence
dataor
linkedmarkers
(AFL
Por
SNPs)
Gaussianclusteringmdashpo
pulatio
nassignment
usingGaussianclustering
Aprogram
forBayesianinferenceof
thegenetic
structurein
apopulatio
nModelgroups
sampleinto
popu
latio
nsusinggeno
typicdata
bysearchingforclusters
that
canbe
attributed
tobeingmixturesof
norm
alallelefrequencydistributio
nsG
aussianclustering
requires
adatasetwhere
thecasesaredefinedby
variable
ofmetricscaleandhasbeen
used
with
geneticenvironmentalandmorphologicaldatasetsindividuallyinadditio
nto
integrated
datasets
Limita
tions
Tem
poraldivergence
andrelatio
nships
amongputativ
egroups
arenot
explicitlyestim
atedE
quivalence
togenetic
clusters
tospecies-levelg
roupsisuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
Implem
entedin
Rusingtheprabclus
(HausdorfandHennig20
10)andmclust
packages
(FraleyandRaftery
2007)
Genotyp
icdata
(flexible)
(con
tinued)
18 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
Guillo
trsquosUnified
Mod
elmdashpo
pulatio
nassignmentusingBayesianclustering
Thisapproach
provides
astatistical
model
that
allowsoneto
analyzegenetic
and
phenotyp
ticdata
with
inaun
ified
mod
elandinferencefram
eworkandop
tionally
incorporateinform
ationaboutthespatialdistributio
nof
samplesA
Bayesianclustering
algorithm
assumes
that
each
clusterin
ageographical
domaincanbe
approxim
ated
bypo
lygons
that
arecentered
around
pointsgeneratedby
aPo
issonprocessGuillo
trsquos
Unified
Mod
elisflexiblein
term
sof
thegenetic
data
that
itcanutilize
andcapableof
accurately
delim
iting
species
Limita
tions
Genetic
andphenotypic
data
cantracedifferentevolutionary
historiesfor
instancephylogenetic
divergence
forneutralgenetic
markers
andadaptatio
nfora
morphological
structure
Source
Availableas
anextensionof
theRGENELAND
package(G
uillo
tet
al20
05
2012)(http
www2im
mdtudk
gigu
Geneland)
Genotyp
icandno
n-genetic
(eg
phenotypicalecolog
icalgeograph
ical
behavioral)data
STRUCTUREmdash
populatio
nassignment
usingBayesianclustering
Amodel-based
clustering
methodusingmultilocus
genotype
data
toinferpopulatio
nstructureandassign
individualsto
populatio
nsIndividualsin
thesampleareassigned
probabilistically
topopulatio
nso
rjointly
totwoor
morepopulatio
nsiftheirgenotypes
indicatethatthey
areadmixedT
hemodeldoes
notassum
eaparticular
mutationprocess
anditcanbe
appliedto
mosto
fthecommonly
used
genetic
markersp
rovidedthat
they
areno
tcloselylin
ked
The
methodcanproducehigh
lyaccurate
assignmentsusing
modestn
umbersof
loci(Pritchard
etal2
000)T
hemostappropriatelevelo
fpopulatio
nstructurecanbe
inferred
byassessinglik
elihoodscores
orthead
hocΔKstatistic
(Evann
oet
al20
05)
Limita
tions
Identifying
themostappropriatenumberof
genetic
clusters
ischallenging
clusters
produced
byST
RUCTUREcanbe
strongly
influenced
byvariationin
sample
sizeclusters
createdby
STRUCTUREmay
notbe
consistent
with
theevolutionary
historyof
thepopulatio
nswhentherearerelativ
elylong
divergence
times
with
inevolutionary
lineagesTem
poraldivergence
andrelatio
nships
amongputativ
egroups
isnotexplicitlyestim
atedEquivalence
togenetic
clusters
tospecies-levelgroups
isuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
httppritchardlabstanfordedustructurehtm
ldescribedin
Falush
etal(200
3)andPritchard
etal(200
0)
Genotyp
icdata
(con
tinued)
2 The Dynamic Discipline of Species Delimitation hellip 19
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
Structuram
amdashpo
pulatio
nassignmentusing
Bayesianclustering
Implem
entstheclustering
algorithm
used
inST
RUCTURE(see
above)
that
clusters
samples
into
populatio
nsby
minim
izingHardyndashWeinb
ergdisequ
ilibrium
foragiven
partitioninglevelHow
everStructuram
aalso
includes
theadditio
nof
reversible-jum
pMCMCto
identifytheop
timalpartition
inglevelNearlyanytype
ofgenetic
datacanbe
inpu
tinto
Structuram
aandtheprog
ram
canassign
individu
alsto
populatio
nwith
orwith
outtheadmixture
Limita
tions
Tem
poraldivergence
andrelatio
nships
amongputativ
egroups
isnot
explicitlyestim
atedE
quivalence
togenetic
clusters
tospecies-levelg
roupsisuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
httpctegberkeleyedu
structuram
aindexhtmldescribedin
Huelsenbeck
etal(201
1)
Genotyp
icdata
ABGDmdashbarcodegapusinggenetic
distances
ldquoAutom
aticBarcode
Gap
Discoveryrdquosortssequencesinto
hypotheticalspeciesbasedon
thebarcodegap
The
methoduses
arecursiveapproach
topartition
thedata
andtestfor
sign
ificant
gapsA
BGDisfastsim
plemethodto
split
asequence
alignm
entd
atasetinto
candidatespeciesthat
should
becomplem
entedwith
otherevidence
inan
integrative
taxono
mic
approach
Limita
tions
Single-locus
data
aloneshould
only
beused
toprovideaprelim
inary
perspectiveof
speciesboundaries
andnotas
thesole
evidence
inspecies
circum
scriptions
Source
httpwwwabisnv
jussieufrpublicabg
ddescribedin
Puillandreet
al(201
2)
Single-locus
sequence
alignm
ent
GMYCampbG
MYCmdashgene
tree
The
GMYC
approach
combinesacoalescent
model
ofintraspecificbranchingwith
aYulemod
elforinterspecificbranching
which
isthen
fitto
aninferred
sing
le-gene
topology
toestim
atespeciesboundaries
andastatistical
measure
ofconfi
denceforthe
inferred
boundariesA
san
inputtheGMYCapproach
requires
anultram
etricgene
tree
andrecent
refinementscanaccountforuncertaintyin
phylogenetic
relatio
nships
and
parametersof
theGMYCmod
elT
heGMYCisgenerally
stable
across
awiderangeof
circum
stancesincludingvariousmethods
ofphylogeneticreconstructio
nthepresence
ofahigh
numbersingletonshigh
numbers
ofsampled
speciesandgaps
inintraspecific
sampling
theaccuracy
oftheGMYCismostsign
ificantly
affected
bythemean
populatio
nsize
relativ
eto
divergence
times
betweenthem
Limita
tions
The
GMYCmay
delim
itwell-supportedclades
ofhaplotypes
asindependent
lineagesandas
such
may
beproneto
over-delim
itatio
nSingle-locus
data
aloneshould
only
beused
toprov
ideaprelim
inaryperspectiveof
speciesbo
undaries
andno
tas
the
sole
evidence
inspeciescircum
scriptions
Source
(http
r-forger-projecto
rgprojectssplitshttpssitesgooglecom
site
noahmreidhom
esoftware)describedin
Monaghanet
al(200
9)Fu
jisaw
aandBarrac-
loug
h(201
3)andPo
nset
al(200
6)
Sing
le-locus
ultram
etricgene
tree (con
tinued)
20 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
PTPamp
bPTPmdash
gene
tree
The
Poissontree
processes(PTP)
modelcanbe
used
toinferputativ
especiesboundaries
onagivenphylogenetic
inputtreePT
Pcaninferputativ
especiesboundaries
that
are
consistent
with
thePS
CAnim
portantadvantageof
thismethodisthat
itmod
els
speciatio
nin
term
sof
thenumberof
substitutionsthereby
circum
ventingthepotentially
error-proneandcompute-intensive
processof
generatin
gultram
etrictreeswhich
are
required
asan
inpu
tfor
GMYCmod
el(see
abov
e)F
urthermoreitappearsthatthePT
Pmodel
may
outperform
theGMYCandotherOTU-picking
methods
whenevolutionary
distancesaresm
all
Limita
tions
Single-locus
data
aloneshould
only
beused
toprovideaprelim
inary
perspectiveof
speciesboundaries
andnotas
thesole
evidence
inspecies
circum
scriptions
Source
httpscoh-itsorgexelix
iswebsoftwarePT
Pindexhtmldescribedin
Zhang
etal(201
3)
Sing
le-locus
gene
tree
GeneiousSp
eciesDelim
itatio
nplug
-inmdash
gene
tree
Aplug-into
theGeneioussoftwareprovides
anexploratorytool
allowingtheuser
toassess
phylog
enetic
supportanddiagno
sabilityof
speciesdefinedas
user-selected
collections
oftaxa
onuser-sup
pliedtreesThe
plug
-incompu
tesstatisticsrelatin
gto
the
probability
oftheob
served
mon
ophyly
orexclusivity
having
occurred
bychance
ina
coalescent
processandassesses
thewith
in-andbetween-speciesgenetic
distancesto
infertheprobability
with
which
mem
bers
ofaputativ
especiesmight
beidentifi
edsuccessfully
with
tree-based
methods
Limita
tions
The
plug-insummarizes
measuresof
phylogenetic
supportand
diagnosabilityof
speciesdefinedas
user-selectedcollections
oftaxabutitdoes
not
providedefinitiv
esupportforspeciesgroupsItassumes
speciesaremonophyletic
Source
Implem
entedas
aplug-into
Geneious(geneiouscom)describedin
Mastersetal
(201
1)
Sing
le-locus
gene
tree
BPamp
Pmdashmultispecies
coalescent
model
for
speciesvalid
ation
Thisapproach
tospeciesdelim
itatio
nuses
aBayesianmodelingapproach
togeneratethe
posteriorprobabilitiesof
speciesassignmentstaking
accountof
uncertaintiesdueto
unknow
ngene
treesandtheancestralcoalescent
processThe
methodrelieson
auser-
specified
guidetreeimplem
entin
gareversible-jum
pMarkovchainMonte
Carlo
search
ofparameter
spacethatincludes
θpopulatio
ndivergenceand
estim
ated
distributio
nsof
gene
treesfrom
multip
leloci
Limita
tions
Misspecificatio
nsof
priorsandthegu
idetree
canresultin
inflatedspeciatio
nprobabilitiesitassumes
norecombinatio
nandcomputatio
nallim
itatio
nsrestrict
itsutility
with
next-generationdatasetswith
100s
ofloci
Source
httpabacusgeneuclacuksoftwarehtmldescribedin
YangandRannala
(201
0)andRannala
andYang(200
3)
Multilocus
sequence
alignm
entsandgroup
mem
bership
(con
tinued)
2 The Dynamic Discipline of Species Delimitation hellip 21
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
DISSE
CTmdashmultispecies
coalescent
model
forspeciesdelim
itatio
nDISSE
CTexplores
thefullspaceof
possible
clusteringsof
individualsandspeciestree
topologies
inaBayesianfram
ework
Toavoidtheneed
forreversible-jum
pMCMCit
uses
anapproxim
ationin
theform
ofapriorthatisamodificatio
nof
thebirthndashdeathprior
forthespeciestreeItisim
plem
entedas
partof
BEAST
andrequires
only
afewchanges
from
astandard
BEAST
analysisA
nalysesof
simulated
andem
piricald
atasuggestthat
themethodisshow
nto
beinsensitive
tothedegree
ofapproxim
ation
butq
uitesensitive
tootherparameters
Limita
tions
Recently
describedmethodlackingathorough
theoretical
andem
pirical
evaluatio
nItappearsthat
alargenu
mberof
sequencesarerequ
ired
todraw
firm
conclusion
sSo
urce
httpcodegooglecompbeast-m
cmcamphttpwwwin
driid
com
dissectinbeast
htmldescribedin
JonesandOxelm
an(201
4)
Multilocus
sequence
data
SpeD
eSTEMmdashmultispecies
coalescent
mod
elfordiscov
ery
valid
ation
andjoint
estim
ation
Thismaxim
umlik
elihoo
dandorinform
ationtheory-based
methodwas
developedto
test
speciesboundaries
inasystem
with
existin
gsubspecies
taxonomy(CarstensandDew
ey20
10)andcomputestheprobability
ofthegene
treesgiventhespeciestree
forall
hierarchical
perm
utations
oflin
eage
grou
pingSp
eciesbo
undaries
arecomparedusing
Akaikeinform
ationcriteria
andphylogenetic
uncertaintyin
thespeciestree
topologies
does
notaffect
speciesdelim
itatio
nsLimita
tions
Accuracyisdependenton
quality
ofthegene
tree
estim
ates
Source
(http
carstenslaborgohio-stateedusoftwarehtml)criteria
describedin
Ence
andCarstens(201
1)
Multilocus
sequence
alignm
entsandgroup
mem
bership
Browniemdash
multispecies
coalescent
model
forspeciesdelim
itatio
nThe
nonp
aram
etricheuristic
speciesdelim
itatio
napproach
implem
entedin
theprog
ram
Brownie(O
rsquoMeara
2010)jointly
sortsanonym
oussamples
into
speciesandinfers
aspeciestree
from
inputg
enetreesfrom
differentlociassumingthatforaspeciatio
neven
thecorrespondingnodeson
gene
treeswill
bemoreconsistent
with
each
than
the
divergenceswith
inspecies
Limita
tions
Findingboth
theoptim
umspeciestree
andspeciesboundaries
remains
compu
tatio
nally
challenging
andBrowniehasbeen
show
nto
frequently
yieldincorrect
resultsT
heaccuracy
ofthemethodislik
elycorrelated
with
nodalsupportvalues
inthe
individu
algene
topo
logies
Source
httpwwwbrianom
earain
fobrownie
describedin
OrsquoM
eara
(201
0)
Individual
gene
trees
(con
tinued)
22 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
BFD
mdashmultispecies
coalescent
model
for
speciesdelim
itatio
nThe
recently
developedmethod
Bayes
factor
delim
itatio
n(with
genomicdataB
FD)
combinesady
namic
programmingalgorithm
forestim
atingspeciestreesthat
bypasses
thecompu
tatio
nally
intensiveMCMCintegrationov
ergene
treesto
prov
idearigorous
techniqueforspeciesdelim
itatio
nstudiesusinggenome-wideSN
PdataCom
petin
gspeciesdelim
itatio
nmodelsarecomparedusingBayes
factorsanditappearsthat
this
approach
isrobustto
samplesizes(iefew
loci
andlim
itedsamples
perspecies)
and
misspecificatio
nof
thepriorforpo
pulatio
nsize
(θ)
Limita
tions
Recently
describedmethodlackingathorough
theoretical
andem
pirical
evaluatio
nSo
urce
httpwwwbeast2orgwikiindexphpBFD
describedin
Leacheacuteet
al(201
4)
Genom
e-wideSN
Pdata
2 The Dynamic Discipline of Species Delimitation hellip 23
distances among organisms belonging to thesame species are smaller than distances amongorganisms from different species (Puillandreet al 2012 Hebert et al 2003) Genetic distanceapproaches hold particular promise as an identi-fication tool shortcutting the difficulties of mor-phology-based identification (Hebert et al 2004)although in practice a barcode gap may not existfor many groups (Wiemers and Fiedler 2007)Furthermore the role of distance-based approa-ches using a single genetic marker for speciesdiscovery remains more controversial (Rubinoff2006) and without other corroborating evidenceOTUs inferred from single-locus dataset shouldonly be considered ldquocandidaterdquo species Tree-based methods aim to detect monophyletic cladescorresponding to species-level diversity bydetecting discontinuities associated within inter-and intraspecific branching patterns (Fujisawaand Barraclough 2013 Zhang et al 2013Monaghan et al 2009 Pons et al 2006) Tree-based species delimitation methods can also beused on the basis of other properties related tophylogenetic tree topologies (monophyly con-cordance with geography etc (Sites and Marshall2003 2004) Both distance- and tree-basedmethods have been applied for assessing speciesboundaries in lichen-forming fungi (Leavitt et al2012c 2014 Parnmen et al 2012 Del-Pradoet al 2010 2011 Molina et al 2011)
A number of tree-based methods partiallyautomate the species delimitation process withspecific bioinformatical analyses (Table 21)including the general mixed Yule coalescent(GMYC) approach (Fujisawa and Barraclough2013 Monaghan et al 2009 Pons et al 2006)and the Poisson tree processes (PTP) model(Zhang et al 2013) These methods provide rel-atively straightforward and objective pipelinesfor delimiting putative species-level lineagesfrom inferred gene trees by fitting within- andbetween-species branching models to an inferredsingle-locus topology A number of other tree-based methods for species delimitation areeffectively summarized in Sites and Marshall(2004)
When only single-locus data are available theGMYC has been shown to be a relatively robust
tool for species delimitation The GMYCapproach combines a coalescent model of intra-specific branching with a Yule model for inter-specific branching which is then fit to an inferredsingle-gene topology to estimate species bound-aries and a statistical measure of confidence for theinferred boundaries As an input the GMYCapproach requires an ultrametric gene tree andrecent refinements can account for uncertainty inphylogenetic relationships and parameters of theGMYC model (Fujisawa and Barraclough 2013Reid and Carstens 2012) Regardless of theseimprovements it may be difficult to accuratelyinfer an adequate ultrametric tree for large data-sets Although it appears that the GMYC isgenerally stable across a wide range of circum-stances including various methods of phyloge-netic reconstruction the presence of a highnumber singletons high numbers of sampledspecies and gaps in intraspecific sampling theaccuracy of the GMYC is most significantlyaffected by the mean population size relative todivergence times between them (Fujisawa andBarraclough 2013 Talavera et al 2013) Fur-thermore research suggests that the so-calledsingle-threshold version of the GMYC methodlikely outperforms the multiple-thresholdapproach (Fujisawa and Barraclough 2013Monaghan et al 2009) However other studiessuggest that theGMYCmethodmay often providehigher estimates for the total number ofOTUs thanother molecular species delimitation methods(Hamilton et al 2014 Miralles and Vences 2013Talavera et al 2013) warranting a cautiousinterpretation of results from GMYC analyses
Compared to the GMYC approach therecently introduced PTP method for speciesdelimitation has been suggested to be moreaccurate for preliminary species delimitation(Zhang et al 2013) Relative to the GMYC PTPoffers a more straightforward implementationrequiring a simple phylogenetic tree rather thanan ultrametric chronogram However moreresearch is required to assess the general perfor-mance of PTP across wide range of empirical andsimulated species delimitation studies At thispoint data support the use of the GMYC andPTP methods as objective and reasonable starting
24 SD Leavitt et al
points for species delimitation using single-genetopologies
The ldquoSpecies Delimitationrdquo plug-in to theGeneious software provides statistical approachesfor exploring species boundaries in single-genetopologies (Masters et al 2011) Using a priorispecimen assignments to putative species theldquoSpecies Delimitationrdquo plug-in computes statis-tics relating to the probability of the observedmonophyly or exclusivity having occurred bychance in a coalescent process and assesses thewithin- and between-species genetic distances inorder to infer the probability with which membersof a putative species might be identified suc-cessfully with tree-based methods An importantcontribution of the ldquoSpecies Delimitationrdquo plug-in is that it provides an objective means for usersto assess putative species within an empiricalstatistic-based framework rather than qualitativeevaluations of what level of hierarchy constitutesa species in a phylogeny
In contrast to simple sequence similaritythresholds (OTU-picking) for delimiting putativeevolutionarily significant units the AutomaticBarcode Gap Discovery (ABGD) method is anautomated procedure that sorts sequences intohypothetical species based on the existence of abarcode gap observed whenever intraspecificgenetic distances are less than those amongorganisms from different species (Puillandreet al 2012) Ultimately ABGD is a fast simplemethod that can be used to group individualsrepresented in a single-locus sequence alignmentinto candidate species that should be comple-mented with other lines of evidence in an inte-grative taxonomic approach (Kekkonen andHebert 2014 Puillandre et al 2012)
In general the ABGD GMYC and PTP ana-lytical protocols using single-locus data arerepeatable and computationally relatively fastproviding a valuable starting point for a pre-liminary perspective into species boundaries inunderstudied groups that can be validated withsubsequent studies (Kekkonen and Hebert 2014)Similarly analyses of single-locus data can beused to corroborate traditional phenotype-basedspecies boundaries and identify candidate speciesthat have previously been hidden within nominal
species However single-locus species delimita-tionmethodsmaybeparticularly prone to failure inrecognizing significant proportions of species-level biodiversity due to the strict criterion forreciprocal monophyly or alternatively providespurious inflations of estimated species diversitybased on genetic differences that donot correspondto species-level lineages For example recentestimates suggest that the incidence of species-level gene tree paraphyly is approximately 20 (Ross 2014) suggesting that analyses of single-locus datasets would likely fail to accurately deli-mit species in one in every five cases In contrastrecent empirical studies suggest that in some casesthe GMYC provides a striking overestimate ofspecies diversity (Miralles and Vences 2013)
The internal transcribed spacer region (ITS)has played a central role in molecular phyloge-netic studies of lichenized fungi and has recentlybeen adopted as the official barcoding marker forfungi (Schoch et al 2012) In many cases the ITSis sufficiently variable to resolve species bound-aries for lichenized fungi (Schoch et al 2012Kelly et al 2011) although accurate specimenidentification using sequence-based identificationapproaches requires a thoroughly curated refer-ence database (Leavitt et al 2014 Orock et al2012 Kelly et al 2011) In spite of the generalutility of the ITS marker a number of issues maypotentially limit its effective use in speciesdelimitation studies including the potential forintragenomic variation of the nuclear ribosomaltandem repeat and difficulties in aligning ITSsequences from divergent taxa (Kiss 2012)Because the ITS has been formally adopted as theofficial barcoding marker for fungi we recom-mend that species delimitation studies attempt toinclude this region for consistency across studiesHowever we recognize that in some taxonomicgroups the inclusion of the ITS may be prob-lematic and therefore suggest a careful screeningof other markers to identify appropriate loci forresolving species-level relationships
Ultimately the success of any single-locusspecies delimitation method largely depends onthe evolutionary history of the species groupunder study and the variability of the selectedmarker (Fig 22) Species delimitation will be
2 The Dynamic Discipline of Species Delimitation hellip 25
more difficult in more recently diverged lineagesand in cases with some level of interspecific geneflow relative to older well-diverged lineages(eg Leavitt et al 2012c 2013e) In the end wereemphasize that single-locus data alone shouldonly be used to provide a preliminary perspectiveof species boundaries and not as the sole evi-dence in species circumscriptions
222 Sampling Across the GenomeMultilocus Sequence Dataand Genome-Wide Markers
Although single-locus methods can provide anefficient approach for preliminary large-scaleassessments of species diversity there are
significant limitations particularly in recentlydiverged species groups where retention ofancestral polymorphisms and incomplete lineagesorting will likely result in different neutral locihaving unique gene topologies that do not mirrorthe speciation process (Knowles and Carstens2007 Heled and Drummond 2010 Taylor et al2000) In contrast to single-locus and strictlyphenotype-based approaches for species delimi-tation analyses of genetic data collected fromindependent genomic regions can provide robusthypotheses of species boundaries with increasingconfidence (Satler et al 2013 Zhang et al 2011Yang and Rannala 2010) Sequences from mul-tiple independent loci provide an importantsource of data for species delimitation studiesincluding recently developed models that
Fig 22 A simplified diagram illustrating the process ofspeciation through time in a single gene history andresulting gene topologies sampled at two points in thespeciation history (modified from Leliaert et al 2014)aEach dot represents a distinct gene copy and each row onenon-overlapping generation with lines connecting genecopies to their ancestors in the previous generation (onerow below) dashed diagonal lines represent reproductivebarriers two hypothetical sampling intervals at differentpoints in the speciation history are shown Tx and Ty
species delimitation methods using single-locus data areeffective only when species are reciprocally monophyleticin the sampled gene tree genetic clustering and coalescent-based species delimitation methods can circumscribespecies when species may not be monophyletic in sampledgenetic loci b Gene topology representing sampledhaplotypes at time Tx (shown in panel a) hypotheticalspecies are reciprocally monophyletic c Gene topologyrepresenting sampled haplotypes at time Ty (shown in panela) hypothetical species are para- and polyphyletic
26 SD Leavitt et al
combine individual gene genealogies and speciesphylogenies via modeling the coalescent historyof markers (Yang and Rannala 2010 Edwards2009 Knowles and Carstens 2007) As aresponse to advances in sequencing technologiesbioinformatical approaches for multimarker spe-cies delimitation analyses continue to be devel-oped (Table 21 Camargo and Sites 2013)
Long-term reproductive isolation of candidatespecies can be assessed with multilocus sequencedata by evaluating genealogical concordance ofunlinked markers (Baum and Shaw 1995 Aviseand Ball 1990) Within species the mixingeffects of recombination cause unlinked loci tohave distinct genealogical histories but geneticdrift and long-term divergence leads to concor-dant genealogical histories at loci across thegenome Relationships of individuals belongingto distinct candidate species can be evaluatedusing gene genealogies (eg haplotype networksor single-gene topologies) to identify lineagesthat exhibit genealogical exclusivity acrossunlinked neutral loci (Hudson and Coyne 2002Dettman et al 2003b Avise and Ball 1990) Thepresence of the same clades in the majority ofsingle-locus genealogies is taken as evidence thatthe clades represent reproductively isolated lin-eages (Dettman et al 2003a Pringle et al 2005)In practice the criteria of reciprocal monophylyand genealogical concordance of unlinked lociprovide a conservative approach for assessingspecies boundaries due to the fact that a sub-stantial amount of time is required after the initialdivergence of species until ancestral polymor-phisms have fully sorted (Knowles and Carstens2007 Hudson and Coyne 2002) Consequentlygroups with recent divergence histories willlikely go undiscovered under a genealogicalconcordance criterion due to the fact that speciesboundaries likely are not reflected in genegenealogies For example it would take morethan 1 million years after speciation before spe-cies would be delimited if 15 loci were sampledin species with an effective population size (Ne)of 100000 assuming one generation a yearunder a strict reciprocal monophyly criterion(Knowles and Carstens 2007) The amount oftime required for a species to be recognized using
a criterion of reciprocal monophyly increasesproportionally with increasing population sizes(Hudson and Coyne 2002) In a number ofstudies of lichen-forming fungi a genealogicalconcordance criterion has been used to bothdelimit previously unrecognized species-levellineages and validate some conventional pheno-type-based species (Leavitt et al 2012a c2013b Molina et al 2011 Kroken and Taylor2001)
Although analyses of molecular sequence datarelying on reciprocal monophyly or fixed char-acter differences for species delimitation canserve as important criteria for identifying speciesthese criteria are not commonly met acrossmultiple loci particularly in recent speciationhistories (Fig 22) To accommodate theobserved conflict among genealogies from mul-tiple loci with the underlying speciation historythe recent merge of coalescent theory with phy-logenetics has driven a major paradigm shift inspecies delimitation and molecular systematics ingeneral (Fujita et al 2012 Edwards 2009) Themultispecies coalescent model can be applied togenealogical histories from multiple independentloci to assemble separate coalescent processesoccurring in populations into a species tree(Degnan and Rosenberg 2009 Rannala andYang 2003) Within this coalescent-basedframework multiple individuals can be assignedto a single speciespopulation and the speciationhistory of ancestral and descendant species-levellineages can be inferred as a ldquospecies treerdquo incontrast to estimating gene genealogies fromindividual samples (Degnan and Rosenberg2006 2009 Rannala and Yang 2003)
A number of multispecies coalescent-basedspecies delimitation methods have recently beenintroduced offering an exciting framework forempirically assessing species boundaries byselecting the best species tree model from a set ofalternative models representing differenthypotheses of species boundaries (Table 21)For example SpeDeSTEM (Ence and Carstens2011) finds the maximum likelihood values for aspecies tree assuming all putative species areseparate lineages and for alternative species treeswhere two or more species are collapsed into a
2 The Dynamic Discipline of Species Delimitation hellip 27
single lineage The best fitting model is thenselected using the Akaike information criterionunder the assumption of constant populationsizes and fixed gene topologies across the speciestree The species delimitation program BayesianPhylogenetic and Phylogeography (BPampPYangand Rannala 2010) accommodates gene treeuncertainty and variable population sizes andsamples from the Bayesian posterior distributionof species delimitation models using reversible-jump Markov chain Monte Carlo (rjMCMC)sampling BPampP requires a user-provided guidetree with resolution finer than the species leveland evaluates alternative modes derived from allpossible subtrees that are generated by collapsingor splitting nodes on the guide tree (Yang andRannala 2010 Rannala and Yang 2003) Theproportion of time spent on each model is pro-portional to the posterior probability of themodel ie ldquospeciation probabilitiesrdquo WhileBPampP ranks among the most popular coalescent-based species delimitation programs (Fujita et al2012) misspecifications of the guide tree andorthe prior distribution for population size (θ) canresult in strong support for models containing aninflated number of species (Leacheacute and Fujita2010 Zhang et al 2011) The nonparametricheuristic species delimitation approach imple-mented in the program Brownie (OrsquoMeara 2010)jointly sorts anonymous samples into species andinfers a species tree from input gene trees fromdifferent loci assuming that for a speciationevent the corresponding nodes on gene trees willbe more consistent with each than the diver-gences within species However finding both theoptimum species tree and species boundariesremains computationally challenging andBrownie has been shown to frequently yieldincorrect results (OrsquoMeara 2010)
Most recently available coalescent-basedspecies delimitation methods require individualassignments to a species or population a priori Ina number of scenarios correct assignments ofsamples to species may be difficult includingpresence of cryptic species incongruencebetween conventional species and moleculardata or simply the fact that accurate specimenidentification in complex groups with subtle or
difficult to discern diagnostic characters is anontrivial task (Leavitt et al 2013e) Statisticalmethods for assessing individual assignment andspecies detection prior to coalescent-based spe-cies delimitation and species tree reconstructionand species provide a more objective approachfor understanding species boundaries
Population assignment tests using a variety ofclustering algorithms using genetic data offer auseful approach for grouping individuals intoputative reproductively isolated groups (Table 21Hausdorf and Hennig 2010 Corander et al2004 2008 Falush et al 2003 Pritchard et al2000) particularly for species delimitation prob-lems that exist at the interface of traditionalpopulation genetic and phylogenetic analyses(Carstens et al 2013 Weisrock et al 2010Knowles and Carstens 2007)
The programs STRUCTURE (Falush et al2003 Pritchard et al 2000) and STRUCTURA-MA (Huelsenbeck et al 2011) cluster samplesinto populations by minimizing HardyndashWeinbergdisequilibrium for a given number of populationclusters using unlinked genetic markers In gen-eral unlinked markers are not available for mostgroups of lichen-forming fungi and a numberof studies have used information from multilocussequence data for STRUCTURE analyses(Leavitt et al 2011a b c 2013e Fernaacutendez-Mendoza and Printzen 2013 Fernaacutendez-Mendoza et al 2011) Varying approaches havebeen implemented to convert DNA sequencedata to allelic data for Bayesian clustering (seeOrsquoNeill et al 2013) STRUCTURE is expectedto perform well when there is sufficient inde-pendence across regions such that linkage dis-equilibrium within regions does not dominate thedata (STRUCTURE manual) but can also beeffective using multilocus sequence data andtreating all SNPs as independent loci regardlessof physical linkage within each locus (OrsquoNeillet al 2013 Falush et al 2003) A recent study ofthe lichen-forming genus Letharia showed thatBayesian clustering implemented in the programSTRUCTURE was generally able to recover thesame putative Letharia lineages circumscribedemploying a gene genealogical approach inKroken and Taylorrsquos iconic species delimitation
28 SD Leavitt et al
study (Altermann et al 2014 Kroken and Taylor2001) Altermann et al (2014) show that popu-lation assignments were largely consistent acrossa range of scenarios including extensiveamounts of missing data the exclusion of SNPsfrom variable markers and inferences based onSNPs from as few as three gene regions Incontrast to STRUCTURE and STRUCTURA-MA the program BAPS (Corander et al 2008)can explicitly infer genetic structure using hap-loid sequence data or other linked geneticmarkers in the ldquoclustering with linked locirdquomodel Another advantage of BAPS is that theprogram is much more computationally efficientthan STRUCTURE or STRUCTURAMA Sim-ulation studies indicate that both BAPS andSTRUCTURE perform well at low levels ofpopulation differentiation and when clusters arenot well differentiated (Latch et al 2006)
In practice inferring the most appropriatelevel of population structure using Bayesianclustering algorithms remains challenging (Latchet al 2006 Evanno et al 2005) BAPS andSTRUCTURAMA can infer both individualassignments and the most likely number ofgenetic clusters (Corander et al 2008 Huelsen-beck et al 2011) and the ad hoc ΔK statisticproposed by Evanno et al (2005) provides anobjective approach for identifying the uppermosthierarchical level of structure However carefulconsideration of other levels of populationstructure may ultimately reveal more biologicallymeaning results and researchers should examineindividual assignments across a range of geneticgroupings Some markers generated fromindependent genomic regions such as SNPs andfast-evolving microsatellites can be used to dis-tinguish fine-scale population structuring on thebasis of allele frequencies and species delimita-tion analyses based on these markers may implythe risk of severe taxonomic oversplitting Inthese cases validation approaches such asBPampP and SpeDeSTEM and corroborationamong different species delimitation approachescan provide perspective between intraspecificpopulation structure and species-level clusters
Another limitation of clustering approaches isthat they do not assess or take into account
evolutionarily divergences and relationshipsamong population clusters Coalescent-basedspecies tree methods (discussed below) provide ameans to assess the diversification histories ofpopulations inferred from clustering analysesTherefore a potential working protocol for aninformed species delimitation study that takesinto account population structure could consist offirst applying a genetic clustering analysis under apopulation genetics criterion (eg BAPSSTRUCTURE STRUCTURAMA) to identifygenetically distinct population clusters that can beconsidered ldquocandidate speciesrdquo From these can-didate species a species tree can be inferred forfocal group using coalescent-based species treereconstruction methods (eg BEAST) Subse-quently a coalescent-based validation methodcan be applied to assess whether the distinctpopulation clusters represent independent evolu-tionary lineages (eg BPampP) This multistepapproach would provide a consistent and standardcriterion for distinguishing between population-and species-level lineages (Camargo and Sites2013) and has been applied to a number of casesincluding lichen-forming fungi (Leavitt et al2011a b c 2013e Leacheacute and Fujita 2010)
High-throughput sequencing methods providethe means to effectively sample hundreds tothousands of loci from across a species genome fora large number of species and continue to revolu-tionize studies that can be performed even in non-model organisms Targeted high-throughputsequencing approaches such as anchored phy-logenomics transcriptome sequencing reduced-representation genomic library sequencing(restriction-site-associated DNA sequencingRAD-Seq and genotype-by-sequencing GBS)and high-throughput amplicon sequencing pro-vide important insight into species boundaries fora number groups although none of these approa-ches has yet been applied to studies of lichenizedfungi
For example restriction-site-associated(RAD-Seq) DNA can simultaneously detect andgenotype thousands of genome-wide SNPs byfocusing the sequencing effort on a reducedrepresentation of the entire genome (Baird et al2008) and has been successfully applied to
2 The Dynamic Discipline of Species Delimitation hellip 29
intraspecies (Lewis et al 2007 Miller et al 2007Emerson et al 2010) and interspecies studies(Rubin et al 2012 Eaton and Ree 2013 Wagneret al 2013) This approach has provided strikinginsight into the recent adaption radiation of LakeVictoria cichlids (Wagner et al 2013) Analternative approach targeting the sequencing ofspecific loci using next-generation sequencingplatforms provides an efficient means of gener-ating data for loci of known genomic locationorthology size and expected level of variation(OrsquoNeill et al 2013) Markers can be targetedusing well-established PCR techniques or newerhybridization techniques and subsequentlypooled for high-throughput sequencing usingparallel-tagged sequencing of multiple individualsamples within a single sequencing run (OrsquoNeillet al 2013) A recent study of North Americantiger salamanders (Ambystoma tigrinum)demonstrated the potential for amplicon-basedparallel-tagged sequencing to rapidly generatelarge-scale genomic data for species delimitationand species tree research (OrsquoNeill et al 2013)
Although these methods provide an enormousamount of data and insight into diversification atan unprecedented scale computational limita-tions restrict the applicable analytical methodsfor large datasets although computational andanalytical advances are happening rapidly Mostrecent coalescent-based species delimitation andspecies tree models using gene trees have beenlimited to handle tens of loci for multiple indi-viduals and combining hundreds or thousands ofgene trees into a single species delimitationframework presents considerable computationalchallenges (Camargo and Sites 2013) Therecently developed method Bayes factor delim-itation (with genomic data BFD) combines adynamic programming algorithm for estimatingspecies trees that bypasses the computationallyintensive MCMC integration over gene trees toprovide a rigorous technique for species delimi-tation studies using genome-wide SNP data(Leacheacute et al 2014) Competing species delimi-tation models are compared using Bayes factorsand it appears that this approach is robust tosample sizes (ie few loci and limited samples
per species) and misspecification of the prior forpopulation size (θ) (Leacheacute et al 2014)
Fungi are an ideal model for assessing diver-gence in eukaryotes due to their simple mor-phologies small genomes broad ecological rolesand diverse lifestyles (Gladieux et al 2014)However the use of genomic data from high-throughput sequencing methods have not yet beenincluded in species delimitation studies of lichen-forming fungi This is due in part to challenges indealing with metagenomic data containing geno-mic information from a plethora of symbiontsassociated with a lichen thallus scant genomicresources and discipline-specific inertia A num-ber of lichen-forming fungal genomes are cur-rently available including Endocarponpusillum(Wang et al 2014) Cladonia spp (Park et al2013a 2014) Caloplaca flavorubescens (Parket al 2013b) and Cladonia greyi and Xanthoriaparientina through the Fungal Genomics Programat Joint Genome Institute of the United StatesDepartment of Energy (httpwwwjgidoegov)The foreseeable ongoing expansion of genomicresources for lichen-forming fungi will be centralto developing approaches for delimiting speciesusing high-throughput sequencing Specificallygenomic resources will be crucial in identifyingnovel markers (variable genes regions SNPsmicrosatellites etc) identifying conserved fungalmarkers for targeted high-throughput sequencingapproaches and references for entire genomecomparisons (Devkota et al 2014 Werth et al2013)
23 Can We Make SpeciesDelimitation in Lichen-FormingFungi Truly Integrative
The widespread availability of genetic data hascreated a biased viewpoint that only genetic datashould be used for statistical species delimitationHowever an ongoing appeal to researchers toassess species boundaries from multiple andcomplementary perspectives (phylogeneticspopulation genetics comparative morphologydevelopment ecology etc) has resulted in an
30 SD Leavitt et al
integrative taxonomic framework bringing theseconceptual and methodological perspectivestogether (Hamilton et al 2014 Fujita et al 2012Salicini et al 2011 Yeates et al 2011 Padialet al 2010 Dayrat 2005 Will et al 2005 Wiensand Penkrot 2002) In practice any study linkingdifferent kinds of data to support hypotheses ofspecies boundaries including mapping morpho-logical characters onto a molecular phylogenycan be considered integrative Integrative meth-ods for species delimitation fall across a broadspectrum ranging from verbal and qualitativeassessments of data classes to quantitative meth-ods that allow different data types to contribute tostatistical species delimitation (Yeates et al2011) In most taxonomic studies utilizing bothmolecular and morphological data expert opinionis eventually used to some degree to evaluate thefinal status of a candidate species (eg Bond andStockman 2008) In this sense many studiesusing evidence for independent data sources fordelimiting species boundaries use an iterativeapproach (Yeates et al 2011) where speciesboundaries can be tested within a hypothetico-deductive framework with diverse datasets
From a practical perspective we advocate aprocess of iterative taxonomy (sensu Yeates et al2011) to circumscribe and refine species limitsusing multiple lines of evidence This iterativeprocess involves comparisons of morphologicaldata with a phylogenetic hypothesis (ie single-locus gene tree or concatenated multilocusphylogeny) to identify the least inclusive mono-phyletic clade in the topology characterized by atleast one unambiguously diagnostic morphologi-cal character (Miralles and Vences 2013) Thisphylogenetic tree-informed approach to assessingspecies boundaries represents a common practicein studies of lichen-forming fungi Previouslyunrecognized species-level clades with corre-sponding subtle or overlooked phenotypiccharacters have been commonly observed in bothcrustose lichens [eg Acarospraceae (Wedin et al2009) Graphidaceae (Rivas Plata and Lumbsch2011 Papong et al 2009) Lecanoraceae (Leavittet al 2011a) Lecideaceae (Ruprecht et al 2010)Mycoblastaceae (Spribille et al 2011) andTeloschistaceae (Vondraacutek et al 2009 Muggia
et al 2008)] and foliose and fruticose lichens [egLobariaceae (Moncada et al 2014 McDonaldet al 2003) Parmeliaceae (Leavitt et al 2013aWirtz et al 2012 Divakar et al 2005 Molinaet al 2004 Kroken and Taylor 2001) Peltigera-ceae (OrsquoBrien et al 2009 Goffinet et al 2003)Physciaceae (Elix et al 2009 Divakar et al2007) and Sphaerophoraceae (Houmlgnabba andWedin 2003)] As a specific example Luumlckinget al (2008) used a combination of medullarychemistry and underside pigmentation in speci-mens from the Heterodermia obscurata group inCosta Rica to corroborate monophyletic clades inan ITS gene tree as species-level lineages
While this iterative approach to speciesdelimitation and taxonomy has proven valuablefor understanding species boundaries anddescribing new taxa in some groups of lichenizedfungi a posteriori examination of morphologicaland chemical features has failed to reveal diag-nostic phenotypic characters in a number ofstudies (Muggia et al 2014 Leavitt et al 2011aOtaacutelora et al 2010 OrsquoBrien et al 2009) Fur-thermore a study of widespread species in thegenus Melanelixia (Parmeliaceae) indicated thatphenotypically cryptic lichen-forming fungalspecies-level lineages may be relatively ancientand diagnosable phenotypic differences may beabsent even millions of years after the initialdivergence (Leavitt et al 2012b) The latter studyhighlights the fact that species-level lineages maycommonly exist without any observable diag-nostic phenotypic characters calling into ques-tion the impetus for a universal application ofintegrative taxonomy
In keeping with the principle that as manylines of evidence as available should be com-bined to delimit species (Dayrat 2005) the for-malized integrative taxonomic approach (ITAXMiralles and Vences 2013) uses a mtDNA guidetree and observations from different types of datathat might provide conclusive evidence for theindependence of lineages and thus their identityas different species Miralles and Vences (2013)provide a non-exhaustive list of species delimi-tation criteria to be integrated in the ITAXapproach including (a) sympatric occurrencewithout admixture as revealed by consistent
2 The Dynamic Discipline of Species Delimitation hellip 31
differences in morphological or molecular char-acters at the same geographic location (b) strongdifferences in a behavioral morphological orgenetic character known to mediate prematingisolation (c) unviability or infertility of hybrids(d) lack of gene flow across a geographicalhybrid zone (e) congruent diagnostic differencesbetween sister lineages in various unlinkedmorphological character (f) absence of haplo-type sharing in several unlinked nuclear loci and(g) a combination of criteria endashf Speciesboundaries are based on seeking the least inclu-sive monophyletic group in the mtDNA treewhich fulfills at least one of the criteria listedabove Clearly the ITAX approach is sensitive tosample size and in order to reliably support thedistinctiveness of a given species it has beenrecommended that the sampling strategy includesat least five individuals per species (Miralles andVences 2013) A similar approach could beadopted for lichenized fungi using an ITS genetopology as guide tree rather than mtDNA
A total evidence approach including concat-enation has been a common approach for inte-grating information from different sourcesincluding independent genetic markers in phy-logenetic reconstructions (Kluge 1989 Wiens1998 de Queiroz et al 1995) Proponents forconcatenation of independent data in phyloge-netic analyses argue that when combining alldata the underlying signal of speciation mayemerge even if weakly contradictory signalis contained in the individual data partitions(Gatesy et al 1999) For example establishing apreliminary perspective of species boundariesfrom multilocus sequence data using concatena-tion may provide a reasonable starting point forscreening for cryptic species and species treeinference (Šlapeta et al 2006 Leavitt et al2011a Le Gac et al 2007 Leacheacute 2009)However concatenation and consensus methodsimply a risk of obtaining inflated support forincorrect relationships and information aboutvariance in gene coalescence is lost (Degnan andRosenberg 2006 2009 Kubatko and Degnan2007 Edwards 2009) In spite of the impetus forintegrating evidence from independent data
sources into empirical species delimitation stud-ies and taxonomy (Fujita et al 2012 Padial et al2009) most currently available species delimi-tation methods are unable to accommodate non-genetic data sources in a statistical framework
231 Selecting the Appropriate Data
In the face of increasing availability of geneticdata and associated bioinformatical approachesfor delimiting species researchers should care-fully consider what information is being sacri-ficed by the failure to consider non-genetic datain species delimitation studies and whetheraccuracy could be improved by the addition ofmultiple data types Morphological data havehistorically served as a proxy to identify repro-ductively isolated groups (ie ldquospeciesrdquo) (Ray1686 Fujita et al 2012) Current methods fordelimiting species using non-genetic data (egchemistry morphology and ecology) remainwoefully understudied For example morphol-ogy-based species circumscriptions are generallybased on one or more qualitative (or quantitative)morphological characters that do not appear tooverlap with other species However ascertain-ing that a given trait is truly fixed within apopulation with statistical confidence requiresunrealistic sample sizes even when allowing forsome level of polymorphism in the diagnosticcharacter (Wiens and Servedio 2000) Now var-ious combinations of datamdashfrom morphologygenetics geography and ecologymdashare acceptedas standard information for species delimitationstudies (Ruiz-Sanchez and Sosa 2010 Ross et al2010 Edwards and Knowles 2014)
Below we briefly discuss appropriate datasources for species delimitation studies of lichen-forming fungi However homoplastic characters(similar traits that are not derived from a com-mon ancestor) are common among many traitscommonly used to circumscribe fungal taxa thebiological significance of secondary metabolitevariation remains largely unknown (Lawrey1986) and ecological niches may be difficultto adequately characterize and model due to
32 SD Leavitt et al
microhabitat requirements and data resolutionTherefore we advocate a cautious approach toselecting appropriate and relevant data forassessing species boundaries
In spite of the potential challenges and limi-tations of using phenotypic data these traits haveprovided a plethora of valuable information forunderstanding species boundaries For examplein the Melanelixia fuliginosa group (Parmelia-ceae) a morphometric analysis using color isi-dia and marginal zone free of isidia ascharacters revealed a general pattern of differ-entiation between material formerly recognizedas subspecies fuliginosa and glabratula in spiteof the fact that considerable overlap betweengroups occurs in some characters individually(Arup and Berlin 2011) Furthermore the dis-tinction of the two groups was supported by aphylogenetic analysis of the ITS marker andecological differences with M fuliginosaoccurring predominantly on rock and M glab-ratula on bark (Arup and Berlin 2011) Thisstudy of the M fuliginosa group provides a fit-ting example of using multiple independentsuites of data including ecology morphologyand genetic information to establish a robusthypothesis of species boundaries
In lichen systematics phenotypic dataincluding thallus organization secondary metab-olites mode of reproduction ascoma-type andontogeny ascus and ascospore characters havehistorically played a prominent role (Printzen2009) Ascomatal characters have traditionallyheld a major role in higher-level classification(Printzen 2009) in contrast to species-levelclassification which also tends to include a widearray of vegetative and chemical charactersCommonly assessed morphological charactersinclude thallus form and size cortical features(eg maculae and pseudocyphellae) presenceformcolor of attachment structure (eg rhizines)and reproductive mode (ascomata vs vegetativediaspores) (Printzen 2009) Different morpho-logical types of vegetative diasporesmdashcorticatedisidia and ecorticated sorediamdashand their locationare commonly used to distinguish speciesAscomatal characters including morphologylocation (laminal vs marginal) position (sessile
vs immersed) presence of thalline margins colorof an apothecial disc and presence or color ofpruina are also commonly used in speciesdelimitations of lichen-forming fungi (Printzen2009) Other important characters may includethalline characters form color size and septationof ascospores size and form and structure of ascithe hamathecium type of epihymenium andhypothecium and the type of excipulum orperidium conidiomatal characters etc (Printzen2009)
Assessments of secondary metabolites hasplayed an important role in lichen taxonomybeginning with the introduction of simple spottests by Nylander (1866a b) The use of chem-istry in lichen taxonomy has been discussed indetail in numerous reviews (Lumbsch 1998a bRogers 1989 Brodo 1986 Egan 1986 Leuckert1985 Brodo 1978 Hawksworth 1976 Culber-son 1969 1970) and we refer readers to thesevaluable sources for a more comprehensive per-spective on lichen chemistry In short extrolites(secondary metabolites) belong to various clas-ses the most common and diverse include dep-sides depsidones chlorinated xanthones andanthraquinones (Lumbsch 2002 Culberson1969) The presence or absence of specificextrolites or their replacements by another sub-stance is widely used to distinguish speciesparticularly when correlated with differences ingeographic distributions However if morpho-logical or geographical differences betweenpopulations containing different extrolites are notapparent the taxonomic significance has beendisputed with some authors distinguishing themas species and others preferring to regard them aschemical races within a species In addition tosimply using the presence or absence of extro-lites Culberson and Culberson (1976) proposedto arrange lichen substances into chemosyn-dromes of closely related substances The pres-ence or absence of these chemosyndromes maypotentially be used as characters to delimit spe-cies regarding differences involving the samechemosyndromes as intraspecific variation anddistinct chemosyndromes as evidence for inter-specific populations (Lumbsch 1994 Gowan1986)
2 The Dynamic Discipline of Species Delimitation hellip 33
Due to the fact that species delimitationstudies often incorporate a substantial biogeo-graphical or ecological component ecologicalniche modeling plays an increasing role in phy-logenetic and taxonomic research Niche mod-eling can provide evidence for ecologicalisolation between populations based on eitherconserved or divergent ecological niches andtherefore can provide additional evidence sup-porting lineage independence between putativespecies The application of ecological nichemodeling has been applied to species delimita-tion studies in a number of cases (Ruiz-Sanchezand Sosa 2010 Leacheacute et al 2009 Raxworthyet al 2007 Rissler and Apodaca 2007) althoughit has not been explicitly applied to assess speciesboundaries in lichen-forming fungi By mappingthe spatial distribution of environmental suit-ability of climatic variables for candidate speciesthe application of ecological niche modeling canbe particularly important in cases where specieshave allopatric distributions (Raxworthy et al2007)
Ecological niche modeling utilizes knownassociations between a speciesrsquo occurrencelocalities and environmental variables to defineabiotic conditions within which populations canbe maintained (Guisan and Thuiller 2005) Themethodological approach for modeling is basedon four general properties (i) The current knownspeciesrsquo localities is the dependent variable (ii)the distribution is modeled as a map composed ofgrid cells at a specified resolution (iii) a range ofenvironmental variables (eg temperature pre-cipitation and solar exposure) are collected todescribe the characteristics of each cell and (iv)classifying the degree to which each cell is eithersuitable or unsuitable for each species under arange of models (Guisan and Thuiller 2005)
Ecological data also have the potential to playan important role in understanding speciesboundaries in lichen-forming fungi For exampleNash and Zavada (1977) demonstrated that Xant-hoparmelia populations with distinct chemistriesoccurring in the northern portion of the SonoranDesert exhibit habitat selection among differentrock substrates within a region with relativelyuniform climate and topography In another case
the parmelioid species Parmelia mayi is mor-phologically indistinguishable from P saxatilisbut can be separated by bioclimatic features andgenetic and chemical characters (Molina et al2011) McCune and Printzen(2011) assess distri-butions and climatic niches of species in theLecanora varia group inwesternUSAandprovidea model that uses continental influence and annualtemperature as themajor factors predicting speciesdistributions The distribution of Usnea hirta alichen commonly used in air quality biomonitor-ing research was modeled for a section of theWhite River National Forest in central Coloradobased on the presence of U hirta at 72 biomoni-toring reference sites distributed in the inter-mountain western United States (Shrestha et al2012) The best model for predicting U hirtadistribution included four variablesmdashsolar radia-tion average monthly precipitation and averagemonthly minimum and maximum temperatures(Shrestha et al 2012) These studies supportthe potential use of ecological niche modelingmethods in species delimitation studies of lichen-forming fungi
24 Conclusions What AboutTaxonomy
In most cases species circumscription and tax-onomy requires some degree of qualitativejudgment and individual interpretation Integrat-ing multiple types of data into an empiricalframework for delimiting species boundarieswhere species boundaries can be tested within ahypothetico-deductive framework with diversedatasets can provide robust hypotheses of speciesboundaries and taxonomic stability (Yeates et al2011) However it has long been known toevolutionary biologists that distinct species donot need to have diagnosable morphologicaldifferences (Mayr 1963) and increasing avail-ability of genetic data has allowed researchers toidentify species and to rigorously test speciesboundaries with a level of precision that wasunimaginable a decade ago While analyticaladvances in statistical species delimitation havebeen largely based on genetic data the utility of
34 SD Leavitt et al
these approaches to formal taxonomy remainselusive Due to these challenges an eclecticapproach to delimiting species and cautionagainst the reliance on any single dataset ormethod is required when delimiting species
While results from many recent studies maycontradict traditional species boundaries acrossmany groups of lichen-forming fungi we areoptimistic that this research represents substantialprogress toward a more accurate perspective onspecies boundaries and diversity in fungi As aresult of ongoing research of species boundariesin lichen-forming fungi the taxonomic value ofmany phenotypes is now better understood ourunderstanding of ecological evolutionary andbiogeographic patterns has improved and we canbegin to better understand patterns of symbiontinteractions in lichens Integrating new data(including novel morphological characters andgenetic data) will be essential to accurately rep-resent species-level diversity across all groups oflichenized fungi Hopefully an improved per-spective on lichen diversity also increases ourappreciation of these incredible symbiotic sys-tems While we are strong advocates for theapplication of independent data types in devel-oping an integrative taxonomy there is anincreasing need to formally recognize the exis-tence of phenotypically cryptic species-levellineages in lichen-forming fungi (see Hibbettet al 2011) In some cases a molecular taxon-omy may provide the most practical approach toconsistent treatment of mycobiont species withinlichen groups where diagnostic morphologicalcharacters are unidentifiable or practically notfeasible (Leavitt et al 2013c d)
These are exciting times for taxonomists andphylogeneticists A closer look at lichen taxon-omy with the inclusion of new data will help usto better understand the diversity of these fasci-nating organisms accurately interpret distribu-tion patterns and play a more important role inmeaningful conservation practices Howeversome level of uncertainty will accompany pro-gress In many taxonomic groups our traditionalapproach for species identification will likelyneed to be substantially modified The search forcorroborating morphological support for cryptic
species identified using molecular data willrequire meticulous and creative approaches toassess phenotypic variation in potentially unor-thodox ways We are hopeful that lichenologistswho traditionally have been eager to include newmethods such as chromatography in their rou-tine identifications will be amenable to includemolecular techniques to their routine examina-tion of specimens for identification and classifi-cation Although this may prove difficult toachieve by single individuals especially citizenscientists that traditionally play an important rolein lichen taxonomy the increasing numberof collaborative projects in the discipline (egLumbsch et al 2011 Crespo et al 2010Gueidan et al 2009) make us optimistic thatbroad-scale collaborative approaches will facili-tate the inclusion of molecular data in lichenresearch at all levels
Acknowledgments We are indebted to various col-leagues for valuable thought-provoking discussionnotably Matthew Nelsen (University of Chicago) AnaCrespo (Universidad Complutense de Madrid) PradeepDivakar (Universidad Complutense de Madrid) BeckettSterner (The Field Museum) and Joyce Havstad (TheField Museum) We also thank anonymous reviewers whoprovided valuable comments that improved this chapterSupport by the US National Science Foundation isgratefully acknowledged (ldquoHidden diversity in parmelioidlichensrdquo DEB-0949147)
References
Ahti T Hawksworth DL (2005) Xanthoparmelia steno-phylla the correct name for X somloeumlnsis one of themost widespread usnic acid containing species of thegenus The Lichenologist 37(4)363ndash366 doi101017S0024282905015197
Altermann S Leavitt SD Goward T Nelsen MPLumbsch HT (2014) How do you solve a problemlike Letharia A new look at cryptic species in lichen-forming fungi using Bayesian clustering and SNPSsfrom multilocus sequence data PLoS ONE 9(5)e97556 doi101371journalpone0097556
Amo de Paz G Cubas P Crespo A Elix JA Lumbsch HT(2012) Transoceanic dispersal and subsequent diver-sification on separate continents shaped diversity ofthe Xanthoparmelia pulla group (Ascomycota) PLoSONE 7(6)e39683 doi101371journalpone0039683
Arguumlello A Del Prado R Cubas P Crespo A (2007)Parmelina quercina (Parmeliaceae Lecanorales)
2 The Dynamic Discipline of Species Delimitation hellip 35
includes four phylogenetically supported morphospe-cies Biol J Linn Soc 91(3)455ndash467 doi101111j1095-8312200700810x
Arup U Berlin ES (2011) A taxonomic study ofMelanelixia fuliginosa in Europe The Lichenologist43(02)89ndash97 doi101017S0024282910000678
Arup U Grube M (2000) Is Rhizoplaca (Lecanoraleslichenized Ascomycota) a monophyletic genus Can JBot 78(3)318ndash327 doi101139b00-006
Avise J Ball R (1990) Principles of genealogicalconcordance in species concepts and biological tax-onomy Oxf Surv Evol Biol 745ndash67
Baird NA Etter PD Atwood TS Currey MC Shiver ALLewis ZA Selker EU Cresko WA Johnson EA(2008) Rapid SNP discovery and genetic mappingusing sequenced RAD markers PLoS ONE 3(10)e3376 doi101371journalpone0003376
Baum DA Shaw KL (1995) Genealogical perspectives onthe species problem In Hoch PC Stephenson AG(eds) Experimental and molecular approaches to plantbiosystematics Missouri Botanical Garden St Louispp 289ndash303
Beaumont MA Nielsen R Robert C Hey J Gaggiotti OKnowles L Estoup A Panchal M Corander JHickerson M Sisson SA Fagundes N Chikhi LBeerli P Vitalis R Cornuet J-M Huelsenbeck J FollM Yang Z Rousset F Balding D Excoffier L (2010)In defence of model-based inference in phylogeogra-phy Mol Ecol 19(3)436ndash446 doi101111j1365-294X200904515x
Bickford D Lohman DJ Sodhi NS Ng PKL Meier RWinker K Ingram KK Das I (2007) Cryptic speciesas a window on diversity and conservation TrendsEcol Evol 22(3)148ndash155 doi101016jtree200611004
Bonan GB Shugart HH (1989) Environmental factors andecological processes in boreal forests Annu Rev EcolSyst 20(1989)1ndash28
Bond J Stockman A (2008) An integrative method fordelimiting cohesion species finding the population-species interface in a group of Californian trapdoorspiders with extreme genetic divergence and geo-graphic structuring Syst Biol 57(4)628ndash646 doi10108010635150802302443
Brodo IM (1978) Changing concepts regarding chemicaldiversity in lichens The Lichenologist 10(1)1ndash11doi101017S0024282978000031
Brodo IM (1986) Interpreting chemical variation inlichens for systematic purposes The Bryologist 89(2)132ndash138
Caley MJ Fisher R Mengersen K (2014) Global speciesrichness estimates have not converged Trends EcolEvol 29(4)187ndash188 doi101016jtree201402002
Camargo A Sites JW (2013) Species delimitation adecade after the Renaissance In Pavlinov I (ed) Thespecies problemmdashongoing issues InTech doi10577252664
Camargo A Morando M Avila LJ Sites JW (2012)Species delimitation with ABC and other coalescent-based methods a test of accuracy with simulations and
an empirical example with lizards of the Liolaemusdarwinii complex (Squamata Liolaemidae) Evolution66(9)2834ndash2849 doi101111j1558-5646201201640x
Carstens BC Dewey TA (2010) Species delimitationusing a combined coalescent and information-theoreticapproach an example from North American Myotisbats Syst Biol 59(4)400ndash414 doi101093sysbiosyq024
Carstens BC Pelletier TA Reid NM Satler JD (2013)How to fail at species delimitation Mol Ecol 22(17)4369ndash4383 doi101111mec12413
Corander J Marttinen P (2006) Bayesian identification ofadmixture events using multi-locus molecular mark-ers Mol Ecol 15(10)2833ndash2843 doi101111j1365-294X200602994x
Corander J Waldmann P Marttinen P Sillanpaa M(2004) BAPS 2 enhanced possibilities for the analysisof genetic population structure Bioinformatics 20(15)2363ndash2369 doi101093bioinformaticsbth250
Corander J Marttinen P Mantyniemi S (2006) Bayesianidentification of stock mixtures from molecular markerdata Fish Bull 104550ndash558
Corander J Marttinen P Siren J Tang J (2008) EnhancedBayesian modelling in BAPS software for learninggenetic structures of populations BMC Bioinf 9(1)539 doi1011861471-2105-9-539
Coyne JA Orr HA (2004) Speciation Sinauer AssociatesSunderland
Cracraft J (1983) Species concepts and speciation anal-ysis Curr Ornithol 1159ndash187
Crespo A Lumbsch HT (2010) Cryptic species in lichen-forming fungi IMA Fungus 1167ndash170 doi105598imafungus2010010209
Crespo A Peacuterez-Ortega S (2009) Cryptic species andspecies pairs in lichens a discussion on the relation-ship between molecular phylogenies and morpholog-ical characters Anales del Jardin Botanico de Madrid66(S1)71ndash81 doi103989ajbm2225
Crespo A Kauff F Divakar PK del Prado R Perez-Ortega S Amo de Paz G Ferencova Z Blanco ORoca-Valiente B Nunez-Zapata J Cubas P ArguelloA Elix JA Esslinger TL Hawksworth DL MillanesA Molina MC Wedin M Ahti T Aptroot A et al(2010) Phylogenetic generic classification of parmeli-oid lichens (Parmeliaceae Ascomycota) based onmolecular morphological and chemical evidenceTaxon 59(6)1735ndash1753
Culberson WL (1969) The use of chemistry in thesystematics of the lichens Taxon 18498ndash505
Culberson WL (1970) Chemosystematics and ecology oflichen-forming fungi Annu Rev Ecol Syst 1153ndash170
Culberson CF Culberson WL (1976) Chemosyndromicvariation in lichens Syst Bot 1325ndash339
Darwin C (1859) On the origin of species by means ofnatural selection or the preservation of favoured racesin the struggle for life J Murray London
Dayrat B (2005) Towards integrative taxonomy Biol JLinn Soc 85(3)407ndash415 doi101111j1095-8312200500503x
36 SD Leavitt et al
de Queiroz K (1998) The general lineage concept ofspecies species criteria and the process of speciationa conceptual unification and terminological recom-mendations In Howard DJ Berlocher SH (eds)Endless forms species and speciation Oxford Uni-versity Press Oxford pp 57ndash75
de Queiroz K (1999) The general lineage concept ofspecies and the defining properties of the speciescategory In Wilson RA (ed) Species new interdis-ciplinary essays MIT Press Cambridge pp 49ndash89
de Queiroz K (2007) Species concepts and speciesdelimitation Syst Biol 56(6)879ndash886 doi10108010635150701701083
de Queiroz A Donoghue MJ Kim J (1995) Separateversus combined analysis of phylogenetic evidenceAnnu Rev Ecol Syst 26(1)657ndash681 doi101146annureves26110195003301
Degnan J Rosenberg N (2006) Discordance of speciestrees with their most likely gene trees PLoS Genet 2(5)e68 doi101371journalpgen0020068
Degnan JH Rosenberg NA (2009) Gene tree discordancephylogenetic inference and the multispecies coales-cent Trends Ecol Evol 24(6)332ndash340 doihttpdxdoiorg101016jtree200901009
Del-Prado R Cubas P Lumbsch HT Divakar PK BlancoO de Paz GA Molina MC Crespo A (2010) Geneticdistances within and among species in monophyleticlineages of Parmeliaceae (Ascomycota) as a tool fortaxon delimitation Mol Phylogenet Evol 56(1)125ndash133 doi101016jympev201004014
Del-Prado R Divakar PK Crespo A (2011) Using geneticdistances in addition to ITS molecular phylogeny toidentify potential species in the Parmotrema reticul-atum complex a case study The Lichenologist 43(06)569ndash583 doi101017S0024282911000582
Del-Prado R Blanco O Lumbsch HT Divakar PK ElixJA Molina MC Crespo A (2013) Molecular phylog-eny and historical biogeography of the lichen-formingfungal genus Flavoparmelia (Ascomycota Parmelia-ceae) Taxon 62(5)928ndash939 doi101270562522
Dettman J Jacobson D Taylor J (2003a) A multilocusgenealogical approach to phylogenetic species recog-nition in the model eukaryote Neurospora Evolution57(12)2703ndash2720 doi101111j0014-38202003tb01514x
Dettman JR Jacobson DJ Turner E Pringle A TaylorJW (2003b) Reproductive isolation and phylogeneticdivergence in Neurospora comparing methods ofspecies recognition in a model eukaryote Evolution57(12)2721 doi10155403-074
Devkota S Cornejo C Werth S Chaudhary RP Sche-idegger C (2014) Characterization of microsatelliteloci in the Himalayan lichen fungus Lobaria pindar-ensis (Lobariaceae) Appl Plant Sci 2(5)1300101doi103732apps1300101
Divakar PK Molina MC Lumbsch HT Crespo A (2005)Parmelia barrenoae a new lichen species related toParmelia sulcata (Parmeliaceae) based on molecularand morphological data The Lichenologist 37(01)37ndash46 doi101017S0024282904014641
Divakar PK Amo De paz G del Prado R Esslinger TLCrespo A (2007) Upper cortex anatomy corroboratesphylogenetic hypothesis in species of Physconia(Ascomycota Lecanoromycetes) Mycol Res 111(11)1311ndash1320 doi101016jmycres200708009
Divakar PK Figueras G Hladun N Crespo A (2010)Molecular phylogenetic studies reveal an undescribedspecies within the North American concept of Mela-nelixia glabra (Parmeliaceae) Fungal Divers 42(1)47ndash55 doi101007s13225-010-0027-3
Donoghue MJ Gauthier A (2004) Implementing thephylocode Trends Ecol Evol 19(6)281ndash282 doi101016jtree200404004
Eaton DAR Ree RH (2013) Inferring phylogeny andintrogression using RADseq data an example fromflowering plants (Pedicularis Orobanchaceae) SystBiol 62(5)689ndash706 doi101093sysbiosyt032
Edwards SV (2009) Is a new and general theory ofmolecular systematics emerging Evolution 63(1)1ndash19 doi101111j1558-5646200800549x
Edwards DL Knowles LL (2014) Species detection andindividual assignment in species delimitation canintegrative data increase efficacy Proc R Soc B BiolSci 281(1777) doi101098rspb20132765
Egan RS (1986) Correlations and non-correlations ofchemical variation patterns with lichen morphologyand geography The Bryologist 8999ndash110
Elix JA Corush J Lumbsch HT (2009) Triterpenechemosyndromes and subtle morphological characterscharacterise lineages in the Physcia aipolia group inAustralia (Ascomycota) Syst Biodivers 7(04)479ndash487 doi101017S1477200009990223
Emerson KJ Merz CR Catchen JM Hohenlohe PACresko WA Bradshaw WE Holzapfel CM (2010)Resolving postglacial phylogeography using high-throughput sequencing Proc Natl Acad Sci 107(37)16196ndash16200 doi101073pnas1006538107
Ence DD Carstens BC (2011) SpedeSTEM a rapid andaccurate method for species delimitation Mol EcolResour 11(3)473ndash480 doi101111j1755-0998201002947x
EvannoGRegnaut SGoudet J (2005)Detecting thenumberof clusters of individuals using the software STRUC-TURE a simulation study Mol Ecol 14(8)2611ndash2620doi101111j1365-294X200502553x
Falush D Stephens M Pritchard JK (2003) Inference ofpopulation structure using multilocus genotype datalinked loci and correlated allele frequencies Genetics164(4)1567ndash1587 doi101111j1471-8286200701758x
Fan HH Kubatko LS (2011) Estimating species treesusing approximate Bayesian computation Mol Phylo-genet Evol 59(2)354ndash363 doi101016jympev201102019
Fernaacutendez-Mendoza F Printzen C (2013) Pleistoceneexpansion of the bipolar lichen Cetraria aculeata intothe Southern hemisphere Mol Ecol 22(7)1961ndash1983doi101111mec12210
Fernaacutendez-Mendoza F Domaschke S Garciacutea MA JordanP Martin MP Printzen C (2011) Population structure
2 The Dynamic Discipline of Species Delimitation hellip 37
of mycobionts and photobionts of the widespreadlichen Cetraria aculeata Mol Ecol 20(6)1208ndash1232doi101111j1365-294X201004993x
Fraley C Raftery A (2007) Model-based methods ofclassification Using the mclust software in chemo-metrics J Stat Softw 18i06 [Available online at httpwwwdoajorgdoajfunc=abstractampid=218544]
Fujisawa T Barraclough TG (2013) Delimiting speciesusing single-locus data and the generalized mixed yulecoalescent (GMYC) approach a revised method andevaluation on simulated datasets Syst Biol 62(5)707ndash724 doi101093sysbiosyt033
Fujita MK Leacheacute AD Burbrink FT McGuire JA MoritzC (2012) Coalescent-based species delimitation in anintegrative taxonomy Trends Ecol Evol 27(9)480ndash488 doi101016jtree201204012
Gatesy J OrsquoGrady P Baker RH (1999) Corroborationamong data sets in simultaneous analysis hiddensupport for phylogenetic relationships among higherlevel Artiodactyl taxa Cladistics 15(3)271ndash313doi101111j1096-00311999tb00268x
Gladieux P Ropars J Badouin H Branca A Aguileta Gde Vienne DM Rodriacuteguez de la Vega RC Branco SGiraud T (2014) Fungal evolutionary genomicsprovides insight into the mechanisms of adaptivedivergence in eukaryotes Mol Ecol 23(4)753ndash773doi101111mec12631
Goffinet B Miadlikowska J Goward T (2003) Phyloge-netic inferences based on nrDNA sequences supportfive morphospecies within the Peltigera didactylacomplex (Lichenized Ascomycota) The Bryologist106(3)349ndash364 doi10163901
Gowan SP (1986) Evolution of secondary natural productsin the genus Porpidia (Ascomycata Porpidiaceae) AmJ Bot 73606
Griffin PC Hoffmann AA (2014) Limited genetic diver-gence among Australian alpine Poa tussock grassescoupled with regional structuring points to ongoinggene flow and taxonomic challenges Ann Bot doi101093aobmcu017
Grube M Hawksworth DL (2007) Trouble with lichenthe re-evaluation and re-interpretation of thallus formand fruit body types in the molecular era Mycol Res111(9)1116ndash1132 doi101016jmycres200704008
Gueidan C Savi S Thues H Roux C Keller C Tibell LPrieto M Heimarsson S Breuss O Orange A FrobergL Wynns AA Navarro-Rosines P Krzewicka BPykaelae J Grube M Lutzoni F (2009) Genericclassification of the Verrucariaceae (Ascomycota)based on molecular and morphological evidencerecent progress and remaining challenges Taxon 58(1)184ndash208
Guillot G Mortier F Estoup A (2005) Geneland acomputer package for landscape genetics Mol EcolNotes 5(3)712ndash715 doi101111j1471-8286200501031x
Guillot G Renaud S Ledevin R Michaux J Claude J(2012) A unifying model for the analysis of pheno-typic genetic and geographic data Syst Biol 61(6)897ndash911 doi101093sysbiosys038
Guisan A Thuiller W (2005) Predicting species distribu-tion offering more than simple habitat models EcolLett 8(9)993ndash1009 doi101111j1461-0248200500792x
Hale ME (1990) A synopsis of the lichen genusXanthoparmelia (Vainio) Hale (AscomycotinaParmeliaceae) Smithsonian Institution Press Wash-ington DC
Hamilton CA Hendrixson BE Brewer MS Bond JE(2014) An evaluation of sampling effects on multipleDNA barcoding methods leads to an integrativeapproach for delimiting species a case study ofthe North American tarantula genus Aphonopelma(Araneae Mygalomorphae Theraphosidae) MolPhylogenet Evol 7179ndash93 doi101016jympev201311007
Hausdorf B (2011) Progress toward a general speciesconcept Evolution 65(4)923ndash931 doi101111j1558-5646201101231x
Hausdorf B Hennig C (2010) Species delimitation usingdominant and codominant multilocus markers SystBiol 59(5)491ndash503 doi101093sysbiosyq039
Hawksworth DL (1976) Lichen chemotaxonomy InBailey RH (ed) Lichenology progress and problemsAcademic London pp 139ndash184
Hebert PDN Cywinska A Ball SL deWaard JR (2003)Biological identifications through DNA barcodesProc R Soc Lond B Biol Sci 270(1512)313ndash321doi101098rspb20022218
Hebert PDN Stoeckle MY Zemlak TS Francis CM(2004) Identification of birds through DNA barcodesPLoS Biol 2(10)e312 doi101371journalpbio0020312
Heled J Drummond AJ (2010) Bayesian inference ofspecies trees from multilocus data Mol Biol Evol 27(3)570ndash580 doi101093molbevmsp274
Hey J (2006) On the failure of modern species conceptsTrends Ecol Evol 21(8)447ndash450 doi101016jtree200605011
Hibbett DS OhmanA Glotzer D NuhnMKirk P NilssonRH (2011) Progress in molecular and morphologicaltaxon discovery in Fungi and options for formalclassification on environmental sequences Fungal BiolRev 2538ndash47 doi101016jfbr201101001
Houmlgnabba F Wedin M (2003) Molecular phylogeny ofthe Sphaerophorus globosus species complex Cladis-tics 19(3)224ndash232 doi101111j1096-00312003tb00365x
Hudson RR Coyne JA (2002) Mathematical conse-quences of the genealogical species concept Evolu-tion 56(8)1557 doi101111j0014-38202002tb01467x
Huelsenbeck JP Andolfatto P Huelsenbeck ET (2011)Structurama Bayesian inference of population struc-ture Evol Bioinf Online 7(2011)55ndash59 doi104137EBOS6761
Jones G Oxelman B (2014) DISSECT an assignment-free Bayesian discovery method for species delimita-tion under the multispecies coalescent Bioinformaticsdoi101101003178
38 SD Leavitt et al
Kekkonen M Hebert PD (2014) DNA barcode-baseddelineation of putative species efficient start fortaxonomic workflows Mol Ecol Res doi1011111755-099812233
Kelly LJ Hollingsworth PM Coppins BJ Ellis CJHarrold P Tosh J Yahr R (2011) DNA barcoding oflichenized fungi demonstrates high identification suc-cess in a floristic context New Phytol 191(1)288ndash300doi101111j1469-8137201103677x
Kiss L (2012) Limits of nuclear ribosomal DNA internaltranscribed spacer (ITS) sequences as species barcodesfor fungi Proc Natl Acad Sci 109(27)E1811ndashE1811doi101073pnas1207143109
Kluge AG (1989) A concern for evidence and aphylogenetic hypothesis for relationships among Epi-crates (Boidae Serpentes) Syst Zool 38(1)7ndash25doi101093sysbio3817
Knowles LL Carstens BC (2007) Delimiting specieswithout monophyletic gene trees Syst Biol 56(6)887ndash895 doi10108010635150701701091
Kroken S Taylor JW (2001) A gene genealogical approachto recognize phylogenetic species boundaries in thelichenized fungus Letharia Mycologia 93(1)38ndash53
Kubatko LS Degnan JH (2007) Inconsistency of phylo-genetic estimates from concatenated data under coa-lescence Syst Biol 56(1)17ndash24 doi10108010635150601146041
Latch EK Dharmarajan G Glaubitz JC Rhodes OE Jr(2006) Relative performance of Bayesian clusteringsoftware for inferring population substructure andindividual assignments at low levels of populationdifferentiation Conserv Genet 7(2)295ndash302 doi101007s10592-005-9098-1
Lawrey JD (1986) Biological role of lichen substancesThe Bryologist 89(2)111ndash122
Le Gac M Hood ME Fournier E Giraud T (2007)Phylogenetic evidence of host-specific cryptic speciesin the anther smut fungus Evolution 61(1)15ndash26doi101111j1558-5646200700002x
Leacheacute AD (2009) Species tree discordance traces tophylogeographic clade boundaries in North Americanfence lizards (Sceloporus) Syst Biol 58(6)547ndash559doi101093sysbiosyp057
Leacheacute AD Fujita MK (2010) Bayesian species delim-itation in West African forest geckos (Hemidactylusfasciatus) Proc R Soc B Biol Sci 2773071ndash3077doi101098rspb20100662
Leacheacute AD Koo M Spencer C Papenfuss T Fisher RMcGuire J (2009) Quantifying ecological morpho-logical and genetic variation to delimit species in thecoast horned lizard species complex (Phrynosoma)Proc Natl Acad Sci USA 10612418ndash12423 doi101073pnas0906380106
Leacheacute AD Fujita MK Minin VN Bouckaert RR (2014)Species delimitation using genome-wide SNP dataSystematic Biology doi101093sysbiosyu018
Leavitt SD Fankhauser JD Leavitt DH Porter LDJohnson LA St Clair LL (2011a) Complex patterns ofspeciation in cosmopolitan ldquorock posyrdquo lichensmdashdiscovering and delimiting cryptic fungal species in
the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
Leavitt SD Esslinger TL Lumbsch HT (2012c) Neogene-dominated diversification in neotropical montanelichens dating divergence events in the lichen-form-ing fungal genus Oropogon (Parmeliaceae) Am J Bot99(11)1764ndash1777 doi103732ajb1200146
Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Lumbsch HT St Clair LL(2013c) DNA barcode identification of lichen-formingfungal species in the Rhizoplaca melanophthalmaspecies-complex (Lecanorales Lecanoraceae) includ-ing five new species MycoKeys 71ndash22 doi103897mycokeys74508
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Vondraacutek J Thorsten Lum-bsch H Clair LLS (2013d) Local representation ofglobal diversity in a cosmopolitan lichen-formingfungal species complex (Rhizoplaca Ascomycota)J Biogeogr 40(9)1792ndash1806 doi101111jbi12118
Leavitt SD Lumbsch HT Stenroos S St Clair LL(2013e) Pleistocene speciation in North American
2 The Dynamic Discipline of Species Delimitation hellip 39
lichenized fungi and the impact of alternative speciescircumscriptions and rates of molecular evolution ondivergence estimates PLoS ONE 8(12)e85240doi101371journalpone0085240
Leavitt SD Esslinger TL Hansen ES Divakar PK CrespoA Loomis BF Lumbsch HT (2014) DNA barcoding ofbrown Parmeliae (Parmeliaceae) species a molecularapproach for accurate specimen identification empha-sizing species in Greenland Organ Divers Evol 14(1)11ndash20 doi101007s13127-013-0147-1
Leliaert F Verbruggen H Vanormelingen P Steen FLopez-Bautista JM Zuccarello GC De Clerck O(2014) DNA-based species delimitation in algae Eur JPhycol 49(2) doi101080096702622014904524
Leuckert C (1985) Probleme der Flechten-Chemotaxon-omiemdashStoffkombinationen und ihre taxonomischeWertung Ber Deut Bot Ges 98401ndash408
Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
Lohtander K Kaumlllersjouml M Roland M Tehler A (2000)The family physciaceae in fennoscandia phylogenyinferred from its sequences Mycologia 92(4)728ndash735
Longton RE (1997) The role of bryophytes and lichens inpolar ecosystems In Woodin SJ Marquiss M (eds)Ecology of Arctic Environments Blackwell ScienceOxford pp 69ndash96 (Special publication No 13)
Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
Lumbsch HT (1994) Die Lecanora subfusca-Gruppe inAustralasien J Hattori Bot Lab 771ndash175
Lumbsch HT (1998a) Taxonomic use of metabolic data inlichen-forming fungi In Frisvad JC Bridge PDArora DK (eds) Chemical Fungal Taxonomy MarcelDekker New York pp 345ndash387
Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
Mayden RL (1997) A hierarchy of species concepts thedenouement in the saga of the species problem InClaridge MF Dawah HA Wilson MR (eds) Speciesthe units of biodiversity Chapman amp Hall Londonpp 381ndash424
Mayr E (1963) Animal species and evolution HarvardUniversity Press Cambridge
Mayr E (1970) Populations species and evolutionBelknap Press of Harvard University Press Cambridge
McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
McDonald T Miadlikowska J Lutzoni F (2003) Thelichen genus Sticta in the Great Smoky Mountains a
40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
McKay BD Mays HL Wu Y Li H Yao C-T Nishiumi IZou F (2013) An empirical comparison of character-based and coalescent-based approaches to speciesdelimitation in a young avian complex Mol Ecol 22(19)4943ndash4957 doi101111mec12446
Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
Mishler BD Brandon RN (1987) Individuality pluralismand the phylogenetic species concept Biol Philos 2397
Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
Nash TH Zavada M (1977) Population studies amongSonoran Desert species of Parmelia subg Xanthop-armelia (Parmeliaceae) Am J Bot 64(6)664ndash669
Nylander W (1866a) Circa novum in studio Lichenumcriterium chemicum Flora 49198ndash201
Nylander W (1866b) Hypochlorite of lime and hydrate ofpotash Two new criteria for the study of lichens(Tanslated and communicated by the Rev W ALeighton) Bot J Linn Soc 9358ndash365
OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
OrsquoNeill EM Schwartz R Bullock CT Williams JSShaffer HB Aguilar-Miguel X Parra-Olea G Weis-rock DW (2013) Parallel tagged amplicon sequencingreveals major lineages and phylogenetic structure inthe North American tiger salamander (Ambystomatigrinum) species complex Mol Ecol 22(1)111ndash129doi101111mec12049
Orock EA Leavitt SD Fonge BA St Clair LL LumbschHT (2012) DNA-based identification of lichen-form-ing fungi Can publicly available sequence databasesaid in lichen diversity inventories of Mount Cameroon(West Africa) The Lichenologist 44(6)833ndash839doi101017S0024282912000424
Otaacutelora MAG Martiacutenez I Aragoacuten G Molina MC (2010)Phylogeography and divergence date estimates of alichen species complex with a disjunct distributionpattern Am J Bot 97(2)216ndash223 doi103732ajb0900064
Padial J Castroviejo-Fisher S Kohler J Vila C ChaparroJ De la Riva I (2009) Deciphering the products ofevolution at the species level the need for anintegrative taxonomy Zoolog Scr 38(4)431ndash447doi101111j1463-6409200800381x
Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
Papong K Luumlcking R Thammathaworn A BoonpragobK (2009) Four new taxa of Chroodiscus (thelotremoidGraphidaceae) from Southeast Asia The Bryologist112(1)152ndash163 doi1016390007-2745-1121152
Park S-Y Choi J Kim JA Jeong M-H Kim S Lee Y-HHur J-S (2013a) Draft genome sequence of Cladoniamacilenta KoLRI003786 a lichen-forming fungusproducing biruloquinone Genome Announcements 1(5)e00695-13 doi101128genomeA00695-13
Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
Peacuterez-Ortega S Fernaacutendez-Mendoza F Raggio J VivasM Ascaso C Sancho LG Printzen C de los Riacuteos A(2012) Extreme phenotypic variation in Cetrariaaculeata (lichenized Ascomycota) adaptation or inci-dental modification Ann Bot 109(6)1133ndash1148doi101093aobmcs042
Pino-Bodas R Burgaz A Martiacuten M Lumbsch HT (2011)Phenotypical plasticity and homoplasy complicatespecies delimitation in the Cladonia gracilis group(Cladoniaceae Ascomycota) Organ Divers Evol 11(5)343ndash355 doi101007s13127-011-0062-2
Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
Pino-Bodas R Martiacuten M Burgaz A (2012b) Cladoniasubturgida and C iberica (Cladoniaceae) form asingle morphologically and chemically polymorphicspecies Mycol Prog 11(1)269ndash278 doi101007s11557-011-0746-1
Pino-Bodas R Ahti T Stenroos S Martiacuten MP BurgazAR (2013a) Multilocus approach to species recogni-tion in the Cladonia humilis complex (CladoniaceaeAscomycota) Am J Bot 100(4)664ndash678 doi103732ajb1200162
Pino-Bodas R Martiacuten AP Burgaz AR Lumbsch HT(2013b) Species delimitation in Cladonia (Ascomy-cota) a challenge to the DNA barcoding philosophyMol Ecol Resour 13(6)1058ndash1068 doi1011111755-099812086
Pons J Barraclough TG Gomez-Zurita J Cardoso ADuran DP Hazell S Kamoun S Sumlin WD VoglerAP (2006) Sequence-based species delimitation for theDNA taxonomy of undescribed insects Syst Biol 55(4)595ndash609 doi10108010635150600852011
Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
Pringle A Baker DM Platt JL Wares JP Latgeacute JPTaylor JW (2005) Cryptic speciation in the cosmo-politan and clonal human pathogenic fungus Asper-gillus fumigatus Evolution 59(9)1886ndash1899 doi10155404-2411
Printzen C (2009) Lichen systematics the role ofmorphological and molecular data to reconstructphylogenetic relationships Progress in botany vol71 Springer Berlin pp 233ndash275
Pritchard JK Stephens M Donnelly P (2000) Inference ofpopulation structure using multilocus genotype dataGenetics 155(2)945ndash959
Puillandre N Lambert A Brouillet S Achaz G (2012)ABGD automatic barcode gap discovery for primaryspecies delimitation Mol Ecol 21(8)1864ndash1877doi101111j1365-294X201105239x
Rannala B Yang Z (2003) Bayes estimation of speciesdivergence times and ancestral population sizes usingDNA sequences from multiple loci Genetics1641645ndash1656
Raxworthy CJ Ingram CM Rabibisoa N Pearson RG(2007) Applications of ecological niche modelingfor species delimitation a review and empiricalevaluation using day geckos (Phelsuma) fromMadagascar Syst Biol 56(6)907ndash923 doi10108010635150701775111
Ray J (1686) Historia planarum vol 1 Clark LondonReese Naeligsborg R Ekman S Tibell L (2007) Molecular
phylogeny of the genus Lecania (Ramalinaceaelichenized Ascomycota) Mycol Res 111(5)581ndash591doi101016jmycres200703001
Reid N Carstens B (2012) Phylogenetic estimation errorcan decrease the accuracy of species delimitation aBayesian implementation of the general mixed Yule-coalescent model BMC Evol Biol 12(1)196 doi1011861471-2148-12-196
Rissler L Apodaca J (2007) Adding more ecology intospecies delimitation ecological niche models andphylogeography help define cryptic species in theBlack Salamander (Aneides flavipunctatus) Syst Biol56924ndash942 doi10108010635150701703063
Rivas Plata E Luumlcking R (2013) High diversity ofGraphidaceae (lichenized Ascomycota Ostropales) inAmazonian Peruacute Fungal Divers 58(1)13ndash32 doi101007s13225-012-0172-y
Rivas Plata E Lumbsch HT (2011) Parallel evolution andphenotypic divergence in lichenized fungi a casestudy in the lichen-forming fungal family Graphida-ceae (Ascomycota Lecanoromycetes Ostropales)Mol Phylogenet Evol 61(1)45ndash63 doi101016jympev201104025
Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
Ross KG Gotzek D Ascunce MS Shoemaker DD (2010)Species delimitation a case study in a problematic anttaxon Syst Biol 59(2)162ndash184 doi101093sysbiosyp089
Rubin BER Ree RH Moreau CS (2012) Inferringphylogenies from RAD sequence data PLoS ONE 7(4)e33394 doi101371journalpone0033394
Rubinoff D (2006) Utility of mitochondrial DNA barcodesin species conservation Conserv Biol 20(4)1026ndash1033 doi101111j1523-1739200600372x
Ruiz-Sanchez E Sosa V (2010) Delimiting speciesboundaries within the Neotropical bamboo Otatea(Poaceae Bambusoideae) using molecular morpho-logical and ecological data Mol Phylogenet Evol 54(2)344ndash356 doi101016jympev200910035
Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
morphological characters Antarctic Sci 22 (SpecialIssue 06)727ndash741 doi101017S0954102010000477
Salicini I Ibaacutentildeez C Juste J (2011) Multilocus phylogenyand species delimitation within the Nattererrsquos batspecies complex in the Western Palearctic MolPhylogenet Evol 61(3)888ndash898 doi101016jympev201108010
Satler JD Carstens BC Hedin M (2013) Multilocusspecies delimitation in a complex of morphologicallyconserved trapdoor spiders (Mygalomorphae Antro-diaetidae Aliatypus) Syst Biol 62(6)805ndash823 doi101093sysbiosyt041
Schlick-Steiner BC Steiner FM Seifert B Stauffer CChristian E Crozier RH (2010) Integrative taxonomya multisource approach to exploring biodiversityAnnu Rev Entomol 55(1)421ndash438 doi101146annurev-ento-112408-085432
Schoch CL Seifert KA Huhndorf S Robert V SpougeJL Levesque CA Chen W Fungal Barcoding Con-sortium (2012) Nuclear ribosomal internal transcribedspacer (ITS) region as a universal DNA barcodemarker for Fungi Proc Natl Acad Sci 109(16)6241ndash6246 doi101073pnas1117018109
Seacuterusiaux E Villarreal AJC Wheeler T Goffinet B(2011) Recent origin active speciation and dispersalfor the lichen genus Nephroma (Peltigerales) inMacaronesia J Biogeogr 38(6)1138ndash1151 doi101111j1365-2699201002469x
Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
Simpson GG (1951) The species concept Evolution5285ndash298
Sites JW Marshall JC (2003) Delimiting species a renais-sance issue in systematic biology Trends Ecol Evol18462ndash470 doi101016S0169-5347(03)00184-8
Sites JW Marshall JC (2004) Operational criteria fordelimiting species Annu Rev Ecol Evol Syst 35(1)199ndash227 doi101146annurevecolsys35112202130128
Šlapeta J Loacutepez-Garciacutea P Moreira D (2006) Globaldispersal and ancient cryptic species in the smallestmarine eukaryotes Mol Biol Evol 23(1)23ndash29doi101093molbevmsj001
Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
Talavera G Dincă V Vila R (2013) Factors affectingspecies delimitations with the GMYC model insightsfrom a butterfly survey Methods Ecol Evol 4(12)1101ndash1110 doi1011112041-210X12107
Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
Tewksbury J Anderson JGT Bakker JD Billo TJDunwiddie PW Groom MJ Hampton SE HermanSG Levey DJ Machnicki NJ del Rio CM PowerME Rowell K Salomon AK Stacey L TrombulakSC Wheeler TA (2014) Natural historyrsquos place inscience and society BioScience doi101093bioscibiu032
Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
Werth S Cornejo C Scheidegger C (2013) Characteriza-tion of microsatellite loci in the lichen fungus Lobariapulmonaria (Lobariaceae) Appl Plant Sci 1(2)1200290 doi103732apps1200290
Westberg M Arup U Kaumlrnefelt I (2007) Phylogeneticstudies in the Candelariaceae (lichenized Ascomy-cota) based on nuclear ITS DNA sequence dataMycol Res 111(11)1277ndash1284 doi101017S0953756204002102
Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
2 The Dynamic Discipline of Species Delimitation hellip 43
(Lepidoptera Lycaenidae) Front Zoo 4(1)8 doi1011861742-9994-4-8
Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
ambiguous due to the occurrence of phenotypi-cally similar lichens occurring across broadintercontinental distributions and uncertainty ofappropriate species circumscriptions While anumber of lichen-forming fungal species havebeen found to be truly widespread (eg Lindblomand Soslashchting 2008 Fernaacutendez-Mendoza et al2011 Ahti and Hawksworth 2005 Del-Pradoet al 2013) improved recognition of speciesboundaries has provided insight into importantbiogeographical patterns in lichens previouslyassumed to have cosmopolitan distribution pat-terns (Leavitt et al 2013d Del-Prado et al 2013Amo de Paz et al 2012 Seacuterusiaux et al 2011Otaacutelora et al 2010 Elix et al 2009) Crypticdiversity and complex biogeographic patterns arehighlighted in the Rhizoplaca melanophthalmaspecies group (Fig 21 Leavitt et al 2013d)Analyses of R melanophthalma sensu lato col-lected from five continents supported the presenceof cryptic species within this complex Two ofthese lineages were found to have broad inter-continental distributions while the other four werelimited to western North America (Leavitt et al2013d) Most strikingly of the six lineages fivewere found on a single mountain in the westernUSA and the sixth occurred no more than 200 kmaway from this mountain A recent study ofPseudocyphellaria (Lobariaceae) sensu lato inHawaii revealed a surprising number of previouslyunrecognized species hidden within nominal taxawith putative broad geographic distributionssuggesting a high degree of endemism in Hawaii(Moncada et al 2014) These studies providecrucial impetus to reevaluate species boundaries inorder to improve our understanding of diversitydistributions and evolution in lichenized fungi
22 A Practical Guideto Contemporary SpeciesDelimitation
As it has become clear that conventional phe-notype-based criteria for species circumscriptionsare often problematic (Bickford et al 2007)molecular data are thereby particularly valuablefor assessing traditional species boundaries and
for species delimitation in general (Lumbsch andLeavitt 2011 Fujita et al 2012) Below weprovide an overview of relevant operationalspecies delimitation methods the majority ofwhich are reliant on molecular data coupled withmolecular phylogenetic and population geneticanalyses Assuming that species do in fact rep-resent ldquosegments of metapopulation lineagesrdquo (deQueiroz 1998) direct genetic evidence of lineagestatus is particularly relevant to species delimi-tation studies when analyzed within a rigorousstatistical framework regardless of whether lin-eages differ in phenotypic characters that areapparent to human observers (Fujita et al 2012)This perspective should not be taken as supportfor disregarding phenotypic characters in speciesdelimitation studies (Edwards and Knowles2014 Fujita et al 2012 Yeates et al 2011)Rather hypotheses of species boundaries shouldbe considered more robust with increasing cor-roboration from independent data sources (iemolecular chemistry morphology ecologyetc) and the integration of independent data forempirical species delimitation studies should be amajor focus of species delimitation research(Fujita et al 2012)
Themajority of molecular phylogenetic studiesof lichenized fungi focus on generating sequencedata from a number of specimens representing thefocal group inferring a gene tree from the geneticdata matrix and assessing the monophyly of thesampled taxa This approach has provided valu-able acumen into evolutionary relationships thevalue of morphological characters for taxonomyand insight into diversity of lichenized fungi(Thell et al 2009 Reese Naeligsborg et al 2007Westberg et al 2007 Martiacuten et al 2003 Arup andGrube 2000 Lohtander et al 2000 Stenroos andDePriest 1998) However simply assessing themonophyly of traditional phenotype-based spe-cies often offers an incomplete perspective onspecies boundaries Studies explicitly designed forempirically delimiting species are pivotal toadvancing our understanding of speciation andspecies diversity in lichens For example althougha nominal species may be recovered as mono-phyletic in a gene tree intraspecific phylogeneticsubstructure may correspond to evolutionarily
16 SD Leavitt et al
independent lineages (eg Leavitt et al 2011a)As a construct of taxonomy recognizing amonophyletic clade comprised of multiple mor-phological indistinguishable species-level lin-eages as a single species may hold some appealhowever failing to formally recognize this diver-sity can have far-reaching implications in ourbiological (eg ecology evolution reproduction)interpretation of the group Alternatively well-supported intraspecific phylogenetic substructuremay be the result of stochastic evolutionary pro-cesses uniparental inheritance etc rather thanevolutionary independence Interpreting this typeof phylogenetic substructure as species-leveldiversity can introduce detrimental bias
Empirical species delimitation methods havebroadly focused on four main areas detectingputative species individual specimen assignmentto a species group or operational taxonomic unit(OTU) validation of candidate species or OTUsas evolutionarily distinct lineages and inferringspecies relationships (ie species tree inference)Ideally operational species delimitation criteriashould be based on explicit statistical protocolsin order to objectively assess species boundariesand minimize the need of subjective interpreta-tions or taxonomic expertise In this section weprovide a brief overview on a number of empir-ical species delimitation methods that to varyingdegrees fit these criteria For convenience wedivide these methods into three general catego-ries (i) species discovery methods for assigningsamples to populations without a priori infor-mation (ii) species validation approaches andcoestimating individual assignments and speciestrees and (iii) species delimitation using geno-mic data (Table 21)
Empirical species delimitation has receivedincreasing attention including ongoing develop-ment of bioinformatical approaches and themethods and programs provided in this chapterare by no means intended to be a comprehensivelist of all available analytical approaches Ratherthe methods provided here have been shown tobe useful in a number of previous studies orshow particular promise for future speciesdelimitation studies Our aim is that these can
serve as a starting point when designing studiesto assess species limits Not surprisingly variousapproaches to species delimitation may yielddifferent estimates of species boundaries and theresearcher may be required to make some degreeof subjective interpretation of the most biologi-cally appropriate species boundaries Comple-mentarily recently developed metrics forquantifying the congruence between two taxo-nomies (Ctax) and the potential for an approach tocapture a high number of species boundaries(Rtax) provide a means to objectively assess dis-crepancies among species delimitation methods(Miralles and Vences 2013) More sophisticatedapproaches including selection of speciesdelimitation models using approximate Bayesiancomputing (Camargo et al 2012 Fan andKubatko 2011 Beaumont et al 2010) anddesigning and conducting a simulation study thatmatches the characteristics of the empirical study(Carstens et al 2013 Camargo et al 2012) canbe used to more objectively evaluate competinghypotheses of species boundaries In most con-texts it is likely better to fail to delimit speciesthan it is to falsely circumscribe entities that donot represent actual species and therefore theinferences drawn from species delimitation stud-ies should generally be conservative (Carstenset al 2013 Miralles and Vences 2013)
221 Corroborating TraditionalTaxonomy and DiscoveringCryptic Species Using Single-Locus Data
Objectively defining a threshold separatingintraspecific population substructure from inter-specific divergence is the general aim of speciesdelimitation studies using single-marker datasetsMost species delimitation methods based onsingle-locus sequence data fall under two generalcategories either genetic distance or tree-basedapproaches (Sites and Marshall 2004) Distance-based approaches attempt to detect a differencebetween intra- and interspecific distances (ieldquobarcode gaprdquo) where the pairwise genetic
2 The Dynamic Discipline of Species Delimitation hellip 17
Table
21
Somemetho
dsused
forspeciesdelim
itatio
ninclud
ingspeciesdiscov
erymetho
dsforassign
ingsamples
topo
pulatio
nswith
outapriori
inform
ation(BAPS
Gaussianclustering
Guillo
trsquosUnified
Mod
elS
TRUCTURES
TRUCTURAMA)genetic
distance-based
metho
dforsortingsequ
encesinto
hypo
theticalspecies(A
BGD)tree-
basedspeciesdiscov
erymetho
ds(bGMYCG
MYCb
PTP
PTP
ldquoSpecies
Delim
itatio
nrdquoplug
-inforGeneiou
s)and
jointdiscov
eryandvalid
ationmetho
ds(BPamp
PBrownie
DISSE
CTSp
eDeStem)
Metho
dDescriptio
nInpu
tdata
BAPS
mdashpo
pulatio
nassignmentusing
Bayesianclustering
Aprogram
forBayesianinferenceof
thegenetic
structurein
apopulatio
nclustering
molecular
data
andperformingadmixture
analysesGenetic
mixture
analyses
canbe
performed
atbo
thgroupandindividu
allevelsusingeither
ano
n-spatialo
rspatialm
odel
BAPS
treatsboth
theallele
frequenciesof
themolecular
markers
(ornucleotid
efrequenciesforDNA
sequ
ence
data)andthenu
mberof
genetically
diverged
groups
inpopulatio
nas
random
variablesIn
theldquoclusteringwith
linkedlocirdquomod
elagenetic
mixture
analysiscanbe
done
usinghaploidsequ
ence
data
orotherlin
kedgenetic
markersA
nalysesandmodel
comparisons
canalso
beperformed
usingafixednu
mber
ofgenetically
diverged
grou
psor
prespecified
populatio
nstructures
Limita
tions
Tem
poraldivergence
andrelatio
nships
amongputativ
egroups
arenot
explicitlyestim
atedE
quivalence
togenetic
clusters
tospecies-levelg
roupsisuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
Availablefrom
httpwwwhelsinkifi
bsgsoftwareBAPS
describedin
Coran-
deret
al(200
420
0620
08)CoranderandMarttinen20
06)
Genotyp
icdatahaploidsequence
dataor
linkedmarkers
(AFL
Por
SNPs)
Gaussianclusteringmdashpo
pulatio
nassignment
usingGaussianclustering
Aprogram
forBayesianinferenceof
thegenetic
structurein
apopulatio
nModelgroups
sampleinto
popu
latio
nsusinggeno
typicdata
bysearchingforclusters
that
canbe
attributed
tobeingmixturesof
norm
alallelefrequencydistributio
nsG
aussianclustering
requires
adatasetwhere
thecasesaredefinedby
variable
ofmetricscaleandhasbeen
used
with
geneticenvironmentalandmorphologicaldatasetsindividuallyinadditio
nto
integrated
datasets
Limita
tions
Tem
poraldivergence
andrelatio
nships
amongputativ
egroups
arenot
explicitlyestim
atedE
quivalence
togenetic
clusters
tospecies-levelg
roupsisuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
Implem
entedin
Rusingtheprabclus
(HausdorfandHennig20
10)andmclust
packages
(FraleyandRaftery
2007)
Genotyp
icdata
(flexible)
(con
tinued)
18 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
Guillo
trsquosUnified
Mod
elmdashpo
pulatio
nassignmentusingBayesianclustering
Thisapproach
provides
astatistical
model
that
allowsoneto
analyzegenetic
and
phenotyp
ticdata
with
inaun
ified
mod
elandinferencefram
eworkandop
tionally
incorporateinform
ationaboutthespatialdistributio
nof
samplesA
Bayesianclustering
algorithm
assumes
that
each
clusterin
ageographical
domaincanbe
approxim
ated
bypo
lygons
that
arecentered
around
pointsgeneratedby
aPo
issonprocessGuillo
trsquos
Unified
Mod
elisflexiblein
term
sof
thegenetic
data
that
itcanutilize
andcapableof
accurately
delim
iting
species
Limita
tions
Genetic
andphenotypic
data
cantracedifferentevolutionary
historiesfor
instancephylogenetic
divergence
forneutralgenetic
markers
andadaptatio
nfora
morphological
structure
Source
Availableas
anextensionof
theRGENELAND
package(G
uillo
tet
al20
05
2012)(http
www2im
mdtudk
gigu
Geneland)
Genotyp
icandno
n-genetic
(eg
phenotypicalecolog
icalgeograph
ical
behavioral)data
STRUCTUREmdash
populatio
nassignment
usingBayesianclustering
Amodel-based
clustering
methodusingmultilocus
genotype
data
toinferpopulatio
nstructureandassign
individualsto
populatio
nsIndividualsin
thesampleareassigned
probabilistically
topopulatio
nso
rjointly
totwoor
morepopulatio
nsiftheirgenotypes
indicatethatthey
areadmixedT
hemodeldoes
notassum
eaparticular
mutationprocess
anditcanbe
appliedto
mosto
fthecommonly
used
genetic
markersp
rovidedthat
they
areno
tcloselylin
ked
The
methodcanproducehigh
lyaccurate
assignmentsusing
modestn
umbersof
loci(Pritchard
etal2
000)T
hemostappropriatelevelo
fpopulatio
nstructurecanbe
inferred
byassessinglik
elihoodscores
orthead
hocΔKstatistic
(Evann
oet
al20
05)
Limita
tions
Identifying
themostappropriatenumberof
genetic
clusters
ischallenging
clusters
produced
byST
RUCTUREcanbe
strongly
influenced
byvariationin
sample
sizeclusters
createdby
STRUCTUREmay
notbe
consistent
with
theevolutionary
historyof
thepopulatio
nswhentherearerelativ
elylong
divergence
times
with
inevolutionary
lineagesTem
poraldivergence
andrelatio
nships
amongputativ
egroups
isnotexplicitlyestim
atedEquivalence
togenetic
clusters
tospecies-levelgroups
isuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
httppritchardlabstanfordedustructurehtm
ldescribedin
Falush
etal(200
3)andPritchard
etal(200
0)
Genotyp
icdata
(con
tinued)
2 The Dynamic Discipline of Species Delimitation hellip 19
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
Structuram
amdashpo
pulatio
nassignmentusing
Bayesianclustering
Implem
entstheclustering
algorithm
used
inST
RUCTURE(see
above)
that
clusters
samples
into
populatio
nsby
minim
izingHardyndashWeinb
ergdisequ
ilibrium
foragiven
partitioninglevelHow
everStructuram
aalso
includes
theadditio
nof
reversible-jum
pMCMCto
identifytheop
timalpartition
inglevelNearlyanytype
ofgenetic
datacanbe
inpu
tinto
Structuram
aandtheprog
ram
canassign
individu
alsto
populatio
nwith
orwith
outtheadmixture
Limita
tions
Tem
poraldivergence
andrelatio
nships
amongputativ
egroups
isnot
explicitlyestim
atedE
quivalence
togenetic
clusters
tospecies-levelg
roupsisuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
httpctegberkeleyedu
structuram
aindexhtmldescribedin
Huelsenbeck
etal(201
1)
Genotyp
icdata
ABGDmdashbarcodegapusinggenetic
distances
ldquoAutom
aticBarcode
Gap
Discoveryrdquosortssequencesinto
hypotheticalspeciesbasedon
thebarcodegap
The
methoduses
arecursiveapproach
topartition
thedata
andtestfor
sign
ificant
gapsA
BGDisfastsim
plemethodto
split
asequence
alignm
entd
atasetinto
candidatespeciesthat
should
becomplem
entedwith
otherevidence
inan
integrative
taxono
mic
approach
Limita
tions
Single-locus
data
aloneshould
only
beused
toprovideaprelim
inary
perspectiveof
speciesboundaries
andnotas
thesole
evidence
inspecies
circum
scriptions
Source
httpwwwabisnv
jussieufrpublicabg
ddescribedin
Puillandreet
al(201
2)
Single-locus
sequence
alignm
ent
GMYCampbG
MYCmdashgene
tree
The
GMYC
approach
combinesacoalescent
model
ofintraspecificbranchingwith
aYulemod
elforinterspecificbranching
which
isthen
fitto
aninferred
sing
le-gene
topology
toestim
atespeciesboundaries
andastatistical
measure
ofconfi
denceforthe
inferred
boundariesA
san
inputtheGMYCapproach
requires
anultram
etricgene
tree
andrecent
refinementscanaccountforuncertaintyin
phylogenetic
relatio
nships
and
parametersof
theGMYCmod
elT
heGMYCisgenerally
stable
across
awiderangeof
circum
stancesincludingvariousmethods
ofphylogeneticreconstructio
nthepresence
ofahigh
numbersingletonshigh
numbers
ofsampled
speciesandgaps
inintraspecific
sampling
theaccuracy
oftheGMYCismostsign
ificantly
affected
bythemean
populatio
nsize
relativ
eto
divergence
times
betweenthem
Limita
tions
The
GMYCmay
delim
itwell-supportedclades
ofhaplotypes
asindependent
lineagesandas
such
may
beproneto
over-delim
itatio
nSingle-locus
data
aloneshould
only
beused
toprov
ideaprelim
inaryperspectiveof
speciesbo
undaries
andno
tas
the
sole
evidence
inspeciescircum
scriptions
Source
(http
r-forger-projecto
rgprojectssplitshttpssitesgooglecom
site
noahmreidhom
esoftware)describedin
Monaghanet
al(200
9)Fu
jisaw
aandBarrac-
loug
h(201
3)andPo
nset
al(200
6)
Sing
le-locus
ultram
etricgene
tree (con
tinued)
20 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
PTPamp
bPTPmdash
gene
tree
The
Poissontree
processes(PTP)
modelcanbe
used
toinferputativ
especiesboundaries
onagivenphylogenetic
inputtreePT
Pcaninferputativ
especiesboundaries
that
are
consistent
with
thePS
CAnim
portantadvantageof
thismethodisthat
itmod
els
speciatio
nin
term
sof
thenumberof
substitutionsthereby
circum
ventingthepotentially
error-proneandcompute-intensive
processof
generatin
gultram
etrictreeswhich
are
required
asan
inpu
tfor
GMYCmod
el(see
abov
e)F
urthermoreitappearsthatthePT
Pmodel
may
outperform
theGMYCandotherOTU-picking
methods
whenevolutionary
distancesaresm
all
Limita
tions
Single-locus
data
aloneshould
only
beused
toprovideaprelim
inary
perspectiveof
speciesboundaries
andnotas
thesole
evidence
inspecies
circum
scriptions
Source
httpscoh-itsorgexelix
iswebsoftwarePT
Pindexhtmldescribedin
Zhang
etal(201
3)
Sing
le-locus
gene
tree
GeneiousSp
eciesDelim
itatio
nplug
-inmdash
gene
tree
Aplug-into
theGeneioussoftwareprovides
anexploratorytool
allowingtheuser
toassess
phylog
enetic
supportanddiagno
sabilityof
speciesdefinedas
user-selected
collections
oftaxa
onuser-sup
pliedtreesThe
plug
-incompu
tesstatisticsrelatin
gto
the
probability
oftheob
served
mon
ophyly
orexclusivity
having
occurred
bychance
ina
coalescent
processandassesses
thewith
in-andbetween-speciesgenetic
distancesto
infertheprobability
with
which
mem
bers
ofaputativ
especiesmight
beidentifi
edsuccessfully
with
tree-based
methods
Limita
tions
The
plug-insummarizes
measuresof
phylogenetic
supportand
diagnosabilityof
speciesdefinedas
user-selectedcollections
oftaxabutitdoes
not
providedefinitiv
esupportforspeciesgroupsItassumes
speciesaremonophyletic
Source
Implem
entedas
aplug-into
Geneious(geneiouscom)describedin
Mastersetal
(201
1)
Sing
le-locus
gene
tree
BPamp
Pmdashmultispecies
coalescent
model
for
speciesvalid
ation
Thisapproach
tospeciesdelim
itatio
nuses
aBayesianmodelingapproach
togeneratethe
posteriorprobabilitiesof
speciesassignmentstaking
accountof
uncertaintiesdueto
unknow
ngene
treesandtheancestralcoalescent
processThe
methodrelieson
auser-
specified
guidetreeimplem
entin
gareversible-jum
pMarkovchainMonte
Carlo
search
ofparameter
spacethatincludes
θpopulatio
ndivergenceand
estim
ated
distributio
nsof
gene
treesfrom
multip
leloci
Limita
tions
Misspecificatio
nsof
priorsandthegu
idetree
canresultin
inflatedspeciatio
nprobabilitiesitassumes
norecombinatio
nandcomputatio
nallim
itatio
nsrestrict
itsutility
with
next-generationdatasetswith
100s
ofloci
Source
httpabacusgeneuclacuksoftwarehtmldescribedin
YangandRannala
(201
0)andRannala
andYang(200
3)
Multilocus
sequence
alignm
entsandgroup
mem
bership
(con
tinued)
2 The Dynamic Discipline of Species Delimitation hellip 21
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
DISSE
CTmdashmultispecies
coalescent
model
forspeciesdelim
itatio
nDISSE
CTexplores
thefullspaceof
possible
clusteringsof
individualsandspeciestree
topologies
inaBayesianfram
ework
Toavoidtheneed
forreversible-jum
pMCMCit
uses
anapproxim
ationin
theform
ofapriorthatisamodificatio
nof
thebirthndashdeathprior
forthespeciestreeItisim
plem
entedas
partof
BEAST
andrequires
only
afewchanges
from
astandard
BEAST
analysisA
nalysesof
simulated
andem
piricald
atasuggestthat
themethodisshow
nto
beinsensitive
tothedegree
ofapproxim
ation
butq
uitesensitive
tootherparameters
Limita
tions
Recently
describedmethodlackingathorough
theoretical
andem
pirical
evaluatio
nItappearsthat
alargenu
mberof
sequencesarerequ
ired
todraw
firm
conclusion
sSo
urce
httpcodegooglecompbeast-m
cmcamphttpwwwin
driid
com
dissectinbeast
htmldescribedin
JonesandOxelm
an(201
4)
Multilocus
sequence
data
SpeD
eSTEMmdashmultispecies
coalescent
mod
elfordiscov
ery
valid
ation
andjoint
estim
ation
Thismaxim
umlik
elihoo
dandorinform
ationtheory-based
methodwas
developedto
test
speciesboundaries
inasystem
with
existin
gsubspecies
taxonomy(CarstensandDew
ey20
10)andcomputestheprobability
ofthegene
treesgiventhespeciestree
forall
hierarchical
perm
utations
oflin
eage
grou
pingSp
eciesbo
undaries
arecomparedusing
Akaikeinform
ationcriteria
andphylogenetic
uncertaintyin
thespeciestree
topologies
does
notaffect
speciesdelim
itatio
nsLimita
tions
Accuracyisdependenton
quality
ofthegene
tree
estim
ates
Source
(http
carstenslaborgohio-stateedusoftwarehtml)criteria
describedin
Ence
andCarstens(201
1)
Multilocus
sequence
alignm
entsandgroup
mem
bership
Browniemdash
multispecies
coalescent
model
forspeciesdelim
itatio
nThe
nonp
aram
etricheuristic
speciesdelim
itatio
napproach
implem
entedin
theprog
ram
Brownie(O
rsquoMeara
2010)jointly
sortsanonym
oussamples
into
speciesandinfers
aspeciestree
from
inputg
enetreesfrom
differentlociassumingthatforaspeciatio
neven
thecorrespondingnodeson
gene
treeswill
bemoreconsistent
with
each
than
the
divergenceswith
inspecies
Limita
tions
Findingboth
theoptim
umspeciestree
andspeciesboundaries
remains
compu
tatio
nally
challenging
andBrowniehasbeen
show
nto
frequently
yieldincorrect
resultsT
heaccuracy
ofthemethodislik
elycorrelated
with
nodalsupportvalues
inthe
individu
algene
topo
logies
Source
httpwwwbrianom
earain
fobrownie
describedin
OrsquoM
eara
(201
0)
Individual
gene
trees
(con
tinued)
22 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
BFD
mdashmultispecies
coalescent
model
for
speciesdelim
itatio
nThe
recently
developedmethod
Bayes
factor
delim
itatio
n(with
genomicdataB
FD)
combinesady
namic
programmingalgorithm
forestim
atingspeciestreesthat
bypasses
thecompu
tatio
nally
intensiveMCMCintegrationov
ergene
treesto
prov
idearigorous
techniqueforspeciesdelim
itatio
nstudiesusinggenome-wideSN
PdataCom
petin
gspeciesdelim
itatio
nmodelsarecomparedusingBayes
factorsanditappearsthat
this
approach
isrobustto
samplesizes(iefew
loci
andlim
itedsamples
perspecies)
and
misspecificatio
nof
thepriorforpo
pulatio
nsize
(θ)
Limita
tions
Recently
describedmethodlackingathorough
theoretical
andem
pirical
evaluatio
nSo
urce
httpwwwbeast2orgwikiindexphpBFD
describedin
Leacheacuteet
al(201
4)
Genom
e-wideSN
Pdata
2 The Dynamic Discipline of Species Delimitation hellip 23
distances among organisms belonging to thesame species are smaller than distances amongorganisms from different species (Puillandreet al 2012 Hebert et al 2003) Genetic distanceapproaches hold particular promise as an identi-fication tool shortcutting the difficulties of mor-phology-based identification (Hebert et al 2004)although in practice a barcode gap may not existfor many groups (Wiemers and Fiedler 2007)Furthermore the role of distance-based approa-ches using a single genetic marker for speciesdiscovery remains more controversial (Rubinoff2006) and without other corroborating evidenceOTUs inferred from single-locus dataset shouldonly be considered ldquocandidaterdquo species Tree-based methods aim to detect monophyletic cladescorresponding to species-level diversity bydetecting discontinuities associated within inter-and intraspecific branching patterns (Fujisawaand Barraclough 2013 Zhang et al 2013Monaghan et al 2009 Pons et al 2006) Tree-based species delimitation methods can also beused on the basis of other properties related tophylogenetic tree topologies (monophyly con-cordance with geography etc (Sites and Marshall2003 2004) Both distance- and tree-basedmethods have been applied for assessing speciesboundaries in lichen-forming fungi (Leavitt et al2012c 2014 Parnmen et al 2012 Del-Pradoet al 2010 2011 Molina et al 2011)
A number of tree-based methods partiallyautomate the species delimitation process withspecific bioinformatical analyses (Table 21)including the general mixed Yule coalescent(GMYC) approach (Fujisawa and Barraclough2013 Monaghan et al 2009 Pons et al 2006)and the Poisson tree processes (PTP) model(Zhang et al 2013) These methods provide rel-atively straightforward and objective pipelinesfor delimiting putative species-level lineagesfrom inferred gene trees by fitting within- andbetween-species branching models to an inferredsingle-locus topology A number of other tree-based methods for species delimitation areeffectively summarized in Sites and Marshall(2004)
When only single-locus data are available theGMYC has been shown to be a relatively robust
tool for species delimitation The GMYCapproach combines a coalescent model of intra-specific branching with a Yule model for inter-specific branching which is then fit to an inferredsingle-gene topology to estimate species bound-aries and a statistical measure of confidence for theinferred boundaries As an input the GMYCapproach requires an ultrametric gene tree andrecent refinements can account for uncertainty inphylogenetic relationships and parameters of theGMYC model (Fujisawa and Barraclough 2013Reid and Carstens 2012) Regardless of theseimprovements it may be difficult to accuratelyinfer an adequate ultrametric tree for large data-sets Although it appears that the GMYC isgenerally stable across a wide range of circum-stances including various methods of phyloge-netic reconstruction the presence of a highnumber singletons high numbers of sampledspecies and gaps in intraspecific sampling theaccuracy of the GMYC is most significantlyaffected by the mean population size relative todivergence times between them (Fujisawa andBarraclough 2013 Talavera et al 2013) Fur-thermore research suggests that the so-calledsingle-threshold version of the GMYC methodlikely outperforms the multiple-thresholdapproach (Fujisawa and Barraclough 2013Monaghan et al 2009) However other studiessuggest that theGMYCmethodmay often providehigher estimates for the total number ofOTUs thanother molecular species delimitation methods(Hamilton et al 2014 Miralles and Vences 2013Talavera et al 2013) warranting a cautiousinterpretation of results from GMYC analyses
Compared to the GMYC approach therecently introduced PTP method for speciesdelimitation has been suggested to be moreaccurate for preliminary species delimitation(Zhang et al 2013) Relative to the GMYC PTPoffers a more straightforward implementationrequiring a simple phylogenetic tree rather thanan ultrametric chronogram However moreresearch is required to assess the general perfor-mance of PTP across wide range of empirical andsimulated species delimitation studies At thispoint data support the use of the GMYC andPTP methods as objective and reasonable starting
24 SD Leavitt et al
points for species delimitation using single-genetopologies
The ldquoSpecies Delimitationrdquo plug-in to theGeneious software provides statistical approachesfor exploring species boundaries in single-genetopologies (Masters et al 2011) Using a priorispecimen assignments to putative species theldquoSpecies Delimitationrdquo plug-in computes statis-tics relating to the probability of the observedmonophyly or exclusivity having occurred bychance in a coalescent process and assesses thewithin- and between-species genetic distances inorder to infer the probability with which membersof a putative species might be identified suc-cessfully with tree-based methods An importantcontribution of the ldquoSpecies Delimitationrdquo plug-in is that it provides an objective means for usersto assess putative species within an empiricalstatistic-based framework rather than qualitativeevaluations of what level of hierarchy constitutesa species in a phylogeny
In contrast to simple sequence similaritythresholds (OTU-picking) for delimiting putativeevolutionarily significant units the AutomaticBarcode Gap Discovery (ABGD) method is anautomated procedure that sorts sequences intohypothetical species based on the existence of abarcode gap observed whenever intraspecificgenetic distances are less than those amongorganisms from different species (Puillandreet al 2012) Ultimately ABGD is a fast simplemethod that can be used to group individualsrepresented in a single-locus sequence alignmentinto candidate species that should be comple-mented with other lines of evidence in an inte-grative taxonomic approach (Kekkonen andHebert 2014 Puillandre et al 2012)
In general the ABGD GMYC and PTP ana-lytical protocols using single-locus data arerepeatable and computationally relatively fastproviding a valuable starting point for a pre-liminary perspective into species boundaries inunderstudied groups that can be validated withsubsequent studies (Kekkonen and Hebert 2014)Similarly analyses of single-locus data can beused to corroborate traditional phenotype-basedspecies boundaries and identify candidate speciesthat have previously been hidden within nominal
species However single-locus species delimita-tionmethodsmaybeparticularly prone to failure inrecognizing significant proportions of species-level biodiversity due to the strict criterion forreciprocal monophyly or alternatively providespurious inflations of estimated species diversitybased on genetic differences that donot correspondto species-level lineages For example recentestimates suggest that the incidence of species-level gene tree paraphyly is approximately 20 (Ross 2014) suggesting that analyses of single-locus datasets would likely fail to accurately deli-mit species in one in every five cases In contrastrecent empirical studies suggest that in some casesthe GMYC provides a striking overestimate ofspecies diversity (Miralles and Vences 2013)
The internal transcribed spacer region (ITS)has played a central role in molecular phyloge-netic studies of lichenized fungi and has recentlybeen adopted as the official barcoding marker forfungi (Schoch et al 2012) In many cases the ITSis sufficiently variable to resolve species bound-aries for lichenized fungi (Schoch et al 2012Kelly et al 2011) although accurate specimenidentification using sequence-based identificationapproaches requires a thoroughly curated refer-ence database (Leavitt et al 2014 Orock et al2012 Kelly et al 2011) In spite of the generalutility of the ITS marker a number of issues maypotentially limit its effective use in speciesdelimitation studies including the potential forintragenomic variation of the nuclear ribosomaltandem repeat and difficulties in aligning ITSsequences from divergent taxa (Kiss 2012)Because the ITS has been formally adopted as theofficial barcoding marker for fungi we recom-mend that species delimitation studies attempt toinclude this region for consistency across studiesHowever we recognize that in some taxonomicgroups the inclusion of the ITS may be prob-lematic and therefore suggest a careful screeningof other markers to identify appropriate loci forresolving species-level relationships
Ultimately the success of any single-locusspecies delimitation method largely depends onthe evolutionary history of the species groupunder study and the variability of the selectedmarker (Fig 22) Species delimitation will be
2 The Dynamic Discipline of Species Delimitation hellip 25
more difficult in more recently diverged lineagesand in cases with some level of interspecific geneflow relative to older well-diverged lineages(eg Leavitt et al 2012c 2013e) In the end wereemphasize that single-locus data alone shouldonly be used to provide a preliminary perspectiveof species boundaries and not as the sole evi-dence in species circumscriptions
222 Sampling Across the GenomeMultilocus Sequence Dataand Genome-Wide Markers
Although single-locus methods can provide anefficient approach for preliminary large-scaleassessments of species diversity there are
significant limitations particularly in recentlydiverged species groups where retention ofancestral polymorphisms and incomplete lineagesorting will likely result in different neutral locihaving unique gene topologies that do not mirrorthe speciation process (Knowles and Carstens2007 Heled and Drummond 2010 Taylor et al2000) In contrast to single-locus and strictlyphenotype-based approaches for species delimi-tation analyses of genetic data collected fromindependent genomic regions can provide robusthypotheses of species boundaries with increasingconfidence (Satler et al 2013 Zhang et al 2011Yang and Rannala 2010) Sequences from mul-tiple independent loci provide an importantsource of data for species delimitation studiesincluding recently developed models that
Fig 22 A simplified diagram illustrating the process ofspeciation through time in a single gene history andresulting gene topologies sampled at two points in thespeciation history (modified from Leliaert et al 2014)aEach dot represents a distinct gene copy and each row onenon-overlapping generation with lines connecting genecopies to their ancestors in the previous generation (onerow below) dashed diagonal lines represent reproductivebarriers two hypothetical sampling intervals at differentpoints in the speciation history are shown Tx and Ty
species delimitation methods using single-locus data areeffective only when species are reciprocally monophyleticin the sampled gene tree genetic clustering and coalescent-based species delimitation methods can circumscribespecies when species may not be monophyletic in sampledgenetic loci b Gene topology representing sampledhaplotypes at time Tx (shown in panel a) hypotheticalspecies are reciprocally monophyletic c Gene topologyrepresenting sampled haplotypes at time Ty (shown in panela) hypothetical species are para- and polyphyletic
26 SD Leavitt et al
combine individual gene genealogies and speciesphylogenies via modeling the coalescent historyof markers (Yang and Rannala 2010 Edwards2009 Knowles and Carstens 2007) As aresponse to advances in sequencing technologiesbioinformatical approaches for multimarker spe-cies delimitation analyses continue to be devel-oped (Table 21 Camargo and Sites 2013)
Long-term reproductive isolation of candidatespecies can be assessed with multilocus sequencedata by evaluating genealogical concordance ofunlinked markers (Baum and Shaw 1995 Aviseand Ball 1990) Within species the mixingeffects of recombination cause unlinked loci tohave distinct genealogical histories but geneticdrift and long-term divergence leads to concor-dant genealogical histories at loci across thegenome Relationships of individuals belongingto distinct candidate species can be evaluatedusing gene genealogies (eg haplotype networksor single-gene topologies) to identify lineagesthat exhibit genealogical exclusivity acrossunlinked neutral loci (Hudson and Coyne 2002Dettman et al 2003b Avise and Ball 1990) Thepresence of the same clades in the majority ofsingle-locus genealogies is taken as evidence thatthe clades represent reproductively isolated lin-eages (Dettman et al 2003a Pringle et al 2005)In practice the criteria of reciprocal monophylyand genealogical concordance of unlinked lociprovide a conservative approach for assessingspecies boundaries due to the fact that a sub-stantial amount of time is required after the initialdivergence of species until ancestral polymor-phisms have fully sorted (Knowles and Carstens2007 Hudson and Coyne 2002) Consequentlygroups with recent divergence histories willlikely go undiscovered under a genealogicalconcordance criterion due to the fact that speciesboundaries likely are not reflected in genegenealogies For example it would take morethan 1 million years after speciation before spe-cies would be delimited if 15 loci were sampledin species with an effective population size (Ne)of 100000 assuming one generation a yearunder a strict reciprocal monophyly criterion(Knowles and Carstens 2007) The amount oftime required for a species to be recognized using
a criterion of reciprocal monophyly increasesproportionally with increasing population sizes(Hudson and Coyne 2002) In a number ofstudies of lichen-forming fungi a genealogicalconcordance criterion has been used to bothdelimit previously unrecognized species-levellineages and validate some conventional pheno-type-based species (Leavitt et al 2012a c2013b Molina et al 2011 Kroken and Taylor2001)
Although analyses of molecular sequence datarelying on reciprocal monophyly or fixed char-acter differences for species delimitation canserve as important criteria for identifying speciesthese criteria are not commonly met acrossmultiple loci particularly in recent speciationhistories (Fig 22) To accommodate theobserved conflict among genealogies from mul-tiple loci with the underlying speciation historythe recent merge of coalescent theory with phy-logenetics has driven a major paradigm shift inspecies delimitation and molecular systematics ingeneral (Fujita et al 2012 Edwards 2009) Themultispecies coalescent model can be applied togenealogical histories from multiple independentloci to assemble separate coalescent processesoccurring in populations into a species tree(Degnan and Rosenberg 2009 Rannala andYang 2003) Within this coalescent-basedframework multiple individuals can be assignedto a single speciespopulation and the speciationhistory of ancestral and descendant species-levellineages can be inferred as a ldquospecies treerdquo incontrast to estimating gene genealogies fromindividual samples (Degnan and Rosenberg2006 2009 Rannala and Yang 2003)
A number of multispecies coalescent-basedspecies delimitation methods have recently beenintroduced offering an exciting framework forempirically assessing species boundaries byselecting the best species tree model from a set ofalternative models representing differenthypotheses of species boundaries (Table 21)For example SpeDeSTEM (Ence and Carstens2011) finds the maximum likelihood values for aspecies tree assuming all putative species areseparate lineages and for alternative species treeswhere two or more species are collapsed into a
2 The Dynamic Discipline of Species Delimitation hellip 27
single lineage The best fitting model is thenselected using the Akaike information criterionunder the assumption of constant populationsizes and fixed gene topologies across the speciestree The species delimitation program BayesianPhylogenetic and Phylogeography (BPampPYangand Rannala 2010) accommodates gene treeuncertainty and variable population sizes andsamples from the Bayesian posterior distributionof species delimitation models using reversible-jump Markov chain Monte Carlo (rjMCMC)sampling BPampP requires a user-provided guidetree with resolution finer than the species leveland evaluates alternative modes derived from allpossible subtrees that are generated by collapsingor splitting nodes on the guide tree (Yang andRannala 2010 Rannala and Yang 2003) Theproportion of time spent on each model is pro-portional to the posterior probability of themodel ie ldquospeciation probabilitiesrdquo WhileBPampP ranks among the most popular coalescent-based species delimitation programs (Fujita et al2012) misspecifications of the guide tree andorthe prior distribution for population size (θ) canresult in strong support for models containing aninflated number of species (Leacheacute and Fujita2010 Zhang et al 2011) The nonparametricheuristic species delimitation approach imple-mented in the program Brownie (OrsquoMeara 2010)jointly sorts anonymous samples into species andinfers a species tree from input gene trees fromdifferent loci assuming that for a speciationevent the corresponding nodes on gene trees willbe more consistent with each than the diver-gences within species However finding both theoptimum species tree and species boundariesremains computationally challenging andBrownie has been shown to frequently yieldincorrect results (OrsquoMeara 2010)
Most recently available coalescent-basedspecies delimitation methods require individualassignments to a species or population a priori Ina number of scenarios correct assignments ofsamples to species may be difficult includingpresence of cryptic species incongruencebetween conventional species and moleculardata or simply the fact that accurate specimenidentification in complex groups with subtle or
difficult to discern diagnostic characters is anontrivial task (Leavitt et al 2013e) Statisticalmethods for assessing individual assignment andspecies detection prior to coalescent-based spe-cies delimitation and species tree reconstructionand species provide a more objective approachfor understanding species boundaries
Population assignment tests using a variety ofclustering algorithms using genetic data offer auseful approach for grouping individuals intoputative reproductively isolated groups (Table 21Hausdorf and Hennig 2010 Corander et al2004 2008 Falush et al 2003 Pritchard et al2000) particularly for species delimitation prob-lems that exist at the interface of traditionalpopulation genetic and phylogenetic analyses(Carstens et al 2013 Weisrock et al 2010Knowles and Carstens 2007)
The programs STRUCTURE (Falush et al2003 Pritchard et al 2000) and STRUCTURA-MA (Huelsenbeck et al 2011) cluster samplesinto populations by minimizing HardyndashWeinbergdisequilibrium for a given number of populationclusters using unlinked genetic markers In gen-eral unlinked markers are not available for mostgroups of lichen-forming fungi and a numberof studies have used information from multilocussequence data for STRUCTURE analyses(Leavitt et al 2011a b c 2013e Fernaacutendez-Mendoza and Printzen 2013 Fernaacutendez-Mendoza et al 2011) Varying approaches havebeen implemented to convert DNA sequencedata to allelic data for Bayesian clustering (seeOrsquoNeill et al 2013) STRUCTURE is expectedto perform well when there is sufficient inde-pendence across regions such that linkage dis-equilibrium within regions does not dominate thedata (STRUCTURE manual) but can also beeffective using multilocus sequence data andtreating all SNPs as independent loci regardlessof physical linkage within each locus (OrsquoNeillet al 2013 Falush et al 2003) A recent study ofthe lichen-forming genus Letharia showed thatBayesian clustering implemented in the programSTRUCTURE was generally able to recover thesame putative Letharia lineages circumscribedemploying a gene genealogical approach inKroken and Taylorrsquos iconic species delimitation
28 SD Leavitt et al
study (Altermann et al 2014 Kroken and Taylor2001) Altermann et al (2014) show that popu-lation assignments were largely consistent acrossa range of scenarios including extensiveamounts of missing data the exclusion of SNPsfrom variable markers and inferences based onSNPs from as few as three gene regions Incontrast to STRUCTURE and STRUCTURA-MA the program BAPS (Corander et al 2008)can explicitly infer genetic structure using hap-loid sequence data or other linked geneticmarkers in the ldquoclustering with linked locirdquomodel Another advantage of BAPS is that theprogram is much more computationally efficientthan STRUCTURE or STRUCTURAMA Sim-ulation studies indicate that both BAPS andSTRUCTURE perform well at low levels ofpopulation differentiation and when clusters arenot well differentiated (Latch et al 2006)
In practice inferring the most appropriatelevel of population structure using Bayesianclustering algorithms remains challenging (Latchet al 2006 Evanno et al 2005) BAPS andSTRUCTURAMA can infer both individualassignments and the most likely number ofgenetic clusters (Corander et al 2008 Huelsen-beck et al 2011) and the ad hoc ΔK statisticproposed by Evanno et al (2005) provides anobjective approach for identifying the uppermosthierarchical level of structure However carefulconsideration of other levels of populationstructure may ultimately reveal more biologicallymeaning results and researchers should examineindividual assignments across a range of geneticgroupings Some markers generated fromindependent genomic regions such as SNPs andfast-evolving microsatellites can be used to dis-tinguish fine-scale population structuring on thebasis of allele frequencies and species delimita-tion analyses based on these markers may implythe risk of severe taxonomic oversplitting Inthese cases validation approaches such asBPampP and SpeDeSTEM and corroborationamong different species delimitation approachescan provide perspective between intraspecificpopulation structure and species-level clusters
Another limitation of clustering approaches isthat they do not assess or take into account
evolutionarily divergences and relationshipsamong population clusters Coalescent-basedspecies tree methods (discussed below) provide ameans to assess the diversification histories ofpopulations inferred from clustering analysesTherefore a potential working protocol for aninformed species delimitation study that takesinto account population structure could consist offirst applying a genetic clustering analysis under apopulation genetics criterion (eg BAPSSTRUCTURE STRUCTURAMA) to identifygenetically distinct population clusters that can beconsidered ldquocandidate speciesrdquo From these can-didate species a species tree can be inferred forfocal group using coalescent-based species treereconstruction methods (eg BEAST) Subse-quently a coalescent-based validation methodcan be applied to assess whether the distinctpopulation clusters represent independent evolu-tionary lineages (eg BPampP) This multistepapproach would provide a consistent and standardcriterion for distinguishing between population-and species-level lineages (Camargo and Sites2013) and has been applied to a number of casesincluding lichen-forming fungi (Leavitt et al2011a b c 2013e Leacheacute and Fujita 2010)
High-throughput sequencing methods providethe means to effectively sample hundreds tothousands of loci from across a species genome fora large number of species and continue to revolu-tionize studies that can be performed even in non-model organisms Targeted high-throughputsequencing approaches such as anchored phy-logenomics transcriptome sequencing reduced-representation genomic library sequencing(restriction-site-associated DNA sequencingRAD-Seq and genotype-by-sequencing GBS)and high-throughput amplicon sequencing pro-vide important insight into species boundaries fora number groups although none of these approa-ches has yet been applied to studies of lichenizedfungi
For example restriction-site-associated(RAD-Seq) DNA can simultaneously detect andgenotype thousands of genome-wide SNPs byfocusing the sequencing effort on a reducedrepresentation of the entire genome (Baird et al2008) and has been successfully applied to
2 The Dynamic Discipline of Species Delimitation hellip 29
intraspecies (Lewis et al 2007 Miller et al 2007Emerson et al 2010) and interspecies studies(Rubin et al 2012 Eaton and Ree 2013 Wagneret al 2013) This approach has provided strikinginsight into the recent adaption radiation of LakeVictoria cichlids (Wagner et al 2013) Analternative approach targeting the sequencing ofspecific loci using next-generation sequencingplatforms provides an efficient means of gener-ating data for loci of known genomic locationorthology size and expected level of variation(OrsquoNeill et al 2013) Markers can be targetedusing well-established PCR techniques or newerhybridization techniques and subsequentlypooled for high-throughput sequencing usingparallel-tagged sequencing of multiple individualsamples within a single sequencing run (OrsquoNeillet al 2013) A recent study of North Americantiger salamanders (Ambystoma tigrinum)demonstrated the potential for amplicon-basedparallel-tagged sequencing to rapidly generatelarge-scale genomic data for species delimitationand species tree research (OrsquoNeill et al 2013)
Although these methods provide an enormousamount of data and insight into diversification atan unprecedented scale computational limita-tions restrict the applicable analytical methodsfor large datasets although computational andanalytical advances are happening rapidly Mostrecent coalescent-based species delimitation andspecies tree models using gene trees have beenlimited to handle tens of loci for multiple indi-viduals and combining hundreds or thousands ofgene trees into a single species delimitationframework presents considerable computationalchallenges (Camargo and Sites 2013) Therecently developed method Bayes factor delim-itation (with genomic data BFD) combines adynamic programming algorithm for estimatingspecies trees that bypasses the computationallyintensive MCMC integration over gene trees toprovide a rigorous technique for species delimi-tation studies using genome-wide SNP data(Leacheacute et al 2014) Competing species delimi-tation models are compared using Bayes factorsand it appears that this approach is robust tosample sizes (ie few loci and limited samples
per species) and misspecification of the prior forpopulation size (θ) (Leacheacute et al 2014)
Fungi are an ideal model for assessing diver-gence in eukaryotes due to their simple mor-phologies small genomes broad ecological rolesand diverse lifestyles (Gladieux et al 2014)However the use of genomic data from high-throughput sequencing methods have not yet beenincluded in species delimitation studies of lichen-forming fungi This is due in part to challenges indealing with metagenomic data containing geno-mic information from a plethora of symbiontsassociated with a lichen thallus scant genomicresources and discipline-specific inertia A num-ber of lichen-forming fungal genomes are cur-rently available including Endocarponpusillum(Wang et al 2014) Cladonia spp (Park et al2013a 2014) Caloplaca flavorubescens (Parket al 2013b) and Cladonia greyi and Xanthoriaparientina through the Fungal Genomics Programat Joint Genome Institute of the United StatesDepartment of Energy (httpwwwjgidoegov)The foreseeable ongoing expansion of genomicresources for lichen-forming fungi will be centralto developing approaches for delimiting speciesusing high-throughput sequencing Specificallygenomic resources will be crucial in identifyingnovel markers (variable genes regions SNPsmicrosatellites etc) identifying conserved fungalmarkers for targeted high-throughput sequencingapproaches and references for entire genomecomparisons (Devkota et al 2014 Werth et al2013)
23 Can We Make SpeciesDelimitation in Lichen-FormingFungi Truly Integrative
The widespread availability of genetic data hascreated a biased viewpoint that only genetic datashould be used for statistical species delimitationHowever an ongoing appeal to researchers toassess species boundaries from multiple andcomplementary perspectives (phylogeneticspopulation genetics comparative morphologydevelopment ecology etc) has resulted in an
30 SD Leavitt et al
integrative taxonomic framework bringing theseconceptual and methodological perspectivestogether (Hamilton et al 2014 Fujita et al 2012Salicini et al 2011 Yeates et al 2011 Padialet al 2010 Dayrat 2005 Will et al 2005 Wiensand Penkrot 2002) In practice any study linkingdifferent kinds of data to support hypotheses ofspecies boundaries including mapping morpho-logical characters onto a molecular phylogenycan be considered integrative Integrative meth-ods for species delimitation fall across a broadspectrum ranging from verbal and qualitativeassessments of data classes to quantitative meth-ods that allow different data types to contribute tostatistical species delimitation (Yeates et al2011) In most taxonomic studies utilizing bothmolecular and morphological data expert opinionis eventually used to some degree to evaluate thefinal status of a candidate species (eg Bond andStockman 2008) In this sense many studiesusing evidence for independent data sources fordelimiting species boundaries use an iterativeapproach (Yeates et al 2011) where speciesboundaries can be tested within a hypothetico-deductive framework with diverse datasets
From a practical perspective we advocate aprocess of iterative taxonomy (sensu Yeates et al2011) to circumscribe and refine species limitsusing multiple lines of evidence This iterativeprocess involves comparisons of morphologicaldata with a phylogenetic hypothesis (ie single-locus gene tree or concatenated multilocusphylogeny) to identify the least inclusive mono-phyletic clade in the topology characterized by atleast one unambiguously diagnostic morphologi-cal character (Miralles and Vences 2013) Thisphylogenetic tree-informed approach to assessingspecies boundaries represents a common practicein studies of lichen-forming fungi Previouslyunrecognized species-level clades with corre-sponding subtle or overlooked phenotypiccharacters have been commonly observed in bothcrustose lichens [eg Acarospraceae (Wedin et al2009) Graphidaceae (Rivas Plata and Lumbsch2011 Papong et al 2009) Lecanoraceae (Leavittet al 2011a) Lecideaceae (Ruprecht et al 2010)Mycoblastaceae (Spribille et al 2011) andTeloschistaceae (Vondraacutek et al 2009 Muggia
et al 2008)] and foliose and fruticose lichens [egLobariaceae (Moncada et al 2014 McDonaldet al 2003) Parmeliaceae (Leavitt et al 2013aWirtz et al 2012 Divakar et al 2005 Molinaet al 2004 Kroken and Taylor 2001) Peltigera-ceae (OrsquoBrien et al 2009 Goffinet et al 2003)Physciaceae (Elix et al 2009 Divakar et al2007) and Sphaerophoraceae (Houmlgnabba andWedin 2003)] As a specific example Luumlckinget al (2008) used a combination of medullarychemistry and underside pigmentation in speci-mens from the Heterodermia obscurata group inCosta Rica to corroborate monophyletic clades inan ITS gene tree as species-level lineages
While this iterative approach to speciesdelimitation and taxonomy has proven valuablefor understanding species boundaries anddescribing new taxa in some groups of lichenizedfungi a posteriori examination of morphologicaland chemical features has failed to reveal diag-nostic phenotypic characters in a number ofstudies (Muggia et al 2014 Leavitt et al 2011aOtaacutelora et al 2010 OrsquoBrien et al 2009) Fur-thermore a study of widespread species in thegenus Melanelixia (Parmeliaceae) indicated thatphenotypically cryptic lichen-forming fungalspecies-level lineages may be relatively ancientand diagnosable phenotypic differences may beabsent even millions of years after the initialdivergence (Leavitt et al 2012b) The latter studyhighlights the fact that species-level lineages maycommonly exist without any observable diag-nostic phenotypic characters calling into ques-tion the impetus for a universal application ofintegrative taxonomy
In keeping with the principle that as manylines of evidence as available should be com-bined to delimit species (Dayrat 2005) the for-malized integrative taxonomic approach (ITAXMiralles and Vences 2013) uses a mtDNA guidetree and observations from different types of datathat might provide conclusive evidence for theindependence of lineages and thus their identityas different species Miralles and Vences (2013)provide a non-exhaustive list of species delimi-tation criteria to be integrated in the ITAXapproach including (a) sympatric occurrencewithout admixture as revealed by consistent
2 The Dynamic Discipline of Species Delimitation hellip 31
differences in morphological or molecular char-acters at the same geographic location (b) strongdifferences in a behavioral morphological orgenetic character known to mediate prematingisolation (c) unviability or infertility of hybrids(d) lack of gene flow across a geographicalhybrid zone (e) congruent diagnostic differencesbetween sister lineages in various unlinkedmorphological character (f) absence of haplo-type sharing in several unlinked nuclear loci and(g) a combination of criteria endashf Speciesboundaries are based on seeking the least inclu-sive monophyletic group in the mtDNA treewhich fulfills at least one of the criteria listedabove Clearly the ITAX approach is sensitive tosample size and in order to reliably support thedistinctiveness of a given species it has beenrecommended that the sampling strategy includesat least five individuals per species (Miralles andVences 2013) A similar approach could beadopted for lichenized fungi using an ITS genetopology as guide tree rather than mtDNA
A total evidence approach including concat-enation has been a common approach for inte-grating information from different sourcesincluding independent genetic markers in phy-logenetic reconstructions (Kluge 1989 Wiens1998 de Queiroz et al 1995) Proponents forconcatenation of independent data in phyloge-netic analyses argue that when combining alldata the underlying signal of speciation mayemerge even if weakly contradictory signalis contained in the individual data partitions(Gatesy et al 1999) For example establishing apreliminary perspective of species boundariesfrom multilocus sequence data using concatena-tion may provide a reasonable starting point forscreening for cryptic species and species treeinference (Šlapeta et al 2006 Leavitt et al2011a Le Gac et al 2007 Leacheacute 2009)However concatenation and consensus methodsimply a risk of obtaining inflated support forincorrect relationships and information aboutvariance in gene coalescence is lost (Degnan andRosenberg 2006 2009 Kubatko and Degnan2007 Edwards 2009) In spite of the impetus forintegrating evidence from independent data
sources into empirical species delimitation stud-ies and taxonomy (Fujita et al 2012 Padial et al2009) most currently available species delimi-tation methods are unable to accommodate non-genetic data sources in a statistical framework
231 Selecting the Appropriate Data
In the face of increasing availability of geneticdata and associated bioinformatical approachesfor delimiting species researchers should care-fully consider what information is being sacri-ficed by the failure to consider non-genetic datain species delimitation studies and whetheraccuracy could be improved by the addition ofmultiple data types Morphological data havehistorically served as a proxy to identify repro-ductively isolated groups (ie ldquospeciesrdquo) (Ray1686 Fujita et al 2012) Current methods fordelimiting species using non-genetic data (egchemistry morphology and ecology) remainwoefully understudied For example morphol-ogy-based species circumscriptions are generallybased on one or more qualitative (or quantitative)morphological characters that do not appear tooverlap with other species However ascertain-ing that a given trait is truly fixed within apopulation with statistical confidence requiresunrealistic sample sizes even when allowing forsome level of polymorphism in the diagnosticcharacter (Wiens and Servedio 2000) Now var-ious combinations of datamdashfrom morphologygenetics geography and ecologymdashare acceptedas standard information for species delimitationstudies (Ruiz-Sanchez and Sosa 2010 Ross et al2010 Edwards and Knowles 2014)
Below we briefly discuss appropriate datasources for species delimitation studies of lichen-forming fungi However homoplastic characters(similar traits that are not derived from a com-mon ancestor) are common among many traitscommonly used to circumscribe fungal taxa thebiological significance of secondary metabolitevariation remains largely unknown (Lawrey1986) and ecological niches may be difficultto adequately characterize and model due to
32 SD Leavitt et al
microhabitat requirements and data resolutionTherefore we advocate a cautious approach toselecting appropriate and relevant data forassessing species boundaries
In spite of the potential challenges and limi-tations of using phenotypic data these traits haveprovided a plethora of valuable information forunderstanding species boundaries For examplein the Melanelixia fuliginosa group (Parmelia-ceae) a morphometric analysis using color isi-dia and marginal zone free of isidia ascharacters revealed a general pattern of differ-entiation between material formerly recognizedas subspecies fuliginosa and glabratula in spiteof the fact that considerable overlap betweengroups occurs in some characters individually(Arup and Berlin 2011) Furthermore the dis-tinction of the two groups was supported by aphylogenetic analysis of the ITS marker andecological differences with M fuliginosaoccurring predominantly on rock and M glab-ratula on bark (Arup and Berlin 2011) Thisstudy of the M fuliginosa group provides a fit-ting example of using multiple independentsuites of data including ecology morphologyand genetic information to establish a robusthypothesis of species boundaries
In lichen systematics phenotypic dataincluding thallus organization secondary metab-olites mode of reproduction ascoma-type andontogeny ascus and ascospore characters havehistorically played a prominent role (Printzen2009) Ascomatal characters have traditionallyheld a major role in higher-level classification(Printzen 2009) in contrast to species-levelclassification which also tends to include a widearray of vegetative and chemical charactersCommonly assessed morphological charactersinclude thallus form and size cortical features(eg maculae and pseudocyphellae) presenceformcolor of attachment structure (eg rhizines)and reproductive mode (ascomata vs vegetativediaspores) (Printzen 2009) Different morpho-logical types of vegetative diasporesmdashcorticatedisidia and ecorticated sorediamdashand their locationare commonly used to distinguish speciesAscomatal characters including morphologylocation (laminal vs marginal) position (sessile
vs immersed) presence of thalline margins colorof an apothecial disc and presence or color ofpruina are also commonly used in speciesdelimitations of lichen-forming fungi (Printzen2009) Other important characters may includethalline characters form color size and septationof ascospores size and form and structure of ascithe hamathecium type of epihymenium andhypothecium and the type of excipulum orperidium conidiomatal characters etc (Printzen2009)
Assessments of secondary metabolites hasplayed an important role in lichen taxonomybeginning with the introduction of simple spottests by Nylander (1866a b) The use of chem-istry in lichen taxonomy has been discussed indetail in numerous reviews (Lumbsch 1998a bRogers 1989 Brodo 1986 Egan 1986 Leuckert1985 Brodo 1978 Hawksworth 1976 Culber-son 1969 1970) and we refer readers to thesevaluable sources for a more comprehensive per-spective on lichen chemistry In short extrolites(secondary metabolites) belong to various clas-ses the most common and diverse include dep-sides depsidones chlorinated xanthones andanthraquinones (Lumbsch 2002 Culberson1969) The presence or absence of specificextrolites or their replacements by another sub-stance is widely used to distinguish speciesparticularly when correlated with differences ingeographic distributions However if morpho-logical or geographical differences betweenpopulations containing different extrolites are notapparent the taxonomic significance has beendisputed with some authors distinguishing themas species and others preferring to regard them aschemical races within a species In addition tosimply using the presence or absence of extro-lites Culberson and Culberson (1976) proposedto arrange lichen substances into chemosyn-dromes of closely related substances The pres-ence or absence of these chemosyndromes maypotentially be used as characters to delimit spe-cies regarding differences involving the samechemosyndromes as intraspecific variation anddistinct chemosyndromes as evidence for inter-specific populations (Lumbsch 1994 Gowan1986)
2 The Dynamic Discipline of Species Delimitation hellip 33
Due to the fact that species delimitationstudies often incorporate a substantial biogeo-graphical or ecological component ecologicalniche modeling plays an increasing role in phy-logenetic and taxonomic research Niche mod-eling can provide evidence for ecologicalisolation between populations based on eitherconserved or divergent ecological niches andtherefore can provide additional evidence sup-porting lineage independence between putativespecies The application of ecological nichemodeling has been applied to species delimita-tion studies in a number of cases (Ruiz-Sanchezand Sosa 2010 Leacheacute et al 2009 Raxworthyet al 2007 Rissler and Apodaca 2007) althoughit has not been explicitly applied to assess speciesboundaries in lichen-forming fungi By mappingthe spatial distribution of environmental suit-ability of climatic variables for candidate speciesthe application of ecological niche modeling canbe particularly important in cases where specieshave allopatric distributions (Raxworthy et al2007)
Ecological niche modeling utilizes knownassociations between a speciesrsquo occurrencelocalities and environmental variables to defineabiotic conditions within which populations canbe maintained (Guisan and Thuiller 2005) Themethodological approach for modeling is basedon four general properties (i) The current knownspeciesrsquo localities is the dependent variable (ii)the distribution is modeled as a map composed ofgrid cells at a specified resolution (iii) a range ofenvironmental variables (eg temperature pre-cipitation and solar exposure) are collected todescribe the characteristics of each cell and (iv)classifying the degree to which each cell is eithersuitable or unsuitable for each species under arange of models (Guisan and Thuiller 2005)
Ecological data also have the potential to playan important role in understanding speciesboundaries in lichen-forming fungi For exampleNash and Zavada (1977) demonstrated that Xant-hoparmelia populations with distinct chemistriesoccurring in the northern portion of the SonoranDesert exhibit habitat selection among differentrock substrates within a region with relativelyuniform climate and topography In another case
the parmelioid species Parmelia mayi is mor-phologically indistinguishable from P saxatilisbut can be separated by bioclimatic features andgenetic and chemical characters (Molina et al2011) McCune and Printzen(2011) assess distri-butions and climatic niches of species in theLecanora varia group inwesternUSAandprovidea model that uses continental influence and annualtemperature as themajor factors predicting speciesdistributions The distribution of Usnea hirta alichen commonly used in air quality biomonitor-ing research was modeled for a section of theWhite River National Forest in central Coloradobased on the presence of U hirta at 72 biomoni-toring reference sites distributed in the inter-mountain western United States (Shrestha et al2012) The best model for predicting U hirtadistribution included four variablesmdashsolar radia-tion average monthly precipitation and averagemonthly minimum and maximum temperatures(Shrestha et al 2012) These studies supportthe potential use of ecological niche modelingmethods in species delimitation studies of lichen-forming fungi
24 Conclusions What AboutTaxonomy
In most cases species circumscription and tax-onomy requires some degree of qualitativejudgment and individual interpretation Integrat-ing multiple types of data into an empiricalframework for delimiting species boundarieswhere species boundaries can be tested within ahypothetico-deductive framework with diversedatasets can provide robust hypotheses of speciesboundaries and taxonomic stability (Yeates et al2011) However it has long been known toevolutionary biologists that distinct species donot need to have diagnosable morphologicaldifferences (Mayr 1963) and increasing avail-ability of genetic data has allowed researchers toidentify species and to rigorously test speciesboundaries with a level of precision that wasunimaginable a decade ago While analyticaladvances in statistical species delimitation havebeen largely based on genetic data the utility of
34 SD Leavitt et al
these approaches to formal taxonomy remainselusive Due to these challenges an eclecticapproach to delimiting species and cautionagainst the reliance on any single dataset ormethod is required when delimiting species
While results from many recent studies maycontradict traditional species boundaries acrossmany groups of lichen-forming fungi we areoptimistic that this research represents substantialprogress toward a more accurate perspective onspecies boundaries and diversity in fungi As aresult of ongoing research of species boundariesin lichen-forming fungi the taxonomic value ofmany phenotypes is now better understood ourunderstanding of ecological evolutionary andbiogeographic patterns has improved and we canbegin to better understand patterns of symbiontinteractions in lichens Integrating new data(including novel morphological characters andgenetic data) will be essential to accurately rep-resent species-level diversity across all groups oflichenized fungi Hopefully an improved per-spective on lichen diversity also increases ourappreciation of these incredible symbiotic sys-tems While we are strong advocates for theapplication of independent data types in devel-oping an integrative taxonomy there is anincreasing need to formally recognize the exis-tence of phenotypically cryptic species-levellineages in lichen-forming fungi (see Hibbettet al 2011) In some cases a molecular taxon-omy may provide the most practical approach toconsistent treatment of mycobiont species withinlichen groups where diagnostic morphologicalcharacters are unidentifiable or practically notfeasible (Leavitt et al 2013c d)
These are exciting times for taxonomists andphylogeneticists A closer look at lichen taxon-omy with the inclusion of new data will help usto better understand the diversity of these fasci-nating organisms accurately interpret distribu-tion patterns and play a more important role inmeaningful conservation practices Howeversome level of uncertainty will accompany pro-gress In many taxonomic groups our traditionalapproach for species identification will likelyneed to be substantially modified The search forcorroborating morphological support for cryptic
species identified using molecular data willrequire meticulous and creative approaches toassess phenotypic variation in potentially unor-thodox ways We are hopeful that lichenologistswho traditionally have been eager to include newmethods such as chromatography in their rou-tine identifications will be amenable to includemolecular techniques to their routine examina-tion of specimens for identification and classifi-cation Although this may prove difficult toachieve by single individuals especially citizenscientists that traditionally play an important rolein lichen taxonomy the increasing numberof collaborative projects in the discipline (egLumbsch et al 2011 Crespo et al 2010Gueidan et al 2009) make us optimistic thatbroad-scale collaborative approaches will facili-tate the inclusion of molecular data in lichenresearch at all levels
Acknowledgments We are indebted to various col-leagues for valuable thought-provoking discussionnotably Matthew Nelsen (University of Chicago) AnaCrespo (Universidad Complutense de Madrid) PradeepDivakar (Universidad Complutense de Madrid) BeckettSterner (The Field Museum) and Joyce Havstad (TheField Museum) We also thank anonymous reviewers whoprovided valuable comments that improved this chapterSupport by the US National Science Foundation isgratefully acknowledged (ldquoHidden diversity in parmelioidlichensrdquo DEB-0949147)
References
Ahti T Hawksworth DL (2005) Xanthoparmelia steno-phylla the correct name for X somloeumlnsis one of themost widespread usnic acid containing species of thegenus The Lichenologist 37(4)363ndash366 doi101017S0024282905015197
Altermann S Leavitt SD Goward T Nelsen MPLumbsch HT (2014) How do you solve a problemlike Letharia A new look at cryptic species in lichen-forming fungi using Bayesian clustering and SNPSsfrom multilocus sequence data PLoS ONE 9(5)e97556 doi101371journalpone0097556
Amo de Paz G Cubas P Crespo A Elix JA Lumbsch HT(2012) Transoceanic dispersal and subsequent diver-sification on separate continents shaped diversity ofthe Xanthoparmelia pulla group (Ascomycota) PLoSONE 7(6)e39683 doi101371journalpone0039683
Arguumlello A Del Prado R Cubas P Crespo A (2007)Parmelina quercina (Parmeliaceae Lecanorales)
2 The Dynamic Discipline of Species Delimitation hellip 35
includes four phylogenetically supported morphospe-cies Biol J Linn Soc 91(3)455ndash467 doi101111j1095-8312200700810x
Arup U Berlin ES (2011) A taxonomic study ofMelanelixia fuliginosa in Europe The Lichenologist43(02)89ndash97 doi101017S0024282910000678
Arup U Grube M (2000) Is Rhizoplaca (Lecanoraleslichenized Ascomycota) a monophyletic genus Can JBot 78(3)318ndash327 doi101139b00-006
Avise J Ball R (1990) Principles of genealogicalconcordance in species concepts and biological tax-onomy Oxf Surv Evol Biol 745ndash67
Baird NA Etter PD Atwood TS Currey MC Shiver ALLewis ZA Selker EU Cresko WA Johnson EA(2008) Rapid SNP discovery and genetic mappingusing sequenced RAD markers PLoS ONE 3(10)e3376 doi101371journalpone0003376
Baum DA Shaw KL (1995) Genealogical perspectives onthe species problem In Hoch PC Stephenson AG(eds) Experimental and molecular approaches to plantbiosystematics Missouri Botanical Garden St Louispp 289ndash303
Beaumont MA Nielsen R Robert C Hey J Gaggiotti OKnowles L Estoup A Panchal M Corander JHickerson M Sisson SA Fagundes N Chikhi LBeerli P Vitalis R Cornuet J-M Huelsenbeck J FollM Yang Z Rousset F Balding D Excoffier L (2010)In defence of model-based inference in phylogeogra-phy Mol Ecol 19(3)436ndash446 doi101111j1365-294X200904515x
Bickford D Lohman DJ Sodhi NS Ng PKL Meier RWinker K Ingram KK Das I (2007) Cryptic speciesas a window on diversity and conservation TrendsEcol Evol 22(3)148ndash155 doi101016jtree200611004
Bonan GB Shugart HH (1989) Environmental factors andecological processes in boreal forests Annu Rev EcolSyst 20(1989)1ndash28
Bond J Stockman A (2008) An integrative method fordelimiting cohesion species finding the population-species interface in a group of Californian trapdoorspiders with extreme genetic divergence and geo-graphic structuring Syst Biol 57(4)628ndash646 doi10108010635150802302443
Brodo IM (1978) Changing concepts regarding chemicaldiversity in lichens The Lichenologist 10(1)1ndash11doi101017S0024282978000031
Brodo IM (1986) Interpreting chemical variation inlichens for systematic purposes The Bryologist 89(2)132ndash138
Caley MJ Fisher R Mengersen K (2014) Global speciesrichness estimates have not converged Trends EcolEvol 29(4)187ndash188 doi101016jtree201402002
Camargo A Sites JW (2013) Species delimitation adecade after the Renaissance In Pavlinov I (ed) Thespecies problemmdashongoing issues InTech doi10577252664
Camargo A Morando M Avila LJ Sites JW (2012)Species delimitation with ABC and other coalescent-based methods a test of accuracy with simulations and
an empirical example with lizards of the Liolaemusdarwinii complex (Squamata Liolaemidae) Evolution66(9)2834ndash2849 doi101111j1558-5646201201640x
Carstens BC Dewey TA (2010) Species delimitationusing a combined coalescent and information-theoreticapproach an example from North American Myotisbats Syst Biol 59(4)400ndash414 doi101093sysbiosyq024
Carstens BC Pelletier TA Reid NM Satler JD (2013)How to fail at species delimitation Mol Ecol 22(17)4369ndash4383 doi101111mec12413
Corander J Marttinen P (2006) Bayesian identification ofadmixture events using multi-locus molecular mark-ers Mol Ecol 15(10)2833ndash2843 doi101111j1365-294X200602994x
Corander J Waldmann P Marttinen P Sillanpaa M(2004) BAPS 2 enhanced possibilities for the analysisof genetic population structure Bioinformatics 20(15)2363ndash2369 doi101093bioinformaticsbth250
Corander J Marttinen P Mantyniemi S (2006) Bayesianidentification of stock mixtures from molecular markerdata Fish Bull 104550ndash558
Corander J Marttinen P Siren J Tang J (2008) EnhancedBayesian modelling in BAPS software for learninggenetic structures of populations BMC Bioinf 9(1)539 doi1011861471-2105-9-539
Coyne JA Orr HA (2004) Speciation Sinauer AssociatesSunderland
Cracraft J (1983) Species concepts and speciation anal-ysis Curr Ornithol 1159ndash187
Crespo A Lumbsch HT (2010) Cryptic species in lichen-forming fungi IMA Fungus 1167ndash170 doi105598imafungus2010010209
Crespo A Peacuterez-Ortega S (2009) Cryptic species andspecies pairs in lichens a discussion on the relation-ship between molecular phylogenies and morpholog-ical characters Anales del Jardin Botanico de Madrid66(S1)71ndash81 doi103989ajbm2225
Crespo A Kauff F Divakar PK del Prado R Perez-Ortega S Amo de Paz G Ferencova Z Blanco ORoca-Valiente B Nunez-Zapata J Cubas P ArguelloA Elix JA Esslinger TL Hawksworth DL MillanesA Molina MC Wedin M Ahti T Aptroot A et al(2010) Phylogenetic generic classification of parmeli-oid lichens (Parmeliaceae Ascomycota) based onmolecular morphological and chemical evidenceTaxon 59(6)1735ndash1753
Culberson WL (1969) The use of chemistry in thesystematics of the lichens Taxon 18498ndash505
Culberson WL (1970) Chemosystematics and ecology oflichen-forming fungi Annu Rev Ecol Syst 1153ndash170
Culberson CF Culberson WL (1976) Chemosyndromicvariation in lichens Syst Bot 1325ndash339
Darwin C (1859) On the origin of species by means ofnatural selection or the preservation of favoured racesin the struggle for life J Murray London
Dayrat B (2005) Towards integrative taxonomy Biol JLinn Soc 85(3)407ndash415 doi101111j1095-8312200500503x
36 SD Leavitt et al
de Queiroz K (1998) The general lineage concept ofspecies species criteria and the process of speciationa conceptual unification and terminological recom-mendations In Howard DJ Berlocher SH (eds)Endless forms species and speciation Oxford Uni-versity Press Oxford pp 57ndash75
de Queiroz K (1999) The general lineage concept ofspecies and the defining properties of the speciescategory In Wilson RA (ed) Species new interdis-ciplinary essays MIT Press Cambridge pp 49ndash89
de Queiroz K (2007) Species concepts and speciesdelimitation Syst Biol 56(6)879ndash886 doi10108010635150701701083
de Queiroz A Donoghue MJ Kim J (1995) Separateversus combined analysis of phylogenetic evidenceAnnu Rev Ecol Syst 26(1)657ndash681 doi101146annureves26110195003301
Degnan J Rosenberg N (2006) Discordance of speciestrees with their most likely gene trees PLoS Genet 2(5)e68 doi101371journalpgen0020068
Degnan JH Rosenberg NA (2009) Gene tree discordancephylogenetic inference and the multispecies coales-cent Trends Ecol Evol 24(6)332ndash340 doihttpdxdoiorg101016jtree200901009
Del-Prado R Cubas P Lumbsch HT Divakar PK BlancoO de Paz GA Molina MC Crespo A (2010) Geneticdistances within and among species in monophyleticlineages of Parmeliaceae (Ascomycota) as a tool fortaxon delimitation Mol Phylogenet Evol 56(1)125ndash133 doi101016jympev201004014
Del-Prado R Divakar PK Crespo A (2011) Using geneticdistances in addition to ITS molecular phylogeny toidentify potential species in the Parmotrema reticul-atum complex a case study The Lichenologist 43(06)569ndash583 doi101017S0024282911000582
Del-Prado R Blanco O Lumbsch HT Divakar PK ElixJA Molina MC Crespo A (2013) Molecular phylog-eny and historical biogeography of the lichen-formingfungal genus Flavoparmelia (Ascomycota Parmelia-ceae) Taxon 62(5)928ndash939 doi101270562522
Dettman J Jacobson D Taylor J (2003a) A multilocusgenealogical approach to phylogenetic species recog-nition in the model eukaryote Neurospora Evolution57(12)2703ndash2720 doi101111j0014-38202003tb01514x
Dettman JR Jacobson DJ Turner E Pringle A TaylorJW (2003b) Reproductive isolation and phylogeneticdivergence in Neurospora comparing methods ofspecies recognition in a model eukaryote Evolution57(12)2721 doi10155403-074
Devkota S Cornejo C Werth S Chaudhary RP Sche-idegger C (2014) Characterization of microsatelliteloci in the Himalayan lichen fungus Lobaria pindar-ensis (Lobariaceae) Appl Plant Sci 2(5)1300101doi103732apps1300101
Divakar PK Molina MC Lumbsch HT Crespo A (2005)Parmelia barrenoae a new lichen species related toParmelia sulcata (Parmeliaceae) based on molecularand morphological data The Lichenologist 37(01)37ndash46 doi101017S0024282904014641
Divakar PK Amo De paz G del Prado R Esslinger TLCrespo A (2007) Upper cortex anatomy corroboratesphylogenetic hypothesis in species of Physconia(Ascomycota Lecanoromycetes) Mycol Res 111(11)1311ndash1320 doi101016jmycres200708009
Divakar PK Figueras G Hladun N Crespo A (2010)Molecular phylogenetic studies reveal an undescribedspecies within the North American concept of Mela-nelixia glabra (Parmeliaceae) Fungal Divers 42(1)47ndash55 doi101007s13225-010-0027-3
Donoghue MJ Gauthier A (2004) Implementing thephylocode Trends Ecol Evol 19(6)281ndash282 doi101016jtree200404004
Eaton DAR Ree RH (2013) Inferring phylogeny andintrogression using RADseq data an example fromflowering plants (Pedicularis Orobanchaceae) SystBiol 62(5)689ndash706 doi101093sysbiosyt032
Edwards SV (2009) Is a new and general theory ofmolecular systematics emerging Evolution 63(1)1ndash19 doi101111j1558-5646200800549x
Edwards DL Knowles LL (2014) Species detection andindividual assignment in species delimitation canintegrative data increase efficacy Proc R Soc B BiolSci 281(1777) doi101098rspb20132765
Egan RS (1986) Correlations and non-correlations ofchemical variation patterns with lichen morphologyand geography The Bryologist 8999ndash110
Elix JA Corush J Lumbsch HT (2009) Triterpenechemosyndromes and subtle morphological characterscharacterise lineages in the Physcia aipolia group inAustralia (Ascomycota) Syst Biodivers 7(04)479ndash487 doi101017S1477200009990223
Emerson KJ Merz CR Catchen JM Hohenlohe PACresko WA Bradshaw WE Holzapfel CM (2010)Resolving postglacial phylogeography using high-throughput sequencing Proc Natl Acad Sci 107(37)16196ndash16200 doi101073pnas1006538107
Ence DD Carstens BC (2011) SpedeSTEM a rapid andaccurate method for species delimitation Mol EcolResour 11(3)473ndash480 doi101111j1755-0998201002947x
EvannoGRegnaut SGoudet J (2005)Detecting thenumberof clusters of individuals using the software STRUC-TURE a simulation study Mol Ecol 14(8)2611ndash2620doi101111j1365-294X200502553x
Falush D Stephens M Pritchard JK (2003) Inference ofpopulation structure using multilocus genotype datalinked loci and correlated allele frequencies Genetics164(4)1567ndash1587 doi101111j1471-8286200701758x
Fan HH Kubatko LS (2011) Estimating species treesusing approximate Bayesian computation Mol Phylo-genet Evol 59(2)354ndash363 doi101016jympev201102019
Fernaacutendez-Mendoza F Printzen C (2013) Pleistoceneexpansion of the bipolar lichen Cetraria aculeata intothe Southern hemisphere Mol Ecol 22(7)1961ndash1983doi101111mec12210
Fernaacutendez-Mendoza F Domaschke S Garciacutea MA JordanP Martin MP Printzen C (2011) Population structure
2 The Dynamic Discipline of Species Delimitation hellip 37
of mycobionts and photobionts of the widespreadlichen Cetraria aculeata Mol Ecol 20(6)1208ndash1232doi101111j1365-294X201004993x
Fraley C Raftery A (2007) Model-based methods ofclassification Using the mclust software in chemo-metrics J Stat Softw 18i06 [Available online at httpwwwdoajorgdoajfunc=abstractampid=218544]
Fujisawa T Barraclough TG (2013) Delimiting speciesusing single-locus data and the generalized mixed yulecoalescent (GMYC) approach a revised method andevaluation on simulated datasets Syst Biol 62(5)707ndash724 doi101093sysbiosyt033
Fujita MK Leacheacute AD Burbrink FT McGuire JA MoritzC (2012) Coalescent-based species delimitation in anintegrative taxonomy Trends Ecol Evol 27(9)480ndash488 doi101016jtree201204012
Gatesy J OrsquoGrady P Baker RH (1999) Corroborationamong data sets in simultaneous analysis hiddensupport for phylogenetic relationships among higherlevel Artiodactyl taxa Cladistics 15(3)271ndash313doi101111j1096-00311999tb00268x
Gladieux P Ropars J Badouin H Branca A Aguileta Gde Vienne DM Rodriacuteguez de la Vega RC Branco SGiraud T (2014) Fungal evolutionary genomicsprovides insight into the mechanisms of adaptivedivergence in eukaryotes Mol Ecol 23(4)753ndash773doi101111mec12631
Goffinet B Miadlikowska J Goward T (2003) Phyloge-netic inferences based on nrDNA sequences supportfive morphospecies within the Peltigera didactylacomplex (Lichenized Ascomycota) The Bryologist106(3)349ndash364 doi10163901
Gowan SP (1986) Evolution of secondary natural productsin the genus Porpidia (Ascomycata Porpidiaceae) AmJ Bot 73606
Griffin PC Hoffmann AA (2014) Limited genetic diver-gence among Australian alpine Poa tussock grassescoupled with regional structuring points to ongoinggene flow and taxonomic challenges Ann Bot doi101093aobmcu017
Grube M Hawksworth DL (2007) Trouble with lichenthe re-evaluation and re-interpretation of thallus formand fruit body types in the molecular era Mycol Res111(9)1116ndash1132 doi101016jmycres200704008
Gueidan C Savi S Thues H Roux C Keller C Tibell LPrieto M Heimarsson S Breuss O Orange A FrobergL Wynns AA Navarro-Rosines P Krzewicka BPykaelae J Grube M Lutzoni F (2009) Genericclassification of the Verrucariaceae (Ascomycota)based on molecular and morphological evidencerecent progress and remaining challenges Taxon 58(1)184ndash208
Guillot G Mortier F Estoup A (2005) Geneland acomputer package for landscape genetics Mol EcolNotes 5(3)712ndash715 doi101111j1471-8286200501031x
Guillot G Renaud S Ledevin R Michaux J Claude J(2012) A unifying model for the analysis of pheno-typic genetic and geographic data Syst Biol 61(6)897ndash911 doi101093sysbiosys038
Guisan A Thuiller W (2005) Predicting species distribu-tion offering more than simple habitat models EcolLett 8(9)993ndash1009 doi101111j1461-0248200500792x
Hale ME (1990) A synopsis of the lichen genusXanthoparmelia (Vainio) Hale (AscomycotinaParmeliaceae) Smithsonian Institution Press Wash-ington DC
Hamilton CA Hendrixson BE Brewer MS Bond JE(2014) An evaluation of sampling effects on multipleDNA barcoding methods leads to an integrativeapproach for delimiting species a case study ofthe North American tarantula genus Aphonopelma(Araneae Mygalomorphae Theraphosidae) MolPhylogenet Evol 7179ndash93 doi101016jympev201311007
Hausdorf B (2011) Progress toward a general speciesconcept Evolution 65(4)923ndash931 doi101111j1558-5646201101231x
Hausdorf B Hennig C (2010) Species delimitation usingdominant and codominant multilocus markers SystBiol 59(5)491ndash503 doi101093sysbiosyq039
Hawksworth DL (1976) Lichen chemotaxonomy InBailey RH (ed) Lichenology progress and problemsAcademic London pp 139ndash184
Hebert PDN Cywinska A Ball SL deWaard JR (2003)Biological identifications through DNA barcodesProc R Soc Lond B Biol Sci 270(1512)313ndash321doi101098rspb20022218
Hebert PDN Stoeckle MY Zemlak TS Francis CM(2004) Identification of birds through DNA barcodesPLoS Biol 2(10)e312 doi101371journalpbio0020312
Heled J Drummond AJ (2010) Bayesian inference ofspecies trees from multilocus data Mol Biol Evol 27(3)570ndash580 doi101093molbevmsp274
Hey J (2006) On the failure of modern species conceptsTrends Ecol Evol 21(8)447ndash450 doi101016jtree200605011
Hibbett DS OhmanA Glotzer D NuhnMKirk P NilssonRH (2011) Progress in molecular and morphologicaltaxon discovery in Fungi and options for formalclassification on environmental sequences Fungal BiolRev 2538ndash47 doi101016jfbr201101001
Houmlgnabba F Wedin M (2003) Molecular phylogeny ofthe Sphaerophorus globosus species complex Cladis-tics 19(3)224ndash232 doi101111j1096-00312003tb00365x
Hudson RR Coyne JA (2002) Mathematical conse-quences of the genealogical species concept Evolu-tion 56(8)1557 doi101111j0014-38202002tb01467x
Huelsenbeck JP Andolfatto P Huelsenbeck ET (2011)Structurama Bayesian inference of population struc-ture Evol Bioinf Online 7(2011)55ndash59 doi104137EBOS6761
Jones G Oxelman B (2014) DISSECT an assignment-free Bayesian discovery method for species delimita-tion under the multispecies coalescent Bioinformaticsdoi101101003178
38 SD Leavitt et al
Kekkonen M Hebert PD (2014) DNA barcode-baseddelineation of putative species efficient start fortaxonomic workflows Mol Ecol Res doi1011111755-099812233
Kelly LJ Hollingsworth PM Coppins BJ Ellis CJHarrold P Tosh J Yahr R (2011) DNA barcoding oflichenized fungi demonstrates high identification suc-cess in a floristic context New Phytol 191(1)288ndash300doi101111j1469-8137201103677x
Kiss L (2012) Limits of nuclear ribosomal DNA internaltranscribed spacer (ITS) sequences as species barcodesfor fungi Proc Natl Acad Sci 109(27)E1811ndashE1811doi101073pnas1207143109
Kluge AG (1989) A concern for evidence and aphylogenetic hypothesis for relationships among Epi-crates (Boidae Serpentes) Syst Zool 38(1)7ndash25doi101093sysbio3817
Knowles LL Carstens BC (2007) Delimiting specieswithout monophyletic gene trees Syst Biol 56(6)887ndash895 doi10108010635150701701091
Kroken S Taylor JW (2001) A gene genealogical approachto recognize phylogenetic species boundaries in thelichenized fungus Letharia Mycologia 93(1)38ndash53
Kubatko LS Degnan JH (2007) Inconsistency of phylo-genetic estimates from concatenated data under coa-lescence Syst Biol 56(1)17ndash24 doi10108010635150601146041
Latch EK Dharmarajan G Glaubitz JC Rhodes OE Jr(2006) Relative performance of Bayesian clusteringsoftware for inferring population substructure andindividual assignments at low levels of populationdifferentiation Conserv Genet 7(2)295ndash302 doi101007s10592-005-9098-1
Lawrey JD (1986) Biological role of lichen substancesThe Bryologist 89(2)111ndash122
Le Gac M Hood ME Fournier E Giraud T (2007)Phylogenetic evidence of host-specific cryptic speciesin the anther smut fungus Evolution 61(1)15ndash26doi101111j1558-5646200700002x
Leacheacute AD (2009) Species tree discordance traces tophylogeographic clade boundaries in North Americanfence lizards (Sceloporus) Syst Biol 58(6)547ndash559doi101093sysbiosyp057
Leacheacute AD Fujita MK (2010) Bayesian species delim-itation in West African forest geckos (Hemidactylusfasciatus) Proc R Soc B Biol Sci 2773071ndash3077doi101098rspb20100662
Leacheacute AD Koo M Spencer C Papenfuss T Fisher RMcGuire J (2009) Quantifying ecological morpho-logical and genetic variation to delimit species in thecoast horned lizard species complex (Phrynosoma)Proc Natl Acad Sci USA 10612418ndash12423 doi101073pnas0906380106
Leacheacute AD Fujita MK Minin VN Bouckaert RR (2014)Species delimitation using genome-wide SNP dataSystematic Biology doi101093sysbiosyu018
Leavitt SD Fankhauser JD Leavitt DH Porter LDJohnson LA St Clair LL (2011a) Complex patterns ofspeciation in cosmopolitan ldquorock posyrdquo lichensmdashdiscovering and delimiting cryptic fungal species in
the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
Leavitt SD Esslinger TL Lumbsch HT (2012c) Neogene-dominated diversification in neotropical montanelichens dating divergence events in the lichen-form-ing fungal genus Oropogon (Parmeliaceae) Am J Bot99(11)1764ndash1777 doi103732ajb1200146
Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Lumbsch HT St Clair LL(2013c) DNA barcode identification of lichen-formingfungal species in the Rhizoplaca melanophthalmaspecies-complex (Lecanorales Lecanoraceae) includ-ing five new species MycoKeys 71ndash22 doi103897mycokeys74508
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Vondraacutek J Thorsten Lum-bsch H Clair LLS (2013d) Local representation ofglobal diversity in a cosmopolitan lichen-formingfungal species complex (Rhizoplaca Ascomycota)J Biogeogr 40(9)1792ndash1806 doi101111jbi12118
Leavitt SD Lumbsch HT Stenroos S St Clair LL(2013e) Pleistocene speciation in North American
2 The Dynamic Discipline of Species Delimitation hellip 39
lichenized fungi and the impact of alternative speciescircumscriptions and rates of molecular evolution ondivergence estimates PLoS ONE 8(12)e85240doi101371journalpone0085240
Leavitt SD Esslinger TL Hansen ES Divakar PK CrespoA Loomis BF Lumbsch HT (2014) DNA barcoding ofbrown Parmeliae (Parmeliaceae) species a molecularapproach for accurate specimen identification empha-sizing species in Greenland Organ Divers Evol 14(1)11ndash20 doi101007s13127-013-0147-1
Leliaert F Verbruggen H Vanormelingen P Steen FLopez-Bautista JM Zuccarello GC De Clerck O(2014) DNA-based species delimitation in algae Eur JPhycol 49(2) doi101080096702622014904524
Leuckert C (1985) Probleme der Flechten-Chemotaxon-omiemdashStoffkombinationen und ihre taxonomischeWertung Ber Deut Bot Ges 98401ndash408
Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
Lohtander K Kaumlllersjouml M Roland M Tehler A (2000)The family physciaceae in fennoscandia phylogenyinferred from its sequences Mycologia 92(4)728ndash735
Longton RE (1997) The role of bryophytes and lichens inpolar ecosystems In Woodin SJ Marquiss M (eds)Ecology of Arctic Environments Blackwell ScienceOxford pp 69ndash96 (Special publication No 13)
Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
Lumbsch HT (1994) Die Lecanora subfusca-Gruppe inAustralasien J Hattori Bot Lab 771ndash175
Lumbsch HT (1998a) Taxonomic use of metabolic data inlichen-forming fungi In Frisvad JC Bridge PDArora DK (eds) Chemical Fungal Taxonomy MarcelDekker New York pp 345ndash387
Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
Mayden RL (1997) A hierarchy of species concepts thedenouement in the saga of the species problem InClaridge MF Dawah HA Wilson MR (eds) Speciesthe units of biodiversity Chapman amp Hall Londonpp 381ndash424
Mayr E (1963) Animal species and evolution HarvardUniversity Press Cambridge
Mayr E (1970) Populations species and evolutionBelknap Press of Harvard University Press Cambridge
McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
McDonald T Miadlikowska J Lutzoni F (2003) Thelichen genus Sticta in the Great Smoky Mountains a
40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
McKay BD Mays HL Wu Y Li H Yao C-T Nishiumi IZou F (2013) An empirical comparison of character-based and coalescent-based approaches to speciesdelimitation in a young avian complex Mol Ecol 22(19)4943ndash4957 doi101111mec12446
Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
Mishler BD Brandon RN (1987) Individuality pluralismand the phylogenetic species concept Biol Philos 2397
Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
Nash TH Zavada M (1977) Population studies amongSonoran Desert species of Parmelia subg Xanthop-armelia (Parmeliaceae) Am J Bot 64(6)664ndash669
Nylander W (1866a) Circa novum in studio Lichenumcriterium chemicum Flora 49198ndash201
Nylander W (1866b) Hypochlorite of lime and hydrate ofpotash Two new criteria for the study of lichens(Tanslated and communicated by the Rev W ALeighton) Bot J Linn Soc 9358ndash365
OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
OrsquoNeill EM Schwartz R Bullock CT Williams JSShaffer HB Aguilar-Miguel X Parra-Olea G Weis-rock DW (2013) Parallel tagged amplicon sequencingreveals major lineages and phylogenetic structure inthe North American tiger salamander (Ambystomatigrinum) species complex Mol Ecol 22(1)111ndash129doi101111mec12049
Orock EA Leavitt SD Fonge BA St Clair LL LumbschHT (2012) DNA-based identification of lichen-form-ing fungi Can publicly available sequence databasesaid in lichen diversity inventories of Mount Cameroon(West Africa) The Lichenologist 44(6)833ndash839doi101017S0024282912000424
Otaacutelora MAG Martiacutenez I Aragoacuten G Molina MC (2010)Phylogeography and divergence date estimates of alichen species complex with a disjunct distributionpattern Am J Bot 97(2)216ndash223 doi103732ajb0900064
Padial J Castroviejo-Fisher S Kohler J Vila C ChaparroJ De la Riva I (2009) Deciphering the products ofevolution at the species level the need for anintegrative taxonomy Zoolog Scr 38(4)431ndash447doi101111j1463-6409200800381x
Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
Papong K Luumlcking R Thammathaworn A BoonpragobK (2009) Four new taxa of Chroodiscus (thelotremoidGraphidaceae) from Southeast Asia The Bryologist112(1)152ndash163 doi1016390007-2745-1121152
Park S-Y Choi J Kim JA Jeong M-H Kim S Lee Y-HHur J-S (2013a) Draft genome sequence of Cladoniamacilenta KoLRI003786 a lichen-forming fungusproducing biruloquinone Genome Announcements 1(5)e00695-13 doi101128genomeA00695-13
Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
Peacuterez-Ortega S Fernaacutendez-Mendoza F Raggio J VivasM Ascaso C Sancho LG Printzen C de los Riacuteos A(2012) Extreme phenotypic variation in Cetrariaaculeata (lichenized Ascomycota) adaptation or inci-dental modification Ann Bot 109(6)1133ndash1148doi101093aobmcs042
Pino-Bodas R Burgaz A Martiacuten M Lumbsch HT (2011)Phenotypical plasticity and homoplasy complicatespecies delimitation in the Cladonia gracilis group(Cladoniaceae Ascomycota) Organ Divers Evol 11(5)343ndash355 doi101007s13127-011-0062-2
Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
Pino-Bodas R Martiacuten M Burgaz A (2012b) Cladoniasubturgida and C iberica (Cladoniaceae) form asingle morphologically and chemically polymorphicspecies Mycol Prog 11(1)269ndash278 doi101007s11557-011-0746-1
Pino-Bodas R Ahti T Stenroos S Martiacuten MP BurgazAR (2013a) Multilocus approach to species recogni-tion in the Cladonia humilis complex (CladoniaceaeAscomycota) Am J Bot 100(4)664ndash678 doi103732ajb1200162
Pino-Bodas R Martiacuten AP Burgaz AR Lumbsch HT(2013b) Species delimitation in Cladonia (Ascomy-cota) a challenge to the DNA barcoding philosophyMol Ecol Resour 13(6)1058ndash1068 doi1011111755-099812086
Pons J Barraclough TG Gomez-Zurita J Cardoso ADuran DP Hazell S Kamoun S Sumlin WD VoglerAP (2006) Sequence-based species delimitation for theDNA taxonomy of undescribed insects Syst Biol 55(4)595ndash609 doi10108010635150600852011
Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
Pringle A Baker DM Platt JL Wares JP Latgeacute JPTaylor JW (2005) Cryptic speciation in the cosmo-politan and clonal human pathogenic fungus Asper-gillus fumigatus Evolution 59(9)1886ndash1899 doi10155404-2411
Printzen C (2009) Lichen systematics the role ofmorphological and molecular data to reconstructphylogenetic relationships Progress in botany vol71 Springer Berlin pp 233ndash275
Pritchard JK Stephens M Donnelly P (2000) Inference ofpopulation structure using multilocus genotype dataGenetics 155(2)945ndash959
Puillandre N Lambert A Brouillet S Achaz G (2012)ABGD automatic barcode gap discovery for primaryspecies delimitation Mol Ecol 21(8)1864ndash1877doi101111j1365-294X201105239x
Rannala B Yang Z (2003) Bayes estimation of speciesdivergence times and ancestral population sizes usingDNA sequences from multiple loci Genetics1641645ndash1656
Raxworthy CJ Ingram CM Rabibisoa N Pearson RG(2007) Applications of ecological niche modelingfor species delimitation a review and empiricalevaluation using day geckos (Phelsuma) fromMadagascar Syst Biol 56(6)907ndash923 doi10108010635150701775111
Ray J (1686) Historia planarum vol 1 Clark LondonReese Naeligsborg R Ekman S Tibell L (2007) Molecular
phylogeny of the genus Lecania (Ramalinaceaelichenized Ascomycota) Mycol Res 111(5)581ndash591doi101016jmycres200703001
Reid N Carstens B (2012) Phylogenetic estimation errorcan decrease the accuracy of species delimitation aBayesian implementation of the general mixed Yule-coalescent model BMC Evol Biol 12(1)196 doi1011861471-2148-12-196
Rissler L Apodaca J (2007) Adding more ecology intospecies delimitation ecological niche models andphylogeography help define cryptic species in theBlack Salamander (Aneides flavipunctatus) Syst Biol56924ndash942 doi10108010635150701703063
Rivas Plata E Luumlcking R (2013) High diversity ofGraphidaceae (lichenized Ascomycota Ostropales) inAmazonian Peruacute Fungal Divers 58(1)13ndash32 doi101007s13225-012-0172-y
Rivas Plata E Lumbsch HT (2011) Parallel evolution andphenotypic divergence in lichenized fungi a casestudy in the lichen-forming fungal family Graphida-ceae (Ascomycota Lecanoromycetes Ostropales)Mol Phylogenet Evol 61(1)45ndash63 doi101016jympev201104025
Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
Ross KG Gotzek D Ascunce MS Shoemaker DD (2010)Species delimitation a case study in a problematic anttaxon Syst Biol 59(2)162ndash184 doi101093sysbiosyp089
Rubin BER Ree RH Moreau CS (2012) Inferringphylogenies from RAD sequence data PLoS ONE 7(4)e33394 doi101371journalpone0033394
Rubinoff D (2006) Utility of mitochondrial DNA barcodesin species conservation Conserv Biol 20(4)1026ndash1033 doi101111j1523-1739200600372x
Ruiz-Sanchez E Sosa V (2010) Delimiting speciesboundaries within the Neotropical bamboo Otatea(Poaceae Bambusoideae) using molecular morpho-logical and ecological data Mol Phylogenet Evol 54(2)344ndash356 doi101016jympev200910035
Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
morphological characters Antarctic Sci 22 (SpecialIssue 06)727ndash741 doi101017S0954102010000477
Salicini I Ibaacutentildeez C Juste J (2011) Multilocus phylogenyand species delimitation within the Nattererrsquos batspecies complex in the Western Palearctic MolPhylogenet Evol 61(3)888ndash898 doi101016jympev201108010
Satler JD Carstens BC Hedin M (2013) Multilocusspecies delimitation in a complex of morphologicallyconserved trapdoor spiders (Mygalomorphae Antro-diaetidae Aliatypus) Syst Biol 62(6)805ndash823 doi101093sysbiosyt041
Schlick-Steiner BC Steiner FM Seifert B Stauffer CChristian E Crozier RH (2010) Integrative taxonomya multisource approach to exploring biodiversityAnnu Rev Entomol 55(1)421ndash438 doi101146annurev-ento-112408-085432
Schoch CL Seifert KA Huhndorf S Robert V SpougeJL Levesque CA Chen W Fungal Barcoding Con-sortium (2012) Nuclear ribosomal internal transcribedspacer (ITS) region as a universal DNA barcodemarker for Fungi Proc Natl Acad Sci 109(16)6241ndash6246 doi101073pnas1117018109
Seacuterusiaux E Villarreal AJC Wheeler T Goffinet B(2011) Recent origin active speciation and dispersalfor the lichen genus Nephroma (Peltigerales) inMacaronesia J Biogeogr 38(6)1138ndash1151 doi101111j1365-2699201002469x
Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
Simpson GG (1951) The species concept Evolution5285ndash298
Sites JW Marshall JC (2003) Delimiting species a renais-sance issue in systematic biology Trends Ecol Evol18462ndash470 doi101016S0169-5347(03)00184-8
Sites JW Marshall JC (2004) Operational criteria fordelimiting species Annu Rev Ecol Evol Syst 35(1)199ndash227 doi101146annurevecolsys35112202130128
Šlapeta J Loacutepez-Garciacutea P Moreira D (2006) Globaldispersal and ancient cryptic species in the smallestmarine eukaryotes Mol Biol Evol 23(1)23ndash29doi101093molbevmsj001
Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
Talavera G Dincă V Vila R (2013) Factors affectingspecies delimitations with the GMYC model insightsfrom a butterfly survey Methods Ecol Evol 4(12)1101ndash1110 doi1011112041-210X12107
Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
Tewksbury J Anderson JGT Bakker JD Billo TJDunwiddie PW Groom MJ Hampton SE HermanSG Levey DJ Machnicki NJ del Rio CM PowerME Rowell K Salomon AK Stacey L TrombulakSC Wheeler TA (2014) Natural historyrsquos place inscience and society BioScience doi101093bioscibiu032
Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
Werth S Cornejo C Scheidegger C (2013) Characteriza-tion of microsatellite loci in the lichen fungus Lobariapulmonaria (Lobariaceae) Appl Plant Sci 1(2)1200290 doi103732apps1200290
Westberg M Arup U Kaumlrnefelt I (2007) Phylogeneticstudies in the Candelariaceae (lichenized Ascomy-cota) based on nuclear ITS DNA sequence dataMycol Res 111(11)1277ndash1284 doi101017S0953756204002102
Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
2 The Dynamic Discipline of Species Delimitation hellip 43
(Lepidoptera Lycaenidae) Front Zoo 4(1)8 doi1011861742-9994-4-8
Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
independent lineages (eg Leavitt et al 2011a)As a construct of taxonomy recognizing amonophyletic clade comprised of multiple mor-phological indistinguishable species-level lin-eages as a single species may hold some appealhowever failing to formally recognize this diver-sity can have far-reaching implications in ourbiological (eg ecology evolution reproduction)interpretation of the group Alternatively well-supported intraspecific phylogenetic substructuremay be the result of stochastic evolutionary pro-cesses uniparental inheritance etc rather thanevolutionary independence Interpreting this typeof phylogenetic substructure as species-leveldiversity can introduce detrimental bias
Empirical species delimitation methods havebroadly focused on four main areas detectingputative species individual specimen assignmentto a species group or operational taxonomic unit(OTU) validation of candidate species or OTUsas evolutionarily distinct lineages and inferringspecies relationships (ie species tree inference)Ideally operational species delimitation criteriashould be based on explicit statistical protocolsin order to objectively assess species boundariesand minimize the need of subjective interpreta-tions or taxonomic expertise In this section weprovide a brief overview on a number of empir-ical species delimitation methods that to varyingdegrees fit these criteria For convenience wedivide these methods into three general catego-ries (i) species discovery methods for assigningsamples to populations without a priori infor-mation (ii) species validation approaches andcoestimating individual assignments and speciestrees and (iii) species delimitation using geno-mic data (Table 21)
Empirical species delimitation has receivedincreasing attention including ongoing develop-ment of bioinformatical approaches and themethods and programs provided in this chapterare by no means intended to be a comprehensivelist of all available analytical approaches Ratherthe methods provided here have been shown tobe useful in a number of previous studies orshow particular promise for future speciesdelimitation studies Our aim is that these can
serve as a starting point when designing studiesto assess species limits Not surprisingly variousapproaches to species delimitation may yielddifferent estimates of species boundaries and theresearcher may be required to make some degreeof subjective interpretation of the most biologi-cally appropriate species boundaries Comple-mentarily recently developed metrics forquantifying the congruence between two taxo-nomies (Ctax) and the potential for an approach tocapture a high number of species boundaries(Rtax) provide a means to objectively assess dis-crepancies among species delimitation methods(Miralles and Vences 2013) More sophisticatedapproaches including selection of speciesdelimitation models using approximate Bayesiancomputing (Camargo et al 2012 Fan andKubatko 2011 Beaumont et al 2010) anddesigning and conducting a simulation study thatmatches the characteristics of the empirical study(Carstens et al 2013 Camargo et al 2012) canbe used to more objectively evaluate competinghypotheses of species boundaries In most con-texts it is likely better to fail to delimit speciesthan it is to falsely circumscribe entities that donot represent actual species and therefore theinferences drawn from species delimitation stud-ies should generally be conservative (Carstenset al 2013 Miralles and Vences 2013)
221 Corroborating TraditionalTaxonomy and DiscoveringCryptic Species Using Single-Locus Data
Objectively defining a threshold separatingintraspecific population substructure from inter-specific divergence is the general aim of speciesdelimitation studies using single-marker datasetsMost species delimitation methods based onsingle-locus sequence data fall under two generalcategories either genetic distance or tree-basedapproaches (Sites and Marshall 2004) Distance-based approaches attempt to detect a differencebetween intra- and interspecific distances (ieldquobarcode gaprdquo) where the pairwise genetic
2 The Dynamic Discipline of Species Delimitation hellip 17
Table
21
Somemetho
dsused
forspeciesdelim
itatio
ninclud
ingspeciesdiscov
erymetho
dsforassign
ingsamples
topo
pulatio
nswith
outapriori
inform
ation(BAPS
Gaussianclustering
Guillo
trsquosUnified
Mod
elS
TRUCTURES
TRUCTURAMA)genetic
distance-based
metho
dforsortingsequ
encesinto
hypo
theticalspecies(A
BGD)tree-
basedspeciesdiscov
erymetho
ds(bGMYCG
MYCb
PTP
PTP
ldquoSpecies
Delim
itatio
nrdquoplug
-inforGeneiou
s)and
jointdiscov
eryandvalid
ationmetho
ds(BPamp
PBrownie
DISSE
CTSp
eDeStem)
Metho
dDescriptio
nInpu
tdata
BAPS
mdashpo
pulatio
nassignmentusing
Bayesianclustering
Aprogram
forBayesianinferenceof
thegenetic
structurein
apopulatio
nclustering
molecular
data
andperformingadmixture
analysesGenetic
mixture
analyses
canbe
performed
atbo
thgroupandindividu
allevelsusingeither
ano
n-spatialo
rspatialm
odel
BAPS
treatsboth
theallele
frequenciesof
themolecular
markers
(ornucleotid
efrequenciesforDNA
sequ
ence
data)andthenu
mberof
genetically
diverged
groups
inpopulatio
nas
random
variablesIn
theldquoclusteringwith
linkedlocirdquomod
elagenetic
mixture
analysiscanbe
done
usinghaploidsequ
ence
data
orotherlin
kedgenetic
markersA
nalysesandmodel
comparisons
canalso
beperformed
usingafixednu
mber
ofgenetically
diverged
grou
psor
prespecified
populatio
nstructures
Limita
tions
Tem
poraldivergence
andrelatio
nships
amongputativ
egroups
arenot
explicitlyestim
atedE
quivalence
togenetic
clusters
tospecies-levelg
roupsisuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
Availablefrom
httpwwwhelsinkifi
bsgsoftwareBAPS
describedin
Coran-
deret
al(200
420
0620
08)CoranderandMarttinen20
06)
Genotyp
icdatahaploidsequence
dataor
linkedmarkers
(AFL
Por
SNPs)
Gaussianclusteringmdashpo
pulatio
nassignment
usingGaussianclustering
Aprogram
forBayesianinferenceof
thegenetic
structurein
apopulatio
nModelgroups
sampleinto
popu
latio
nsusinggeno
typicdata
bysearchingforclusters
that
canbe
attributed
tobeingmixturesof
norm
alallelefrequencydistributio
nsG
aussianclustering
requires
adatasetwhere
thecasesaredefinedby
variable
ofmetricscaleandhasbeen
used
with
geneticenvironmentalandmorphologicaldatasetsindividuallyinadditio
nto
integrated
datasets
Limita
tions
Tem
poraldivergence
andrelatio
nships
amongputativ
egroups
arenot
explicitlyestim
atedE
quivalence
togenetic
clusters
tospecies-levelg
roupsisuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
Implem
entedin
Rusingtheprabclus
(HausdorfandHennig20
10)andmclust
packages
(FraleyandRaftery
2007)
Genotyp
icdata
(flexible)
(con
tinued)
18 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
Guillo
trsquosUnified
Mod
elmdashpo
pulatio
nassignmentusingBayesianclustering
Thisapproach
provides
astatistical
model
that
allowsoneto
analyzegenetic
and
phenotyp
ticdata
with
inaun
ified
mod
elandinferencefram
eworkandop
tionally
incorporateinform
ationaboutthespatialdistributio
nof
samplesA
Bayesianclustering
algorithm
assumes
that
each
clusterin
ageographical
domaincanbe
approxim
ated
bypo
lygons
that
arecentered
around
pointsgeneratedby
aPo
issonprocessGuillo
trsquos
Unified
Mod
elisflexiblein
term
sof
thegenetic
data
that
itcanutilize
andcapableof
accurately
delim
iting
species
Limita
tions
Genetic
andphenotypic
data
cantracedifferentevolutionary
historiesfor
instancephylogenetic
divergence
forneutralgenetic
markers
andadaptatio
nfora
morphological
structure
Source
Availableas
anextensionof
theRGENELAND
package(G
uillo
tet
al20
05
2012)(http
www2im
mdtudk
gigu
Geneland)
Genotyp
icandno
n-genetic
(eg
phenotypicalecolog
icalgeograph
ical
behavioral)data
STRUCTUREmdash
populatio
nassignment
usingBayesianclustering
Amodel-based
clustering
methodusingmultilocus
genotype
data
toinferpopulatio
nstructureandassign
individualsto
populatio
nsIndividualsin
thesampleareassigned
probabilistically
topopulatio
nso
rjointly
totwoor
morepopulatio
nsiftheirgenotypes
indicatethatthey
areadmixedT
hemodeldoes
notassum
eaparticular
mutationprocess
anditcanbe
appliedto
mosto
fthecommonly
used
genetic
markersp
rovidedthat
they
areno
tcloselylin
ked
The
methodcanproducehigh
lyaccurate
assignmentsusing
modestn
umbersof
loci(Pritchard
etal2
000)T
hemostappropriatelevelo
fpopulatio
nstructurecanbe
inferred
byassessinglik
elihoodscores
orthead
hocΔKstatistic
(Evann
oet
al20
05)
Limita
tions
Identifying
themostappropriatenumberof
genetic
clusters
ischallenging
clusters
produced
byST
RUCTUREcanbe
strongly
influenced
byvariationin
sample
sizeclusters
createdby
STRUCTUREmay
notbe
consistent
with
theevolutionary
historyof
thepopulatio
nswhentherearerelativ
elylong
divergence
times
with
inevolutionary
lineagesTem
poraldivergence
andrelatio
nships
amongputativ
egroups
isnotexplicitlyestim
atedEquivalence
togenetic
clusters
tospecies-levelgroups
isuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
httppritchardlabstanfordedustructurehtm
ldescribedin
Falush
etal(200
3)andPritchard
etal(200
0)
Genotyp
icdata
(con
tinued)
2 The Dynamic Discipline of Species Delimitation hellip 19
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
Structuram
amdashpo
pulatio
nassignmentusing
Bayesianclustering
Implem
entstheclustering
algorithm
used
inST
RUCTURE(see
above)
that
clusters
samples
into
populatio
nsby
minim
izingHardyndashWeinb
ergdisequ
ilibrium
foragiven
partitioninglevelHow
everStructuram
aalso
includes
theadditio
nof
reversible-jum
pMCMCto
identifytheop
timalpartition
inglevelNearlyanytype
ofgenetic
datacanbe
inpu
tinto
Structuram
aandtheprog
ram
canassign
individu
alsto
populatio
nwith
orwith
outtheadmixture
Limita
tions
Tem
poraldivergence
andrelatio
nships
amongputativ
egroups
isnot
explicitlyestim
atedE
quivalence
togenetic
clusters
tospecies-levelg
roupsisuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
httpctegberkeleyedu
structuram
aindexhtmldescribedin
Huelsenbeck
etal(201
1)
Genotyp
icdata
ABGDmdashbarcodegapusinggenetic
distances
ldquoAutom
aticBarcode
Gap
Discoveryrdquosortssequencesinto
hypotheticalspeciesbasedon
thebarcodegap
The
methoduses
arecursiveapproach
topartition
thedata
andtestfor
sign
ificant
gapsA
BGDisfastsim
plemethodto
split
asequence
alignm
entd
atasetinto
candidatespeciesthat
should
becomplem
entedwith
otherevidence
inan
integrative
taxono
mic
approach
Limita
tions
Single-locus
data
aloneshould
only
beused
toprovideaprelim
inary
perspectiveof
speciesboundaries
andnotas
thesole
evidence
inspecies
circum
scriptions
Source
httpwwwabisnv
jussieufrpublicabg
ddescribedin
Puillandreet
al(201
2)
Single-locus
sequence
alignm
ent
GMYCampbG
MYCmdashgene
tree
The
GMYC
approach
combinesacoalescent
model
ofintraspecificbranchingwith
aYulemod
elforinterspecificbranching
which
isthen
fitto
aninferred
sing
le-gene
topology
toestim
atespeciesboundaries
andastatistical
measure
ofconfi
denceforthe
inferred
boundariesA
san
inputtheGMYCapproach
requires
anultram
etricgene
tree
andrecent
refinementscanaccountforuncertaintyin
phylogenetic
relatio
nships
and
parametersof
theGMYCmod
elT
heGMYCisgenerally
stable
across
awiderangeof
circum
stancesincludingvariousmethods
ofphylogeneticreconstructio
nthepresence
ofahigh
numbersingletonshigh
numbers
ofsampled
speciesandgaps
inintraspecific
sampling
theaccuracy
oftheGMYCismostsign
ificantly
affected
bythemean
populatio
nsize
relativ
eto
divergence
times
betweenthem
Limita
tions
The
GMYCmay
delim
itwell-supportedclades
ofhaplotypes
asindependent
lineagesandas
such
may
beproneto
over-delim
itatio
nSingle-locus
data
aloneshould
only
beused
toprov
ideaprelim
inaryperspectiveof
speciesbo
undaries
andno
tas
the
sole
evidence
inspeciescircum
scriptions
Source
(http
r-forger-projecto
rgprojectssplitshttpssitesgooglecom
site
noahmreidhom
esoftware)describedin
Monaghanet
al(200
9)Fu
jisaw
aandBarrac-
loug
h(201
3)andPo
nset
al(200
6)
Sing
le-locus
ultram
etricgene
tree (con
tinued)
20 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
PTPamp
bPTPmdash
gene
tree
The
Poissontree
processes(PTP)
modelcanbe
used
toinferputativ
especiesboundaries
onagivenphylogenetic
inputtreePT
Pcaninferputativ
especiesboundaries
that
are
consistent
with
thePS
CAnim
portantadvantageof
thismethodisthat
itmod
els
speciatio
nin
term
sof
thenumberof
substitutionsthereby
circum
ventingthepotentially
error-proneandcompute-intensive
processof
generatin
gultram
etrictreeswhich
are
required
asan
inpu
tfor
GMYCmod
el(see
abov
e)F
urthermoreitappearsthatthePT
Pmodel
may
outperform
theGMYCandotherOTU-picking
methods
whenevolutionary
distancesaresm
all
Limita
tions
Single-locus
data
aloneshould
only
beused
toprovideaprelim
inary
perspectiveof
speciesboundaries
andnotas
thesole
evidence
inspecies
circum
scriptions
Source
httpscoh-itsorgexelix
iswebsoftwarePT
Pindexhtmldescribedin
Zhang
etal(201
3)
Sing
le-locus
gene
tree
GeneiousSp
eciesDelim
itatio
nplug
-inmdash
gene
tree
Aplug-into
theGeneioussoftwareprovides
anexploratorytool
allowingtheuser
toassess
phylog
enetic
supportanddiagno
sabilityof
speciesdefinedas
user-selected
collections
oftaxa
onuser-sup
pliedtreesThe
plug
-incompu
tesstatisticsrelatin
gto
the
probability
oftheob
served
mon
ophyly
orexclusivity
having
occurred
bychance
ina
coalescent
processandassesses
thewith
in-andbetween-speciesgenetic
distancesto
infertheprobability
with
which
mem
bers
ofaputativ
especiesmight
beidentifi
edsuccessfully
with
tree-based
methods
Limita
tions
The
plug-insummarizes
measuresof
phylogenetic
supportand
diagnosabilityof
speciesdefinedas
user-selectedcollections
oftaxabutitdoes
not
providedefinitiv
esupportforspeciesgroupsItassumes
speciesaremonophyletic
Source
Implem
entedas
aplug-into
Geneious(geneiouscom)describedin
Mastersetal
(201
1)
Sing
le-locus
gene
tree
BPamp
Pmdashmultispecies
coalescent
model
for
speciesvalid
ation
Thisapproach
tospeciesdelim
itatio
nuses
aBayesianmodelingapproach
togeneratethe
posteriorprobabilitiesof
speciesassignmentstaking
accountof
uncertaintiesdueto
unknow
ngene
treesandtheancestralcoalescent
processThe
methodrelieson
auser-
specified
guidetreeimplem
entin
gareversible-jum
pMarkovchainMonte
Carlo
search
ofparameter
spacethatincludes
θpopulatio
ndivergenceand
estim
ated
distributio
nsof
gene
treesfrom
multip
leloci
Limita
tions
Misspecificatio
nsof
priorsandthegu
idetree
canresultin
inflatedspeciatio
nprobabilitiesitassumes
norecombinatio
nandcomputatio
nallim
itatio
nsrestrict
itsutility
with
next-generationdatasetswith
100s
ofloci
Source
httpabacusgeneuclacuksoftwarehtmldescribedin
YangandRannala
(201
0)andRannala
andYang(200
3)
Multilocus
sequence
alignm
entsandgroup
mem
bership
(con
tinued)
2 The Dynamic Discipline of Species Delimitation hellip 21
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
DISSE
CTmdashmultispecies
coalescent
model
forspeciesdelim
itatio
nDISSE
CTexplores
thefullspaceof
possible
clusteringsof
individualsandspeciestree
topologies
inaBayesianfram
ework
Toavoidtheneed
forreversible-jum
pMCMCit
uses
anapproxim
ationin
theform
ofapriorthatisamodificatio
nof
thebirthndashdeathprior
forthespeciestreeItisim
plem
entedas
partof
BEAST
andrequires
only
afewchanges
from
astandard
BEAST
analysisA
nalysesof
simulated
andem
piricald
atasuggestthat
themethodisshow
nto
beinsensitive
tothedegree
ofapproxim
ation
butq
uitesensitive
tootherparameters
Limita
tions
Recently
describedmethodlackingathorough
theoretical
andem
pirical
evaluatio
nItappearsthat
alargenu
mberof
sequencesarerequ
ired
todraw
firm
conclusion
sSo
urce
httpcodegooglecompbeast-m
cmcamphttpwwwin
driid
com
dissectinbeast
htmldescribedin
JonesandOxelm
an(201
4)
Multilocus
sequence
data
SpeD
eSTEMmdashmultispecies
coalescent
mod
elfordiscov
ery
valid
ation
andjoint
estim
ation
Thismaxim
umlik
elihoo
dandorinform
ationtheory-based
methodwas
developedto
test
speciesboundaries
inasystem
with
existin
gsubspecies
taxonomy(CarstensandDew
ey20
10)andcomputestheprobability
ofthegene
treesgiventhespeciestree
forall
hierarchical
perm
utations
oflin
eage
grou
pingSp
eciesbo
undaries
arecomparedusing
Akaikeinform
ationcriteria
andphylogenetic
uncertaintyin
thespeciestree
topologies
does
notaffect
speciesdelim
itatio
nsLimita
tions
Accuracyisdependenton
quality
ofthegene
tree
estim
ates
Source
(http
carstenslaborgohio-stateedusoftwarehtml)criteria
describedin
Ence
andCarstens(201
1)
Multilocus
sequence
alignm
entsandgroup
mem
bership
Browniemdash
multispecies
coalescent
model
forspeciesdelim
itatio
nThe
nonp
aram
etricheuristic
speciesdelim
itatio
napproach
implem
entedin
theprog
ram
Brownie(O
rsquoMeara
2010)jointly
sortsanonym
oussamples
into
speciesandinfers
aspeciestree
from
inputg
enetreesfrom
differentlociassumingthatforaspeciatio
neven
thecorrespondingnodeson
gene
treeswill
bemoreconsistent
with
each
than
the
divergenceswith
inspecies
Limita
tions
Findingboth
theoptim
umspeciestree
andspeciesboundaries
remains
compu
tatio
nally
challenging
andBrowniehasbeen
show
nto
frequently
yieldincorrect
resultsT
heaccuracy
ofthemethodislik
elycorrelated
with
nodalsupportvalues
inthe
individu
algene
topo
logies
Source
httpwwwbrianom
earain
fobrownie
describedin
OrsquoM
eara
(201
0)
Individual
gene
trees
(con
tinued)
22 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
BFD
mdashmultispecies
coalescent
model
for
speciesdelim
itatio
nThe
recently
developedmethod
Bayes
factor
delim
itatio
n(with
genomicdataB
FD)
combinesady
namic
programmingalgorithm
forestim
atingspeciestreesthat
bypasses
thecompu
tatio
nally
intensiveMCMCintegrationov
ergene
treesto
prov
idearigorous
techniqueforspeciesdelim
itatio
nstudiesusinggenome-wideSN
PdataCom
petin
gspeciesdelim
itatio
nmodelsarecomparedusingBayes
factorsanditappearsthat
this
approach
isrobustto
samplesizes(iefew
loci
andlim
itedsamples
perspecies)
and
misspecificatio
nof
thepriorforpo
pulatio
nsize
(θ)
Limita
tions
Recently
describedmethodlackingathorough
theoretical
andem
pirical
evaluatio
nSo
urce
httpwwwbeast2orgwikiindexphpBFD
describedin
Leacheacuteet
al(201
4)
Genom
e-wideSN
Pdata
2 The Dynamic Discipline of Species Delimitation hellip 23
distances among organisms belonging to thesame species are smaller than distances amongorganisms from different species (Puillandreet al 2012 Hebert et al 2003) Genetic distanceapproaches hold particular promise as an identi-fication tool shortcutting the difficulties of mor-phology-based identification (Hebert et al 2004)although in practice a barcode gap may not existfor many groups (Wiemers and Fiedler 2007)Furthermore the role of distance-based approa-ches using a single genetic marker for speciesdiscovery remains more controversial (Rubinoff2006) and without other corroborating evidenceOTUs inferred from single-locus dataset shouldonly be considered ldquocandidaterdquo species Tree-based methods aim to detect monophyletic cladescorresponding to species-level diversity bydetecting discontinuities associated within inter-and intraspecific branching patterns (Fujisawaand Barraclough 2013 Zhang et al 2013Monaghan et al 2009 Pons et al 2006) Tree-based species delimitation methods can also beused on the basis of other properties related tophylogenetic tree topologies (monophyly con-cordance with geography etc (Sites and Marshall2003 2004) Both distance- and tree-basedmethods have been applied for assessing speciesboundaries in lichen-forming fungi (Leavitt et al2012c 2014 Parnmen et al 2012 Del-Pradoet al 2010 2011 Molina et al 2011)
A number of tree-based methods partiallyautomate the species delimitation process withspecific bioinformatical analyses (Table 21)including the general mixed Yule coalescent(GMYC) approach (Fujisawa and Barraclough2013 Monaghan et al 2009 Pons et al 2006)and the Poisson tree processes (PTP) model(Zhang et al 2013) These methods provide rel-atively straightforward and objective pipelinesfor delimiting putative species-level lineagesfrom inferred gene trees by fitting within- andbetween-species branching models to an inferredsingle-locus topology A number of other tree-based methods for species delimitation areeffectively summarized in Sites and Marshall(2004)
When only single-locus data are available theGMYC has been shown to be a relatively robust
tool for species delimitation The GMYCapproach combines a coalescent model of intra-specific branching with a Yule model for inter-specific branching which is then fit to an inferredsingle-gene topology to estimate species bound-aries and a statistical measure of confidence for theinferred boundaries As an input the GMYCapproach requires an ultrametric gene tree andrecent refinements can account for uncertainty inphylogenetic relationships and parameters of theGMYC model (Fujisawa and Barraclough 2013Reid and Carstens 2012) Regardless of theseimprovements it may be difficult to accuratelyinfer an adequate ultrametric tree for large data-sets Although it appears that the GMYC isgenerally stable across a wide range of circum-stances including various methods of phyloge-netic reconstruction the presence of a highnumber singletons high numbers of sampledspecies and gaps in intraspecific sampling theaccuracy of the GMYC is most significantlyaffected by the mean population size relative todivergence times between them (Fujisawa andBarraclough 2013 Talavera et al 2013) Fur-thermore research suggests that the so-calledsingle-threshold version of the GMYC methodlikely outperforms the multiple-thresholdapproach (Fujisawa and Barraclough 2013Monaghan et al 2009) However other studiessuggest that theGMYCmethodmay often providehigher estimates for the total number ofOTUs thanother molecular species delimitation methods(Hamilton et al 2014 Miralles and Vences 2013Talavera et al 2013) warranting a cautiousinterpretation of results from GMYC analyses
Compared to the GMYC approach therecently introduced PTP method for speciesdelimitation has been suggested to be moreaccurate for preliminary species delimitation(Zhang et al 2013) Relative to the GMYC PTPoffers a more straightforward implementationrequiring a simple phylogenetic tree rather thanan ultrametric chronogram However moreresearch is required to assess the general perfor-mance of PTP across wide range of empirical andsimulated species delimitation studies At thispoint data support the use of the GMYC andPTP methods as objective and reasonable starting
24 SD Leavitt et al
points for species delimitation using single-genetopologies
The ldquoSpecies Delimitationrdquo plug-in to theGeneious software provides statistical approachesfor exploring species boundaries in single-genetopologies (Masters et al 2011) Using a priorispecimen assignments to putative species theldquoSpecies Delimitationrdquo plug-in computes statis-tics relating to the probability of the observedmonophyly or exclusivity having occurred bychance in a coalescent process and assesses thewithin- and between-species genetic distances inorder to infer the probability with which membersof a putative species might be identified suc-cessfully with tree-based methods An importantcontribution of the ldquoSpecies Delimitationrdquo plug-in is that it provides an objective means for usersto assess putative species within an empiricalstatistic-based framework rather than qualitativeevaluations of what level of hierarchy constitutesa species in a phylogeny
In contrast to simple sequence similaritythresholds (OTU-picking) for delimiting putativeevolutionarily significant units the AutomaticBarcode Gap Discovery (ABGD) method is anautomated procedure that sorts sequences intohypothetical species based on the existence of abarcode gap observed whenever intraspecificgenetic distances are less than those amongorganisms from different species (Puillandreet al 2012) Ultimately ABGD is a fast simplemethod that can be used to group individualsrepresented in a single-locus sequence alignmentinto candidate species that should be comple-mented with other lines of evidence in an inte-grative taxonomic approach (Kekkonen andHebert 2014 Puillandre et al 2012)
In general the ABGD GMYC and PTP ana-lytical protocols using single-locus data arerepeatable and computationally relatively fastproviding a valuable starting point for a pre-liminary perspective into species boundaries inunderstudied groups that can be validated withsubsequent studies (Kekkonen and Hebert 2014)Similarly analyses of single-locus data can beused to corroborate traditional phenotype-basedspecies boundaries and identify candidate speciesthat have previously been hidden within nominal
species However single-locus species delimita-tionmethodsmaybeparticularly prone to failure inrecognizing significant proportions of species-level biodiversity due to the strict criterion forreciprocal monophyly or alternatively providespurious inflations of estimated species diversitybased on genetic differences that donot correspondto species-level lineages For example recentestimates suggest that the incidence of species-level gene tree paraphyly is approximately 20 (Ross 2014) suggesting that analyses of single-locus datasets would likely fail to accurately deli-mit species in one in every five cases In contrastrecent empirical studies suggest that in some casesthe GMYC provides a striking overestimate ofspecies diversity (Miralles and Vences 2013)
The internal transcribed spacer region (ITS)has played a central role in molecular phyloge-netic studies of lichenized fungi and has recentlybeen adopted as the official barcoding marker forfungi (Schoch et al 2012) In many cases the ITSis sufficiently variable to resolve species bound-aries for lichenized fungi (Schoch et al 2012Kelly et al 2011) although accurate specimenidentification using sequence-based identificationapproaches requires a thoroughly curated refer-ence database (Leavitt et al 2014 Orock et al2012 Kelly et al 2011) In spite of the generalutility of the ITS marker a number of issues maypotentially limit its effective use in speciesdelimitation studies including the potential forintragenomic variation of the nuclear ribosomaltandem repeat and difficulties in aligning ITSsequences from divergent taxa (Kiss 2012)Because the ITS has been formally adopted as theofficial barcoding marker for fungi we recom-mend that species delimitation studies attempt toinclude this region for consistency across studiesHowever we recognize that in some taxonomicgroups the inclusion of the ITS may be prob-lematic and therefore suggest a careful screeningof other markers to identify appropriate loci forresolving species-level relationships
Ultimately the success of any single-locusspecies delimitation method largely depends onthe evolutionary history of the species groupunder study and the variability of the selectedmarker (Fig 22) Species delimitation will be
2 The Dynamic Discipline of Species Delimitation hellip 25
more difficult in more recently diverged lineagesand in cases with some level of interspecific geneflow relative to older well-diverged lineages(eg Leavitt et al 2012c 2013e) In the end wereemphasize that single-locus data alone shouldonly be used to provide a preliminary perspectiveof species boundaries and not as the sole evi-dence in species circumscriptions
222 Sampling Across the GenomeMultilocus Sequence Dataand Genome-Wide Markers
Although single-locus methods can provide anefficient approach for preliminary large-scaleassessments of species diversity there are
significant limitations particularly in recentlydiverged species groups where retention ofancestral polymorphisms and incomplete lineagesorting will likely result in different neutral locihaving unique gene topologies that do not mirrorthe speciation process (Knowles and Carstens2007 Heled and Drummond 2010 Taylor et al2000) In contrast to single-locus and strictlyphenotype-based approaches for species delimi-tation analyses of genetic data collected fromindependent genomic regions can provide robusthypotheses of species boundaries with increasingconfidence (Satler et al 2013 Zhang et al 2011Yang and Rannala 2010) Sequences from mul-tiple independent loci provide an importantsource of data for species delimitation studiesincluding recently developed models that
Fig 22 A simplified diagram illustrating the process ofspeciation through time in a single gene history andresulting gene topologies sampled at two points in thespeciation history (modified from Leliaert et al 2014)aEach dot represents a distinct gene copy and each row onenon-overlapping generation with lines connecting genecopies to their ancestors in the previous generation (onerow below) dashed diagonal lines represent reproductivebarriers two hypothetical sampling intervals at differentpoints in the speciation history are shown Tx and Ty
species delimitation methods using single-locus data areeffective only when species are reciprocally monophyleticin the sampled gene tree genetic clustering and coalescent-based species delimitation methods can circumscribespecies when species may not be monophyletic in sampledgenetic loci b Gene topology representing sampledhaplotypes at time Tx (shown in panel a) hypotheticalspecies are reciprocally monophyletic c Gene topologyrepresenting sampled haplotypes at time Ty (shown in panela) hypothetical species are para- and polyphyletic
26 SD Leavitt et al
combine individual gene genealogies and speciesphylogenies via modeling the coalescent historyof markers (Yang and Rannala 2010 Edwards2009 Knowles and Carstens 2007) As aresponse to advances in sequencing technologiesbioinformatical approaches for multimarker spe-cies delimitation analyses continue to be devel-oped (Table 21 Camargo and Sites 2013)
Long-term reproductive isolation of candidatespecies can be assessed with multilocus sequencedata by evaluating genealogical concordance ofunlinked markers (Baum and Shaw 1995 Aviseand Ball 1990) Within species the mixingeffects of recombination cause unlinked loci tohave distinct genealogical histories but geneticdrift and long-term divergence leads to concor-dant genealogical histories at loci across thegenome Relationships of individuals belongingto distinct candidate species can be evaluatedusing gene genealogies (eg haplotype networksor single-gene topologies) to identify lineagesthat exhibit genealogical exclusivity acrossunlinked neutral loci (Hudson and Coyne 2002Dettman et al 2003b Avise and Ball 1990) Thepresence of the same clades in the majority ofsingle-locus genealogies is taken as evidence thatthe clades represent reproductively isolated lin-eages (Dettman et al 2003a Pringle et al 2005)In practice the criteria of reciprocal monophylyand genealogical concordance of unlinked lociprovide a conservative approach for assessingspecies boundaries due to the fact that a sub-stantial amount of time is required after the initialdivergence of species until ancestral polymor-phisms have fully sorted (Knowles and Carstens2007 Hudson and Coyne 2002) Consequentlygroups with recent divergence histories willlikely go undiscovered under a genealogicalconcordance criterion due to the fact that speciesboundaries likely are not reflected in genegenealogies For example it would take morethan 1 million years after speciation before spe-cies would be delimited if 15 loci were sampledin species with an effective population size (Ne)of 100000 assuming one generation a yearunder a strict reciprocal monophyly criterion(Knowles and Carstens 2007) The amount oftime required for a species to be recognized using
a criterion of reciprocal monophyly increasesproportionally with increasing population sizes(Hudson and Coyne 2002) In a number ofstudies of lichen-forming fungi a genealogicalconcordance criterion has been used to bothdelimit previously unrecognized species-levellineages and validate some conventional pheno-type-based species (Leavitt et al 2012a c2013b Molina et al 2011 Kroken and Taylor2001)
Although analyses of molecular sequence datarelying on reciprocal monophyly or fixed char-acter differences for species delimitation canserve as important criteria for identifying speciesthese criteria are not commonly met acrossmultiple loci particularly in recent speciationhistories (Fig 22) To accommodate theobserved conflict among genealogies from mul-tiple loci with the underlying speciation historythe recent merge of coalescent theory with phy-logenetics has driven a major paradigm shift inspecies delimitation and molecular systematics ingeneral (Fujita et al 2012 Edwards 2009) Themultispecies coalescent model can be applied togenealogical histories from multiple independentloci to assemble separate coalescent processesoccurring in populations into a species tree(Degnan and Rosenberg 2009 Rannala andYang 2003) Within this coalescent-basedframework multiple individuals can be assignedto a single speciespopulation and the speciationhistory of ancestral and descendant species-levellineages can be inferred as a ldquospecies treerdquo incontrast to estimating gene genealogies fromindividual samples (Degnan and Rosenberg2006 2009 Rannala and Yang 2003)
A number of multispecies coalescent-basedspecies delimitation methods have recently beenintroduced offering an exciting framework forempirically assessing species boundaries byselecting the best species tree model from a set ofalternative models representing differenthypotheses of species boundaries (Table 21)For example SpeDeSTEM (Ence and Carstens2011) finds the maximum likelihood values for aspecies tree assuming all putative species areseparate lineages and for alternative species treeswhere two or more species are collapsed into a
2 The Dynamic Discipline of Species Delimitation hellip 27
single lineage The best fitting model is thenselected using the Akaike information criterionunder the assumption of constant populationsizes and fixed gene topologies across the speciestree The species delimitation program BayesianPhylogenetic and Phylogeography (BPampPYangand Rannala 2010) accommodates gene treeuncertainty and variable population sizes andsamples from the Bayesian posterior distributionof species delimitation models using reversible-jump Markov chain Monte Carlo (rjMCMC)sampling BPampP requires a user-provided guidetree with resolution finer than the species leveland evaluates alternative modes derived from allpossible subtrees that are generated by collapsingor splitting nodes on the guide tree (Yang andRannala 2010 Rannala and Yang 2003) Theproportion of time spent on each model is pro-portional to the posterior probability of themodel ie ldquospeciation probabilitiesrdquo WhileBPampP ranks among the most popular coalescent-based species delimitation programs (Fujita et al2012) misspecifications of the guide tree andorthe prior distribution for population size (θ) canresult in strong support for models containing aninflated number of species (Leacheacute and Fujita2010 Zhang et al 2011) The nonparametricheuristic species delimitation approach imple-mented in the program Brownie (OrsquoMeara 2010)jointly sorts anonymous samples into species andinfers a species tree from input gene trees fromdifferent loci assuming that for a speciationevent the corresponding nodes on gene trees willbe more consistent with each than the diver-gences within species However finding both theoptimum species tree and species boundariesremains computationally challenging andBrownie has been shown to frequently yieldincorrect results (OrsquoMeara 2010)
Most recently available coalescent-basedspecies delimitation methods require individualassignments to a species or population a priori Ina number of scenarios correct assignments ofsamples to species may be difficult includingpresence of cryptic species incongruencebetween conventional species and moleculardata or simply the fact that accurate specimenidentification in complex groups with subtle or
difficult to discern diagnostic characters is anontrivial task (Leavitt et al 2013e) Statisticalmethods for assessing individual assignment andspecies detection prior to coalescent-based spe-cies delimitation and species tree reconstructionand species provide a more objective approachfor understanding species boundaries
Population assignment tests using a variety ofclustering algorithms using genetic data offer auseful approach for grouping individuals intoputative reproductively isolated groups (Table 21Hausdorf and Hennig 2010 Corander et al2004 2008 Falush et al 2003 Pritchard et al2000) particularly for species delimitation prob-lems that exist at the interface of traditionalpopulation genetic and phylogenetic analyses(Carstens et al 2013 Weisrock et al 2010Knowles and Carstens 2007)
The programs STRUCTURE (Falush et al2003 Pritchard et al 2000) and STRUCTURA-MA (Huelsenbeck et al 2011) cluster samplesinto populations by minimizing HardyndashWeinbergdisequilibrium for a given number of populationclusters using unlinked genetic markers In gen-eral unlinked markers are not available for mostgroups of lichen-forming fungi and a numberof studies have used information from multilocussequence data for STRUCTURE analyses(Leavitt et al 2011a b c 2013e Fernaacutendez-Mendoza and Printzen 2013 Fernaacutendez-Mendoza et al 2011) Varying approaches havebeen implemented to convert DNA sequencedata to allelic data for Bayesian clustering (seeOrsquoNeill et al 2013) STRUCTURE is expectedto perform well when there is sufficient inde-pendence across regions such that linkage dis-equilibrium within regions does not dominate thedata (STRUCTURE manual) but can also beeffective using multilocus sequence data andtreating all SNPs as independent loci regardlessof physical linkage within each locus (OrsquoNeillet al 2013 Falush et al 2003) A recent study ofthe lichen-forming genus Letharia showed thatBayesian clustering implemented in the programSTRUCTURE was generally able to recover thesame putative Letharia lineages circumscribedemploying a gene genealogical approach inKroken and Taylorrsquos iconic species delimitation
28 SD Leavitt et al
study (Altermann et al 2014 Kroken and Taylor2001) Altermann et al (2014) show that popu-lation assignments were largely consistent acrossa range of scenarios including extensiveamounts of missing data the exclusion of SNPsfrom variable markers and inferences based onSNPs from as few as three gene regions Incontrast to STRUCTURE and STRUCTURA-MA the program BAPS (Corander et al 2008)can explicitly infer genetic structure using hap-loid sequence data or other linked geneticmarkers in the ldquoclustering with linked locirdquomodel Another advantage of BAPS is that theprogram is much more computationally efficientthan STRUCTURE or STRUCTURAMA Sim-ulation studies indicate that both BAPS andSTRUCTURE perform well at low levels ofpopulation differentiation and when clusters arenot well differentiated (Latch et al 2006)
In practice inferring the most appropriatelevel of population structure using Bayesianclustering algorithms remains challenging (Latchet al 2006 Evanno et al 2005) BAPS andSTRUCTURAMA can infer both individualassignments and the most likely number ofgenetic clusters (Corander et al 2008 Huelsen-beck et al 2011) and the ad hoc ΔK statisticproposed by Evanno et al (2005) provides anobjective approach for identifying the uppermosthierarchical level of structure However carefulconsideration of other levels of populationstructure may ultimately reveal more biologicallymeaning results and researchers should examineindividual assignments across a range of geneticgroupings Some markers generated fromindependent genomic regions such as SNPs andfast-evolving microsatellites can be used to dis-tinguish fine-scale population structuring on thebasis of allele frequencies and species delimita-tion analyses based on these markers may implythe risk of severe taxonomic oversplitting Inthese cases validation approaches such asBPampP and SpeDeSTEM and corroborationamong different species delimitation approachescan provide perspective between intraspecificpopulation structure and species-level clusters
Another limitation of clustering approaches isthat they do not assess or take into account
evolutionarily divergences and relationshipsamong population clusters Coalescent-basedspecies tree methods (discussed below) provide ameans to assess the diversification histories ofpopulations inferred from clustering analysesTherefore a potential working protocol for aninformed species delimitation study that takesinto account population structure could consist offirst applying a genetic clustering analysis under apopulation genetics criterion (eg BAPSSTRUCTURE STRUCTURAMA) to identifygenetically distinct population clusters that can beconsidered ldquocandidate speciesrdquo From these can-didate species a species tree can be inferred forfocal group using coalescent-based species treereconstruction methods (eg BEAST) Subse-quently a coalescent-based validation methodcan be applied to assess whether the distinctpopulation clusters represent independent evolu-tionary lineages (eg BPampP) This multistepapproach would provide a consistent and standardcriterion for distinguishing between population-and species-level lineages (Camargo and Sites2013) and has been applied to a number of casesincluding lichen-forming fungi (Leavitt et al2011a b c 2013e Leacheacute and Fujita 2010)
High-throughput sequencing methods providethe means to effectively sample hundreds tothousands of loci from across a species genome fora large number of species and continue to revolu-tionize studies that can be performed even in non-model organisms Targeted high-throughputsequencing approaches such as anchored phy-logenomics transcriptome sequencing reduced-representation genomic library sequencing(restriction-site-associated DNA sequencingRAD-Seq and genotype-by-sequencing GBS)and high-throughput amplicon sequencing pro-vide important insight into species boundaries fora number groups although none of these approa-ches has yet been applied to studies of lichenizedfungi
For example restriction-site-associated(RAD-Seq) DNA can simultaneously detect andgenotype thousands of genome-wide SNPs byfocusing the sequencing effort on a reducedrepresentation of the entire genome (Baird et al2008) and has been successfully applied to
2 The Dynamic Discipline of Species Delimitation hellip 29
intraspecies (Lewis et al 2007 Miller et al 2007Emerson et al 2010) and interspecies studies(Rubin et al 2012 Eaton and Ree 2013 Wagneret al 2013) This approach has provided strikinginsight into the recent adaption radiation of LakeVictoria cichlids (Wagner et al 2013) Analternative approach targeting the sequencing ofspecific loci using next-generation sequencingplatforms provides an efficient means of gener-ating data for loci of known genomic locationorthology size and expected level of variation(OrsquoNeill et al 2013) Markers can be targetedusing well-established PCR techniques or newerhybridization techniques and subsequentlypooled for high-throughput sequencing usingparallel-tagged sequencing of multiple individualsamples within a single sequencing run (OrsquoNeillet al 2013) A recent study of North Americantiger salamanders (Ambystoma tigrinum)demonstrated the potential for amplicon-basedparallel-tagged sequencing to rapidly generatelarge-scale genomic data for species delimitationand species tree research (OrsquoNeill et al 2013)
Although these methods provide an enormousamount of data and insight into diversification atan unprecedented scale computational limita-tions restrict the applicable analytical methodsfor large datasets although computational andanalytical advances are happening rapidly Mostrecent coalescent-based species delimitation andspecies tree models using gene trees have beenlimited to handle tens of loci for multiple indi-viduals and combining hundreds or thousands ofgene trees into a single species delimitationframework presents considerable computationalchallenges (Camargo and Sites 2013) Therecently developed method Bayes factor delim-itation (with genomic data BFD) combines adynamic programming algorithm for estimatingspecies trees that bypasses the computationallyintensive MCMC integration over gene trees toprovide a rigorous technique for species delimi-tation studies using genome-wide SNP data(Leacheacute et al 2014) Competing species delimi-tation models are compared using Bayes factorsand it appears that this approach is robust tosample sizes (ie few loci and limited samples
per species) and misspecification of the prior forpopulation size (θ) (Leacheacute et al 2014)
Fungi are an ideal model for assessing diver-gence in eukaryotes due to their simple mor-phologies small genomes broad ecological rolesand diverse lifestyles (Gladieux et al 2014)However the use of genomic data from high-throughput sequencing methods have not yet beenincluded in species delimitation studies of lichen-forming fungi This is due in part to challenges indealing with metagenomic data containing geno-mic information from a plethora of symbiontsassociated with a lichen thallus scant genomicresources and discipline-specific inertia A num-ber of lichen-forming fungal genomes are cur-rently available including Endocarponpusillum(Wang et al 2014) Cladonia spp (Park et al2013a 2014) Caloplaca flavorubescens (Parket al 2013b) and Cladonia greyi and Xanthoriaparientina through the Fungal Genomics Programat Joint Genome Institute of the United StatesDepartment of Energy (httpwwwjgidoegov)The foreseeable ongoing expansion of genomicresources for lichen-forming fungi will be centralto developing approaches for delimiting speciesusing high-throughput sequencing Specificallygenomic resources will be crucial in identifyingnovel markers (variable genes regions SNPsmicrosatellites etc) identifying conserved fungalmarkers for targeted high-throughput sequencingapproaches and references for entire genomecomparisons (Devkota et al 2014 Werth et al2013)
23 Can We Make SpeciesDelimitation in Lichen-FormingFungi Truly Integrative
The widespread availability of genetic data hascreated a biased viewpoint that only genetic datashould be used for statistical species delimitationHowever an ongoing appeal to researchers toassess species boundaries from multiple andcomplementary perspectives (phylogeneticspopulation genetics comparative morphologydevelopment ecology etc) has resulted in an
30 SD Leavitt et al
integrative taxonomic framework bringing theseconceptual and methodological perspectivestogether (Hamilton et al 2014 Fujita et al 2012Salicini et al 2011 Yeates et al 2011 Padialet al 2010 Dayrat 2005 Will et al 2005 Wiensand Penkrot 2002) In practice any study linkingdifferent kinds of data to support hypotheses ofspecies boundaries including mapping morpho-logical characters onto a molecular phylogenycan be considered integrative Integrative meth-ods for species delimitation fall across a broadspectrum ranging from verbal and qualitativeassessments of data classes to quantitative meth-ods that allow different data types to contribute tostatistical species delimitation (Yeates et al2011) In most taxonomic studies utilizing bothmolecular and morphological data expert opinionis eventually used to some degree to evaluate thefinal status of a candidate species (eg Bond andStockman 2008) In this sense many studiesusing evidence for independent data sources fordelimiting species boundaries use an iterativeapproach (Yeates et al 2011) where speciesboundaries can be tested within a hypothetico-deductive framework with diverse datasets
From a practical perspective we advocate aprocess of iterative taxonomy (sensu Yeates et al2011) to circumscribe and refine species limitsusing multiple lines of evidence This iterativeprocess involves comparisons of morphologicaldata with a phylogenetic hypothesis (ie single-locus gene tree or concatenated multilocusphylogeny) to identify the least inclusive mono-phyletic clade in the topology characterized by atleast one unambiguously diagnostic morphologi-cal character (Miralles and Vences 2013) Thisphylogenetic tree-informed approach to assessingspecies boundaries represents a common practicein studies of lichen-forming fungi Previouslyunrecognized species-level clades with corre-sponding subtle or overlooked phenotypiccharacters have been commonly observed in bothcrustose lichens [eg Acarospraceae (Wedin et al2009) Graphidaceae (Rivas Plata and Lumbsch2011 Papong et al 2009) Lecanoraceae (Leavittet al 2011a) Lecideaceae (Ruprecht et al 2010)Mycoblastaceae (Spribille et al 2011) andTeloschistaceae (Vondraacutek et al 2009 Muggia
et al 2008)] and foliose and fruticose lichens [egLobariaceae (Moncada et al 2014 McDonaldet al 2003) Parmeliaceae (Leavitt et al 2013aWirtz et al 2012 Divakar et al 2005 Molinaet al 2004 Kroken and Taylor 2001) Peltigera-ceae (OrsquoBrien et al 2009 Goffinet et al 2003)Physciaceae (Elix et al 2009 Divakar et al2007) and Sphaerophoraceae (Houmlgnabba andWedin 2003)] As a specific example Luumlckinget al (2008) used a combination of medullarychemistry and underside pigmentation in speci-mens from the Heterodermia obscurata group inCosta Rica to corroborate monophyletic clades inan ITS gene tree as species-level lineages
While this iterative approach to speciesdelimitation and taxonomy has proven valuablefor understanding species boundaries anddescribing new taxa in some groups of lichenizedfungi a posteriori examination of morphologicaland chemical features has failed to reveal diag-nostic phenotypic characters in a number ofstudies (Muggia et al 2014 Leavitt et al 2011aOtaacutelora et al 2010 OrsquoBrien et al 2009) Fur-thermore a study of widespread species in thegenus Melanelixia (Parmeliaceae) indicated thatphenotypically cryptic lichen-forming fungalspecies-level lineages may be relatively ancientand diagnosable phenotypic differences may beabsent even millions of years after the initialdivergence (Leavitt et al 2012b) The latter studyhighlights the fact that species-level lineages maycommonly exist without any observable diag-nostic phenotypic characters calling into ques-tion the impetus for a universal application ofintegrative taxonomy
In keeping with the principle that as manylines of evidence as available should be com-bined to delimit species (Dayrat 2005) the for-malized integrative taxonomic approach (ITAXMiralles and Vences 2013) uses a mtDNA guidetree and observations from different types of datathat might provide conclusive evidence for theindependence of lineages and thus their identityas different species Miralles and Vences (2013)provide a non-exhaustive list of species delimi-tation criteria to be integrated in the ITAXapproach including (a) sympatric occurrencewithout admixture as revealed by consistent
2 The Dynamic Discipline of Species Delimitation hellip 31
differences in morphological or molecular char-acters at the same geographic location (b) strongdifferences in a behavioral morphological orgenetic character known to mediate prematingisolation (c) unviability or infertility of hybrids(d) lack of gene flow across a geographicalhybrid zone (e) congruent diagnostic differencesbetween sister lineages in various unlinkedmorphological character (f) absence of haplo-type sharing in several unlinked nuclear loci and(g) a combination of criteria endashf Speciesboundaries are based on seeking the least inclu-sive monophyletic group in the mtDNA treewhich fulfills at least one of the criteria listedabove Clearly the ITAX approach is sensitive tosample size and in order to reliably support thedistinctiveness of a given species it has beenrecommended that the sampling strategy includesat least five individuals per species (Miralles andVences 2013) A similar approach could beadopted for lichenized fungi using an ITS genetopology as guide tree rather than mtDNA
A total evidence approach including concat-enation has been a common approach for inte-grating information from different sourcesincluding independent genetic markers in phy-logenetic reconstructions (Kluge 1989 Wiens1998 de Queiroz et al 1995) Proponents forconcatenation of independent data in phyloge-netic analyses argue that when combining alldata the underlying signal of speciation mayemerge even if weakly contradictory signalis contained in the individual data partitions(Gatesy et al 1999) For example establishing apreliminary perspective of species boundariesfrom multilocus sequence data using concatena-tion may provide a reasonable starting point forscreening for cryptic species and species treeinference (Šlapeta et al 2006 Leavitt et al2011a Le Gac et al 2007 Leacheacute 2009)However concatenation and consensus methodsimply a risk of obtaining inflated support forincorrect relationships and information aboutvariance in gene coalescence is lost (Degnan andRosenberg 2006 2009 Kubatko and Degnan2007 Edwards 2009) In spite of the impetus forintegrating evidence from independent data
sources into empirical species delimitation stud-ies and taxonomy (Fujita et al 2012 Padial et al2009) most currently available species delimi-tation methods are unable to accommodate non-genetic data sources in a statistical framework
231 Selecting the Appropriate Data
In the face of increasing availability of geneticdata and associated bioinformatical approachesfor delimiting species researchers should care-fully consider what information is being sacri-ficed by the failure to consider non-genetic datain species delimitation studies and whetheraccuracy could be improved by the addition ofmultiple data types Morphological data havehistorically served as a proxy to identify repro-ductively isolated groups (ie ldquospeciesrdquo) (Ray1686 Fujita et al 2012) Current methods fordelimiting species using non-genetic data (egchemistry morphology and ecology) remainwoefully understudied For example morphol-ogy-based species circumscriptions are generallybased on one or more qualitative (or quantitative)morphological characters that do not appear tooverlap with other species However ascertain-ing that a given trait is truly fixed within apopulation with statistical confidence requiresunrealistic sample sizes even when allowing forsome level of polymorphism in the diagnosticcharacter (Wiens and Servedio 2000) Now var-ious combinations of datamdashfrom morphologygenetics geography and ecologymdashare acceptedas standard information for species delimitationstudies (Ruiz-Sanchez and Sosa 2010 Ross et al2010 Edwards and Knowles 2014)
Below we briefly discuss appropriate datasources for species delimitation studies of lichen-forming fungi However homoplastic characters(similar traits that are not derived from a com-mon ancestor) are common among many traitscommonly used to circumscribe fungal taxa thebiological significance of secondary metabolitevariation remains largely unknown (Lawrey1986) and ecological niches may be difficultto adequately characterize and model due to
32 SD Leavitt et al
microhabitat requirements and data resolutionTherefore we advocate a cautious approach toselecting appropriate and relevant data forassessing species boundaries
In spite of the potential challenges and limi-tations of using phenotypic data these traits haveprovided a plethora of valuable information forunderstanding species boundaries For examplein the Melanelixia fuliginosa group (Parmelia-ceae) a morphometric analysis using color isi-dia and marginal zone free of isidia ascharacters revealed a general pattern of differ-entiation between material formerly recognizedas subspecies fuliginosa and glabratula in spiteof the fact that considerable overlap betweengroups occurs in some characters individually(Arup and Berlin 2011) Furthermore the dis-tinction of the two groups was supported by aphylogenetic analysis of the ITS marker andecological differences with M fuliginosaoccurring predominantly on rock and M glab-ratula on bark (Arup and Berlin 2011) Thisstudy of the M fuliginosa group provides a fit-ting example of using multiple independentsuites of data including ecology morphologyand genetic information to establish a robusthypothesis of species boundaries
In lichen systematics phenotypic dataincluding thallus organization secondary metab-olites mode of reproduction ascoma-type andontogeny ascus and ascospore characters havehistorically played a prominent role (Printzen2009) Ascomatal characters have traditionallyheld a major role in higher-level classification(Printzen 2009) in contrast to species-levelclassification which also tends to include a widearray of vegetative and chemical charactersCommonly assessed morphological charactersinclude thallus form and size cortical features(eg maculae and pseudocyphellae) presenceformcolor of attachment structure (eg rhizines)and reproductive mode (ascomata vs vegetativediaspores) (Printzen 2009) Different morpho-logical types of vegetative diasporesmdashcorticatedisidia and ecorticated sorediamdashand their locationare commonly used to distinguish speciesAscomatal characters including morphologylocation (laminal vs marginal) position (sessile
vs immersed) presence of thalline margins colorof an apothecial disc and presence or color ofpruina are also commonly used in speciesdelimitations of lichen-forming fungi (Printzen2009) Other important characters may includethalline characters form color size and septationof ascospores size and form and structure of ascithe hamathecium type of epihymenium andhypothecium and the type of excipulum orperidium conidiomatal characters etc (Printzen2009)
Assessments of secondary metabolites hasplayed an important role in lichen taxonomybeginning with the introduction of simple spottests by Nylander (1866a b) The use of chem-istry in lichen taxonomy has been discussed indetail in numerous reviews (Lumbsch 1998a bRogers 1989 Brodo 1986 Egan 1986 Leuckert1985 Brodo 1978 Hawksworth 1976 Culber-son 1969 1970) and we refer readers to thesevaluable sources for a more comprehensive per-spective on lichen chemistry In short extrolites(secondary metabolites) belong to various clas-ses the most common and diverse include dep-sides depsidones chlorinated xanthones andanthraquinones (Lumbsch 2002 Culberson1969) The presence or absence of specificextrolites or their replacements by another sub-stance is widely used to distinguish speciesparticularly when correlated with differences ingeographic distributions However if morpho-logical or geographical differences betweenpopulations containing different extrolites are notapparent the taxonomic significance has beendisputed with some authors distinguishing themas species and others preferring to regard them aschemical races within a species In addition tosimply using the presence or absence of extro-lites Culberson and Culberson (1976) proposedto arrange lichen substances into chemosyn-dromes of closely related substances The pres-ence or absence of these chemosyndromes maypotentially be used as characters to delimit spe-cies regarding differences involving the samechemosyndromes as intraspecific variation anddistinct chemosyndromes as evidence for inter-specific populations (Lumbsch 1994 Gowan1986)
2 The Dynamic Discipline of Species Delimitation hellip 33
Due to the fact that species delimitationstudies often incorporate a substantial biogeo-graphical or ecological component ecologicalniche modeling plays an increasing role in phy-logenetic and taxonomic research Niche mod-eling can provide evidence for ecologicalisolation between populations based on eitherconserved or divergent ecological niches andtherefore can provide additional evidence sup-porting lineage independence between putativespecies The application of ecological nichemodeling has been applied to species delimita-tion studies in a number of cases (Ruiz-Sanchezand Sosa 2010 Leacheacute et al 2009 Raxworthyet al 2007 Rissler and Apodaca 2007) althoughit has not been explicitly applied to assess speciesboundaries in lichen-forming fungi By mappingthe spatial distribution of environmental suit-ability of climatic variables for candidate speciesthe application of ecological niche modeling canbe particularly important in cases where specieshave allopatric distributions (Raxworthy et al2007)
Ecological niche modeling utilizes knownassociations between a speciesrsquo occurrencelocalities and environmental variables to defineabiotic conditions within which populations canbe maintained (Guisan and Thuiller 2005) Themethodological approach for modeling is basedon four general properties (i) The current knownspeciesrsquo localities is the dependent variable (ii)the distribution is modeled as a map composed ofgrid cells at a specified resolution (iii) a range ofenvironmental variables (eg temperature pre-cipitation and solar exposure) are collected todescribe the characteristics of each cell and (iv)classifying the degree to which each cell is eithersuitable or unsuitable for each species under arange of models (Guisan and Thuiller 2005)
Ecological data also have the potential to playan important role in understanding speciesboundaries in lichen-forming fungi For exampleNash and Zavada (1977) demonstrated that Xant-hoparmelia populations with distinct chemistriesoccurring in the northern portion of the SonoranDesert exhibit habitat selection among differentrock substrates within a region with relativelyuniform climate and topography In another case
the parmelioid species Parmelia mayi is mor-phologically indistinguishable from P saxatilisbut can be separated by bioclimatic features andgenetic and chemical characters (Molina et al2011) McCune and Printzen(2011) assess distri-butions and climatic niches of species in theLecanora varia group inwesternUSAandprovidea model that uses continental influence and annualtemperature as themajor factors predicting speciesdistributions The distribution of Usnea hirta alichen commonly used in air quality biomonitor-ing research was modeled for a section of theWhite River National Forest in central Coloradobased on the presence of U hirta at 72 biomoni-toring reference sites distributed in the inter-mountain western United States (Shrestha et al2012) The best model for predicting U hirtadistribution included four variablesmdashsolar radia-tion average monthly precipitation and averagemonthly minimum and maximum temperatures(Shrestha et al 2012) These studies supportthe potential use of ecological niche modelingmethods in species delimitation studies of lichen-forming fungi
24 Conclusions What AboutTaxonomy
In most cases species circumscription and tax-onomy requires some degree of qualitativejudgment and individual interpretation Integrat-ing multiple types of data into an empiricalframework for delimiting species boundarieswhere species boundaries can be tested within ahypothetico-deductive framework with diversedatasets can provide robust hypotheses of speciesboundaries and taxonomic stability (Yeates et al2011) However it has long been known toevolutionary biologists that distinct species donot need to have diagnosable morphologicaldifferences (Mayr 1963) and increasing avail-ability of genetic data has allowed researchers toidentify species and to rigorously test speciesboundaries with a level of precision that wasunimaginable a decade ago While analyticaladvances in statistical species delimitation havebeen largely based on genetic data the utility of
34 SD Leavitt et al
these approaches to formal taxonomy remainselusive Due to these challenges an eclecticapproach to delimiting species and cautionagainst the reliance on any single dataset ormethod is required when delimiting species
While results from many recent studies maycontradict traditional species boundaries acrossmany groups of lichen-forming fungi we areoptimistic that this research represents substantialprogress toward a more accurate perspective onspecies boundaries and diversity in fungi As aresult of ongoing research of species boundariesin lichen-forming fungi the taxonomic value ofmany phenotypes is now better understood ourunderstanding of ecological evolutionary andbiogeographic patterns has improved and we canbegin to better understand patterns of symbiontinteractions in lichens Integrating new data(including novel morphological characters andgenetic data) will be essential to accurately rep-resent species-level diversity across all groups oflichenized fungi Hopefully an improved per-spective on lichen diversity also increases ourappreciation of these incredible symbiotic sys-tems While we are strong advocates for theapplication of independent data types in devel-oping an integrative taxonomy there is anincreasing need to formally recognize the exis-tence of phenotypically cryptic species-levellineages in lichen-forming fungi (see Hibbettet al 2011) In some cases a molecular taxon-omy may provide the most practical approach toconsistent treatment of mycobiont species withinlichen groups where diagnostic morphologicalcharacters are unidentifiable or practically notfeasible (Leavitt et al 2013c d)
These are exciting times for taxonomists andphylogeneticists A closer look at lichen taxon-omy with the inclusion of new data will help usto better understand the diversity of these fasci-nating organisms accurately interpret distribu-tion patterns and play a more important role inmeaningful conservation practices Howeversome level of uncertainty will accompany pro-gress In many taxonomic groups our traditionalapproach for species identification will likelyneed to be substantially modified The search forcorroborating morphological support for cryptic
species identified using molecular data willrequire meticulous and creative approaches toassess phenotypic variation in potentially unor-thodox ways We are hopeful that lichenologistswho traditionally have been eager to include newmethods such as chromatography in their rou-tine identifications will be amenable to includemolecular techniques to their routine examina-tion of specimens for identification and classifi-cation Although this may prove difficult toachieve by single individuals especially citizenscientists that traditionally play an important rolein lichen taxonomy the increasing numberof collaborative projects in the discipline (egLumbsch et al 2011 Crespo et al 2010Gueidan et al 2009) make us optimistic thatbroad-scale collaborative approaches will facili-tate the inclusion of molecular data in lichenresearch at all levels
Acknowledgments We are indebted to various col-leagues for valuable thought-provoking discussionnotably Matthew Nelsen (University of Chicago) AnaCrespo (Universidad Complutense de Madrid) PradeepDivakar (Universidad Complutense de Madrid) BeckettSterner (The Field Museum) and Joyce Havstad (TheField Museum) We also thank anonymous reviewers whoprovided valuable comments that improved this chapterSupport by the US National Science Foundation isgratefully acknowledged (ldquoHidden diversity in parmelioidlichensrdquo DEB-0949147)
References
Ahti T Hawksworth DL (2005) Xanthoparmelia steno-phylla the correct name for X somloeumlnsis one of themost widespread usnic acid containing species of thegenus The Lichenologist 37(4)363ndash366 doi101017S0024282905015197
Altermann S Leavitt SD Goward T Nelsen MPLumbsch HT (2014) How do you solve a problemlike Letharia A new look at cryptic species in lichen-forming fungi using Bayesian clustering and SNPSsfrom multilocus sequence data PLoS ONE 9(5)e97556 doi101371journalpone0097556
Amo de Paz G Cubas P Crespo A Elix JA Lumbsch HT(2012) Transoceanic dispersal and subsequent diver-sification on separate continents shaped diversity ofthe Xanthoparmelia pulla group (Ascomycota) PLoSONE 7(6)e39683 doi101371journalpone0039683
Arguumlello A Del Prado R Cubas P Crespo A (2007)Parmelina quercina (Parmeliaceae Lecanorales)
2 The Dynamic Discipline of Species Delimitation hellip 35
includes four phylogenetically supported morphospe-cies Biol J Linn Soc 91(3)455ndash467 doi101111j1095-8312200700810x
Arup U Berlin ES (2011) A taxonomic study ofMelanelixia fuliginosa in Europe The Lichenologist43(02)89ndash97 doi101017S0024282910000678
Arup U Grube M (2000) Is Rhizoplaca (Lecanoraleslichenized Ascomycota) a monophyletic genus Can JBot 78(3)318ndash327 doi101139b00-006
Avise J Ball R (1990) Principles of genealogicalconcordance in species concepts and biological tax-onomy Oxf Surv Evol Biol 745ndash67
Baird NA Etter PD Atwood TS Currey MC Shiver ALLewis ZA Selker EU Cresko WA Johnson EA(2008) Rapid SNP discovery and genetic mappingusing sequenced RAD markers PLoS ONE 3(10)e3376 doi101371journalpone0003376
Baum DA Shaw KL (1995) Genealogical perspectives onthe species problem In Hoch PC Stephenson AG(eds) Experimental and molecular approaches to plantbiosystematics Missouri Botanical Garden St Louispp 289ndash303
Beaumont MA Nielsen R Robert C Hey J Gaggiotti OKnowles L Estoup A Panchal M Corander JHickerson M Sisson SA Fagundes N Chikhi LBeerli P Vitalis R Cornuet J-M Huelsenbeck J FollM Yang Z Rousset F Balding D Excoffier L (2010)In defence of model-based inference in phylogeogra-phy Mol Ecol 19(3)436ndash446 doi101111j1365-294X200904515x
Bickford D Lohman DJ Sodhi NS Ng PKL Meier RWinker K Ingram KK Das I (2007) Cryptic speciesas a window on diversity and conservation TrendsEcol Evol 22(3)148ndash155 doi101016jtree200611004
Bonan GB Shugart HH (1989) Environmental factors andecological processes in boreal forests Annu Rev EcolSyst 20(1989)1ndash28
Bond J Stockman A (2008) An integrative method fordelimiting cohesion species finding the population-species interface in a group of Californian trapdoorspiders with extreme genetic divergence and geo-graphic structuring Syst Biol 57(4)628ndash646 doi10108010635150802302443
Brodo IM (1978) Changing concepts regarding chemicaldiversity in lichens The Lichenologist 10(1)1ndash11doi101017S0024282978000031
Brodo IM (1986) Interpreting chemical variation inlichens for systematic purposes The Bryologist 89(2)132ndash138
Caley MJ Fisher R Mengersen K (2014) Global speciesrichness estimates have not converged Trends EcolEvol 29(4)187ndash188 doi101016jtree201402002
Camargo A Sites JW (2013) Species delimitation adecade after the Renaissance In Pavlinov I (ed) Thespecies problemmdashongoing issues InTech doi10577252664
Camargo A Morando M Avila LJ Sites JW (2012)Species delimitation with ABC and other coalescent-based methods a test of accuracy with simulations and
an empirical example with lizards of the Liolaemusdarwinii complex (Squamata Liolaemidae) Evolution66(9)2834ndash2849 doi101111j1558-5646201201640x
Carstens BC Dewey TA (2010) Species delimitationusing a combined coalescent and information-theoreticapproach an example from North American Myotisbats Syst Biol 59(4)400ndash414 doi101093sysbiosyq024
Carstens BC Pelletier TA Reid NM Satler JD (2013)How to fail at species delimitation Mol Ecol 22(17)4369ndash4383 doi101111mec12413
Corander J Marttinen P (2006) Bayesian identification ofadmixture events using multi-locus molecular mark-ers Mol Ecol 15(10)2833ndash2843 doi101111j1365-294X200602994x
Corander J Waldmann P Marttinen P Sillanpaa M(2004) BAPS 2 enhanced possibilities for the analysisof genetic population structure Bioinformatics 20(15)2363ndash2369 doi101093bioinformaticsbth250
Corander J Marttinen P Mantyniemi S (2006) Bayesianidentification of stock mixtures from molecular markerdata Fish Bull 104550ndash558
Corander J Marttinen P Siren J Tang J (2008) EnhancedBayesian modelling in BAPS software for learninggenetic structures of populations BMC Bioinf 9(1)539 doi1011861471-2105-9-539
Coyne JA Orr HA (2004) Speciation Sinauer AssociatesSunderland
Cracraft J (1983) Species concepts and speciation anal-ysis Curr Ornithol 1159ndash187
Crespo A Lumbsch HT (2010) Cryptic species in lichen-forming fungi IMA Fungus 1167ndash170 doi105598imafungus2010010209
Crespo A Peacuterez-Ortega S (2009) Cryptic species andspecies pairs in lichens a discussion on the relation-ship between molecular phylogenies and morpholog-ical characters Anales del Jardin Botanico de Madrid66(S1)71ndash81 doi103989ajbm2225
Crespo A Kauff F Divakar PK del Prado R Perez-Ortega S Amo de Paz G Ferencova Z Blanco ORoca-Valiente B Nunez-Zapata J Cubas P ArguelloA Elix JA Esslinger TL Hawksworth DL MillanesA Molina MC Wedin M Ahti T Aptroot A et al(2010) Phylogenetic generic classification of parmeli-oid lichens (Parmeliaceae Ascomycota) based onmolecular morphological and chemical evidenceTaxon 59(6)1735ndash1753
Culberson WL (1969) The use of chemistry in thesystematics of the lichens Taxon 18498ndash505
Culberson WL (1970) Chemosystematics and ecology oflichen-forming fungi Annu Rev Ecol Syst 1153ndash170
Culberson CF Culberson WL (1976) Chemosyndromicvariation in lichens Syst Bot 1325ndash339
Darwin C (1859) On the origin of species by means ofnatural selection or the preservation of favoured racesin the struggle for life J Murray London
Dayrat B (2005) Towards integrative taxonomy Biol JLinn Soc 85(3)407ndash415 doi101111j1095-8312200500503x
36 SD Leavitt et al
de Queiroz K (1998) The general lineage concept ofspecies species criteria and the process of speciationa conceptual unification and terminological recom-mendations In Howard DJ Berlocher SH (eds)Endless forms species and speciation Oxford Uni-versity Press Oxford pp 57ndash75
de Queiroz K (1999) The general lineage concept ofspecies and the defining properties of the speciescategory In Wilson RA (ed) Species new interdis-ciplinary essays MIT Press Cambridge pp 49ndash89
de Queiroz K (2007) Species concepts and speciesdelimitation Syst Biol 56(6)879ndash886 doi10108010635150701701083
de Queiroz A Donoghue MJ Kim J (1995) Separateversus combined analysis of phylogenetic evidenceAnnu Rev Ecol Syst 26(1)657ndash681 doi101146annureves26110195003301
Degnan J Rosenberg N (2006) Discordance of speciestrees with their most likely gene trees PLoS Genet 2(5)e68 doi101371journalpgen0020068
Degnan JH Rosenberg NA (2009) Gene tree discordancephylogenetic inference and the multispecies coales-cent Trends Ecol Evol 24(6)332ndash340 doihttpdxdoiorg101016jtree200901009
Del-Prado R Cubas P Lumbsch HT Divakar PK BlancoO de Paz GA Molina MC Crespo A (2010) Geneticdistances within and among species in monophyleticlineages of Parmeliaceae (Ascomycota) as a tool fortaxon delimitation Mol Phylogenet Evol 56(1)125ndash133 doi101016jympev201004014
Del-Prado R Divakar PK Crespo A (2011) Using geneticdistances in addition to ITS molecular phylogeny toidentify potential species in the Parmotrema reticul-atum complex a case study The Lichenologist 43(06)569ndash583 doi101017S0024282911000582
Del-Prado R Blanco O Lumbsch HT Divakar PK ElixJA Molina MC Crespo A (2013) Molecular phylog-eny and historical biogeography of the lichen-formingfungal genus Flavoparmelia (Ascomycota Parmelia-ceae) Taxon 62(5)928ndash939 doi101270562522
Dettman J Jacobson D Taylor J (2003a) A multilocusgenealogical approach to phylogenetic species recog-nition in the model eukaryote Neurospora Evolution57(12)2703ndash2720 doi101111j0014-38202003tb01514x
Dettman JR Jacobson DJ Turner E Pringle A TaylorJW (2003b) Reproductive isolation and phylogeneticdivergence in Neurospora comparing methods ofspecies recognition in a model eukaryote Evolution57(12)2721 doi10155403-074
Devkota S Cornejo C Werth S Chaudhary RP Sche-idegger C (2014) Characterization of microsatelliteloci in the Himalayan lichen fungus Lobaria pindar-ensis (Lobariaceae) Appl Plant Sci 2(5)1300101doi103732apps1300101
Divakar PK Molina MC Lumbsch HT Crespo A (2005)Parmelia barrenoae a new lichen species related toParmelia sulcata (Parmeliaceae) based on molecularand morphological data The Lichenologist 37(01)37ndash46 doi101017S0024282904014641
Divakar PK Amo De paz G del Prado R Esslinger TLCrespo A (2007) Upper cortex anatomy corroboratesphylogenetic hypothesis in species of Physconia(Ascomycota Lecanoromycetes) Mycol Res 111(11)1311ndash1320 doi101016jmycres200708009
Divakar PK Figueras G Hladun N Crespo A (2010)Molecular phylogenetic studies reveal an undescribedspecies within the North American concept of Mela-nelixia glabra (Parmeliaceae) Fungal Divers 42(1)47ndash55 doi101007s13225-010-0027-3
Donoghue MJ Gauthier A (2004) Implementing thephylocode Trends Ecol Evol 19(6)281ndash282 doi101016jtree200404004
Eaton DAR Ree RH (2013) Inferring phylogeny andintrogression using RADseq data an example fromflowering plants (Pedicularis Orobanchaceae) SystBiol 62(5)689ndash706 doi101093sysbiosyt032
Edwards SV (2009) Is a new and general theory ofmolecular systematics emerging Evolution 63(1)1ndash19 doi101111j1558-5646200800549x
Edwards DL Knowles LL (2014) Species detection andindividual assignment in species delimitation canintegrative data increase efficacy Proc R Soc B BiolSci 281(1777) doi101098rspb20132765
Egan RS (1986) Correlations and non-correlations ofchemical variation patterns with lichen morphologyand geography The Bryologist 8999ndash110
Elix JA Corush J Lumbsch HT (2009) Triterpenechemosyndromes and subtle morphological characterscharacterise lineages in the Physcia aipolia group inAustralia (Ascomycota) Syst Biodivers 7(04)479ndash487 doi101017S1477200009990223
Emerson KJ Merz CR Catchen JM Hohenlohe PACresko WA Bradshaw WE Holzapfel CM (2010)Resolving postglacial phylogeography using high-throughput sequencing Proc Natl Acad Sci 107(37)16196ndash16200 doi101073pnas1006538107
Ence DD Carstens BC (2011) SpedeSTEM a rapid andaccurate method for species delimitation Mol EcolResour 11(3)473ndash480 doi101111j1755-0998201002947x
EvannoGRegnaut SGoudet J (2005)Detecting thenumberof clusters of individuals using the software STRUC-TURE a simulation study Mol Ecol 14(8)2611ndash2620doi101111j1365-294X200502553x
Falush D Stephens M Pritchard JK (2003) Inference ofpopulation structure using multilocus genotype datalinked loci and correlated allele frequencies Genetics164(4)1567ndash1587 doi101111j1471-8286200701758x
Fan HH Kubatko LS (2011) Estimating species treesusing approximate Bayesian computation Mol Phylo-genet Evol 59(2)354ndash363 doi101016jympev201102019
Fernaacutendez-Mendoza F Printzen C (2013) Pleistoceneexpansion of the bipolar lichen Cetraria aculeata intothe Southern hemisphere Mol Ecol 22(7)1961ndash1983doi101111mec12210
Fernaacutendez-Mendoza F Domaschke S Garciacutea MA JordanP Martin MP Printzen C (2011) Population structure
2 The Dynamic Discipline of Species Delimitation hellip 37
of mycobionts and photobionts of the widespreadlichen Cetraria aculeata Mol Ecol 20(6)1208ndash1232doi101111j1365-294X201004993x
Fraley C Raftery A (2007) Model-based methods ofclassification Using the mclust software in chemo-metrics J Stat Softw 18i06 [Available online at httpwwwdoajorgdoajfunc=abstractampid=218544]
Fujisawa T Barraclough TG (2013) Delimiting speciesusing single-locus data and the generalized mixed yulecoalescent (GMYC) approach a revised method andevaluation on simulated datasets Syst Biol 62(5)707ndash724 doi101093sysbiosyt033
Fujita MK Leacheacute AD Burbrink FT McGuire JA MoritzC (2012) Coalescent-based species delimitation in anintegrative taxonomy Trends Ecol Evol 27(9)480ndash488 doi101016jtree201204012
Gatesy J OrsquoGrady P Baker RH (1999) Corroborationamong data sets in simultaneous analysis hiddensupport for phylogenetic relationships among higherlevel Artiodactyl taxa Cladistics 15(3)271ndash313doi101111j1096-00311999tb00268x
Gladieux P Ropars J Badouin H Branca A Aguileta Gde Vienne DM Rodriacuteguez de la Vega RC Branco SGiraud T (2014) Fungal evolutionary genomicsprovides insight into the mechanisms of adaptivedivergence in eukaryotes Mol Ecol 23(4)753ndash773doi101111mec12631
Goffinet B Miadlikowska J Goward T (2003) Phyloge-netic inferences based on nrDNA sequences supportfive morphospecies within the Peltigera didactylacomplex (Lichenized Ascomycota) The Bryologist106(3)349ndash364 doi10163901
Gowan SP (1986) Evolution of secondary natural productsin the genus Porpidia (Ascomycata Porpidiaceae) AmJ Bot 73606
Griffin PC Hoffmann AA (2014) Limited genetic diver-gence among Australian alpine Poa tussock grassescoupled with regional structuring points to ongoinggene flow and taxonomic challenges Ann Bot doi101093aobmcu017
Grube M Hawksworth DL (2007) Trouble with lichenthe re-evaluation and re-interpretation of thallus formand fruit body types in the molecular era Mycol Res111(9)1116ndash1132 doi101016jmycres200704008
Gueidan C Savi S Thues H Roux C Keller C Tibell LPrieto M Heimarsson S Breuss O Orange A FrobergL Wynns AA Navarro-Rosines P Krzewicka BPykaelae J Grube M Lutzoni F (2009) Genericclassification of the Verrucariaceae (Ascomycota)based on molecular and morphological evidencerecent progress and remaining challenges Taxon 58(1)184ndash208
Guillot G Mortier F Estoup A (2005) Geneland acomputer package for landscape genetics Mol EcolNotes 5(3)712ndash715 doi101111j1471-8286200501031x
Guillot G Renaud S Ledevin R Michaux J Claude J(2012) A unifying model for the analysis of pheno-typic genetic and geographic data Syst Biol 61(6)897ndash911 doi101093sysbiosys038
Guisan A Thuiller W (2005) Predicting species distribu-tion offering more than simple habitat models EcolLett 8(9)993ndash1009 doi101111j1461-0248200500792x
Hale ME (1990) A synopsis of the lichen genusXanthoparmelia (Vainio) Hale (AscomycotinaParmeliaceae) Smithsonian Institution Press Wash-ington DC
Hamilton CA Hendrixson BE Brewer MS Bond JE(2014) An evaluation of sampling effects on multipleDNA barcoding methods leads to an integrativeapproach for delimiting species a case study ofthe North American tarantula genus Aphonopelma(Araneae Mygalomorphae Theraphosidae) MolPhylogenet Evol 7179ndash93 doi101016jympev201311007
Hausdorf B (2011) Progress toward a general speciesconcept Evolution 65(4)923ndash931 doi101111j1558-5646201101231x
Hausdorf B Hennig C (2010) Species delimitation usingdominant and codominant multilocus markers SystBiol 59(5)491ndash503 doi101093sysbiosyq039
Hawksworth DL (1976) Lichen chemotaxonomy InBailey RH (ed) Lichenology progress and problemsAcademic London pp 139ndash184
Hebert PDN Cywinska A Ball SL deWaard JR (2003)Biological identifications through DNA barcodesProc R Soc Lond B Biol Sci 270(1512)313ndash321doi101098rspb20022218
Hebert PDN Stoeckle MY Zemlak TS Francis CM(2004) Identification of birds through DNA barcodesPLoS Biol 2(10)e312 doi101371journalpbio0020312
Heled J Drummond AJ (2010) Bayesian inference ofspecies trees from multilocus data Mol Biol Evol 27(3)570ndash580 doi101093molbevmsp274
Hey J (2006) On the failure of modern species conceptsTrends Ecol Evol 21(8)447ndash450 doi101016jtree200605011
Hibbett DS OhmanA Glotzer D NuhnMKirk P NilssonRH (2011) Progress in molecular and morphologicaltaxon discovery in Fungi and options for formalclassification on environmental sequences Fungal BiolRev 2538ndash47 doi101016jfbr201101001
Houmlgnabba F Wedin M (2003) Molecular phylogeny ofthe Sphaerophorus globosus species complex Cladis-tics 19(3)224ndash232 doi101111j1096-00312003tb00365x
Hudson RR Coyne JA (2002) Mathematical conse-quences of the genealogical species concept Evolu-tion 56(8)1557 doi101111j0014-38202002tb01467x
Huelsenbeck JP Andolfatto P Huelsenbeck ET (2011)Structurama Bayesian inference of population struc-ture Evol Bioinf Online 7(2011)55ndash59 doi104137EBOS6761
Jones G Oxelman B (2014) DISSECT an assignment-free Bayesian discovery method for species delimita-tion under the multispecies coalescent Bioinformaticsdoi101101003178
38 SD Leavitt et al
Kekkonen M Hebert PD (2014) DNA barcode-baseddelineation of putative species efficient start fortaxonomic workflows Mol Ecol Res doi1011111755-099812233
Kelly LJ Hollingsworth PM Coppins BJ Ellis CJHarrold P Tosh J Yahr R (2011) DNA barcoding oflichenized fungi demonstrates high identification suc-cess in a floristic context New Phytol 191(1)288ndash300doi101111j1469-8137201103677x
Kiss L (2012) Limits of nuclear ribosomal DNA internaltranscribed spacer (ITS) sequences as species barcodesfor fungi Proc Natl Acad Sci 109(27)E1811ndashE1811doi101073pnas1207143109
Kluge AG (1989) A concern for evidence and aphylogenetic hypothesis for relationships among Epi-crates (Boidae Serpentes) Syst Zool 38(1)7ndash25doi101093sysbio3817
Knowles LL Carstens BC (2007) Delimiting specieswithout monophyletic gene trees Syst Biol 56(6)887ndash895 doi10108010635150701701091
Kroken S Taylor JW (2001) A gene genealogical approachto recognize phylogenetic species boundaries in thelichenized fungus Letharia Mycologia 93(1)38ndash53
Kubatko LS Degnan JH (2007) Inconsistency of phylo-genetic estimates from concatenated data under coa-lescence Syst Biol 56(1)17ndash24 doi10108010635150601146041
Latch EK Dharmarajan G Glaubitz JC Rhodes OE Jr(2006) Relative performance of Bayesian clusteringsoftware for inferring population substructure andindividual assignments at low levels of populationdifferentiation Conserv Genet 7(2)295ndash302 doi101007s10592-005-9098-1
Lawrey JD (1986) Biological role of lichen substancesThe Bryologist 89(2)111ndash122
Le Gac M Hood ME Fournier E Giraud T (2007)Phylogenetic evidence of host-specific cryptic speciesin the anther smut fungus Evolution 61(1)15ndash26doi101111j1558-5646200700002x
Leacheacute AD (2009) Species tree discordance traces tophylogeographic clade boundaries in North Americanfence lizards (Sceloporus) Syst Biol 58(6)547ndash559doi101093sysbiosyp057
Leacheacute AD Fujita MK (2010) Bayesian species delim-itation in West African forest geckos (Hemidactylusfasciatus) Proc R Soc B Biol Sci 2773071ndash3077doi101098rspb20100662
Leacheacute AD Koo M Spencer C Papenfuss T Fisher RMcGuire J (2009) Quantifying ecological morpho-logical and genetic variation to delimit species in thecoast horned lizard species complex (Phrynosoma)Proc Natl Acad Sci USA 10612418ndash12423 doi101073pnas0906380106
Leacheacute AD Fujita MK Minin VN Bouckaert RR (2014)Species delimitation using genome-wide SNP dataSystematic Biology doi101093sysbiosyu018
Leavitt SD Fankhauser JD Leavitt DH Porter LDJohnson LA St Clair LL (2011a) Complex patterns ofspeciation in cosmopolitan ldquorock posyrdquo lichensmdashdiscovering and delimiting cryptic fungal species in
the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
Leavitt SD Esslinger TL Lumbsch HT (2012c) Neogene-dominated diversification in neotropical montanelichens dating divergence events in the lichen-form-ing fungal genus Oropogon (Parmeliaceae) Am J Bot99(11)1764ndash1777 doi103732ajb1200146
Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Lumbsch HT St Clair LL(2013c) DNA barcode identification of lichen-formingfungal species in the Rhizoplaca melanophthalmaspecies-complex (Lecanorales Lecanoraceae) includ-ing five new species MycoKeys 71ndash22 doi103897mycokeys74508
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Vondraacutek J Thorsten Lum-bsch H Clair LLS (2013d) Local representation ofglobal diversity in a cosmopolitan lichen-formingfungal species complex (Rhizoplaca Ascomycota)J Biogeogr 40(9)1792ndash1806 doi101111jbi12118
Leavitt SD Lumbsch HT Stenroos S St Clair LL(2013e) Pleistocene speciation in North American
2 The Dynamic Discipline of Species Delimitation hellip 39
lichenized fungi and the impact of alternative speciescircumscriptions and rates of molecular evolution ondivergence estimates PLoS ONE 8(12)e85240doi101371journalpone0085240
Leavitt SD Esslinger TL Hansen ES Divakar PK CrespoA Loomis BF Lumbsch HT (2014) DNA barcoding ofbrown Parmeliae (Parmeliaceae) species a molecularapproach for accurate specimen identification empha-sizing species in Greenland Organ Divers Evol 14(1)11ndash20 doi101007s13127-013-0147-1
Leliaert F Verbruggen H Vanormelingen P Steen FLopez-Bautista JM Zuccarello GC De Clerck O(2014) DNA-based species delimitation in algae Eur JPhycol 49(2) doi101080096702622014904524
Leuckert C (1985) Probleme der Flechten-Chemotaxon-omiemdashStoffkombinationen und ihre taxonomischeWertung Ber Deut Bot Ges 98401ndash408
Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
Lohtander K Kaumlllersjouml M Roland M Tehler A (2000)The family physciaceae in fennoscandia phylogenyinferred from its sequences Mycologia 92(4)728ndash735
Longton RE (1997) The role of bryophytes and lichens inpolar ecosystems In Woodin SJ Marquiss M (eds)Ecology of Arctic Environments Blackwell ScienceOxford pp 69ndash96 (Special publication No 13)
Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
Lumbsch HT (1994) Die Lecanora subfusca-Gruppe inAustralasien J Hattori Bot Lab 771ndash175
Lumbsch HT (1998a) Taxonomic use of metabolic data inlichen-forming fungi In Frisvad JC Bridge PDArora DK (eds) Chemical Fungal Taxonomy MarcelDekker New York pp 345ndash387
Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
Mayden RL (1997) A hierarchy of species concepts thedenouement in the saga of the species problem InClaridge MF Dawah HA Wilson MR (eds) Speciesthe units of biodiversity Chapman amp Hall Londonpp 381ndash424
Mayr E (1963) Animal species and evolution HarvardUniversity Press Cambridge
Mayr E (1970) Populations species and evolutionBelknap Press of Harvard University Press Cambridge
McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
McDonald T Miadlikowska J Lutzoni F (2003) Thelichen genus Sticta in the Great Smoky Mountains a
40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
McKay BD Mays HL Wu Y Li H Yao C-T Nishiumi IZou F (2013) An empirical comparison of character-based and coalescent-based approaches to speciesdelimitation in a young avian complex Mol Ecol 22(19)4943ndash4957 doi101111mec12446
Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
Mishler BD Brandon RN (1987) Individuality pluralismand the phylogenetic species concept Biol Philos 2397
Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
Nash TH Zavada M (1977) Population studies amongSonoran Desert species of Parmelia subg Xanthop-armelia (Parmeliaceae) Am J Bot 64(6)664ndash669
Nylander W (1866a) Circa novum in studio Lichenumcriterium chemicum Flora 49198ndash201
Nylander W (1866b) Hypochlorite of lime and hydrate ofpotash Two new criteria for the study of lichens(Tanslated and communicated by the Rev W ALeighton) Bot J Linn Soc 9358ndash365
OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
OrsquoNeill EM Schwartz R Bullock CT Williams JSShaffer HB Aguilar-Miguel X Parra-Olea G Weis-rock DW (2013) Parallel tagged amplicon sequencingreveals major lineages and phylogenetic structure inthe North American tiger salamander (Ambystomatigrinum) species complex Mol Ecol 22(1)111ndash129doi101111mec12049
Orock EA Leavitt SD Fonge BA St Clair LL LumbschHT (2012) DNA-based identification of lichen-form-ing fungi Can publicly available sequence databasesaid in lichen diversity inventories of Mount Cameroon(West Africa) The Lichenologist 44(6)833ndash839doi101017S0024282912000424
Otaacutelora MAG Martiacutenez I Aragoacuten G Molina MC (2010)Phylogeography and divergence date estimates of alichen species complex with a disjunct distributionpattern Am J Bot 97(2)216ndash223 doi103732ajb0900064
Padial J Castroviejo-Fisher S Kohler J Vila C ChaparroJ De la Riva I (2009) Deciphering the products ofevolution at the species level the need for anintegrative taxonomy Zoolog Scr 38(4)431ndash447doi101111j1463-6409200800381x
Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
Papong K Luumlcking R Thammathaworn A BoonpragobK (2009) Four new taxa of Chroodiscus (thelotremoidGraphidaceae) from Southeast Asia The Bryologist112(1)152ndash163 doi1016390007-2745-1121152
Park S-Y Choi J Kim JA Jeong M-H Kim S Lee Y-HHur J-S (2013a) Draft genome sequence of Cladoniamacilenta KoLRI003786 a lichen-forming fungusproducing biruloquinone Genome Announcements 1(5)e00695-13 doi101128genomeA00695-13
Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
Peacuterez-Ortega S Fernaacutendez-Mendoza F Raggio J VivasM Ascaso C Sancho LG Printzen C de los Riacuteos A(2012) Extreme phenotypic variation in Cetrariaaculeata (lichenized Ascomycota) adaptation or inci-dental modification Ann Bot 109(6)1133ndash1148doi101093aobmcs042
Pino-Bodas R Burgaz A Martiacuten M Lumbsch HT (2011)Phenotypical plasticity and homoplasy complicatespecies delimitation in the Cladonia gracilis group(Cladoniaceae Ascomycota) Organ Divers Evol 11(5)343ndash355 doi101007s13127-011-0062-2
Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
Pino-Bodas R Martiacuten M Burgaz A (2012b) Cladoniasubturgida and C iberica (Cladoniaceae) form asingle morphologically and chemically polymorphicspecies Mycol Prog 11(1)269ndash278 doi101007s11557-011-0746-1
Pino-Bodas R Ahti T Stenroos S Martiacuten MP BurgazAR (2013a) Multilocus approach to species recogni-tion in the Cladonia humilis complex (CladoniaceaeAscomycota) Am J Bot 100(4)664ndash678 doi103732ajb1200162
Pino-Bodas R Martiacuten AP Burgaz AR Lumbsch HT(2013b) Species delimitation in Cladonia (Ascomy-cota) a challenge to the DNA barcoding philosophyMol Ecol Resour 13(6)1058ndash1068 doi1011111755-099812086
Pons J Barraclough TG Gomez-Zurita J Cardoso ADuran DP Hazell S Kamoun S Sumlin WD VoglerAP (2006) Sequence-based species delimitation for theDNA taxonomy of undescribed insects Syst Biol 55(4)595ndash609 doi10108010635150600852011
Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
Pringle A Baker DM Platt JL Wares JP Latgeacute JPTaylor JW (2005) Cryptic speciation in the cosmo-politan and clonal human pathogenic fungus Asper-gillus fumigatus Evolution 59(9)1886ndash1899 doi10155404-2411
Printzen C (2009) Lichen systematics the role ofmorphological and molecular data to reconstructphylogenetic relationships Progress in botany vol71 Springer Berlin pp 233ndash275
Pritchard JK Stephens M Donnelly P (2000) Inference ofpopulation structure using multilocus genotype dataGenetics 155(2)945ndash959
Puillandre N Lambert A Brouillet S Achaz G (2012)ABGD automatic barcode gap discovery for primaryspecies delimitation Mol Ecol 21(8)1864ndash1877doi101111j1365-294X201105239x
Rannala B Yang Z (2003) Bayes estimation of speciesdivergence times and ancestral population sizes usingDNA sequences from multiple loci Genetics1641645ndash1656
Raxworthy CJ Ingram CM Rabibisoa N Pearson RG(2007) Applications of ecological niche modelingfor species delimitation a review and empiricalevaluation using day geckos (Phelsuma) fromMadagascar Syst Biol 56(6)907ndash923 doi10108010635150701775111
Ray J (1686) Historia planarum vol 1 Clark LondonReese Naeligsborg R Ekman S Tibell L (2007) Molecular
phylogeny of the genus Lecania (Ramalinaceaelichenized Ascomycota) Mycol Res 111(5)581ndash591doi101016jmycres200703001
Reid N Carstens B (2012) Phylogenetic estimation errorcan decrease the accuracy of species delimitation aBayesian implementation of the general mixed Yule-coalescent model BMC Evol Biol 12(1)196 doi1011861471-2148-12-196
Rissler L Apodaca J (2007) Adding more ecology intospecies delimitation ecological niche models andphylogeography help define cryptic species in theBlack Salamander (Aneides flavipunctatus) Syst Biol56924ndash942 doi10108010635150701703063
Rivas Plata E Luumlcking R (2013) High diversity ofGraphidaceae (lichenized Ascomycota Ostropales) inAmazonian Peruacute Fungal Divers 58(1)13ndash32 doi101007s13225-012-0172-y
Rivas Plata E Lumbsch HT (2011) Parallel evolution andphenotypic divergence in lichenized fungi a casestudy in the lichen-forming fungal family Graphida-ceae (Ascomycota Lecanoromycetes Ostropales)Mol Phylogenet Evol 61(1)45ndash63 doi101016jympev201104025
Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
Ross KG Gotzek D Ascunce MS Shoemaker DD (2010)Species delimitation a case study in a problematic anttaxon Syst Biol 59(2)162ndash184 doi101093sysbiosyp089
Rubin BER Ree RH Moreau CS (2012) Inferringphylogenies from RAD sequence data PLoS ONE 7(4)e33394 doi101371journalpone0033394
Rubinoff D (2006) Utility of mitochondrial DNA barcodesin species conservation Conserv Biol 20(4)1026ndash1033 doi101111j1523-1739200600372x
Ruiz-Sanchez E Sosa V (2010) Delimiting speciesboundaries within the Neotropical bamboo Otatea(Poaceae Bambusoideae) using molecular morpho-logical and ecological data Mol Phylogenet Evol 54(2)344ndash356 doi101016jympev200910035
Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
morphological characters Antarctic Sci 22 (SpecialIssue 06)727ndash741 doi101017S0954102010000477
Salicini I Ibaacutentildeez C Juste J (2011) Multilocus phylogenyand species delimitation within the Nattererrsquos batspecies complex in the Western Palearctic MolPhylogenet Evol 61(3)888ndash898 doi101016jympev201108010
Satler JD Carstens BC Hedin M (2013) Multilocusspecies delimitation in a complex of morphologicallyconserved trapdoor spiders (Mygalomorphae Antro-diaetidae Aliatypus) Syst Biol 62(6)805ndash823 doi101093sysbiosyt041
Schlick-Steiner BC Steiner FM Seifert B Stauffer CChristian E Crozier RH (2010) Integrative taxonomya multisource approach to exploring biodiversityAnnu Rev Entomol 55(1)421ndash438 doi101146annurev-ento-112408-085432
Schoch CL Seifert KA Huhndorf S Robert V SpougeJL Levesque CA Chen W Fungal Barcoding Con-sortium (2012) Nuclear ribosomal internal transcribedspacer (ITS) region as a universal DNA barcodemarker for Fungi Proc Natl Acad Sci 109(16)6241ndash6246 doi101073pnas1117018109
Seacuterusiaux E Villarreal AJC Wheeler T Goffinet B(2011) Recent origin active speciation and dispersalfor the lichen genus Nephroma (Peltigerales) inMacaronesia J Biogeogr 38(6)1138ndash1151 doi101111j1365-2699201002469x
Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
Simpson GG (1951) The species concept Evolution5285ndash298
Sites JW Marshall JC (2003) Delimiting species a renais-sance issue in systematic biology Trends Ecol Evol18462ndash470 doi101016S0169-5347(03)00184-8
Sites JW Marshall JC (2004) Operational criteria fordelimiting species Annu Rev Ecol Evol Syst 35(1)199ndash227 doi101146annurevecolsys35112202130128
Šlapeta J Loacutepez-Garciacutea P Moreira D (2006) Globaldispersal and ancient cryptic species in the smallestmarine eukaryotes Mol Biol Evol 23(1)23ndash29doi101093molbevmsj001
Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
Talavera G Dincă V Vila R (2013) Factors affectingspecies delimitations with the GMYC model insightsfrom a butterfly survey Methods Ecol Evol 4(12)1101ndash1110 doi1011112041-210X12107
Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
Tewksbury J Anderson JGT Bakker JD Billo TJDunwiddie PW Groom MJ Hampton SE HermanSG Levey DJ Machnicki NJ del Rio CM PowerME Rowell K Salomon AK Stacey L TrombulakSC Wheeler TA (2014) Natural historyrsquos place inscience and society BioScience doi101093bioscibiu032
Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
Werth S Cornejo C Scheidegger C (2013) Characteriza-tion of microsatellite loci in the lichen fungus Lobariapulmonaria (Lobariaceae) Appl Plant Sci 1(2)1200290 doi103732apps1200290
Westberg M Arup U Kaumlrnefelt I (2007) Phylogeneticstudies in the Candelariaceae (lichenized Ascomy-cota) based on nuclear ITS DNA sequence dataMycol Res 111(11)1277ndash1284 doi101017S0953756204002102
Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
2 The Dynamic Discipline of Species Delimitation hellip 43
(Lepidoptera Lycaenidae) Front Zoo 4(1)8 doi1011861742-9994-4-8
Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
Table
21
Somemetho
dsused
forspeciesdelim
itatio
ninclud
ingspeciesdiscov
erymetho
dsforassign
ingsamples
topo
pulatio
nswith
outapriori
inform
ation(BAPS
Gaussianclustering
Guillo
trsquosUnified
Mod
elS
TRUCTURES
TRUCTURAMA)genetic
distance-based
metho
dforsortingsequ
encesinto
hypo
theticalspecies(A
BGD)tree-
basedspeciesdiscov
erymetho
ds(bGMYCG
MYCb
PTP
PTP
ldquoSpecies
Delim
itatio
nrdquoplug
-inforGeneiou
s)and
jointdiscov
eryandvalid
ationmetho
ds(BPamp
PBrownie
DISSE
CTSp
eDeStem)
Metho
dDescriptio
nInpu
tdata
BAPS
mdashpo
pulatio
nassignmentusing
Bayesianclustering
Aprogram
forBayesianinferenceof
thegenetic
structurein
apopulatio
nclustering
molecular
data
andperformingadmixture
analysesGenetic
mixture
analyses
canbe
performed
atbo
thgroupandindividu
allevelsusingeither
ano
n-spatialo
rspatialm
odel
BAPS
treatsboth
theallele
frequenciesof
themolecular
markers
(ornucleotid
efrequenciesforDNA
sequ
ence
data)andthenu
mberof
genetically
diverged
groups
inpopulatio
nas
random
variablesIn
theldquoclusteringwith
linkedlocirdquomod
elagenetic
mixture
analysiscanbe
done
usinghaploidsequ
ence
data
orotherlin
kedgenetic
markersA
nalysesandmodel
comparisons
canalso
beperformed
usingafixednu
mber
ofgenetically
diverged
grou
psor
prespecified
populatio
nstructures
Limita
tions
Tem
poraldivergence
andrelatio
nships
amongputativ
egroups
arenot
explicitlyestim
atedE
quivalence
togenetic
clusters
tospecies-levelg
roupsisuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
Availablefrom
httpwwwhelsinkifi
bsgsoftwareBAPS
describedin
Coran-
deret
al(200
420
0620
08)CoranderandMarttinen20
06)
Genotyp
icdatahaploidsequence
dataor
linkedmarkers
(AFL
Por
SNPs)
Gaussianclusteringmdashpo
pulatio
nassignment
usingGaussianclustering
Aprogram
forBayesianinferenceof
thegenetic
structurein
apopulatio
nModelgroups
sampleinto
popu
latio
nsusinggeno
typicdata
bysearchingforclusters
that
canbe
attributed
tobeingmixturesof
norm
alallelefrequencydistributio
nsG
aussianclustering
requires
adatasetwhere
thecasesaredefinedby
variable
ofmetricscaleandhasbeen
used
with
geneticenvironmentalandmorphologicaldatasetsindividuallyinadditio
nto
integrated
datasets
Limita
tions
Tem
poraldivergence
andrelatio
nships
amongputativ
egroups
arenot
explicitlyestim
atedE
quivalence
togenetic
clusters
tospecies-levelg
roupsisuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
Implem
entedin
Rusingtheprabclus
(HausdorfandHennig20
10)andmclust
packages
(FraleyandRaftery
2007)
Genotyp
icdata
(flexible)
(con
tinued)
18 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
Guillo
trsquosUnified
Mod
elmdashpo
pulatio
nassignmentusingBayesianclustering
Thisapproach
provides
astatistical
model
that
allowsoneto
analyzegenetic
and
phenotyp
ticdata
with
inaun
ified
mod
elandinferencefram
eworkandop
tionally
incorporateinform
ationaboutthespatialdistributio
nof
samplesA
Bayesianclustering
algorithm
assumes
that
each
clusterin
ageographical
domaincanbe
approxim
ated
bypo
lygons
that
arecentered
around
pointsgeneratedby
aPo
issonprocessGuillo
trsquos
Unified
Mod
elisflexiblein
term
sof
thegenetic
data
that
itcanutilize
andcapableof
accurately
delim
iting
species
Limita
tions
Genetic
andphenotypic
data
cantracedifferentevolutionary
historiesfor
instancephylogenetic
divergence
forneutralgenetic
markers
andadaptatio
nfora
morphological
structure
Source
Availableas
anextensionof
theRGENELAND
package(G
uillo
tet
al20
05
2012)(http
www2im
mdtudk
gigu
Geneland)
Genotyp
icandno
n-genetic
(eg
phenotypicalecolog
icalgeograph
ical
behavioral)data
STRUCTUREmdash
populatio
nassignment
usingBayesianclustering
Amodel-based
clustering
methodusingmultilocus
genotype
data
toinferpopulatio
nstructureandassign
individualsto
populatio
nsIndividualsin
thesampleareassigned
probabilistically
topopulatio
nso
rjointly
totwoor
morepopulatio
nsiftheirgenotypes
indicatethatthey
areadmixedT
hemodeldoes
notassum
eaparticular
mutationprocess
anditcanbe
appliedto
mosto
fthecommonly
used
genetic
markersp
rovidedthat
they
areno
tcloselylin
ked
The
methodcanproducehigh
lyaccurate
assignmentsusing
modestn
umbersof
loci(Pritchard
etal2
000)T
hemostappropriatelevelo
fpopulatio
nstructurecanbe
inferred
byassessinglik
elihoodscores
orthead
hocΔKstatistic
(Evann
oet
al20
05)
Limita
tions
Identifying
themostappropriatenumberof
genetic
clusters
ischallenging
clusters
produced
byST
RUCTUREcanbe
strongly
influenced
byvariationin
sample
sizeclusters
createdby
STRUCTUREmay
notbe
consistent
with
theevolutionary
historyof
thepopulatio
nswhentherearerelativ
elylong
divergence
times
with
inevolutionary
lineagesTem
poraldivergence
andrelatio
nships
amongputativ
egroups
isnotexplicitlyestim
atedEquivalence
togenetic
clusters
tospecies-levelgroups
isuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
httppritchardlabstanfordedustructurehtm
ldescribedin
Falush
etal(200
3)andPritchard
etal(200
0)
Genotyp
icdata
(con
tinued)
2 The Dynamic Discipline of Species Delimitation hellip 19
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
Structuram
amdashpo
pulatio
nassignmentusing
Bayesianclustering
Implem
entstheclustering
algorithm
used
inST
RUCTURE(see
above)
that
clusters
samples
into
populatio
nsby
minim
izingHardyndashWeinb
ergdisequ
ilibrium
foragiven
partitioninglevelHow
everStructuram
aalso
includes
theadditio
nof
reversible-jum
pMCMCto
identifytheop
timalpartition
inglevelNearlyanytype
ofgenetic
datacanbe
inpu
tinto
Structuram
aandtheprog
ram
canassign
individu
alsto
populatio
nwith
orwith
outtheadmixture
Limita
tions
Tem
poraldivergence
andrelatio
nships
amongputativ
egroups
isnot
explicitlyestim
atedE
quivalence
togenetic
clusters
tospecies-levelg
roupsisuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
httpctegberkeleyedu
structuram
aindexhtmldescribedin
Huelsenbeck
etal(201
1)
Genotyp
icdata
ABGDmdashbarcodegapusinggenetic
distances
ldquoAutom
aticBarcode
Gap
Discoveryrdquosortssequencesinto
hypotheticalspeciesbasedon
thebarcodegap
The
methoduses
arecursiveapproach
topartition
thedata
andtestfor
sign
ificant
gapsA
BGDisfastsim
plemethodto
split
asequence
alignm
entd
atasetinto
candidatespeciesthat
should
becomplem
entedwith
otherevidence
inan
integrative
taxono
mic
approach
Limita
tions
Single-locus
data
aloneshould
only
beused
toprovideaprelim
inary
perspectiveof
speciesboundaries
andnotas
thesole
evidence
inspecies
circum
scriptions
Source
httpwwwabisnv
jussieufrpublicabg
ddescribedin
Puillandreet
al(201
2)
Single-locus
sequence
alignm
ent
GMYCampbG
MYCmdashgene
tree
The
GMYC
approach
combinesacoalescent
model
ofintraspecificbranchingwith
aYulemod
elforinterspecificbranching
which
isthen
fitto
aninferred
sing
le-gene
topology
toestim
atespeciesboundaries
andastatistical
measure
ofconfi
denceforthe
inferred
boundariesA
san
inputtheGMYCapproach
requires
anultram
etricgene
tree
andrecent
refinementscanaccountforuncertaintyin
phylogenetic
relatio
nships
and
parametersof
theGMYCmod
elT
heGMYCisgenerally
stable
across
awiderangeof
circum
stancesincludingvariousmethods
ofphylogeneticreconstructio
nthepresence
ofahigh
numbersingletonshigh
numbers
ofsampled
speciesandgaps
inintraspecific
sampling
theaccuracy
oftheGMYCismostsign
ificantly
affected
bythemean
populatio
nsize
relativ
eto
divergence
times
betweenthem
Limita
tions
The
GMYCmay
delim
itwell-supportedclades
ofhaplotypes
asindependent
lineagesandas
such
may
beproneto
over-delim
itatio
nSingle-locus
data
aloneshould
only
beused
toprov
ideaprelim
inaryperspectiveof
speciesbo
undaries
andno
tas
the
sole
evidence
inspeciescircum
scriptions
Source
(http
r-forger-projecto
rgprojectssplitshttpssitesgooglecom
site
noahmreidhom
esoftware)describedin
Monaghanet
al(200
9)Fu
jisaw
aandBarrac-
loug
h(201
3)andPo
nset
al(200
6)
Sing
le-locus
ultram
etricgene
tree (con
tinued)
20 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
PTPamp
bPTPmdash
gene
tree
The
Poissontree
processes(PTP)
modelcanbe
used
toinferputativ
especiesboundaries
onagivenphylogenetic
inputtreePT
Pcaninferputativ
especiesboundaries
that
are
consistent
with
thePS
CAnim
portantadvantageof
thismethodisthat
itmod
els
speciatio
nin
term
sof
thenumberof
substitutionsthereby
circum
ventingthepotentially
error-proneandcompute-intensive
processof
generatin
gultram
etrictreeswhich
are
required
asan
inpu
tfor
GMYCmod
el(see
abov
e)F
urthermoreitappearsthatthePT
Pmodel
may
outperform
theGMYCandotherOTU-picking
methods
whenevolutionary
distancesaresm
all
Limita
tions
Single-locus
data
aloneshould
only
beused
toprovideaprelim
inary
perspectiveof
speciesboundaries
andnotas
thesole
evidence
inspecies
circum
scriptions
Source
httpscoh-itsorgexelix
iswebsoftwarePT
Pindexhtmldescribedin
Zhang
etal(201
3)
Sing
le-locus
gene
tree
GeneiousSp
eciesDelim
itatio
nplug
-inmdash
gene
tree
Aplug-into
theGeneioussoftwareprovides
anexploratorytool
allowingtheuser
toassess
phylog
enetic
supportanddiagno
sabilityof
speciesdefinedas
user-selected
collections
oftaxa
onuser-sup
pliedtreesThe
plug
-incompu
tesstatisticsrelatin
gto
the
probability
oftheob
served
mon
ophyly
orexclusivity
having
occurred
bychance
ina
coalescent
processandassesses
thewith
in-andbetween-speciesgenetic
distancesto
infertheprobability
with
which
mem
bers
ofaputativ
especiesmight
beidentifi
edsuccessfully
with
tree-based
methods
Limita
tions
The
plug-insummarizes
measuresof
phylogenetic
supportand
diagnosabilityof
speciesdefinedas
user-selectedcollections
oftaxabutitdoes
not
providedefinitiv
esupportforspeciesgroupsItassumes
speciesaremonophyletic
Source
Implem
entedas
aplug-into
Geneious(geneiouscom)describedin
Mastersetal
(201
1)
Sing
le-locus
gene
tree
BPamp
Pmdashmultispecies
coalescent
model
for
speciesvalid
ation
Thisapproach
tospeciesdelim
itatio
nuses
aBayesianmodelingapproach
togeneratethe
posteriorprobabilitiesof
speciesassignmentstaking
accountof
uncertaintiesdueto
unknow
ngene
treesandtheancestralcoalescent
processThe
methodrelieson
auser-
specified
guidetreeimplem
entin
gareversible-jum
pMarkovchainMonte
Carlo
search
ofparameter
spacethatincludes
θpopulatio
ndivergenceand
estim
ated
distributio
nsof
gene
treesfrom
multip
leloci
Limita
tions
Misspecificatio
nsof
priorsandthegu
idetree
canresultin
inflatedspeciatio
nprobabilitiesitassumes
norecombinatio
nandcomputatio
nallim
itatio
nsrestrict
itsutility
with
next-generationdatasetswith
100s
ofloci
Source
httpabacusgeneuclacuksoftwarehtmldescribedin
YangandRannala
(201
0)andRannala
andYang(200
3)
Multilocus
sequence
alignm
entsandgroup
mem
bership
(con
tinued)
2 The Dynamic Discipline of Species Delimitation hellip 21
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
DISSE
CTmdashmultispecies
coalescent
model
forspeciesdelim
itatio
nDISSE
CTexplores
thefullspaceof
possible
clusteringsof
individualsandspeciestree
topologies
inaBayesianfram
ework
Toavoidtheneed
forreversible-jum
pMCMCit
uses
anapproxim
ationin
theform
ofapriorthatisamodificatio
nof
thebirthndashdeathprior
forthespeciestreeItisim
plem
entedas
partof
BEAST
andrequires
only
afewchanges
from
astandard
BEAST
analysisA
nalysesof
simulated
andem
piricald
atasuggestthat
themethodisshow
nto
beinsensitive
tothedegree
ofapproxim
ation
butq
uitesensitive
tootherparameters
Limita
tions
Recently
describedmethodlackingathorough
theoretical
andem
pirical
evaluatio
nItappearsthat
alargenu
mberof
sequencesarerequ
ired
todraw
firm
conclusion
sSo
urce
httpcodegooglecompbeast-m
cmcamphttpwwwin
driid
com
dissectinbeast
htmldescribedin
JonesandOxelm
an(201
4)
Multilocus
sequence
data
SpeD
eSTEMmdashmultispecies
coalescent
mod
elfordiscov
ery
valid
ation
andjoint
estim
ation
Thismaxim
umlik
elihoo
dandorinform
ationtheory-based
methodwas
developedto
test
speciesboundaries
inasystem
with
existin
gsubspecies
taxonomy(CarstensandDew
ey20
10)andcomputestheprobability
ofthegene
treesgiventhespeciestree
forall
hierarchical
perm
utations
oflin
eage
grou
pingSp
eciesbo
undaries
arecomparedusing
Akaikeinform
ationcriteria
andphylogenetic
uncertaintyin
thespeciestree
topologies
does
notaffect
speciesdelim
itatio
nsLimita
tions
Accuracyisdependenton
quality
ofthegene
tree
estim
ates
Source
(http
carstenslaborgohio-stateedusoftwarehtml)criteria
describedin
Ence
andCarstens(201
1)
Multilocus
sequence
alignm
entsandgroup
mem
bership
Browniemdash
multispecies
coalescent
model
forspeciesdelim
itatio
nThe
nonp
aram
etricheuristic
speciesdelim
itatio
napproach
implem
entedin
theprog
ram
Brownie(O
rsquoMeara
2010)jointly
sortsanonym
oussamples
into
speciesandinfers
aspeciestree
from
inputg
enetreesfrom
differentlociassumingthatforaspeciatio
neven
thecorrespondingnodeson
gene
treeswill
bemoreconsistent
with
each
than
the
divergenceswith
inspecies
Limita
tions
Findingboth
theoptim
umspeciestree
andspeciesboundaries
remains
compu
tatio
nally
challenging
andBrowniehasbeen
show
nto
frequently
yieldincorrect
resultsT
heaccuracy
ofthemethodislik
elycorrelated
with
nodalsupportvalues
inthe
individu
algene
topo
logies
Source
httpwwwbrianom
earain
fobrownie
describedin
OrsquoM
eara
(201
0)
Individual
gene
trees
(con
tinued)
22 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
BFD
mdashmultispecies
coalescent
model
for
speciesdelim
itatio
nThe
recently
developedmethod
Bayes
factor
delim
itatio
n(with
genomicdataB
FD)
combinesady
namic
programmingalgorithm
forestim
atingspeciestreesthat
bypasses
thecompu
tatio
nally
intensiveMCMCintegrationov
ergene
treesto
prov
idearigorous
techniqueforspeciesdelim
itatio
nstudiesusinggenome-wideSN
PdataCom
petin
gspeciesdelim
itatio
nmodelsarecomparedusingBayes
factorsanditappearsthat
this
approach
isrobustto
samplesizes(iefew
loci
andlim
itedsamples
perspecies)
and
misspecificatio
nof
thepriorforpo
pulatio
nsize
(θ)
Limita
tions
Recently
describedmethodlackingathorough
theoretical
andem
pirical
evaluatio
nSo
urce
httpwwwbeast2orgwikiindexphpBFD
describedin
Leacheacuteet
al(201
4)
Genom
e-wideSN
Pdata
2 The Dynamic Discipline of Species Delimitation hellip 23
distances among organisms belonging to thesame species are smaller than distances amongorganisms from different species (Puillandreet al 2012 Hebert et al 2003) Genetic distanceapproaches hold particular promise as an identi-fication tool shortcutting the difficulties of mor-phology-based identification (Hebert et al 2004)although in practice a barcode gap may not existfor many groups (Wiemers and Fiedler 2007)Furthermore the role of distance-based approa-ches using a single genetic marker for speciesdiscovery remains more controversial (Rubinoff2006) and without other corroborating evidenceOTUs inferred from single-locus dataset shouldonly be considered ldquocandidaterdquo species Tree-based methods aim to detect monophyletic cladescorresponding to species-level diversity bydetecting discontinuities associated within inter-and intraspecific branching patterns (Fujisawaand Barraclough 2013 Zhang et al 2013Monaghan et al 2009 Pons et al 2006) Tree-based species delimitation methods can also beused on the basis of other properties related tophylogenetic tree topologies (monophyly con-cordance with geography etc (Sites and Marshall2003 2004) Both distance- and tree-basedmethods have been applied for assessing speciesboundaries in lichen-forming fungi (Leavitt et al2012c 2014 Parnmen et al 2012 Del-Pradoet al 2010 2011 Molina et al 2011)
A number of tree-based methods partiallyautomate the species delimitation process withspecific bioinformatical analyses (Table 21)including the general mixed Yule coalescent(GMYC) approach (Fujisawa and Barraclough2013 Monaghan et al 2009 Pons et al 2006)and the Poisson tree processes (PTP) model(Zhang et al 2013) These methods provide rel-atively straightforward and objective pipelinesfor delimiting putative species-level lineagesfrom inferred gene trees by fitting within- andbetween-species branching models to an inferredsingle-locus topology A number of other tree-based methods for species delimitation areeffectively summarized in Sites and Marshall(2004)
When only single-locus data are available theGMYC has been shown to be a relatively robust
tool for species delimitation The GMYCapproach combines a coalescent model of intra-specific branching with a Yule model for inter-specific branching which is then fit to an inferredsingle-gene topology to estimate species bound-aries and a statistical measure of confidence for theinferred boundaries As an input the GMYCapproach requires an ultrametric gene tree andrecent refinements can account for uncertainty inphylogenetic relationships and parameters of theGMYC model (Fujisawa and Barraclough 2013Reid and Carstens 2012) Regardless of theseimprovements it may be difficult to accuratelyinfer an adequate ultrametric tree for large data-sets Although it appears that the GMYC isgenerally stable across a wide range of circum-stances including various methods of phyloge-netic reconstruction the presence of a highnumber singletons high numbers of sampledspecies and gaps in intraspecific sampling theaccuracy of the GMYC is most significantlyaffected by the mean population size relative todivergence times between them (Fujisawa andBarraclough 2013 Talavera et al 2013) Fur-thermore research suggests that the so-calledsingle-threshold version of the GMYC methodlikely outperforms the multiple-thresholdapproach (Fujisawa and Barraclough 2013Monaghan et al 2009) However other studiessuggest that theGMYCmethodmay often providehigher estimates for the total number ofOTUs thanother molecular species delimitation methods(Hamilton et al 2014 Miralles and Vences 2013Talavera et al 2013) warranting a cautiousinterpretation of results from GMYC analyses
Compared to the GMYC approach therecently introduced PTP method for speciesdelimitation has been suggested to be moreaccurate for preliminary species delimitation(Zhang et al 2013) Relative to the GMYC PTPoffers a more straightforward implementationrequiring a simple phylogenetic tree rather thanan ultrametric chronogram However moreresearch is required to assess the general perfor-mance of PTP across wide range of empirical andsimulated species delimitation studies At thispoint data support the use of the GMYC andPTP methods as objective and reasonable starting
24 SD Leavitt et al
points for species delimitation using single-genetopologies
The ldquoSpecies Delimitationrdquo plug-in to theGeneious software provides statistical approachesfor exploring species boundaries in single-genetopologies (Masters et al 2011) Using a priorispecimen assignments to putative species theldquoSpecies Delimitationrdquo plug-in computes statis-tics relating to the probability of the observedmonophyly or exclusivity having occurred bychance in a coalescent process and assesses thewithin- and between-species genetic distances inorder to infer the probability with which membersof a putative species might be identified suc-cessfully with tree-based methods An importantcontribution of the ldquoSpecies Delimitationrdquo plug-in is that it provides an objective means for usersto assess putative species within an empiricalstatistic-based framework rather than qualitativeevaluations of what level of hierarchy constitutesa species in a phylogeny
In contrast to simple sequence similaritythresholds (OTU-picking) for delimiting putativeevolutionarily significant units the AutomaticBarcode Gap Discovery (ABGD) method is anautomated procedure that sorts sequences intohypothetical species based on the existence of abarcode gap observed whenever intraspecificgenetic distances are less than those amongorganisms from different species (Puillandreet al 2012) Ultimately ABGD is a fast simplemethod that can be used to group individualsrepresented in a single-locus sequence alignmentinto candidate species that should be comple-mented with other lines of evidence in an inte-grative taxonomic approach (Kekkonen andHebert 2014 Puillandre et al 2012)
In general the ABGD GMYC and PTP ana-lytical protocols using single-locus data arerepeatable and computationally relatively fastproviding a valuable starting point for a pre-liminary perspective into species boundaries inunderstudied groups that can be validated withsubsequent studies (Kekkonen and Hebert 2014)Similarly analyses of single-locus data can beused to corroborate traditional phenotype-basedspecies boundaries and identify candidate speciesthat have previously been hidden within nominal
species However single-locus species delimita-tionmethodsmaybeparticularly prone to failure inrecognizing significant proportions of species-level biodiversity due to the strict criterion forreciprocal monophyly or alternatively providespurious inflations of estimated species diversitybased on genetic differences that donot correspondto species-level lineages For example recentestimates suggest that the incidence of species-level gene tree paraphyly is approximately 20 (Ross 2014) suggesting that analyses of single-locus datasets would likely fail to accurately deli-mit species in one in every five cases In contrastrecent empirical studies suggest that in some casesthe GMYC provides a striking overestimate ofspecies diversity (Miralles and Vences 2013)
The internal transcribed spacer region (ITS)has played a central role in molecular phyloge-netic studies of lichenized fungi and has recentlybeen adopted as the official barcoding marker forfungi (Schoch et al 2012) In many cases the ITSis sufficiently variable to resolve species bound-aries for lichenized fungi (Schoch et al 2012Kelly et al 2011) although accurate specimenidentification using sequence-based identificationapproaches requires a thoroughly curated refer-ence database (Leavitt et al 2014 Orock et al2012 Kelly et al 2011) In spite of the generalutility of the ITS marker a number of issues maypotentially limit its effective use in speciesdelimitation studies including the potential forintragenomic variation of the nuclear ribosomaltandem repeat and difficulties in aligning ITSsequences from divergent taxa (Kiss 2012)Because the ITS has been formally adopted as theofficial barcoding marker for fungi we recom-mend that species delimitation studies attempt toinclude this region for consistency across studiesHowever we recognize that in some taxonomicgroups the inclusion of the ITS may be prob-lematic and therefore suggest a careful screeningof other markers to identify appropriate loci forresolving species-level relationships
Ultimately the success of any single-locusspecies delimitation method largely depends onthe evolutionary history of the species groupunder study and the variability of the selectedmarker (Fig 22) Species delimitation will be
2 The Dynamic Discipline of Species Delimitation hellip 25
more difficult in more recently diverged lineagesand in cases with some level of interspecific geneflow relative to older well-diverged lineages(eg Leavitt et al 2012c 2013e) In the end wereemphasize that single-locus data alone shouldonly be used to provide a preliminary perspectiveof species boundaries and not as the sole evi-dence in species circumscriptions
222 Sampling Across the GenomeMultilocus Sequence Dataand Genome-Wide Markers
Although single-locus methods can provide anefficient approach for preliminary large-scaleassessments of species diversity there are
significant limitations particularly in recentlydiverged species groups where retention ofancestral polymorphisms and incomplete lineagesorting will likely result in different neutral locihaving unique gene topologies that do not mirrorthe speciation process (Knowles and Carstens2007 Heled and Drummond 2010 Taylor et al2000) In contrast to single-locus and strictlyphenotype-based approaches for species delimi-tation analyses of genetic data collected fromindependent genomic regions can provide robusthypotheses of species boundaries with increasingconfidence (Satler et al 2013 Zhang et al 2011Yang and Rannala 2010) Sequences from mul-tiple independent loci provide an importantsource of data for species delimitation studiesincluding recently developed models that
Fig 22 A simplified diagram illustrating the process ofspeciation through time in a single gene history andresulting gene topologies sampled at two points in thespeciation history (modified from Leliaert et al 2014)aEach dot represents a distinct gene copy and each row onenon-overlapping generation with lines connecting genecopies to their ancestors in the previous generation (onerow below) dashed diagonal lines represent reproductivebarriers two hypothetical sampling intervals at differentpoints in the speciation history are shown Tx and Ty
species delimitation methods using single-locus data areeffective only when species are reciprocally monophyleticin the sampled gene tree genetic clustering and coalescent-based species delimitation methods can circumscribespecies when species may not be monophyletic in sampledgenetic loci b Gene topology representing sampledhaplotypes at time Tx (shown in panel a) hypotheticalspecies are reciprocally monophyletic c Gene topologyrepresenting sampled haplotypes at time Ty (shown in panela) hypothetical species are para- and polyphyletic
26 SD Leavitt et al
combine individual gene genealogies and speciesphylogenies via modeling the coalescent historyof markers (Yang and Rannala 2010 Edwards2009 Knowles and Carstens 2007) As aresponse to advances in sequencing technologiesbioinformatical approaches for multimarker spe-cies delimitation analyses continue to be devel-oped (Table 21 Camargo and Sites 2013)
Long-term reproductive isolation of candidatespecies can be assessed with multilocus sequencedata by evaluating genealogical concordance ofunlinked markers (Baum and Shaw 1995 Aviseand Ball 1990) Within species the mixingeffects of recombination cause unlinked loci tohave distinct genealogical histories but geneticdrift and long-term divergence leads to concor-dant genealogical histories at loci across thegenome Relationships of individuals belongingto distinct candidate species can be evaluatedusing gene genealogies (eg haplotype networksor single-gene topologies) to identify lineagesthat exhibit genealogical exclusivity acrossunlinked neutral loci (Hudson and Coyne 2002Dettman et al 2003b Avise and Ball 1990) Thepresence of the same clades in the majority ofsingle-locus genealogies is taken as evidence thatthe clades represent reproductively isolated lin-eages (Dettman et al 2003a Pringle et al 2005)In practice the criteria of reciprocal monophylyand genealogical concordance of unlinked lociprovide a conservative approach for assessingspecies boundaries due to the fact that a sub-stantial amount of time is required after the initialdivergence of species until ancestral polymor-phisms have fully sorted (Knowles and Carstens2007 Hudson and Coyne 2002) Consequentlygroups with recent divergence histories willlikely go undiscovered under a genealogicalconcordance criterion due to the fact that speciesboundaries likely are not reflected in genegenealogies For example it would take morethan 1 million years after speciation before spe-cies would be delimited if 15 loci were sampledin species with an effective population size (Ne)of 100000 assuming one generation a yearunder a strict reciprocal monophyly criterion(Knowles and Carstens 2007) The amount oftime required for a species to be recognized using
a criterion of reciprocal monophyly increasesproportionally with increasing population sizes(Hudson and Coyne 2002) In a number ofstudies of lichen-forming fungi a genealogicalconcordance criterion has been used to bothdelimit previously unrecognized species-levellineages and validate some conventional pheno-type-based species (Leavitt et al 2012a c2013b Molina et al 2011 Kroken and Taylor2001)
Although analyses of molecular sequence datarelying on reciprocal monophyly or fixed char-acter differences for species delimitation canserve as important criteria for identifying speciesthese criteria are not commonly met acrossmultiple loci particularly in recent speciationhistories (Fig 22) To accommodate theobserved conflict among genealogies from mul-tiple loci with the underlying speciation historythe recent merge of coalescent theory with phy-logenetics has driven a major paradigm shift inspecies delimitation and molecular systematics ingeneral (Fujita et al 2012 Edwards 2009) Themultispecies coalescent model can be applied togenealogical histories from multiple independentloci to assemble separate coalescent processesoccurring in populations into a species tree(Degnan and Rosenberg 2009 Rannala andYang 2003) Within this coalescent-basedframework multiple individuals can be assignedto a single speciespopulation and the speciationhistory of ancestral and descendant species-levellineages can be inferred as a ldquospecies treerdquo incontrast to estimating gene genealogies fromindividual samples (Degnan and Rosenberg2006 2009 Rannala and Yang 2003)
A number of multispecies coalescent-basedspecies delimitation methods have recently beenintroduced offering an exciting framework forempirically assessing species boundaries byselecting the best species tree model from a set ofalternative models representing differenthypotheses of species boundaries (Table 21)For example SpeDeSTEM (Ence and Carstens2011) finds the maximum likelihood values for aspecies tree assuming all putative species areseparate lineages and for alternative species treeswhere two or more species are collapsed into a
2 The Dynamic Discipline of Species Delimitation hellip 27
single lineage The best fitting model is thenselected using the Akaike information criterionunder the assumption of constant populationsizes and fixed gene topologies across the speciestree The species delimitation program BayesianPhylogenetic and Phylogeography (BPampPYangand Rannala 2010) accommodates gene treeuncertainty and variable population sizes andsamples from the Bayesian posterior distributionof species delimitation models using reversible-jump Markov chain Monte Carlo (rjMCMC)sampling BPampP requires a user-provided guidetree with resolution finer than the species leveland evaluates alternative modes derived from allpossible subtrees that are generated by collapsingor splitting nodes on the guide tree (Yang andRannala 2010 Rannala and Yang 2003) Theproportion of time spent on each model is pro-portional to the posterior probability of themodel ie ldquospeciation probabilitiesrdquo WhileBPampP ranks among the most popular coalescent-based species delimitation programs (Fujita et al2012) misspecifications of the guide tree andorthe prior distribution for population size (θ) canresult in strong support for models containing aninflated number of species (Leacheacute and Fujita2010 Zhang et al 2011) The nonparametricheuristic species delimitation approach imple-mented in the program Brownie (OrsquoMeara 2010)jointly sorts anonymous samples into species andinfers a species tree from input gene trees fromdifferent loci assuming that for a speciationevent the corresponding nodes on gene trees willbe more consistent with each than the diver-gences within species However finding both theoptimum species tree and species boundariesremains computationally challenging andBrownie has been shown to frequently yieldincorrect results (OrsquoMeara 2010)
Most recently available coalescent-basedspecies delimitation methods require individualassignments to a species or population a priori Ina number of scenarios correct assignments ofsamples to species may be difficult includingpresence of cryptic species incongruencebetween conventional species and moleculardata or simply the fact that accurate specimenidentification in complex groups with subtle or
difficult to discern diagnostic characters is anontrivial task (Leavitt et al 2013e) Statisticalmethods for assessing individual assignment andspecies detection prior to coalescent-based spe-cies delimitation and species tree reconstructionand species provide a more objective approachfor understanding species boundaries
Population assignment tests using a variety ofclustering algorithms using genetic data offer auseful approach for grouping individuals intoputative reproductively isolated groups (Table 21Hausdorf and Hennig 2010 Corander et al2004 2008 Falush et al 2003 Pritchard et al2000) particularly for species delimitation prob-lems that exist at the interface of traditionalpopulation genetic and phylogenetic analyses(Carstens et al 2013 Weisrock et al 2010Knowles and Carstens 2007)
The programs STRUCTURE (Falush et al2003 Pritchard et al 2000) and STRUCTURA-MA (Huelsenbeck et al 2011) cluster samplesinto populations by minimizing HardyndashWeinbergdisequilibrium for a given number of populationclusters using unlinked genetic markers In gen-eral unlinked markers are not available for mostgroups of lichen-forming fungi and a numberof studies have used information from multilocussequence data for STRUCTURE analyses(Leavitt et al 2011a b c 2013e Fernaacutendez-Mendoza and Printzen 2013 Fernaacutendez-Mendoza et al 2011) Varying approaches havebeen implemented to convert DNA sequencedata to allelic data for Bayesian clustering (seeOrsquoNeill et al 2013) STRUCTURE is expectedto perform well when there is sufficient inde-pendence across regions such that linkage dis-equilibrium within regions does not dominate thedata (STRUCTURE manual) but can also beeffective using multilocus sequence data andtreating all SNPs as independent loci regardlessof physical linkage within each locus (OrsquoNeillet al 2013 Falush et al 2003) A recent study ofthe lichen-forming genus Letharia showed thatBayesian clustering implemented in the programSTRUCTURE was generally able to recover thesame putative Letharia lineages circumscribedemploying a gene genealogical approach inKroken and Taylorrsquos iconic species delimitation
28 SD Leavitt et al
study (Altermann et al 2014 Kroken and Taylor2001) Altermann et al (2014) show that popu-lation assignments were largely consistent acrossa range of scenarios including extensiveamounts of missing data the exclusion of SNPsfrom variable markers and inferences based onSNPs from as few as three gene regions Incontrast to STRUCTURE and STRUCTURA-MA the program BAPS (Corander et al 2008)can explicitly infer genetic structure using hap-loid sequence data or other linked geneticmarkers in the ldquoclustering with linked locirdquomodel Another advantage of BAPS is that theprogram is much more computationally efficientthan STRUCTURE or STRUCTURAMA Sim-ulation studies indicate that both BAPS andSTRUCTURE perform well at low levels ofpopulation differentiation and when clusters arenot well differentiated (Latch et al 2006)
In practice inferring the most appropriatelevel of population structure using Bayesianclustering algorithms remains challenging (Latchet al 2006 Evanno et al 2005) BAPS andSTRUCTURAMA can infer both individualassignments and the most likely number ofgenetic clusters (Corander et al 2008 Huelsen-beck et al 2011) and the ad hoc ΔK statisticproposed by Evanno et al (2005) provides anobjective approach for identifying the uppermosthierarchical level of structure However carefulconsideration of other levels of populationstructure may ultimately reveal more biologicallymeaning results and researchers should examineindividual assignments across a range of geneticgroupings Some markers generated fromindependent genomic regions such as SNPs andfast-evolving microsatellites can be used to dis-tinguish fine-scale population structuring on thebasis of allele frequencies and species delimita-tion analyses based on these markers may implythe risk of severe taxonomic oversplitting Inthese cases validation approaches such asBPampP and SpeDeSTEM and corroborationamong different species delimitation approachescan provide perspective between intraspecificpopulation structure and species-level clusters
Another limitation of clustering approaches isthat they do not assess or take into account
evolutionarily divergences and relationshipsamong population clusters Coalescent-basedspecies tree methods (discussed below) provide ameans to assess the diversification histories ofpopulations inferred from clustering analysesTherefore a potential working protocol for aninformed species delimitation study that takesinto account population structure could consist offirst applying a genetic clustering analysis under apopulation genetics criterion (eg BAPSSTRUCTURE STRUCTURAMA) to identifygenetically distinct population clusters that can beconsidered ldquocandidate speciesrdquo From these can-didate species a species tree can be inferred forfocal group using coalescent-based species treereconstruction methods (eg BEAST) Subse-quently a coalescent-based validation methodcan be applied to assess whether the distinctpopulation clusters represent independent evolu-tionary lineages (eg BPampP) This multistepapproach would provide a consistent and standardcriterion for distinguishing between population-and species-level lineages (Camargo and Sites2013) and has been applied to a number of casesincluding lichen-forming fungi (Leavitt et al2011a b c 2013e Leacheacute and Fujita 2010)
High-throughput sequencing methods providethe means to effectively sample hundreds tothousands of loci from across a species genome fora large number of species and continue to revolu-tionize studies that can be performed even in non-model organisms Targeted high-throughputsequencing approaches such as anchored phy-logenomics transcriptome sequencing reduced-representation genomic library sequencing(restriction-site-associated DNA sequencingRAD-Seq and genotype-by-sequencing GBS)and high-throughput amplicon sequencing pro-vide important insight into species boundaries fora number groups although none of these approa-ches has yet been applied to studies of lichenizedfungi
For example restriction-site-associated(RAD-Seq) DNA can simultaneously detect andgenotype thousands of genome-wide SNPs byfocusing the sequencing effort on a reducedrepresentation of the entire genome (Baird et al2008) and has been successfully applied to
2 The Dynamic Discipline of Species Delimitation hellip 29
intraspecies (Lewis et al 2007 Miller et al 2007Emerson et al 2010) and interspecies studies(Rubin et al 2012 Eaton and Ree 2013 Wagneret al 2013) This approach has provided strikinginsight into the recent adaption radiation of LakeVictoria cichlids (Wagner et al 2013) Analternative approach targeting the sequencing ofspecific loci using next-generation sequencingplatforms provides an efficient means of gener-ating data for loci of known genomic locationorthology size and expected level of variation(OrsquoNeill et al 2013) Markers can be targetedusing well-established PCR techniques or newerhybridization techniques and subsequentlypooled for high-throughput sequencing usingparallel-tagged sequencing of multiple individualsamples within a single sequencing run (OrsquoNeillet al 2013) A recent study of North Americantiger salamanders (Ambystoma tigrinum)demonstrated the potential for amplicon-basedparallel-tagged sequencing to rapidly generatelarge-scale genomic data for species delimitationand species tree research (OrsquoNeill et al 2013)
Although these methods provide an enormousamount of data and insight into diversification atan unprecedented scale computational limita-tions restrict the applicable analytical methodsfor large datasets although computational andanalytical advances are happening rapidly Mostrecent coalescent-based species delimitation andspecies tree models using gene trees have beenlimited to handle tens of loci for multiple indi-viduals and combining hundreds or thousands ofgene trees into a single species delimitationframework presents considerable computationalchallenges (Camargo and Sites 2013) Therecently developed method Bayes factor delim-itation (with genomic data BFD) combines adynamic programming algorithm for estimatingspecies trees that bypasses the computationallyintensive MCMC integration over gene trees toprovide a rigorous technique for species delimi-tation studies using genome-wide SNP data(Leacheacute et al 2014) Competing species delimi-tation models are compared using Bayes factorsand it appears that this approach is robust tosample sizes (ie few loci and limited samples
per species) and misspecification of the prior forpopulation size (θ) (Leacheacute et al 2014)
Fungi are an ideal model for assessing diver-gence in eukaryotes due to their simple mor-phologies small genomes broad ecological rolesand diverse lifestyles (Gladieux et al 2014)However the use of genomic data from high-throughput sequencing methods have not yet beenincluded in species delimitation studies of lichen-forming fungi This is due in part to challenges indealing with metagenomic data containing geno-mic information from a plethora of symbiontsassociated with a lichen thallus scant genomicresources and discipline-specific inertia A num-ber of lichen-forming fungal genomes are cur-rently available including Endocarponpusillum(Wang et al 2014) Cladonia spp (Park et al2013a 2014) Caloplaca flavorubescens (Parket al 2013b) and Cladonia greyi and Xanthoriaparientina through the Fungal Genomics Programat Joint Genome Institute of the United StatesDepartment of Energy (httpwwwjgidoegov)The foreseeable ongoing expansion of genomicresources for lichen-forming fungi will be centralto developing approaches for delimiting speciesusing high-throughput sequencing Specificallygenomic resources will be crucial in identifyingnovel markers (variable genes regions SNPsmicrosatellites etc) identifying conserved fungalmarkers for targeted high-throughput sequencingapproaches and references for entire genomecomparisons (Devkota et al 2014 Werth et al2013)
23 Can We Make SpeciesDelimitation in Lichen-FormingFungi Truly Integrative
The widespread availability of genetic data hascreated a biased viewpoint that only genetic datashould be used for statistical species delimitationHowever an ongoing appeal to researchers toassess species boundaries from multiple andcomplementary perspectives (phylogeneticspopulation genetics comparative morphologydevelopment ecology etc) has resulted in an
30 SD Leavitt et al
integrative taxonomic framework bringing theseconceptual and methodological perspectivestogether (Hamilton et al 2014 Fujita et al 2012Salicini et al 2011 Yeates et al 2011 Padialet al 2010 Dayrat 2005 Will et al 2005 Wiensand Penkrot 2002) In practice any study linkingdifferent kinds of data to support hypotheses ofspecies boundaries including mapping morpho-logical characters onto a molecular phylogenycan be considered integrative Integrative meth-ods for species delimitation fall across a broadspectrum ranging from verbal and qualitativeassessments of data classes to quantitative meth-ods that allow different data types to contribute tostatistical species delimitation (Yeates et al2011) In most taxonomic studies utilizing bothmolecular and morphological data expert opinionis eventually used to some degree to evaluate thefinal status of a candidate species (eg Bond andStockman 2008) In this sense many studiesusing evidence for independent data sources fordelimiting species boundaries use an iterativeapproach (Yeates et al 2011) where speciesboundaries can be tested within a hypothetico-deductive framework with diverse datasets
From a practical perspective we advocate aprocess of iterative taxonomy (sensu Yeates et al2011) to circumscribe and refine species limitsusing multiple lines of evidence This iterativeprocess involves comparisons of morphologicaldata with a phylogenetic hypothesis (ie single-locus gene tree or concatenated multilocusphylogeny) to identify the least inclusive mono-phyletic clade in the topology characterized by atleast one unambiguously diagnostic morphologi-cal character (Miralles and Vences 2013) Thisphylogenetic tree-informed approach to assessingspecies boundaries represents a common practicein studies of lichen-forming fungi Previouslyunrecognized species-level clades with corre-sponding subtle or overlooked phenotypiccharacters have been commonly observed in bothcrustose lichens [eg Acarospraceae (Wedin et al2009) Graphidaceae (Rivas Plata and Lumbsch2011 Papong et al 2009) Lecanoraceae (Leavittet al 2011a) Lecideaceae (Ruprecht et al 2010)Mycoblastaceae (Spribille et al 2011) andTeloschistaceae (Vondraacutek et al 2009 Muggia
et al 2008)] and foliose and fruticose lichens [egLobariaceae (Moncada et al 2014 McDonaldet al 2003) Parmeliaceae (Leavitt et al 2013aWirtz et al 2012 Divakar et al 2005 Molinaet al 2004 Kroken and Taylor 2001) Peltigera-ceae (OrsquoBrien et al 2009 Goffinet et al 2003)Physciaceae (Elix et al 2009 Divakar et al2007) and Sphaerophoraceae (Houmlgnabba andWedin 2003)] As a specific example Luumlckinget al (2008) used a combination of medullarychemistry and underside pigmentation in speci-mens from the Heterodermia obscurata group inCosta Rica to corroborate monophyletic clades inan ITS gene tree as species-level lineages
While this iterative approach to speciesdelimitation and taxonomy has proven valuablefor understanding species boundaries anddescribing new taxa in some groups of lichenizedfungi a posteriori examination of morphologicaland chemical features has failed to reveal diag-nostic phenotypic characters in a number ofstudies (Muggia et al 2014 Leavitt et al 2011aOtaacutelora et al 2010 OrsquoBrien et al 2009) Fur-thermore a study of widespread species in thegenus Melanelixia (Parmeliaceae) indicated thatphenotypically cryptic lichen-forming fungalspecies-level lineages may be relatively ancientand diagnosable phenotypic differences may beabsent even millions of years after the initialdivergence (Leavitt et al 2012b) The latter studyhighlights the fact that species-level lineages maycommonly exist without any observable diag-nostic phenotypic characters calling into ques-tion the impetus for a universal application ofintegrative taxonomy
In keeping with the principle that as manylines of evidence as available should be com-bined to delimit species (Dayrat 2005) the for-malized integrative taxonomic approach (ITAXMiralles and Vences 2013) uses a mtDNA guidetree and observations from different types of datathat might provide conclusive evidence for theindependence of lineages and thus their identityas different species Miralles and Vences (2013)provide a non-exhaustive list of species delimi-tation criteria to be integrated in the ITAXapproach including (a) sympatric occurrencewithout admixture as revealed by consistent
2 The Dynamic Discipline of Species Delimitation hellip 31
differences in morphological or molecular char-acters at the same geographic location (b) strongdifferences in a behavioral morphological orgenetic character known to mediate prematingisolation (c) unviability or infertility of hybrids(d) lack of gene flow across a geographicalhybrid zone (e) congruent diagnostic differencesbetween sister lineages in various unlinkedmorphological character (f) absence of haplo-type sharing in several unlinked nuclear loci and(g) a combination of criteria endashf Speciesboundaries are based on seeking the least inclu-sive monophyletic group in the mtDNA treewhich fulfills at least one of the criteria listedabove Clearly the ITAX approach is sensitive tosample size and in order to reliably support thedistinctiveness of a given species it has beenrecommended that the sampling strategy includesat least five individuals per species (Miralles andVences 2013) A similar approach could beadopted for lichenized fungi using an ITS genetopology as guide tree rather than mtDNA
A total evidence approach including concat-enation has been a common approach for inte-grating information from different sourcesincluding independent genetic markers in phy-logenetic reconstructions (Kluge 1989 Wiens1998 de Queiroz et al 1995) Proponents forconcatenation of independent data in phyloge-netic analyses argue that when combining alldata the underlying signal of speciation mayemerge even if weakly contradictory signalis contained in the individual data partitions(Gatesy et al 1999) For example establishing apreliminary perspective of species boundariesfrom multilocus sequence data using concatena-tion may provide a reasonable starting point forscreening for cryptic species and species treeinference (Šlapeta et al 2006 Leavitt et al2011a Le Gac et al 2007 Leacheacute 2009)However concatenation and consensus methodsimply a risk of obtaining inflated support forincorrect relationships and information aboutvariance in gene coalescence is lost (Degnan andRosenberg 2006 2009 Kubatko and Degnan2007 Edwards 2009) In spite of the impetus forintegrating evidence from independent data
sources into empirical species delimitation stud-ies and taxonomy (Fujita et al 2012 Padial et al2009) most currently available species delimi-tation methods are unable to accommodate non-genetic data sources in a statistical framework
231 Selecting the Appropriate Data
In the face of increasing availability of geneticdata and associated bioinformatical approachesfor delimiting species researchers should care-fully consider what information is being sacri-ficed by the failure to consider non-genetic datain species delimitation studies and whetheraccuracy could be improved by the addition ofmultiple data types Morphological data havehistorically served as a proxy to identify repro-ductively isolated groups (ie ldquospeciesrdquo) (Ray1686 Fujita et al 2012) Current methods fordelimiting species using non-genetic data (egchemistry morphology and ecology) remainwoefully understudied For example morphol-ogy-based species circumscriptions are generallybased on one or more qualitative (or quantitative)morphological characters that do not appear tooverlap with other species However ascertain-ing that a given trait is truly fixed within apopulation with statistical confidence requiresunrealistic sample sizes even when allowing forsome level of polymorphism in the diagnosticcharacter (Wiens and Servedio 2000) Now var-ious combinations of datamdashfrom morphologygenetics geography and ecologymdashare acceptedas standard information for species delimitationstudies (Ruiz-Sanchez and Sosa 2010 Ross et al2010 Edwards and Knowles 2014)
Below we briefly discuss appropriate datasources for species delimitation studies of lichen-forming fungi However homoplastic characters(similar traits that are not derived from a com-mon ancestor) are common among many traitscommonly used to circumscribe fungal taxa thebiological significance of secondary metabolitevariation remains largely unknown (Lawrey1986) and ecological niches may be difficultto adequately characterize and model due to
32 SD Leavitt et al
microhabitat requirements and data resolutionTherefore we advocate a cautious approach toselecting appropriate and relevant data forassessing species boundaries
In spite of the potential challenges and limi-tations of using phenotypic data these traits haveprovided a plethora of valuable information forunderstanding species boundaries For examplein the Melanelixia fuliginosa group (Parmelia-ceae) a morphometric analysis using color isi-dia and marginal zone free of isidia ascharacters revealed a general pattern of differ-entiation between material formerly recognizedas subspecies fuliginosa and glabratula in spiteof the fact that considerable overlap betweengroups occurs in some characters individually(Arup and Berlin 2011) Furthermore the dis-tinction of the two groups was supported by aphylogenetic analysis of the ITS marker andecological differences with M fuliginosaoccurring predominantly on rock and M glab-ratula on bark (Arup and Berlin 2011) Thisstudy of the M fuliginosa group provides a fit-ting example of using multiple independentsuites of data including ecology morphologyand genetic information to establish a robusthypothesis of species boundaries
In lichen systematics phenotypic dataincluding thallus organization secondary metab-olites mode of reproduction ascoma-type andontogeny ascus and ascospore characters havehistorically played a prominent role (Printzen2009) Ascomatal characters have traditionallyheld a major role in higher-level classification(Printzen 2009) in contrast to species-levelclassification which also tends to include a widearray of vegetative and chemical charactersCommonly assessed morphological charactersinclude thallus form and size cortical features(eg maculae and pseudocyphellae) presenceformcolor of attachment structure (eg rhizines)and reproductive mode (ascomata vs vegetativediaspores) (Printzen 2009) Different morpho-logical types of vegetative diasporesmdashcorticatedisidia and ecorticated sorediamdashand their locationare commonly used to distinguish speciesAscomatal characters including morphologylocation (laminal vs marginal) position (sessile
vs immersed) presence of thalline margins colorof an apothecial disc and presence or color ofpruina are also commonly used in speciesdelimitations of lichen-forming fungi (Printzen2009) Other important characters may includethalline characters form color size and septationof ascospores size and form and structure of ascithe hamathecium type of epihymenium andhypothecium and the type of excipulum orperidium conidiomatal characters etc (Printzen2009)
Assessments of secondary metabolites hasplayed an important role in lichen taxonomybeginning with the introduction of simple spottests by Nylander (1866a b) The use of chem-istry in lichen taxonomy has been discussed indetail in numerous reviews (Lumbsch 1998a bRogers 1989 Brodo 1986 Egan 1986 Leuckert1985 Brodo 1978 Hawksworth 1976 Culber-son 1969 1970) and we refer readers to thesevaluable sources for a more comprehensive per-spective on lichen chemistry In short extrolites(secondary metabolites) belong to various clas-ses the most common and diverse include dep-sides depsidones chlorinated xanthones andanthraquinones (Lumbsch 2002 Culberson1969) The presence or absence of specificextrolites or their replacements by another sub-stance is widely used to distinguish speciesparticularly when correlated with differences ingeographic distributions However if morpho-logical or geographical differences betweenpopulations containing different extrolites are notapparent the taxonomic significance has beendisputed with some authors distinguishing themas species and others preferring to regard them aschemical races within a species In addition tosimply using the presence or absence of extro-lites Culberson and Culberson (1976) proposedto arrange lichen substances into chemosyn-dromes of closely related substances The pres-ence or absence of these chemosyndromes maypotentially be used as characters to delimit spe-cies regarding differences involving the samechemosyndromes as intraspecific variation anddistinct chemosyndromes as evidence for inter-specific populations (Lumbsch 1994 Gowan1986)
2 The Dynamic Discipline of Species Delimitation hellip 33
Due to the fact that species delimitationstudies often incorporate a substantial biogeo-graphical or ecological component ecologicalniche modeling plays an increasing role in phy-logenetic and taxonomic research Niche mod-eling can provide evidence for ecologicalisolation between populations based on eitherconserved or divergent ecological niches andtherefore can provide additional evidence sup-porting lineage independence between putativespecies The application of ecological nichemodeling has been applied to species delimita-tion studies in a number of cases (Ruiz-Sanchezand Sosa 2010 Leacheacute et al 2009 Raxworthyet al 2007 Rissler and Apodaca 2007) althoughit has not been explicitly applied to assess speciesboundaries in lichen-forming fungi By mappingthe spatial distribution of environmental suit-ability of climatic variables for candidate speciesthe application of ecological niche modeling canbe particularly important in cases where specieshave allopatric distributions (Raxworthy et al2007)
Ecological niche modeling utilizes knownassociations between a speciesrsquo occurrencelocalities and environmental variables to defineabiotic conditions within which populations canbe maintained (Guisan and Thuiller 2005) Themethodological approach for modeling is basedon four general properties (i) The current knownspeciesrsquo localities is the dependent variable (ii)the distribution is modeled as a map composed ofgrid cells at a specified resolution (iii) a range ofenvironmental variables (eg temperature pre-cipitation and solar exposure) are collected todescribe the characteristics of each cell and (iv)classifying the degree to which each cell is eithersuitable or unsuitable for each species under arange of models (Guisan and Thuiller 2005)
Ecological data also have the potential to playan important role in understanding speciesboundaries in lichen-forming fungi For exampleNash and Zavada (1977) demonstrated that Xant-hoparmelia populations with distinct chemistriesoccurring in the northern portion of the SonoranDesert exhibit habitat selection among differentrock substrates within a region with relativelyuniform climate and topography In another case
the parmelioid species Parmelia mayi is mor-phologically indistinguishable from P saxatilisbut can be separated by bioclimatic features andgenetic and chemical characters (Molina et al2011) McCune and Printzen(2011) assess distri-butions and climatic niches of species in theLecanora varia group inwesternUSAandprovidea model that uses continental influence and annualtemperature as themajor factors predicting speciesdistributions The distribution of Usnea hirta alichen commonly used in air quality biomonitor-ing research was modeled for a section of theWhite River National Forest in central Coloradobased on the presence of U hirta at 72 biomoni-toring reference sites distributed in the inter-mountain western United States (Shrestha et al2012) The best model for predicting U hirtadistribution included four variablesmdashsolar radia-tion average monthly precipitation and averagemonthly minimum and maximum temperatures(Shrestha et al 2012) These studies supportthe potential use of ecological niche modelingmethods in species delimitation studies of lichen-forming fungi
24 Conclusions What AboutTaxonomy
In most cases species circumscription and tax-onomy requires some degree of qualitativejudgment and individual interpretation Integrat-ing multiple types of data into an empiricalframework for delimiting species boundarieswhere species boundaries can be tested within ahypothetico-deductive framework with diversedatasets can provide robust hypotheses of speciesboundaries and taxonomic stability (Yeates et al2011) However it has long been known toevolutionary biologists that distinct species donot need to have diagnosable morphologicaldifferences (Mayr 1963) and increasing avail-ability of genetic data has allowed researchers toidentify species and to rigorously test speciesboundaries with a level of precision that wasunimaginable a decade ago While analyticaladvances in statistical species delimitation havebeen largely based on genetic data the utility of
34 SD Leavitt et al
these approaches to formal taxonomy remainselusive Due to these challenges an eclecticapproach to delimiting species and cautionagainst the reliance on any single dataset ormethod is required when delimiting species
While results from many recent studies maycontradict traditional species boundaries acrossmany groups of lichen-forming fungi we areoptimistic that this research represents substantialprogress toward a more accurate perspective onspecies boundaries and diversity in fungi As aresult of ongoing research of species boundariesin lichen-forming fungi the taxonomic value ofmany phenotypes is now better understood ourunderstanding of ecological evolutionary andbiogeographic patterns has improved and we canbegin to better understand patterns of symbiontinteractions in lichens Integrating new data(including novel morphological characters andgenetic data) will be essential to accurately rep-resent species-level diversity across all groups oflichenized fungi Hopefully an improved per-spective on lichen diversity also increases ourappreciation of these incredible symbiotic sys-tems While we are strong advocates for theapplication of independent data types in devel-oping an integrative taxonomy there is anincreasing need to formally recognize the exis-tence of phenotypically cryptic species-levellineages in lichen-forming fungi (see Hibbettet al 2011) In some cases a molecular taxon-omy may provide the most practical approach toconsistent treatment of mycobiont species withinlichen groups where diagnostic morphologicalcharacters are unidentifiable or practically notfeasible (Leavitt et al 2013c d)
These are exciting times for taxonomists andphylogeneticists A closer look at lichen taxon-omy with the inclusion of new data will help usto better understand the diversity of these fasci-nating organisms accurately interpret distribu-tion patterns and play a more important role inmeaningful conservation practices Howeversome level of uncertainty will accompany pro-gress In many taxonomic groups our traditionalapproach for species identification will likelyneed to be substantially modified The search forcorroborating morphological support for cryptic
species identified using molecular data willrequire meticulous and creative approaches toassess phenotypic variation in potentially unor-thodox ways We are hopeful that lichenologistswho traditionally have been eager to include newmethods such as chromatography in their rou-tine identifications will be amenable to includemolecular techniques to their routine examina-tion of specimens for identification and classifi-cation Although this may prove difficult toachieve by single individuals especially citizenscientists that traditionally play an important rolein lichen taxonomy the increasing numberof collaborative projects in the discipline (egLumbsch et al 2011 Crespo et al 2010Gueidan et al 2009) make us optimistic thatbroad-scale collaborative approaches will facili-tate the inclusion of molecular data in lichenresearch at all levels
Acknowledgments We are indebted to various col-leagues for valuable thought-provoking discussionnotably Matthew Nelsen (University of Chicago) AnaCrespo (Universidad Complutense de Madrid) PradeepDivakar (Universidad Complutense de Madrid) BeckettSterner (The Field Museum) and Joyce Havstad (TheField Museum) We also thank anonymous reviewers whoprovided valuable comments that improved this chapterSupport by the US National Science Foundation isgratefully acknowledged (ldquoHidden diversity in parmelioidlichensrdquo DEB-0949147)
References
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Altermann S Leavitt SD Goward T Nelsen MPLumbsch HT (2014) How do you solve a problemlike Letharia A new look at cryptic species in lichen-forming fungi using Bayesian clustering and SNPSsfrom multilocus sequence data PLoS ONE 9(5)e97556 doi101371journalpone0097556
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Arguumlello A Del Prado R Cubas P Crespo A (2007)Parmelina quercina (Parmeliaceae Lecanorales)
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Arup U Berlin ES (2011) A taxonomic study ofMelanelixia fuliginosa in Europe The Lichenologist43(02)89ndash97 doi101017S0024282910000678
Arup U Grube M (2000) Is Rhizoplaca (Lecanoraleslichenized Ascomycota) a monophyletic genus Can JBot 78(3)318ndash327 doi101139b00-006
Avise J Ball R (1990) Principles of genealogicalconcordance in species concepts and biological tax-onomy Oxf Surv Evol Biol 745ndash67
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Bond J Stockman A (2008) An integrative method fordelimiting cohesion species finding the population-species interface in a group of Californian trapdoorspiders with extreme genetic divergence and geo-graphic structuring Syst Biol 57(4)628ndash646 doi10108010635150802302443
Brodo IM (1978) Changing concepts regarding chemicaldiversity in lichens The Lichenologist 10(1)1ndash11doi101017S0024282978000031
Brodo IM (1986) Interpreting chemical variation inlichens for systematic purposes The Bryologist 89(2)132ndash138
Caley MJ Fisher R Mengersen K (2014) Global speciesrichness estimates have not converged Trends EcolEvol 29(4)187ndash188 doi101016jtree201402002
Camargo A Sites JW (2013) Species delimitation adecade after the Renaissance In Pavlinov I (ed) Thespecies problemmdashongoing issues InTech doi10577252664
Camargo A Morando M Avila LJ Sites JW (2012)Species delimitation with ABC and other coalescent-based methods a test of accuracy with simulations and
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Carstens BC Dewey TA (2010) Species delimitationusing a combined coalescent and information-theoreticapproach an example from North American Myotisbats Syst Biol 59(4)400ndash414 doi101093sysbiosyq024
Carstens BC Pelletier TA Reid NM Satler JD (2013)How to fail at species delimitation Mol Ecol 22(17)4369ndash4383 doi101111mec12413
Corander J Marttinen P (2006) Bayesian identification ofadmixture events using multi-locus molecular mark-ers Mol Ecol 15(10)2833ndash2843 doi101111j1365-294X200602994x
Corander J Waldmann P Marttinen P Sillanpaa M(2004) BAPS 2 enhanced possibilities for the analysisof genetic population structure Bioinformatics 20(15)2363ndash2369 doi101093bioinformaticsbth250
Corander J Marttinen P Mantyniemi S (2006) Bayesianidentification of stock mixtures from molecular markerdata Fish Bull 104550ndash558
Corander J Marttinen P Siren J Tang J (2008) EnhancedBayesian modelling in BAPS software for learninggenetic structures of populations BMC Bioinf 9(1)539 doi1011861471-2105-9-539
Coyne JA Orr HA (2004) Speciation Sinauer AssociatesSunderland
Cracraft J (1983) Species concepts and speciation anal-ysis Curr Ornithol 1159ndash187
Crespo A Lumbsch HT (2010) Cryptic species in lichen-forming fungi IMA Fungus 1167ndash170 doi105598imafungus2010010209
Crespo A Peacuterez-Ortega S (2009) Cryptic species andspecies pairs in lichens a discussion on the relation-ship between molecular phylogenies and morpholog-ical characters Anales del Jardin Botanico de Madrid66(S1)71ndash81 doi103989ajbm2225
Crespo A Kauff F Divakar PK del Prado R Perez-Ortega S Amo de Paz G Ferencova Z Blanco ORoca-Valiente B Nunez-Zapata J Cubas P ArguelloA Elix JA Esslinger TL Hawksworth DL MillanesA Molina MC Wedin M Ahti T Aptroot A et al(2010) Phylogenetic generic classification of parmeli-oid lichens (Parmeliaceae Ascomycota) based onmolecular morphological and chemical evidenceTaxon 59(6)1735ndash1753
Culberson WL (1969) The use of chemistry in thesystematics of the lichens Taxon 18498ndash505
Culberson WL (1970) Chemosystematics and ecology oflichen-forming fungi Annu Rev Ecol Syst 1153ndash170
Culberson CF Culberson WL (1976) Chemosyndromicvariation in lichens Syst Bot 1325ndash339
Darwin C (1859) On the origin of species by means ofnatural selection or the preservation of favoured racesin the struggle for life J Murray London
Dayrat B (2005) Towards integrative taxonomy Biol JLinn Soc 85(3)407ndash415 doi101111j1095-8312200500503x
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de Queiroz K (1998) The general lineage concept ofspecies species criteria and the process of speciationa conceptual unification and terminological recom-mendations In Howard DJ Berlocher SH (eds)Endless forms species and speciation Oxford Uni-versity Press Oxford pp 57ndash75
de Queiroz K (1999) The general lineage concept ofspecies and the defining properties of the speciescategory In Wilson RA (ed) Species new interdis-ciplinary essays MIT Press Cambridge pp 49ndash89
de Queiroz K (2007) Species concepts and speciesdelimitation Syst Biol 56(6)879ndash886 doi10108010635150701701083
de Queiroz A Donoghue MJ Kim J (1995) Separateversus combined analysis of phylogenetic evidenceAnnu Rev Ecol Syst 26(1)657ndash681 doi101146annureves26110195003301
Degnan J Rosenberg N (2006) Discordance of speciestrees with their most likely gene trees PLoS Genet 2(5)e68 doi101371journalpgen0020068
Degnan JH Rosenberg NA (2009) Gene tree discordancephylogenetic inference and the multispecies coales-cent Trends Ecol Evol 24(6)332ndash340 doihttpdxdoiorg101016jtree200901009
Del-Prado R Cubas P Lumbsch HT Divakar PK BlancoO de Paz GA Molina MC Crespo A (2010) Geneticdistances within and among species in monophyleticlineages of Parmeliaceae (Ascomycota) as a tool fortaxon delimitation Mol Phylogenet Evol 56(1)125ndash133 doi101016jympev201004014
Del-Prado R Divakar PK Crespo A (2011) Using geneticdistances in addition to ITS molecular phylogeny toidentify potential species in the Parmotrema reticul-atum complex a case study The Lichenologist 43(06)569ndash583 doi101017S0024282911000582
Del-Prado R Blanco O Lumbsch HT Divakar PK ElixJA Molina MC Crespo A (2013) Molecular phylog-eny and historical biogeography of the lichen-formingfungal genus Flavoparmelia (Ascomycota Parmelia-ceae) Taxon 62(5)928ndash939 doi101270562522
Dettman J Jacobson D Taylor J (2003a) A multilocusgenealogical approach to phylogenetic species recog-nition in the model eukaryote Neurospora Evolution57(12)2703ndash2720 doi101111j0014-38202003tb01514x
Dettman JR Jacobson DJ Turner E Pringle A TaylorJW (2003b) Reproductive isolation and phylogeneticdivergence in Neurospora comparing methods ofspecies recognition in a model eukaryote Evolution57(12)2721 doi10155403-074
Devkota S Cornejo C Werth S Chaudhary RP Sche-idegger C (2014) Characterization of microsatelliteloci in the Himalayan lichen fungus Lobaria pindar-ensis (Lobariaceae) Appl Plant Sci 2(5)1300101doi103732apps1300101
Divakar PK Molina MC Lumbsch HT Crespo A (2005)Parmelia barrenoae a new lichen species related toParmelia sulcata (Parmeliaceae) based on molecularand morphological data The Lichenologist 37(01)37ndash46 doi101017S0024282904014641
Divakar PK Amo De paz G del Prado R Esslinger TLCrespo A (2007) Upper cortex anatomy corroboratesphylogenetic hypothesis in species of Physconia(Ascomycota Lecanoromycetes) Mycol Res 111(11)1311ndash1320 doi101016jmycres200708009
Divakar PK Figueras G Hladun N Crespo A (2010)Molecular phylogenetic studies reveal an undescribedspecies within the North American concept of Mela-nelixia glabra (Parmeliaceae) Fungal Divers 42(1)47ndash55 doi101007s13225-010-0027-3
Donoghue MJ Gauthier A (2004) Implementing thephylocode Trends Ecol Evol 19(6)281ndash282 doi101016jtree200404004
Eaton DAR Ree RH (2013) Inferring phylogeny andintrogression using RADseq data an example fromflowering plants (Pedicularis Orobanchaceae) SystBiol 62(5)689ndash706 doi101093sysbiosyt032
Edwards SV (2009) Is a new and general theory ofmolecular systematics emerging Evolution 63(1)1ndash19 doi101111j1558-5646200800549x
Edwards DL Knowles LL (2014) Species detection andindividual assignment in species delimitation canintegrative data increase efficacy Proc R Soc B BiolSci 281(1777) doi101098rspb20132765
Egan RS (1986) Correlations and non-correlations ofchemical variation patterns with lichen morphologyand geography The Bryologist 8999ndash110
Elix JA Corush J Lumbsch HT (2009) Triterpenechemosyndromes and subtle morphological characterscharacterise lineages in the Physcia aipolia group inAustralia (Ascomycota) Syst Biodivers 7(04)479ndash487 doi101017S1477200009990223
Emerson KJ Merz CR Catchen JM Hohenlohe PACresko WA Bradshaw WE Holzapfel CM (2010)Resolving postglacial phylogeography using high-throughput sequencing Proc Natl Acad Sci 107(37)16196ndash16200 doi101073pnas1006538107
Ence DD Carstens BC (2011) SpedeSTEM a rapid andaccurate method for species delimitation Mol EcolResour 11(3)473ndash480 doi101111j1755-0998201002947x
EvannoGRegnaut SGoudet J (2005)Detecting thenumberof clusters of individuals using the software STRUC-TURE a simulation study Mol Ecol 14(8)2611ndash2620doi101111j1365-294X200502553x
Falush D Stephens M Pritchard JK (2003) Inference ofpopulation structure using multilocus genotype datalinked loci and correlated allele frequencies Genetics164(4)1567ndash1587 doi101111j1471-8286200701758x
Fan HH Kubatko LS (2011) Estimating species treesusing approximate Bayesian computation Mol Phylo-genet Evol 59(2)354ndash363 doi101016jympev201102019
Fernaacutendez-Mendoza F Printzen C (2013) Pleistoceneexpansion of the bipolar lichen Cetraria aculeata intothe Southern hemisphere Mol Ecol 22(7)1961ndash1983doi101111mec12210
Fernaacutendez-Mendoza F Domaschke S Garciacutea MA JordanP Martin MP Printzen C (2011) Population structure
2 The Dynamic Discipline of Species Delimitation hellip 37
of mycobionts and photobionts of the widespreadlichen Cetraria aculeata Mol Ecol 20(6)1208ndash1232doi101111j1365-294X201004993x
Fraley C Raftery A (2007) Model-based methods ofclassification Using the mclust software in chemo-metrics J Stat Softw 18i06 [Available online at httpwwwdoajorgdoajfunc=abstractampid=218544]
Fujisawa T Barraclough TG (2013) Delimiting speciesusing single-locus data and the generalized mixed yulecoalescent (GMYC) approach a revised method andevaluation on simulated datasets Syst Biol 62(5)707ndash724 doi101093sysbiosyt033
Fujita MK Leacheacute AD Burbrink FT McGuire JA MoritzC (2012) Coalescent-based species delimitation in anintegrative taxonomy Trends Ecol Evol 27(9)480ndash488 doi101016jtree201204012
Gatesy J OrsquoGrady P Baker RH (1999) Corroborationamong data sets in simultaneous analysis hiddensupport for phylogenetic relationships among higherlevel Artiodactyl taxa Cladistics 15(3)271ndash313doi101111j1096-00311999tb00268x
Gladieux P Ropars J Badouin H Branca A Aguileta Gde Vienne DM Rodriacuteguez de la Vega RC Branco SGiraud T (2014) Fungal evolutionary genomicsprovides insight into the mechanisms of adaptivedivergence in eukaryotes Mol Ecol 23(4)753ndash773doi101111mec12631
Goffinet B Miadlikowska J Goward T (2003) Phyloge-netic inferences based on nrDNA sequences supportfive morphospecies within the Peltigera didactylacomplex (Lichenized Ascomycota) The Bryologist106(3)349ndash364 doi10163901
Gowan SP (1986) Evolution of secondary natural productsin the genus Porpidia (Ascomycata Porpidiaceae) AmJ Bot 73606
Griffin PC Hoffmann AA (2014) Limited genetic diver-gence among Australian alpine Poa tussock grassescoupled with regional structuring points to ongoinggene flow and taxonomic challenges Ann Bot doi101093aobmcu017
Grube M Hawksworth DL (2007) Trouble with lichenthe re-evaluation and re-interpretation of thallus formand fruit body types in the molecular era Mycol Res111(9)1116ndash1132 doi101016jmycres200704008
Gueidan C Savi S Thues H Roux C Keller C Tibell LPrieto M Heimarsson S Breuss O Orange A FrobergL Wynns AA Navarro-Rosines P Krzewicka BPykaelae J Grube M Lutzoni F (2009) Genericclassification of the Verrucariaceae (Ascomycota)based on molecular and morphological evidencerecent progress and remaining challenges Taxon 58(1)184ndash208
Guillot G Mortier F Estoup A (2005) Geneland acomputer package for landscape genetics Mol EcolNotes 5(3)712ndash715 doi101111j1471-8286200501031x
Guillot G Renaud S Ledevin R Michaux J Claude J(2012) A unifying model for the analysis of pheno-typic genetic and geographic data Syst Biol 61(6)897ndash911 doi101093sysbiosys038
Guisan A Thuiller W (2005) Predicting species distribu-tion offering more than simple habitat models EcolLett 8(9)993ndash1009 doi101111j1461-0248200500792x
Hale ME (1990) A synopsis of the lichen genusXanthoparmelia (Vainio) Hale (AscomycotinaParmeliaceae) Smithsonian Institution Press Wash-ington DC
Hamilton CA Hendrixson BE Brewer MS Bond JE(2014) An evaluation of sampling effects on multipleDNA barcoding methods leads to an integrativeapproach for delimiting species a case study ofthe North American tarantula genus Aphonopelma(Araneae Mygalomorphae Theraphosidae) MolPhylogenet Evol 7179ndash93 doi101016jympev201311007
Hausdorf B (2011) Progress toward a general speciesconcept Evolution 65(4)923ndash931 doi101111j1558-5646201101231x
Hausdorf B Hennig C (2010) Species delimitation usingdominant and codominant multilocus markers SystBiol 59(5)491ndash503 doi101093sysbiosyq039
Hawksworth DL (1976) Lichen chemotaxonomy InBailey RH (ed) Lichenology progress and problemsAcademic London pp 139ndash184
Hebert PDN Cywinska A Ball SL deWaard JR (2003)Biological identifications through DNA barcodesProc R Soc Lond B Biol Sci 270(1512)313ndash321doi101098rspb20022218
Hebert PDN Stoeckle MY Zemlak TS Francis CM(2004) Identification of birds through DNA barcodesPLoS Biol 2(10)e312 doi101371journalpbio0020312
Heled J Drummond AJ (2010) Bayesian inference ofspecies trees from multilocus data Mol Biol Evol 27(3)570ndash580 doi101093molbevmsp274
Hey J (2006) On the failure of modern species conceptsTrends Ecol Evol 21(8)447ndash450 doi101016jtree200605011
Hibbett DS OhmanA Glotzer D NuhnMKirk P NilssonRH (2011) Progress in molecular and morphologicaltaxon discovery in Fungi and options for formalclassification on environmental sequences Fungal BiolRev 2538ndash47 doi101016jfbr201101001
Houmlgnabba F Wedin M (2003) Molecular phylogeny ofthe Sphaerophorus globosus species complex Cladis-tics 19(3)224ndash232 doi101111j1096-00312003tb00365x
Hudson RR Coyne JA (2002) Mathematical conse-quences of the genealogical species concept Evolu-tion 56(8)1557 doi101111j0014-38202002tb01467x
Huelsenbeck JP Andolfatto P Huelsenbeck ET (2011)Structurama Bayesian inference of population struc-ture Evol Bioinf Online 7(2011)55ndash59 doi104137EBOS6761
Jones G Oxelman B (2014) DISSECT an assignment-free Bayesian discovery method for species delimita-tion under the multispecies coalescent Bioinformaticsdoi101101003178
38 SD Leavitt et al
Kekkonen M Hebert PD (2014) DNA barcode-baseddelineation of putative species efficient start fortaxonomic workflows Mol Ecol Res doi1011111755-099812233
Kelly LJ Hollingsworth PM Coppins BJ Ellis CJHarrold P Tosh J Yahr R (2011) DNA barcoding oflichenized fungi demonstrates high identification suc-cess in a floristic context New Phytol 191(1)288ndash300doi101111j1469-8137201103677x
Kiss L (2012) Limits of nuclear ribosomal DNA internaltranscribed spacer (ITS) sequences as species barcodesfor fungi Proc Natl Acad Sci 109(27)E1811ndashE1811doi101073pnas1207143109
Kluge AG (1989) A concern for evidence and aphylogenetic hypothesis for relationships among Epi-crates (Boidae Serpentes) Syst Zool 38(1)7ndash25doi101093sysbio3817
Knowles LL Carstens BC (2007) Delimiting specieswithout monophyletic gene trees Syst Biol 56(6)887ndash895 doi10108010635150701701091
Kroken S Taylor JW (2001) A gene genealogical approachto recognize phylogenetic species boundaries in thelichenized fungus Letharia Mycologia 93(1)38ndash53
Kubatko LS Degnan JH (2007) Inconsistency of phylo-genetic estimates from concatenated data under coa-lescence Syst Biol 56(1)17ndash24 doi10108010635150601146041
Latch EK Dharmarajan G Glaubitz JC Rhodes OE Jr(2006) Relative performance of Bayesian clusteringsoftware for inferring population substructure andindividual assignments at low levels of populationdifferentiation Conserv Genet 7(2)295ndash302 doi101007s10592-005-9098-1
Lawrey JD (1986) Biological role of lichen substancesThe Bryologist 89(2)111ndash122
Le Gac M Hood ME Fournier E Giraud T (2007)Phylogenetic evidence of host-specific cryptic speciesin the anther smut fungus Evolution 61(1)15ndash26doi101111j1558-5646200700002x
Leacheacute AD (2009) Species tree discordance traces tophylogeographic clade boundaries in North Americanfence lizards (Sceloporus) Syst Biol 58(6)547ndash559doi101093sysbiosyp057
Leacheacute AD Fujita MK (2010) Bayesian species delim-itation in West African forest geckos (Hemidactylusfasciatus) Proc R Soc B Biol Sci 2773071ndash3077doi101098rspb20100662
Leacheacute AD Koo M Spencer C Papenfuss T Fisher RMcGuire J (2009) Quantifying ecological morpho-logical and genetic variation to delimit species in thecoast horned lizard species complex (Phrynosoma)Proc Natl Acad Sci USA 10612418ndash12423 doi101073pnas0906380106
Leacheacute AD Fujita MK Minin VN Bouckaert RR (2014)Species delimitation using genome-wide SNP dataSystematic Biology doi101093sysbiosyu018
Leavitt SD Fankhauser JD Leavitt DH Porter LDJohnson LA St Clair LL (2011a) Complex patterns ofspeciation in cosmopolitan ldquorock posyrdquo lichensmdashdiscovering and delimiting cryptic fungal species in
the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
Leavitt SD Esslinger TL Lumbsch HT (2012c) Neogene-dominated diversification in neotropical montanelichens dating divergence events in the lichen-form-ing fungal genus Oropogon (Parmeliaceae) Am J Bot99(11)1764ndash1777 doi103732ajb1200146
Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Lumbsch HT St Clair LL(2013c) DNA barcode identification of lichen-formingfungal species in the Rhizoplaca melanophthalmaspecies-complex (Lecanorales Lecanoraceae) includ-ing five new species MycoKeys 71ndash22 doi103897mycokeys74508
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Vondraacutek J Thorsten Lum-bsch H Clair LLS (2013d) Local representation ofglobal diversity in a cosmopolitan lichen-formingfungal species complex (Rhizoplaca Ascomycota)J Biogeogr 40(9)1792ndash1806 doi101111jbi12118
Leavitt SD Lumbsch HT Stenroos S St Clair LL(2013e) Pleistocene speciation in North American
2 The Dynamic Discipline of Species Delimitation hellip 39
lichenized fungi and the impact of alternative speciescircumscriptions and rates of molecular evolution ondivergence estimates PLoS ONE 8(12)e85240doi101371journalpone0085240
Leavitt SD Esslinger TL Hansen ES Divakar PK CrespoA Loomis BF Lumbsch HT (2014) DNA barcoding ofbrown Parmeliae (Parmeliaceae) species a molecularapproach for accurate specimen identification empha-sizing species in Greenland Organ Divers Evol 14(1)11ndash20 doi101007s13127-013-0147-1
Leliaert F Verbruggen H Vanormelingen P Steen FLopez-Bautista JM Zuccarello GC De Clerck O(2014) DNA-based species delimitation in algae Eur JPhycol 49(2) doi101080096702622014904524
Leuckert C (1985) Probleme der Flechten-Chemotaxon-omiemdashStoffkombinationen und ihre taxonomischeWertung Ber Deut Bot Ges 98401ndash408
Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
Lohtander K Kaumlllersjouml M Roland M Tehler A (2000)The family physciaceae in fennoscandia phylogenyinferred from its sequences Mycologia 92(4)728ndash735
Longton RE (1997) The role of bryophytes and lichens inpolar ecosystems In Woodin SJ Marquiss M (eds)Ecology of Arctic Environments Blackwell ScienceOxford pp 69ndash96 (Special publication No 13)
Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
Lumbsch HT (1994) Die Lecanora subfusca-Gruppe inAustralasien J Hattori Bot Lab 771ndash175
Lumbsch HT (1998a) Taxonomic use of metabolic data inlichen-forming fungi In Frisvad JC Bridge PDArora DK (eds) Chemical Fungal Taxonomy MarcelDekker New York pp 345ndash387
Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
Mayden RL (1997) A hierarchy of species concepts thedenouement in the saga of the species problem InClaridge MF Dawah HA Wilson MR (eds) Speciesthe units of biodiversity Chapman amp Hall Londonpp 381ndash424
Mayr E (1963) Animal species and evolution HarvardUniversity Press Cambridge
Mayr E (1970) Populations species and evolutionBelknap Press of Harvard University Press Cambridge
McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
McDonald T Miadlikowska J Lutzoni F (2003) Thelichen genus Sticta in the Great Smoky Mountains a
40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
McKay BD Mays HL Wu Y Li H Yao C-T Nishiumi IZou F (2013) An empirical comparison of character-based and coalescent-based approaches to speciesdelimitation in a young avian complex Mol Ecol 22(19)4943ndash4957 doi101111mec12446
Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
Mishler BD Brandon RN (1987) Individuality pluralismand the phylogenetic species concept Biol Philos 2397
Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
Nash TH Zavada M (1977) Population studies amongSonoran Desert species of Parmelia subg Xanthop-armelia (Parmeliaceae) Am J Bot 64(6)664ndash669
Nylander W (1866a) Circa novum in studio Lichenumcriterium chemicum Flora 49198ndash201
Nylander W (1866b) Hypochlorite of lime and hydrate ofpotash Two new criteria for the study of lichens(Tanslated and communicated by the Rev W ALeighton) Bot J Linn Soc 9358ndash365
OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
OrsquoNeill EM Schwartz R Bullock CT Williams JSShaffer HB Aguilar-Miguel X Parra-Olea G Weis-rock DW (2013) Parallel tagged amplicon sequencingreveals major lineages and phylogenetic structure inthe North American tiger salamander (Ambystomatigrinum) species complex Mol Ecol 22(1)111ndash129doi101111mec12049
Orock EA Leavitt SD Fonge BA St Clair LL LumbschHT (2012) DNA-based identification of lichen-form-ing fungi Can publicly available sequence databasesaid in lichen diversity inventories of Mount Cameroon(West Africa) The Lichenologist 44(6)833ndash839doi101017S0024282912000424
Otaacutelora MAG Martiacutenez I Aragoacuten G Molina MC (2010)Phylogeography and divergence date estimates of alichen species complex with a disjunct distributionpattern Am J Bot 97(2)216ndash223 doi103732ajb0900064
Padial J Castroviejo-Fisher S Kohler J Vila C ChaparroJ De la Riva I (2009) Deciphering the products ofevolution at the species level the need for anintegrative taxonomy Zoolog Scr 38(4)431ndash447doi101111j1463-6409200800381x
Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
Papong K Luumlcking R Thammathaworn A BoonpragobK (2009) Four new taxa of Chroodiscus (thelotremoidGraphidaceae) from Southeast Asia The Bryologist112(1)152ndash163 doi1016390007-2745-1121152
Park S-Y Choi J Kim JA Jeong M-H Kim S Lee Y-HHur J-S (2013a) Draft genome sequence of Cladoniamacilenta KoLRI003786 a lichen-forming fungusproducing biruloquinone Genome Announcements 1(5)e00695-13 doi101128genomeA00695-13
Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
Peacuterez-Ortega S Fernaacutendez-Mendoza F Raggio J VivasM Ascaso C Sancho LG Printzen C de los Riacuteos A(2012) Extreme phenotypic variation in Cetrariaaculeata (lichenized Ascomycota) adaptation or inci-dental modification Ann Bot 109(6)1133ndash1148doi101093aobmcs042
Pino-Bodas R Burgaz A Martiacuten M Lumbsch HT (2011)Phenotypical plasticity and homoplasy complicatespecies delimitation in the Cladonia gracilis group(Cladoniaceae Ascomycota) Organ Divers Evol 11(5)343ndash355 doi101007s13127-011-0062-2
Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
Pino-Bodas R Martiacuten M Burgaz A (2012b) Cladoniasubturgida and C iberica (Cladoniaceae) form asingle morphologically and chemically polymorphicspecies Mycol Prog 11(1)269ndash278 doi101007s11557-011-0746-1
Pino-Bodas R Ahti T Stenroos S Martiacuten MP BurgazAR (2013a) Multilocus approach to species recogni-tion in the Cladonia humilis complex (CladoniaceaeAscomycota) Am J Bot 100(4)664ndash678 doi103732ajb1200162
Pino-Bodas R Martiacuten AP Burgaz AR Lumbsch HT(2013b) Species delimitation in Cladonia (Ascomy-cota) a challenge to the DNA barcoding philosophyMol Ecol Resour 13(6)1058ndash1068 doi1011111755-099812086
Pons J Barraclough TG Gomez-Zurita J Cardoso ADuran DP Hazell S Kamoun S Sumlin WD VoglerAP (2006) Sequence-based species delimitation for theDNA taxonomy of undescribed insects Syst Biol 55(4)595ndash609 doi10108010635150600852011
Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
Pringle A Baker DM Platt JL Wares JP Latgeacute JPTaylor JW (2005) Cryptic speciation in the cosmo-politan and clonal human pathogenic fungus Asper-gillus fumigatus Evolution 59(9)1886ndash1899 doi10155404-2411
Printzen C (2009) Lichen systematics the role ofmorphological and molecular data to reconstructphylogenetic relationships Progress in botany vol71 Springer Berlin pp 233ndash275
Pritchard JK Stephens M Donnelly P (2000) Inference ofpopulation structure using multilocus genotype dataGenetics 155(2)945ndash959
Puillandre N Lambert A Brouillet S Achaz G (2012)ABGD automatic barcode gap discovery for primaryspecies delimitation Mol Ecol 21(8)1864ndash1877doi101111j1365-294X201105239x
Rannala B Yang Z (2003) Bayes estimation of speciesdivergence times and ancestral population sizes usingDNA sequences from multiple loci Genetics1641645ndash1656
Raxworthy CJ Ingram CM Rabibisoa N Pearson RG(2007) Applications of ecological niche modelingfor species delimitation a review and empiricalevaluation using day geckos (Phelsuma) fromMadagascar Syst Biol 56(6)907ndash923 doi10108010635150701775111
Ray J (1686) Historia planarum vol 1 Clark LondonReese Naeligsborg R Ekman S Tibell L (2007) Molecular
phylogeny of the genus Lecania (Ramalinaceaelichenized Ascomycota) Mycol Res 111(5)581ndash591doi101016jmycres200703001
Reid N Carstens B (2012) Phylogenetic estimation errorcan decrease the accuracy of species delimitation aBayesian implementation of the general mixed Yule-coalescent model BMC Evol Biol 12(1)196 doi1011861471-2148-12-196
Rissler L Apodaca J (2007) Adding more ecology intospecies delimitation ecological niche models andphylogeography help define cryptic species in theBlack Salamander (Aneides flavipunctatus) Syst Biol56924ndash942 doi10108010635150701703063
Rivas Plata E Luumlcking R (2013) High diversity ofGraphidaceae (lichenized Ascomycota Ostropales) inAmazonian Peruacute Fungal Divers 58(1)13ndash32 doi101007s13225-012-0172-y
Rivas Plata E Lumbsch HT (2011) Parallel evolution andphenotypic divergence in lichenized fungi a casestudy in the lichen-forming fungal family Graphida-ceae (Ascomycota Lecanoromycetes Ostropales)Mol Phylogenet Evol 61(1)45ndash63 doi101016jympev201104025
Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
Ross KG Gotzek D Ascunce MS Shoemaker DD (2010)Species delimitation a case study in a problematic anttaxon Syst Biol 59(2)162ndash184 doi101093sysbiosyp089
Rubin BER Ree RH Moreau CS (2012) Inferringphylogenies from RAD sequence data PLoS ONE 7(4)e33394 doi101371journalpone0033394
Rubinoff D (2006) Utility of mitochondrial DNA barcodesin species conservation Conserv Biol 20(4)1026ndash1033 doi101111j1523-1739200600372x
Ruiz-Sanchez E Sosa V (2010) Delimiting speciesboundaries within the Neotropical bamboo Otatea(Poaceae Bambusoideae) using molecular morpho-logical and ecological data Mol Phylogenet Evol 54(2)344ndash356 doi101016jympev200910035
Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
morphological characters Antarctic Sci 22 (SpecialIssue 06)727ndash741 doi101017S0954102010000477
Salicini I Ibaacutentildeez C Juste J (2011) Multilocus phylogenyand species delimitation within the Nattererrsquos batspecies complex in the Western Palearctic MolPhylogenet Evol 61(3)888ndash898 doi101016jympev201108010
Satler JD Carstens BC Hedin M (2013) Multilocusspecies delimitation in a complex of morphologicallyconserved trapdoor spiders (Mygalomorphae Antro-diaetidae Aliatypus) Syst Biol 62(6)805ndash823 doi101093sysbiosyt041
Schlick-Steiner BC Steiner FM Seifert B Stauffer CChristian E Crozier RH (2010) Integrative taxonomya multisource approach to exploring biodiversityAnnu Rev Entomol 55(1)421ndash438 doi101146annurev-ento-112408-085432
Schoch CL Seifert KA Huhndorf S Robert V SpougeJL Levesque CA Chen W Fungal Barcoding Con-sortium (2012) Nuclear ribosomal internal transcribedspacer (ITS) region as a universal DNA barcodemarker for Fungi Proc Natl Acad Sci 109(16)6241ndash6246 doi101073pnas1117018109
Seacuterusiaux E Villarreal AJC Wheeler T Goffinet B(2011) Recent origin active speciation and dispersalfor the lichen genus Nephroma (Peltigerales) inMacaronesia J Biogeogr 38(6)1138ndash1151 doi101111j1365-2699201002469x
Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
Simpson GG (1951) The species concept Evolution5285ndash298
Sites JW Marshall JC (2003) Delimiting species a renais-sance issue in systematic biology Trends Ecol Evol18462ndash470 doi101016S0169-5347(03)00184-8
Sites JW Marshall JC (2004) Operational criteria fordelimiting species Annu Rev Ecol Evol Syst 35(1)199ndash227 doi101146annurevecolsys35112202130128
Šlapeta J Loacutepez-Garciacutea P Moreira D (2006) Globaldispersal and ancient cryptic species in the smallestmarine eukaryotes Mol Biol Evol 23(1)23ndash29doi101093molbevmsj001
Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
Talavera G Dincă V Vila R (2013) Factors affectingspecies delimitations with the GMYC model insightsfrom a butterfly survey Methods Ecol Evol 4(12)1101ndash1110 doi1011112041-210X12107
Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
Tewksbury J Anderson JGT Bakker JD Billo TJDunwiddie PW Groom MJ Hampton SE HermanSG Levey DJ Machnicki NJ del Rio CM PowerME Rowell K Salomon AK Stacey L TrombulakSC Wheeler TA (2014) Natural historyrsquos place inscience and society BioScience doi101093bioscibiu032
Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
Werth S Cornejo C Scheidegger C (2013) Characteriza-tion of microsatellite loci in the lichen fungus Lobariapulmonaria (Lobariaceae) Appl Plant Sci 1(2)1200290 doi103732apps1200290
Westberg M Arup U Kaumlrnefelt I (2007) Phylogeneticstudies in the Candelariaceae (lichenized Ascomy-cota) based on nuclear ITS DNA sequence dataMycol Res 111(11)1277ndash1284 doi101017S0953756204002102
Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
2 The Dynamic Discipline of Species Delimitation hellip 43
(Lepidoptera Lycaenidae) Front Zoo 4(1)8 doi1011861742-9994-4-8
Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
Guillo
trsquosUnified
Mod
elmdashpo
pulatio
nassignmentusingBayesianclustering
Thisapproach
provides
astatistical
model
that
allowsoneto
analyzegenetic
and
phenotyp
ticdata
with
inaun
ified
mod
elandinferencefram
eworkandop
tionally
incorporateinform
ationaboutthespatialdistributio
nof
samplesA
Bayesianclustering
algorithm
assumes
that
each
clusterin
ageographical
domaincanbe
approxim
ated
bypo
lygons
that
arecentered
around
pointsgeneratedby
aPo
issonprocessGuillo
trsquos
Unified
Mod
elisflexiblein
term
sof
thegenetic
data
that
itcanutilize
andcapableof
accurately
delim
iting
species
Limita
tions
Genetic
andphenotypic
data
cantracedifferentevolutionary
historiesfor
instancephylogenetic
divergence
forneutralgenetic
markers
andadaptatio
nfora
morphological
structure
Source
Availableas
anextensionof
theRGENELAND
package(G
uillo
tet
al20
05
2012)(http
www2im
mdtudk
gigu
Geneland)
Genotyp
icandno
n-genetic
(eg
phenotypicalecolog
icalgeograph
ical
behavioral)data
STRUCTUREmdash
populatio
nassignment
usingBayesianclustering
Amodel-based
clustering
methodusingmultilocus
genotype
data
toinferpopulatio
nstructureandassign
individualsto
populatio
nsIndividualsin
thesampleareassigned
probabilistically
topopulatio
nso
rjointly
totwoor
morepopulatio
nsiftheirgenotypes
indicatethatthey
areadmixedT
hemodeldoes
notassum
eaparticular
mutationprocess
anditcanbe
appliedto
mosto
fthecommonly
used
genetic
markersp
rovidedthat
they
areno
tcloselylin
ked
The
methodcanproducehigh
lyaccurate
assignmentsusing
modestn
umbersof
loci(Pritchard
etal2
000)T
hemostappropriatelevelo
fpopulatio
nstructurecanbe
inferred
byassessinglik
elihoodscores
orthead
hocΔKstatistic
(Evann
oet
al20
05)
Limita
tions
Identifying
themostappropriatenumberof
genetic
clusters
ischallenging
clusters
produced
byST
RUCTUREcanbe
strongly
influenced
byvariationin
sample
sizeclusters
createdby
STRUCTUREmay
notbe
consistent
with
theevolutionary
historyof
thepopulatio
nswhentherearerelativ
elylong
divergence
times
with
inevolutionary
lineagesTem
poraldivergence
andrelatio
nships
amongputativ
egroups
isnotexplicitlyestim
atedEquivalence
togenetic
clusters
tospecies-levelgroups
isuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
httppritchardlabstanfordedustructurehtm
ldescribedin
Falush
etal(200
3)andPritchard
etal(200
0)
Genotyp
icdata
(con
tinued)
2 The Dynamic Discipline of Species Delimitation hellip 19
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
Structuram
amdashpo
pulatio
nassignmentusing
Bayesianclustering
Implem
entstheclustering
algorithm
used
inST
RUCTURE(see
above)
that
clusters
samples
into
populatio
nsby
minim
izingHardyndashWeinb
ergdisequ
ilibrium
foragiven
partitioninglevelHow
everStructuram
aalso
includes
theadditio
nof
reversible-jum
pMCMCto
identifytheop
timalpartition
inglevelNearlyanytype
ofgenetic
datacanbe
inpu
tinto
Structuram
aandtheprog
ram
canassign
individu
alsto
populatio
nwith
orwith
outtheadmixture
Limita
tions
Tem
poraldivergence
andrelatio
nships
amongputativ
egroups
isnot
explicitlyestim
atedE
quivalence
togenetic
clusters
tospecies-levelg
roupsisuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
httpctegberkeleyedu
structuram
aindexhtmldescribedin
Huelsenbeck
etal(201
1)
Genotyp
icdata
ABGDmdashbarcodegapusinggenetic
distances
ldquoAutom
aticBarcode
Gap
Discoveryrdquosortssequencesinto
hypotheticalspeciesbasedon
thebarcodegap
The
methoduses
arecursiveapproach
topartition
thedata
andtestfor
sign
ificant
gapsA
BGDisfastsim
plemethodto
split
asequence
alignm
entd
atasetinto
candidatespeciesthat
should
becomplem
entedwith
otherevidence
inan
integrative
taxono
mic
approach
Limita
tions
Single-locus
data
aloneshould
only
beused
toprovideaprelim
inary
perspectiveof
speciesboundaries
andnotas
thesole
evidence
inspecies
circum
scriptions
Source
httpwwwabisnv
jussieufrpublicabg
ddescribedin
Puillandreet
al(201
2)
Single-locus
sequence
alignm
ent
GMYCampbG
MYCmdashgene
tree
The
GMYC
approach
combinesacoalescent
model
ofintraspecificbranchingwith
aYulemod
elforinterspecificbranching
which
isthen
fitto
aninferred
sing
le-gene
topology
toestim
atespeciesboundaries
andastatistical
measure
ofconfi
denceforthe
inferred
boundariesA
san
inputtheGMYCapproach
requires
anultram
etricgene
tree
andrecent
refinementscanaccountforuncertaintyin
phylogenetic
relatio
nships
and
parametersof
theGMYCmod
elT
heGMYCisgenerally
stable
across
awiderangeof
circum
stancesincludingvariousmethods
ofphylogeneticreconstructio
nthepresence
ofahigh
numbersingletonshigh
numbers
ofsampled
speciesandgaps
inintraspecific
sampling
theaccuracy
oftheGMYCismostsign
ificantly
affected
bythemean
populatio
nsize
relativ
eto
divergence
times
betweenthem
Limita
tions
The
GMYCmay
delim
itwell-supportedclades
ofhaplotypes
asindependent
lineagesandas
such
may
beproneto
over-delim
itatio
nSingle-locus
data
aloneshould
only
beused
toprov
ideaprelim
inaryperspectiveof
speciesbo
undaries
andno
tas
the
sole
evidence
inspeciescircum
scriptions
Source
(http
r-forger-projecto
rgprojectssplitshttpssitesgooglecom
site
noahmreidhom
esoftware)describedin
Monaghanet
al(200
9)Fu
jisaw
aandBarrac-
loug
h(201
3)andPo
nset
al(200
6)
Sing
le-locus
ultram
etricgene
tree (con
tinued)
20 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
PTPamp
bPTPmdash
gene
tree
The
Poissontree
processes(PTP)
modelcanbe
used
toinferputativ
especiesboundaries
onagivenphylogenetic
inputtreePT
Pcaninferputativ
especiesboundaries
that
are
consistent
with
thePS
CAnim
portantadvantageof
thismethodisthat
itmod
els
speciatio
nin
term
sof
thenumberof
substitutionsthereby
circum
ventingthepotentially
error-proneandcompute-intensive
processof
generatin
gultram
etrictreeswhich
are
required
asan
inpu
tfor
GMYCmod
el(see
abov
e)F
urthermoreitappearsthatthePT
Pmodel
may
outperform
theGMYCandotherOTU-picking
methods
whenevolutionary
distancesaresm
all
Limita
tions
Single-locus
data
aloneshould
only
beused
toprovideaprelim
inary
perspectiveof
speciesboundaries
andnotas
thesole
evidence
inspecies
circum
scriptions
Source
httpscoh-itsorgexelix
iswebsoftwarePT
Pindexhtmldescribedin
Zhang
etal(201
3)
Sing
le-locus
gene
tree
GeneiousSp
eciesDelim
itatio
nplug
-inmdash
gene
tree
Aplug-into
theGeneioussoftwareprovides
anexploratorytool
allowingtheuser
toassess
phylog
enetic
supportanddiagno
sabilityof
speciesdefinedas
user-selected
collections
oftaxa
onuser-sup
pliedtreesThe
plug
-incompu
tesstatisticsrelatin
gto
the
probability
oftheob
served
mon
ophyly
orexclusivity
having
occurred
bychance
ina
coalescent
processandassesses
thewith
in-andbetween-speciesgenetic
distancesto
infertheprobability
with
which
mem
bers
ofaputativ
especiesmight
beidentifi
edsuccessfully
with
tree-based
methods
Limita
tions
The
plug-insummarizes
measuresof
phylogenetic
supportand
diagnosabilityof
speciesdefinedas
user-selectedcollections
oftaxabutitdoes
not
providedefinitiv
esupportforspeciesgroupsItassumes
speciesaremonophyletic
Source
Implem
entedas
aplug-into
Geneious(geneiouscom)describedin
Mastersetal
(201
1)
Sing
le-locus
gene
tree
BPamp
Pmdashmultispecies
coalescent
model
for
speciesvalid
ation
Thisapproach
tospeciesdelim
itatio
nuses
aBayesianmodelingapproach
togeneratethe
posteriorprobabilitiesof
speciesassignmentstaking
accountof
uncertaintiesdueto
unknow
ngene
treesandtheancestralcoalescent
processThe
methodrelieson
auser-
specified
guidetreeimplem
entin
gareversible-jum
pMarkovchainMonte
Carlo
search
ofparameter
spacethatincludes
θpopulatio
ndivergenceand
estim
ated
distributio
nsof
gene
treesfrom
multip
leloci
Limita
tions
Misspecificatio
nsof
priorsandthegu
idetree
canresultin
inflatedspeciatio
nprobabilitiesitassumes
norecombinatio
nandcomputatio
nallim
itatio
nsrestrict
itsutility
with
next-generationdatasetswith
100s
ofloci
Source
httpabacusgeneuclacuksoftwarehtmldescribedin
YangandRannala
(201
0)andRannala
andYang(200
3)
Multilocus
sequence
alignm
entsandgroup
mem
bership
(con
tinued)
2 The Dynamic Discipline of Species Delimitation hellip 21
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
DISSE
CTmdashmultispecies
coalescent
model
forspeciesdelim
itatio
nDISSE
CTexplores
thefullspaceof
possible
clusteringsof
individualsandspeciestree
topologies
inaBayesianfram
ework
Toavoidtheneed
forreversible-jum
pMCMCit
uses
anapproxim
ationin
theform
ofapriorthatisamodificatio
nof
thebirthndashdeathprior
forthespeciestreeItisim
plem
entedas
partof
BEAST
andrequires
only
afewchanges
from
astandard
BEAST
analysisA
nalysesof
simulated
andem
piricald
atasuggestthat
themethodisshow
nto
beinsensitive
tothedegree
ofapproxim
ation
butq
uitesensitive
tootherparameters
Limita
tions
Recently
describedmethodlackingathorough
theoretical
andem
pirical
evaluatio
nItappearsthat
alargenu
mberof
sequencesarerequ
ired
todraw
firm
conclusion
sSo
urce
httpcodegooglecompbeast-m
cmcamphttpwwwin
driid
com
dissectinbeast
htmldescribedin
JonesandOxelm
an(201
4)
Multilocus
sequence
data
SpeD
eSTEMmdashmultispecies
coalescent
mod
elfordiscov
ery
valid
ation
andjoint
estim
ation
Thismaxim
umlik
elihoo
dandorinform
ationtheory-based
methodwas
developedto
test
speciesboundaries
inasystem
with
existin
gsubspecies
taxonomy(CarstensandDew
ey20
10)andcomputestheprobability
ofthegene
treesgiventhespeciestree
forall
hierarchical
perm
utations
oflin
eage
grou
pingSp
eciesbo
undaries
arecomparedusing
Akaikeinform
ationcriteria
andphylogenetic
uncertaintyin
thespeciestree
topologies
does
notaffect
speciesdelim
itatio
nsLimita
tions
Accuracyisdependenton
quality
ofthegene
tree
estim
ates
Source
(http
carstenslaborgohio-stateedusoftwarehtml)criteria
describedin
Ence
andCarstens(201
1)
Multilocus
sequence
alignm
entsandgroup
mem
bership
Browniemdash
multispecies
coalescent
model
forspeciesdelim
itatio
nThe
nonp
aram
etricheuristic
speciesdelim
itatio
napproach
implem
entedin
theprog
ram
Brownie(O
rsquoMeara
2010)jointly
sortsanonym
oussamples
into
speciesandinfers
aspeciestree
from
inputg
enetreesfrom
differentlociassumingthatforaspeciatio
neven
thecorrespondingnodeson
gene
treeswill
bemoreconsistent
with
each
than
the
divergenceswith
inspecies
Limita
tions
Findingboth
theoptim
umspeciestree
andspeciesboundaries
remains
compu
tatio
nally
challenging
andBrowniehasbeen
show
nto
frequently
yieldincorrect
resultsT
heaccuracy
ofthemethodislik
elycorrelated
with
nodalsupportvalues
inthe
individu
algene
topo
logies
Source
httpwwwbrianom
earain
fobrownie
describedin
OrsquoM
eara
(201
0)
Individual
gene
trees
(con
tinued)
22 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
BFD
mdashmultispecies
coalescent
model
for
speciesdelim
itatio
nThe
recently
developedmethod
Bayes
factor
delim
itatio
n(with
genomicdataB
FD)
combinesady
namic
programmingalgorithm
forestim
atingspeciestreesthat
bypasses
thecompu
tatio
nally
intensiveMCMCintegrationov
ergene
treesto
prov
idearigorous
techniqueforspeciesdelim
itatio
nstudiesusinggenome-wideSN
PdataCom
petin
gspeciesdelim
itatio
nmodelsarecomparedusingBayes
factorsanditappearsthat
this
approach
isrobustto
samplesizes(iefew
loci
andlim
itedsamples
perspecies)
and
misspecificatio
nof
thepriorforpo
pulatio
nsize
(θ)
Limita
tions
Recently
describedmethodlackingathorough
theoretical
andem
pirical
evaluatio
nSo
urce
httpwwwbeast2orgwikiindexphpBFD
describedin
Leacheacuteet
al(201
4)
Genom
e-wideSN
Pdata
2 The Dynamic Discipline of Species Delimitation hellip 23
distances among organisms belonging to thesame species are smaller than distances amongorganisms from different species (Puillandreet al 2012 Hebert et al 2003) Genetic distanceapproaches hold particular promise as an identi-fication tool shortcutting the difficulties of mor-phology-based identification (Hebert et al 2004)although in practice a barcode gap may not existfor many groups (Wiemers and Fiedler 2007)Furthermore the role of distance-based approa-ches using a single genetic marker for speciesdiscovery remains more controversial (Rubinoff2006) and without other corroborating evidenceOTUs inferred from single-locus dataset shouldonly be considered ldquocandidaterdquo species Tree-based methods aim to detect monophyletic cladescorresponding to species-level diversity bydetecting discontinuities associated within inter-and intraspecific branching patterns (Fujisawaand Barraclough 2013 Zhang et al 2013Monaghan et al 2009 Pons et al 2006) Tree-based species delimitation methods can also beused on the basis of other properties related tophylogenetic tree topologies (monophyly con-cordance with geography etc (Sites and Marshall2003 2004) Both distance- and tree-basedmethods have been applied for assessing speciesboundaries in lichen-forming fungi (Leavitt et al2012c 2014 Parnmen et al 2012 Del-Pradoet al 2010 2011 Molina et al 2011)
A number of tree-based methods partiallyautomate the species delimitation process withspecific bioinformatical analyses (Table 21)including the general mixed Yule coalescent(GMYC) approach (Fujisawa and Barraclough2013 Monaghan et al 2009 Pons et al 2006)and the Poisson tree processes (PTP) model(Zhang et al 2013) These methods provide rel-atively straightforward and objective pipelinesfor delimiting putative species-level lineagesfrom inferred gene trees by fitting within- andbetween-species branching models to an inferredsingle-locus topology A number of other tree-based methods for species delimitation areeffectively summarized in Sites and Marshall(2004)
When only single-locus data are available theGMYC has been shown to be a relatively robust
tool for species delimitation The GMYCapproach combines a coalescent model of intra-specific branching with a Yule model for inter-specific branching which is then fit to an inferredsingle-gene topology to estimate species bound-aries and a statistical measure of confidence for theinferred boundaries As an input the GMYCapproach requires an ultrametric gene tree andrecent refinements can account for uncertainty inphylogenetic relationships and parameters of theGMYC model (Fujisawa and Barraclough 2013Reid and Carstens 2012) Regardless of theseimprovements it may be difficult to accuratelyinfer an adequate ultrametric tree for large data-sets Although it appears that the GMYC isgenerally stable across a wide range of circum-stances including various methods of phyloge-netic reconstruction the presence of a highnumber singletons high numbers of sampledspecies and gaps in intraspecific sampling theaccuracy of the GMYC is most significantlyaffected by the mean population size relative todivergence times between them (Fujisawa andBarraclough 2013 Talavera et al 2013) Fur-thermore research suggests that the so-calledsingle-threshold version of the GMYC methodlikely outperforms the multiple-thresholdapproach (Fujisawa and Barraclough 2013Monaghan et al 2009) However other studiessuggest that theGMYCmethodmay often providehigher estimates for the total number ofOTUs thanother molecular species delimitation methods(Hamilton et al 2014 Miralles and Vences 2013Talavera et al 2013) warranting a cautiousinterpretation of results from GMYC analyses
Compared to the GMYC approach therecently introduced PTP method for speciesdelimitation has been suggested to be moreaccurate for preliminary species delimitation(Zhang et al 2013) Relative to the GMYC PTPoffers a more straightforward implementationrequiring a simple phylogenetic tree rather thanan ultrametric chronogram However moreresearch is required to assess the general perfor-mance of PTP across wide range of empirical andsimulated species delimitation studies At thispoint data support the use of the GMYC andPTP methods as objective and reasonable starting
24 SD Leavitt et al
points for species delimitation using single-genetopologies
The ldquoSpecies Delimitationrdquo plug-in to theGeneious software provides statistical approachesfor exploring species boundaries in single-genetopologies (Masters et al 2011) Using a priorispecimen assignments to putative species theldquoSpecies Delimitationrdquo plug-in computes statis-tics relating to the probability of the observedmonophyly or exclusivity having occurred bychance in a coalescent process and assesses thewithin- and between-species genetic distances inorder to infer the probability with which membersof a putative species might be identified suc-cessfully with tree-based methods An importantcontribution of the ldquoSpecies Delimitationrdquo plug-in is that it provides an objective means for usersto assess putative species within an empiricalstatistic-based framework rather than qualitativeevaluations of what level of hierarchy constitutesa species in a phylogeny
In contrast to simple sequence similaritythresholds (OTU-picking) for delimiting putativeevolutionarily significant units the AutomaticBarcode Gap Discovery (ABGD) method is anautomated procedure that sorts sequences intohypothetical species based on the existence of abarcode gap observed whenever intraspecificgenetic distances are less than those amongorganisms from different species (Puillandreet al 2012) Ultimately ABGD is a fast simplemethod that can be used to group individualsrepresented in a single-locus sequence alignmentinto candidate species that should be comple-mented with other lines of evidence in an inte-grative taxonomic approach (Kekkonen andHebert 2014 Puillandre et al 2012)
In general the ABGD GMYC and PTP ana-lytical protocols using single-locus data arerepeatable and computationally relatively fastproviding a valuable starting point for a pre-liminary perspective into species boundaries inunderstudied groups that can be validated withsubsequent studies (Kekkonen and Hebert 2014)Similarly analyses of single-locus data can beused to corroborate traditional phenotype-basedspecies boundaries and identify candidate speciesthat have previously been hidden within nominal
species However single-locus species delimita-tionmethodsmaybeparticularly prone to failure inrecognizing significant proportions of species-level biodiversity due to the strict criterion forreciprocal monophyly or alternatively providespurious inflations of estimated species diversitybased on genetic differences that donot correspondto species-level lineages For example recentestimates suggest that the incidence of species-level gene tree paraphyly is approximately 20 (Ross 2014) suggesting that analyses of single-locus datasets would likely fail to accurately deli-mit species in one in every five cases In contrastrecent empirical studies suggest that in some casesthe GMYC provides a striking overestimate ofspecies diversity (Miralles and Vences 2013)
The internal transcribed spacer region (ITS)has played a central role in molecular phyloge-netic studies of lichenized fungi and has recentlybeen adopted as the official barcoding marker forfungi (Schoch et al 2012) In many cases the ITSis sufficiently variable to resolve species bound-aries for lichenized fungi (Schoch et al 2012Kelly et al 2011) although accurate specimenidentification using sequence-based identificationapproaches requires a thoroughly curated refer-ence database (Leavitt et al 2014 Orock et al2012 Kelly et al 2011) In spite of the generalutility of the ITS marker a number of issues maypotentially limit its effective use in speciesdelimitation studies including the potential forintragenomic variation of the nuclear ribosomaltandem repeat and difficulties in aligning ITSsequences from divergent taxa (Kiss 2012)Because the ITS has been formally adopted as theofficial barcoding marker for fungi we recom-mend that species delimitation studies attempt toinclude this region for consistency across studiesHowever we recognize that in some taxonomicgroups the inclusion of the ITS may be prob-lematic and therefore suggest a careful screeningof other markers to identify appropriate loci forresolving species-level relationships
Ultimately the success of any single-locusspecies delimitation method largely depends onthe evolutionary history of the species groupunder study and the variability of the selectedmarker (Fig 22) Species delimitation will be
2 The Dynamic Discipline of Species Delimitation hellip 25
more difficult in more recently diverged lineagesand in cases with some level of interspecific geneflow relative to older well-diverged lineages(eg Leavitt et al 2012c 2013e) In the end wereemphasize that single-locus data alone shouldonly be used to provide a preliminary perspectiveof species boundaries and not as the sole evi-dence in species circumscriptions
222 Sampling Across the GenomeMultilocus Sequence Dataand Genome-Wide Markers
Although single-locus methods can provide anefficient approach for preliminary large-scaleassessments of species diversity there are
significant limitations particularly in recentlydiverged species groups where retention ofancestral polymorphisms and incomplete lineagesorting will likely result in different neutral locihaving unique gene topologies that do not mirrorthe speciation process (Knowles and Carstens2007 Heled and Drummond 2010 Taylor et al2000) In contrast to single-locus and strictlyphenotype-based approaches for species delimi-tation analyses of genetic data collected fromindependent genomic regions can provide robusthypotheses of species boundaries with increasingconfidence (Satler et al 2013 Zhang et al 2011Yang and Rannala 2010) Sequences from mul-tiple independent loci provide an importantsource of data for species delimitation studiesincluding recently developed models that
Fig 22 A simplified diagram illustrating the process ofspeciation through time in a single gene history andresulting gene topologies sampled at two points in thespeciation history (modified from Leliaert et al 2014)aEach dot represents a distinct gene copy and each row onenon-overlapping generation with lines connecting genecopies to their ancestors in the previous generation (onerow below) dashed diagonal lines represent reproductivebarriers two hypothetical sampling intervals at differentpoints in the speciation history are shown Tx and Ty
species delimitation methods using single-locus data areeffective only when species are reciprocally monophyleticin the sampled gene tree genetic clustering and coalescent-based species delimitation methods can circumscribespecies when species may not be monophyletic in sampledgenetic loci b Gene topology representing sampledhaplotypes at time Tx (shown in panel a) hypotheticalspecies are reciprocally monophyletic c Gene topologyrepresenting sampled haplotypes at time Ty (shown in panela) hypothetical species are para- and polyphyletic
26 SD Leavitt et al
combine individual gene genealogies and speciesphylogenies via modeling the coalescent historyof markers (Yang and Rannala 2010 Edwards2009 Knowles and Carstens 2007) As aresponse to advances in sequencing technologiesbioinformatical approaches for multimarker spe-cies delimitation analyses continue to be devel-oped (Table 21 Camargo and Sites 2013)
Long-term reproductive isolation of candidatespecies can be assessed with multilocus sequencedata by evaluating genealogical concordance ofunlinked markers (Baum and Shaw 1995 Aviseand Ball 1990) Within species the mixingeffects of recombination cause unlinked loci tohave distinct genealogical histories but geneticdrift and long-term divergence leads to concor-dant genealogical histories at loci across thegenome Relationships of individuals belongingto distinct candidate species can be evaluatedusing gene genealogies (eg haplotype networksor single-gene topologies) to identify lineagesthat exhibit genealogical exclusivity acrossunlinked neutral loci (Hudson and Coyne 2002Dettman et al 2003b Avise and Ball 1990) Thepresence of the same clades in the majority ofsingle-locus genealogies is taken as evidence thatthe clades represent reproductively isolated lin-eages (Dettman et al 2003a Pringle et al 2005)In practice the criteria of reciprocal monophylyand genealogical concordance of unlinked lociprovide a conservative approach for assessingspecies boundaries due to the fact that a sub-stantial amount of time is required after the initialdivergence of species until ancestral polymor-phisms have fully sorted (Knowles and Carstens2007 Hudson and Coyne 2002) Consequentlygroups with recent divergence histories willlikely go undiscovered under a genealogicalconcordance criterion due to the fact that speciesboundaries likely are not reflected in genegenealogies For example it would take morethan 1 million years after speciation before spe-cies would be delimited if 15 loci were sampledin species with an effective population size (Ne)of 100000 assuming one generation a yearunder a strict reciprocal monophyly criterion(Knowles and Carstens 2007) The amount oftime required for a species to be recognized using
a criterion of reciprocal monophyly increasesproportionally with increasing population sizes(Hudson and Coyne 2002) In a number ofstudies of lichen-forming fungi a genealogicalconcordance criterion has been used to bothdelimit previously unrecognized species-levellineages and validate some conventional pheno-type-based species (Leavitt et al 2012a c2013b Molina et al 2011 Kroken and Taylor2001)
Although analyses of molecular sequence datarelying on reciprocal monophyly or fixed char-acter differences for species delimitation canserve as important criteria for identifying speciesthese criteria are not commonly met acrossmultiple loci particularly in recent speciationhistories (Fig 22) To accommodate theobserved conflict among genealogies from mul-tiple loci with the underlying speciation historythe recent merge of coalescent theory with phy-logenetics has driven a major paradigm shift inspecies delimitation and molecular systematics ingeneral (Fujita et al 2012 Edwards 2009) Themultispecies coalescent model can be applied togenealogical histories from multiple independentloci to assemble separate coalescent processesoccurring in populations into a species tree(Degnan and Rosenberg 2009 Rannala andYang 2003) Within this coalescent-basedframework multiple individuals can be assignedto a single speciespopulation and the speciationhistory of ancestral and descendant species-levellineages can be inferred as a ldquospecies treerdquo incontrast to estimating gene genealogies fromindividual samples (Degnan and Rosenberg2006 2009 Rannala and Yang 2003)
A number of multispecies coalescent-basedspecies delimitation methods have recently beenintroduced offering an exciting framework forempirically assessing species boundaries byselecting the best species tree model from a set ofalternative models representing differenthypotheses of species boundaries (Table 21)For example SpeDeSTEM (Ence and Carstens2011) finds the maximum likelihood values for aspecies tree assuming all putative species areseparate lineages and for alternative species treeswhere two or more species are collapsed into a
2 The Dynamic Discipline of Species Delimitation hellip 27
single lineage The best fitting model is thenselected using the Akaike information criterionunder the assumption of constant populationsizes and fixed gene topologies across the speciestree The species delimitation program BayesianPhylogenetic and Phylogeography (BPampPYangand Rannala 2010) accommodates gene treeuncertainty and variable population sizes andsamples from the Bayesian posterior distributionof species delimitation models using reversible-jump Markov chain Monte Carlo (rjMCMC)sampling BPampP requires a user-provided guidetree with resolution finer than the species leveland evaluates alternative modes derived from allpossible subtrees that are generated by collapsingor splitting nodes on the guide tree (Yang andRannala 2010 Rannala and Yang 2003) Theproportion of time spent on each model is pro-portional to the posterior probability of themodel ie ldquospeciation probabilitiesrdquo WhileBPampP ranks among the most popular coalescent-based species delimitation programs (Fujita et al2012) misspecifications of the guide tree andorthe prior distribution for population size (θ) canresult in strong support for models containing aninflated number of species (Leacheacute and Fujita2010 Zhang et al 2011) The nonparametricheuristic species delimitation approach imple-mented in the program Brownie (OrsquoMeara 2010)jointly sorts anonymous samples into species andinfers a species tree from input gene trees fromdifferent loci assuming that for a speciationevent the corresponding nodes on gene trees willbe more consistent with each than the diver-gences within species However finding both theoptimum species tree and species boundariesremains computationally challenging andBrownie has been shown to frequently yieldincorrect results (OrsquoMeara 2010)
Most recently available coalescent-basedspecies delimitation methods require individualassignments to a species or population a priori Ina number of scenarios correct assignments ofsamples to species may be difficult includingpresence of cryptic species incongruencebetween conventional species and moleculardata or simply the fact that accurate specimenidentification in complex groups with subtle or
difficult to discern diagnostic characters is anontrivial task (Leavitt et al 2013e) Statisticalmethods for assessing individual assignment andspecies detection prior to coalescent-based spe-cies delimitation and species tree reconstructionand species provide a more objective approachfor understanding species boundaries
Population assignment tests using a variety ofclustering algorithms using genetic data offer auseful approach for grouping individuals intoputative reproductively isolated groups (Table 21Hausdorf and Hennig 2010 Corander et al2004 2008 Falush et al 2003 Pritchard et al2000) particularly for species delimitation prob-lems that exist at the interface of traditionalpopulation genetic and phylogenetic analyses(Carstens et al 2013 Weisrock et al 2010Knowles and Carstens 2007)
The programs STRUCTURE (Falush et al2003 Pritchard et al 2000) and STRUCTURA-MA (Huelsenbeck et al 2011) cluster samplesinto populations by minimizing HardyndashWeinbergdisequilibrium for a given number of populationclusters using unlinked genetic markers In gen-eral unlinked markers are not available for mostgroups of lichen-forming fungi and a numberof studies have used information from multilocussequence data for STRUCTURE analyses(Leavitt et al 2011a b c 2013e Fernaacutendez-Mendoza and Printzen 2013 Fernaacutendez-Mendoza et al 2011) Varying approaches havebeen implemented to convert DNA sequencedata to allelic data for Bayesian clustering (seeOrsquoNeill et al 2013) STRUCTURE is expectedto perform well when there is sufficient inde-pendence across regions such that linkage dis-equilibrium within regions does not dominate thedata (STRUCTURE manual) but can also beeffective using multilocus sequence data andtreating all SNPs as independent loci regardlessof physical linkage within each locus (OrsquoNeillet al 2013 Falush et al 2003) A recent study ofthe lichen-forming genus Letharia showed thatBayesian clustering implemented in the programSTRUCTURE was generally able to recover thesame putative Letharia lineages circumscribedemploying a gene genealogical approach inKroken and Taylorrsquos iconic species delimitation
28 SD Leavitt et al
study (Altermann et al 2014 Kroken and Taylor2001) Altermann et al (2014) show that popu-lation assignments were largely consistent acrossa range of scenarios including extensiveamounts of missing data the exclusion of SNPsfrom variable markers and inferences based onSNPs from as few as three gene regions Incontrast to STRUCTURE and STRUCTURA-MA the program BAPS (Corander et al 2008)can explicitly infer genetic structure using hap-loid sequence data or other linked geneticmarkers in the ldquoclustering with linked locirdquomodel Another advantage of BAPS is that theprogram is much more computationally efficientthan STRUCTURE or STRUCTURAMA Sim-ulation studies indicate that both BAPS andSTRUCTURE perform well at low levels ofpopulation differentiation and when clusters arenot well differentiated (Latch et al 2006)
In practice inferring the most appropriatelevel of population structure using Bayesianclustering algorithms remains challenging (Latchet al 2006 Evanno et al 2005) BAPS andSTRUCTURAMA can infer both individualassignments and the most likely number ofgenetic clusters (Corander et al 2008 Huelsen-beck et al 2011) and the ad hoc ΔK statisticproposed by Evanno et al (2005) provides anobjective approach for identifying the uppermosthierarchical level of structure However carefulconsideration of other levels of populationstructure may ultimately reveal more biologicallymeaning results and researchers should examineindividual assignments across a range of geneticgroupings Some markers generated fromindependent genomic regions such as SNPs andfast-evolving microsatellites can be used to dis-tinguish fine-scale population structuring on thebasis of allele frequencies and species delimita-tion analyses based on these markers may implythe risk of severe taxonomic oversplitting Inthese cases validation approaches such asBPampP and SpeDeSTEM and corroborationamong different species delimitation approachescan provide perspective between intraspecificpopulation structure and species-level clusters
Another limitation of clustering approaches isthat they do not assess or take into account
evolutionarily divergences and relationshipsamong population clusters Coalescent-basedspecies tree methods (discussed below) provide ameans to assess the diversification histories ofpopulations inferred from clustering analysesTherefore a potential working protocol for aninformed species delimitation study that takesinto account population structure could consist offirst applying a genetic clustering analysis under apopulation genetics criterion (eg BAPSSTRUCTURE STRUCTURAMA) to identifygenetically distinct population clusters that can beconsidered ldquocandidate speciesrdquo From these can-didate species a species tree can be inferred forfocal group using coalescent-based species treereconstruction methods (eg BEAST) Subse-quently a coalescent-based validation methodcan be applied to assess whether the distinctpopulation clusters represent independent evolu-tionary lineages (eg BPampP) This multistepapproach would provide a consistent and standardcriterion for distinguishing between population-and species-level lineages (Camargo and Sites2013) and has been applied to a number of casesincluding lichen-forming fungi (Leavitt et al2011a b c 2013e Leacheacute and Fujita 2010)
High-throughput sequencing methods providethe means to effectively sample hundreds tothousands of loci from across a species genome fora large number of species and continue to revolu-tionize studies that can be performed even in non-model organisms Targeted high-throughputsequencing approaches such as anchored phy-logenomics transcriptome sequencing reduced-representation genomic library sequencing(restriction-site-associated DNA sequencingRAD-Seq and genotype-by-sequencing GBS)and high-throughput amplicon sequencing pro-vide important insight into species boundaries fora number groups although none of these approa-ches has yet been applied to studies of lichenizedfungi
For example restriction-site-associated(RAD-Seq) DNA can simultaneously detect andgenotype thousands of genome-wide SNPs byfocusing the sequencing effort on a reducedrepresentation of the entire genome (Baird et al2008) and has been successfully applied to
2 The Dynamic Discipline of Species Delimitation hellip 29
intraspecies (Lewis et al 2007 Miller et al 2007Emerson et al 2010) and interspecies studies(Rubin et al 2012 Eaton and Ree 2013 Wagneret al 2013) This approach has provided strikinginsight into the recent adaption radiation of LakeVictoria cichlids (Wagner et al 2013) Analternative approach targeting the sequencing ofspecific loci using next-generation sequencingplatforms provides an efficient means of gener-ating data for loci of known genomic locationorthology size and expected level of variation(OrsquoNeill et al 2013) Markers can be targetedusing well-established PCR techniques or newerhybridization techniques and subsequentlypooled for high-throughput sequencing usingparallel-tagged sequencing of multiple individualsamples within a single sequencing run (OrsquoNeillet al 2013) A recent study of North Americantiger salamanders (Ambystoma tigrinum)demonstrated the potential for amplicon-basedparallel-tagged sequencing to rapidly generatelarge-scale genomic data for species delimitationand species tree research (OrsquoNeill et al 2013)
Although these methods provide an enormousamount of data and insight into diversification atan unprecedented scale computational limita-tions restrict the applicable analytical methodsfor large datasets although computational andanalytical advances are happening rapidly Mostrecent coalescent-based species delimitation andspecies tree models using gene trees have beenlimited to handle tens of loci for multiple indi-viduals and combining hundreds or thousands ofgene trees into a single species delimitationframework presents considerable computationalchallenges (Camargo and Sites 2013) Therecently developed method Bayes factor delim-itation (with genomic data BFD) combines adynamic programming algorithm for estimatingspecies trees that bypasses the computationallyintensive MCMC integration over gene trees toprovide a rigorous technique for species delimi-tation studies using genome-wide SNP data(Leacheacute et al 2014) Competing species delimi-tation models are compared using Bayes factorsand it appears that this approach is robust tosample sizes (ie few loci and limited samples
per species) and misspecification of the prior forpopulation size (θ) (Leacheacute et al 2014)
Fungi are an ideal model for assessing diver-gence in eukaryotes due to their simple mor-phologies small genomes broad ecological rolesand diverse lifestyles (Gladieux et al 2014)However the use of genomic data from high-throughput sequencing methods have not yet beenincluded in species delimitation studies of lichen-forming fungi This is due in part to challenges indealing with metagenomic data containing geno-mic information from a plethora of symbiontsassociated with a lichen thallus scant genomicresources and discipline-specific inertia A num-ber of lichen-forming fungal genomes are cur-rently available including Endocarponpusillum(Wang et al 2014) Cladonia spp (Park et al2013a 2014) Caloplaca flavorubescens (Parket al 2013b) and Cladonia greyi and Xanthoriaparientina through the Fungal Genomics Programat Joint Genome Institute of the United StatesDepartment of Energy (httpwwwjgidoegov)The foreseeable ongoing expansion of genomicresources for lichen-forming fungi will be centralto developing approaches for delimiting speciesusing high-throughput sequencing Specificallygenomic resources will be crucial in identifyingnovel markers (variable genes regions SNPsmicrosatellites etc) identifying conserved fungalmarkers for targeted high-throughput sequencingapproaches and references for entire genomecomparisons (Devkota et al 2014 Werth et al2013)
23 Can We Make SpeciesDelimitation in Lichen-FormingFungi Truly Integrative
The widespread availability of genetic data hascreated a biased viewpoint that only genetic datashould be used for statistical species delimitationHowever an ongoing appeal to researchers toassess species boundaries from multiple andcomplementary perspectives (phylogeneticspopulation genetics comparative morphologydevelopment ecology etc) has resulted in an
30 SD Leavitt et al
integrative taxonomic framework bringing theseconceptual and methodological perspectivestogether (Hamilton et al 2014 Fujita et al 2012Salicini et al 2011 Yeates et al 2011 Padialet al 2010 Dayrat 2005 Will et al 2005 Wiensand Penkrot 2002) In practice any study linkingdifferent kinds of data to support hypotheses ofspecies boundaries including mapping morpho-logical characters onto a molecular phylogenycan be considered integrative Integrative meth-ods for species delimitation fall across a broadspectrum ranging from verbal and qualitativeassessments of data classes to quantitative meth-ods that allow different data types to contribute tostatistical species delimitation (Yeates et al2011) In most taxonomic studies utilizing bothmolecular and morphological data expert opinionis eventually used to some degree to evaluate thefinal status of a candidate species (eg Bond andStockman 2008) In this sense many studiesusing evidence for independent data sources fordelimiting species boundaries use an iterativeapproach (Yeates et al 2011) where speciesboundaries can be tested within a hypothetico-deductive framework with diverse datasets
From a practical perspective we advocate aprocess of iterative taxonomy (sensu Yeates et al2011) to circumscribe and refine species limitsusing multiple lines of evidence This iterativeprocess involves comparisons of morphologicaldata with a phylogenetic hypothesis (ie single-locus gene tree or concatenated multilocusphylogeny) to identify the least inclusive mono-phyletic clade in the topology characterized by atleast one unambiguously diagnostic morphologi-cal character (Miralles and Vences 2013) Thisphylogenetic tree-informed approach to assessingspecies boundaries represents a common practicein studies of lichen-forming fungi Previouslyunrecognized species-level clades with corre-sponding subtle or overlooked phenotypiccharacters have been commonly observed in bothcrustose lichens [eg Acarospraceae (Wedin et al2009) Graphidaceae (Rivas Plata and Lumbsch2011 Papong et al 2009) Lecanoraceae (Leavittet al 2011a) Lecideaceae (Ruprecht et al 2010)Mycoblastaceae (Spribille et al 2011) andTeloschistaceae (Vondraacutek et al 2009 Muggia
et al 2008)] and foliose and fruticose lichens [egLobariaceae (Moncada et al 2014 McDonaldet al 2003) Parmeliaceae (Leavitt et al 2013aWirtz et al 2012 Divakar et al 2005 Molinaet al 2004 Kroken and Taylor 2001) Peltigera-ceae (OrsquoBrien et al 2009 Goffinet et al 2003)Physciaceae (Elix et al 2009 Divakar et al2007) and Sphaerophoraceae (Houmlgnabba andWedin 2003)] As a specific example Luumlckinget al (2008) used a combination of medullarychemistry and underside pigmentation in speci-mens from the Heterodermia obscurata group inCosta Rica to corroborate monophyletic clades inan ITS gene tree as species-level lineages
While this iterative approach to speciesdelimitation and taxonomy has proven valuablefor understanding species boundaries anddescribing new taxa in some groups of lichenizedfungi a posteriori examination of morphologicaland chemical features has failed to reveal diag-nostic phenotypic characters in a number ofstudies (Muggia et al 2014 Leavitt et al 2011aOtaacutelora et al 2010 OrsquoBrien et al 2009) Fur-thermore a study of widespread species in thegenus Melanelixia (Parmeliaceae) indicated thatphenotypically cryptic lichen-forming fungalspecies-level lineages may be relatively ancientand diagnosable phenotypic differences may beabsent even millions of years after the initialdivergence (Leavitt et al 2012b) The latter studyhighlights the fact that species-level lineages maycommonly exist without any observable diag-nostic phenotypic characters calling into ques-tion the impetus for a universal application ofintegrative taxonomy
In keeping with the principle that as manylines of evidence as available should be com-bined to delimit species (Dayrat 2005) the for-malized integrative taxonomic approach (ITAXMiralles and Vences 2013) uses a mtDNA guidetree and observations from different types of datathat might provide conclusive evidence for theindependence of lineages and thus their identityas different species Miralles and Vences (2013)provide a non-exhaustive list of species delimi-tation criteria to be integrated in the ITAXapproach including (a) sympatric occurrencewithout admixture as revealed by consistent
2 The Dynamic Discipline of Species Delimitation hellip 31
differences in morphological or molecular char-acters at the same geographic location (b) strongdifferences in a behavioral morphological orgenetic character known to mediate prematingisolation (c) unviability or infertility of hybrids(d) lack of gene flow across a geographicalhybrid zone (e) congruent diagnostic differencesbetween sister lineages in various unlinkedmorphological character (f) absence of haplo-type sharing in several unlinked nuclear loci and(g) a combination of criteria endashf Speciesboundaries are based on seeking the least inclu-sive monophyletic group in the mtDNA treewhich fulfills at least one of the criteria listedabove Clearly the ITAX approach is sensitive tosample size and in order to reliably support thedistinctiveness of a given species it has beenrecommended that the sampling strategy includesat least five individuals per species (Miralles andVences 2013) A similar approach could beadopted for lichenized fungi using an ITS genetopology as guide tree rather than mtDNA
A total evidence approach including concat-enation has been a common approach for inte-grating information from different sourcesincluding independent genetic markers in phy-logenetic reconstructions (Kluge 1989 Wiens1998 de Queiroz et al 1995) Proponents forconcatenation of independent data in phyloge-netic analyses argue that when combining alldata the underlying signal of speciation mayemerge even if weakly contradictory signalis contained in the individual data partitions(Gatesy et al 1999) For example establishing apreliminary perspective of species boundariesfrom multilocus sequence data using concatena-tion may provide a reasonable starting point forscreening for cryptic species and species treeinference (Šlapeta et al 2006 Leavitt et al2011a Le Gac et al 2007 Leacheacute 2009)However concatenation and consensus methodsimply a risk of obtaining inflated support forincorrect relationships and information aboutvariance in gene coalescence is lost (Degnan andRosenberg 2006 2009 Kubatko and Degnan2007 Edwards 2009) In spite of the impetus forintegrating evidence from independent data
sources into empirical species delimitation stud-ies and taxonomy (Fujita et al 2012 Padial et al2009) most currently available species delimi-tation methods are unable to accommodate non-genetic data sources in a statistical framework
231 Selecting the Appropriate Data
In the face of increasing availability of geneticdata and associated bioinformatical approachesfor delimiting species researchers should care-fully consider what information is being sacri-ficed by the failure to consider non-genetic datain species delimitation studies and whetheraccuracy could be improved by the addition ofmultiple data types Morphological data havehistorically served as a proxy to identify repro-ductively isolated groups (ie ldquospeciesrdquo) (Ray1686 Fujita et al 2012) Current methods fordelimiting species using non-genetic data (egchemistry morphology and ecology) remainwoefully understudied For example morphol-ogy-based species circumscriptions are generallybased on one or more qualitative (or quantitative)morphological characters that do not appear tooverlap with other species However ascertain-ing that a given trait is truly fixed within apopulation with statistical confidence requiresunrealistic sample sizes even when allowing forsome level of polymorphism in the diagnosticcharacter (Wiens and Servedio 2000) Now var-ious combinations of datamdashfrom morphologygenetics geography and ecologymdashare acceptedas standard information for species delimitationstudies (Ruiz-Sanchez and Sosa 2010 Ross et al2010 Edwards and Knowles 2014)
Below we briefly discuss appropriate datasources for species delimitation studies of lichen-forming fungi However homoplastic characters(similar traits that are not derived from a com-mon ancestor) are common among many traitscommonly used to circumscribe fungal taxa thebiological significance of secondary metabolitevariation remains largely unknown (Lawrey1986) and ecological niches may be difficultto adequately characterize and model due to
32 SD Leavitt et al
microhabitat requirements and data resolutionTherefore we advocate a cautious approach toselecting appropriate and relevant data forassessing species boundaries
In spite of the potential challenges and limi-tations of using phenotypic data these traits haveprovided a plethora of valuable information forunderstanding species boundaries For examplein the Melanelixia fuliginosa group (Parmelia-ceae) a morphometric analysis using color isi-dia and marginal zone free of isidia ascharacters revealed a general pattern of differ-entiation between material formerly recognizedas subspecies fuliginosa and glabratula in spiteof the fact that considerable overlap betweengroups occurs in some characters individually(Arup and Berlin 2011) Furthermore the dis-tinction of the two groups was supported by aphylogenetic analysis of the ITS marker andecological differences with M fuliginosaoccurring predominantly on rock and M glab-ratula on bark (Arup and Berlin 2011) Thisstudy of the M fuliginosa group provides a fit-ting example of using multiple independentsuites of data including ecology morphologyand genetic information to establish a robusthypothesis of species boundaries
In lichen systematics phenotypic dataincluding thallus organization secondary metab-olites mode of reproduction ascoma-type andontogeny ascus and ascospore characters havehistorically played a prominent role (Printzen2009) Ascomatal characters have traditionallyheld a major role in higher-level classification(Printzen 2009) in contrast to species-levelclassification which also tends to include a widearray of vegetative and chemical charactersCommonly assessed morphological charactersinclude thallus form and size cortical features(eg maculae and pseudocyphellae) presenceformcolor of attachment structure (eg rhizines)and reproductive mode (ascomata vs vegetativediaspores) (Printzen 2009) Different morpho-logical types of vegetative diasporesmdashcorticatedisidia and ecorticated sorediamdashand their locationare commonly used to distinguish speciesAscomatal characters including morphologylocation (laminal vs marginal) position (sessile
vs immersed) presence of thalline margins colorof an apothecial disc and presence or color ofpruina are also commonly used in speciesdelimitations of lichen-forming fungi (Printzen2009) Other important characters may includethalline characters form color size and septationof ascospores size and form and structure of ascithe hamathecium type of epihymenium andhypothecium and the type of excipulum orperidium conidiomatal characters etc (Printzen2009)
Assessments of secondary metabolites hasplayed an important role in lichen taxonomybeginning with the introduction of simple spottests by Nylander (1866a b) The use of chem-istry in lichen taxonomy has been discussed indetail in numerous reviews (Lumbsch 1998a bRogers 1989 Brodo 1986 Egan 1986 Leuckert1985 Brodo 1978 Hawksworth 1976 Culber-son 1969 1970) and we refer readers to thesevaluable sources for a more comprehensive per-spective on lichen chemistry In short extrolites(secondary metabolites) belong to various clas-ses the most common and diverse include dep-sides depsidones chlorinated xanthones andanthraquinones (Lumbsch 2002 Culberson1969) The presence or absence of specificextrolites or their replacements by another sub-stance is widely used to distinguish speciesparticularly when correlated with differences ingeographic distributions However if morpho-logical or geographical differences betweenpopulations containing different extrolites are notapparent the taxonomic significance has beendisputed with some authors distinguishing themas species and others preferring to regard them aschemical races within a species In addition tosimply using the presence or absence of extro-lites Culberson and Culberson (1976) proposedto arrange lichen substances into chemosyn-dromes of closely related substances The pres-ence or absence of these chemosyndromes maypotentially be used as characters to delimit spe-cies regarding differences involving the samechemosyndromes as intraspecific variation anddistinct chemosyndromes as evidence for inter-specific populations (Lumbsch 1994 Gowan1986)
2 The Dynamic Discipline of Species Delimitation hellip 33
Due to the fact that species delimitationstudies often incorporate a substantial biogeo-graphical or ecological component ecologicalniche modeling plays an increasing role in phy-logenetic and taxonomic research Niche mod-eling can provide evidence for ecologicalisolation between populations based on eitherconserved or divergent ecological niches andtherefore can provide additional evidence sup-porting lineage independence between putativespecies The application of ecological nichemodeling has been applied to species delimita-tion studies in a number of cases (Ruiz-Sanchezand Sosa 2010 Leacheacute et al 2009 Raxworthyet al 2007 Rissler and Apodaca 2007) althoughit has not been explicitly applied to assess speciesboundaries in lichen-forming fungi By mappingthe spatial distribution of environmental suit-ability of climatic variables for candidate speciesthe application of ecological niche modeling canbe particularly important in cases where specieshave allopatric distributions (Raxworthy et al2007)
Ecological niche modeling utilizes knownassociations between a speciesrsquo occurrencelocalities and environmental variables to defineabiotic conditions within which populations canbe maintained (Guisan and Thuiller 2005) Themethodological approach for modeling is basedon four general properties (i) The current knownspeciesrsquo localities is the dependent variable (ii)the distribution is modeled as a map composed ofgrid cells at a specified resolution (iii) a range ofenvironmental variables (eg temperature pre-cipitation and solar exposure) are collected todescribe the characteristics of each cell and (iv)classifying the degree to which each cell is eithersuitable or unsuitable for each species under arange of models (Guisan and Thuiller 2005)
Ecological data also have the potential to playan important role in understanding speciesboundaries in lichen-forming fungi For exampleNash and Zavada (1977) demonstrated that Xant-hoparmelia populations with distinct chemistriesoccurring in the northern portion of the SonoranDesert exhibit habitat selection among differentrock substrates within a region with relativelyuniform climate and topography In another case
the parmelioid species Parmelia mayi is mor-phologically indistinguishable from P saxatilisbut can be separated by bioclimatic features andgenetic and chemical characters (Molina et al2011) McCune and Printzen(2011) assess distri-butions and climatic niches of species in theLecanora varia group inwesternUSAandprovidea model that uses continental influence and annualtemperature as themajor factors predicting speciesdistributions The distribution of Usnea hirta alichen commonly used in air quality biomonitor-ing research was modeled for a section of theWhite River National Forest in central Coloradobased on the presence of U hirta at 72 biomoni-toring reference sites distributed in the inter-mountain western United States (Shrestha et al2012) The best model for predicting U hirtadistribution included four variablesmdashsolar radia-tion average monthly precipitation and averagemonthly minimum and maximum temperatures(Shrestha et al 2012) These studies supportthe potential use of ecological niche modelingmethods in species delimitation studies of lichen-forming fungi
24 Conclusions What AboutTaxonomy
In most cases species circumscription and tax-onomy requires some degree of qualitativejudgment and individual interpretation Integrat-ing multiple types of data into an empiricalframework for delimiting species boundarieswhere species boundaries can be tested within ahypothetico-deductive framework with diversedatasets can provide robust hypotheses of speciesboundaries and taxonomic stability (Yeates et al2011) However it has long been known toevolutionary biologists that distinct species donot need to have diagnosable morphologicaldifferences (Mayr 1963) and increasing avail-ability of genetic data has allowed researchers toidentify species and to rigorously test speciesboundaries with a level of precision that wasunimaginable a decade ago While analyticaladvances in statistical species delimitation havebeen largely based on genetic data the utility of
34 SD Leavitt et al
these approaches to formal taxonomy remainselusive Due to these challenges an eclecticapproach to delimiting species and cautionagainst the reliance on any single dataset ormethod is required when delimiting species
While results from many recent studies maycontradict traditional species boundaries acrossmany groups of lichen-forming fungi we areoptimistic that this research represents substantialprogress toward a more accurate perspective onspecies boundaries and diversity in fungi As aresult of ongoing research of species boundariesin lichen-forming fungi the taxonomic value ofmany phenotypes is now better understood ourunderstanding of ecological evolutionary andbiogeographic patterns has improved and we canbegin to better understand patterns of symbiontinteractions in lichens Integrating new data(including novel morphological characters andgenetic data) will be essential to accurately rep-resent species-level diversity across all groups oflichenized fungi Hopefully an improved per-spective on lichen diversity also increases ourappreciation of these incredible symbiotic sys-tems While we are strong advocates for theapplication of independent data types in devel-oping an integrative taxonomy there is anincreasing need to formally recognize the exis-tence of phenotypically cryptic species-levellineages in lichen-forming fungi (see Hibbettet al 2011) In some cases a molecular taxon-omy may provide the most practical approach toconsistent treatment of mycobiont species withinlichen groups where diagnostic morphologicalcharacters are unidentifiable or practically notfeasible (Leavitt et al 2013c d)
These are exciting times for taxonomists andphylogeneticists A closer look at lichen taxon-omy with the inclusion of new data will help usto better understand the diversity of these fasci-nating organisms accurately interpret distribu-tion patterns and play a more important role inmeaningful conservation practices Howeversome level of uncertainty will accompany pro-gress In many taxonomic groups our traditionalapproach for species identification will likelyneed to be substantially modified The search forcorroborating morphological support for cryptic
species identified using molecular data willrequire meticulous and creative approaches toassess phenotypic variation in potentially unor-thodox ways We are hopeful that lichenologistswho traditionally have been eager to include newmethods such as chromatography in their rou-tine identifications will be amenable to includemolecular techniques to their routine examina-tion of specimens for identification and classifi-cation Although this may prove difficult toachieve by single individuals especially citizenscientists that traditionally play an important rolein lichen taxonomy the increasing numberof collaborative projects in the discipline (egLumbsch et al 2011 Crespo et al 2010Gueidan et al 2009) make us optimistic thatbroad-scale collaborative approaches will facili-tate the inclusion of molecular data in lichenresearch at all levels
Acknowledgments We are indebted to various col-leagues for valuable thought-provoking discussionnotably Matthew Nelsen (University of Chicago) AnaCrespo (Universidad Complutense de Madrid) PradeepDivakar (Universidad Complutense de Madrid) BeckettSterner (The Field Museum) and Joyce Havstad (TheField Museum) We also thank anonymous reviewers whoprovided valuable comments that improved this chapterSupport by the US National Science Foundation isgratefully acknowledged (ldquoHidden diversity in parmelioidlichensrdquo DEB-0949147)
References
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Altermann S Leavitt SD Goward T Nelsen MPLumbsch HT (2014) How do you solve a problemlike Letharia A new look at cryptic species in lichen-forming fungi using Bayesian clustering and SNPSsfrom multilocus sequence data PLoS ONE 9(5)e97556 doi101371journalpone0097556
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2 The Dynamic Discipline of Species Delimitation hellip 35
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Corander J Marttinen P (2006) Bayesian identification ofadmixture events using multi-locus molecular mark-ers Mol Ecol 15(10)2833ndash2843 doi101111j1365-294X200602994x
Corander J Waldmann P Marttinen P Sillanpaa M(2004) BAPS 2 enhanced possibilities for the analysisof genetic population structure Bioinformatics 20(15)2363ndash2369 doi101093bioinformaticsbth250
Corander J Marttinen P Mantyniemi S (2006) Bayesianidentification of stock mixtures from molecular markerdata Fish Bull 104550ndash558
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Cracraft J (1983) Species concepts and speciation anal-ysis Curr Ornithol 1159ndash187
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Culberson WL (1969) The use of chemistry in thesystematics of the lichens Taxon 18498ndash505
Culberson WL (1970) Chemosystematics and ecology oflichen-forming fungi Annu Rev Ecol Syst 1153ndash170
Culberson CF Culberson WL (1976) Chemosyndromicvariation in lichens Syst Bot 1325ndash339
Darwin C (1859) On the origin of species by means ofnatural selection or the preservation of favoured racesin the struggle for life J Murray London
Dayrat B (2005) Towards integrative taxonomy Biol JLinn Soc 85(3)407ndash415 doi101111j1095-8312200500503x
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de Queiroz K (1998) The general lineage concept ofspecies species criteria and the process of speciationa conceptual unification and terminological recom-mendations In Howard DJ Berlocher SH (eds)Endless forms species and speciation Oxford Uni-versity Press Oxford pp 57ndash75
de Queiroz K (1999) The general lineage concept ofspecies and the defining properties of the speciescategory In Wilson RA (ed) Species new interdis-ciplinary essays MIT Press Cambridge pp 49ndash89
de Queiroz K (2007) Species concepts and speciesdelimitation Syst Biol 56(6)879ndash886 doi10108010635150701701083
de Queiroz A Donoghue MJ Kim J (1995) Separateversus combined analysis of phylogenetic evidenceAnnu Rev Ecol Syst 26(1)657ndash681 doi101146annureves26110195003301
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Degnan JH Rosenberg NA (2009) Gene tree discordancephylogenetic inference and the multispecies coales-cent Trends Ecol Evol 24(6)332ndash340 doihttpdxdoiorg101016jtree200901009
Del-Prado R Cubas P Lumbsch HT Divakar PK BlancoO de Paz GA Molina MC Crespo A (2010) Geneticdistances within and among species in monophyleticlineages of Parmeliaceae (Ascomycota) as a tool fortaxon delimitation Mol Phylogenet Evol 56(1)125ndash133 doi101016jympev201004014
Del-Prado R Divakar PK Crespo A (2011) Using geneticdistances in addition to ITS molecular phylogeny toidentify potential species in the Parmotrema reticul-atum complex a case study The Lichenologist 43(06)569ndash583 doi101017S0024282911000582
Del-Prado R Blanco O Lumbsch HT Divakar PK ElixJA Molina MC Crespo A (2013) Molecular phylog-eny and historical biogeography of the lichen-formingfungal genus Flavoparmelia (Ascomycota Parmelia-ceae) Taxon 62(5)928ndash939 doi101270562522
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Dettman JR Jacobson DJ Turner E Pringle A TaylorJW (2003b) Reproductive isolation and phylogeneticdivergence in Neurospora comparing methods ofspecies recognition in a model eukaryote Evolution57(12)2721 doi10155403-074
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Divakar PK Molina MC Lumbsch HT Crespo A (2005)Parmelia barrenoae a new lichen species related toParmelia sulcata (Parmeliaceae) based on molecularand morphological data The Lichenologist 37(01)37ndash46 doi101017S0024282904014641
Divakar PK Amo De paz G del Prado R Esslinger TLCrespo A (2007) Upper cortex anatomy corroboratesphylogenetic hypothesis in species of Physconia(Ascomycota Lecanoromycetes) Mycol Res 111(11)1311ndash1320 doi101016jmycres200708009
Divakar PK Figueras G Hladun N Crespo A (2010)Molecular phylogenetic studies reveal an undescribedspecies within the North American concept of Mela-nelixia glabra (Parmeliaceae) Fungal Divers 42(1)47ndash55 doi101007s13225-010-0027-3
Donoghue MJ Gauthier A (2004) Implementing thephylocode Trends Ecol Evol 19(6)281ndash282 doi101016jtree200404004
Eaton DAR Ree RH (2013) Inferring phylogeny andintrogression using RADseq data an example fromflowering plants (Pedicularis Orobanchaceae) SystBiol 62(5)689ndash706 doi101093sysbiosyt032
Edwards SV (2009) Is a new and general theory ofmolecular systematics emerging Evolution 63(1)1ndash19 doi101111j1558-5646200800549x
Edwards DL Knowles LL (2014) Species detection andindividual assignment in species delimitation canintegrative data increase efficacy Proc R Soc B BiolSci 281(1777) doi101098rspb20132765
Egan RS (1986) Correlations and non-correlations ofchemical variation patterns with lichen morphologyand geography The Bryologist 8999ndash110
Elix JA Corush J Lumbsch HT (2009) Triterpenechemosyndromes and subtle morphological characterscharacterise lineages in the Physcia aipolia group inAustralia (Ascomycota) Syst Biodivers 7(04)479ndash487 doi101017S1477200009990223
Emerson KJ Merz CR Catchen JM Hohenlohe PACresko WA Bradshaw WE Holzapfel CM (2010)Resolving postglacial phylogeography using high-throughput sequencing Proc Natl Acad Sci 107(37)16196ndash16200 doi101073pnas1006538107
Ence DD Carstens BC (2011) SpedeSTEM a rapid andaccurate method for species delimitation Mol EcolResour 11(3)473ndash480 doi101111j1755-0998201002947x
EvannoGRegnaut SGoudet J (2005)Detecting thenumberof clusters of individuals using the software STRUC-TURE a simulation study Mol Ecol 14(8)2611ndash2620doi101111j1365-294X200502553x
Falush D Stephens M Pritchard JK (2003) Inference ofpopulation structure using multilocus genotype datalinked loci and correlated allele frequencies Genetics164(4)1567ndash1587 doi101111j1471-8286200701758x
Fan HH Kubatko LS (2011) Estimating species treesusing approximate Bayesian computation Mol Phylo-genet Evol 59(2)354ndash363 doi101016jympev201102019
Fernaacutendez-Mendoza F Printzen C (2013) Pleistoceneexpansion of the bipolar lichen Cetraria aculeata intothe Southern hemisphere Mol Ecol 22(7)1961ndash1983doi101111mec12210
Fernaacutendez-Mendoza F Domaschke S Garciacutea MA JordanP Martin MP Printzen C (2011) Population structure
2 The Dynamic Discipline of Species Delimitation hellip 37
of mycobionts and photobionts of the widespreadlichen Cetraria aculeata Mol Ecol 20(6)1208ndash1232doi101111j1365-294X201004993x
Fraley C Raftery A (2007) Model-based methods ofclassification Using the mclust software in chemo-metrics J Stat Softw 18i06 [Available online at httpwwwdoajorgdoajfunc=abstractampid=218544]
Fujisawa T Barraclough TG (2013) Delimiting speciesusing single-locus data and the generalized mixed yulecoalescent (GMYC) approach a revised method andevaluation on simulated datasets Syst Biol 62(5)707ndash724 doi101093sysbiosyt033
Fujita MK Leacheacute AD Burbrink FT McGuire JA MoritzC (2012) Coalescent-based species delimitation in anintegrative taxonomy Trends Ecol Evol 27(9)480ndash488 doi101016jtree201204012
Gatesy J OrsquoGrady P Baker RH (1999) Corroborationamong data sets in simultaneous analysis hiddensupport for phylogenetic relationships among higherlevel Artiodactyl taxa Cladistics 15(3)271ndash313doi101111j1096-00311999tb00268x
Gladieux P Ropars J Badouin H Branca A Aguileta Gde Vienne DM Rodriacuteguez de la Vega RC Branco SGiraud T (2014) Fungal evolutionary genomicsprovides insight into the mechanisms of adaptivedivergence in eukaryotes Mol Ecol 23(4)753ndash773doi101111mec12631
Goffinet B Miadlikowska J Goward T (2003) Phyloge-netic inferences based on nrDNA sequences supportfive morphospecies within the Peltigera didactylacomplex (Lichenized Ascomycota) The Bryologist106(3)349ndash364 doi10163901
Gowan SP (1986) Evolution of secondary natural productsin the genus Porpidia (Ascomycata Porpidiaceae) AmJ Bot 73606
Griffin PC Hoffmann AA (2014) Limited genetic diver-gence among Australian alpine Poa tussock grassescoupled with regional structuring points to ongoinggene flow and taxonomic challenges Ann Bot doi101093aobmcu017
Grube M Hawksworth DL (2007) Trouble with lichenthe re-evaluation and re-interpretation of thallus formand fruit body types in the molecular era Mycol Res111(9)1116ndash1132 doi101016jmycres200704008
Gueidan C Savi S Thues H Roux C Keller C Tibell LPrieto M Heimarsson S Breuss O Orange A FrobergL Wynns AA Navarro-Rosines P Krzewicka BPykaelae J Grube M Lutzoni F (2009) Genericclassification of the Verrucariaceae (Ascomycota)based on molecular and morphological evidencerecent progress and remaining challenges Taxon 58(1)184ndash208
Guillot G Mortier F Estoup A (2005) Geneland acomputer package for landscape genetics Mol EcolNotes 5(3)712ndash715 doi101111j1471-8286200501031x
Guillot G Renaud S Ledevin R Michaux J Claude J(2012) A unifying model for the analysis of pheno-typic genetic and geographic data Syst Biol 61(6)897ndash911 doi101093sysbiosys038
Guisan A Thuiller W (2005) Predicting species distribu-tion offering more than simple habitat models EcolLett 8(9)993ndash1009 doi101111j1461-0248200500792x
Hale ME (1990) A synopsis of the lichen genusXanthoparmelia (Vainio) Hale (AscomycotinaParmeliaceae) Smithsonian Institution Press Wash-ington DC
Hamilton CA Hendrixson BE Brewer MS Bond JE(2014) An evaluation of sampling effects on multipleDNA barcoding methods leads to an integrativeapproach for delimiting species a case study ofthe North American tarantula genus Aphonopelma(Araneae Mygalomorphae Theraphosidae) MolPhylogenet Evol 7179ndash93 doi101016jympev201311007
Hausdorf B (2011) Progress toward a general speciesconcept Evolution 65(4)923ndash931 doi101111j1558-5646201101231x
Hausdorf B Hennig C (2010) Species delimitation usingdominant and codominant multilocus markers SystBiol 59(5)491ndash503 doi101093sysbiosyq039
Hawksworth DL (1976) Lichen chemotaxonomy InBailey RH (ed) Lichenology progress and problemsAcademic London pp 139ndash184
Hebert PDN Cywinska A Ball SL deWaard JR (2003)Biological identifications through DNA barcodesProc R Soc Lond B Biol Sci 270(1512)313ndash321doi101098rspb20022218
Hebert PDN Stoeckle MY Zemlak TS Francis CM(2004) Identification of birds through DNA barcodesPLoS Biol 2(10)e312 doi101371journalpbio0020312
Heled J Drummond AJ (2010) Bayesian inference ofspecies trees from multilocus data Mol Biol Evol 27(3)570ndash580 doi101093molbevmsp274
Hey J (2006) On the failure of modern species conceptsTrends Ecol Evol 21(8)447ndash450 doi101016jtree200605011
Hibbett DS OhmanA Glotzer D NuhnMKirk P NilssonRH (2011) Progress in molecular and morphologicaltaxon discovery in Fungi and options for formalclassification on environmental sequences Fungal BiolRev 2538ndash47 doi101016jfbr201101001
Houmlgnabba F Wedin M (2003) Molecular phylogeny ofthe Sphaerophorus globosus species complex Cladis-tics 19(3)224ndash232 doi101111j1096-00312003tb00365x
Hudson RR Coyne JA (2002) Mathematical conse-quences of the genealogical species concept Evolu-tion 56(8)1557 doi101111j0014-38202002tb01467x
Huelsenbeck JP Andolfatto P Huelsenbeck ET (2011)Structurama Bayesian inference of population struc-ture Evol Bioinf Online 7(2011)55ndash59 doi104137EBOS6761
Jones G Oxelman B (2014) DISSECT an assignment-free Bayesian discovery method for species delimita-tion under the multispecies coalescent Bioinformaticsdoi101101003178
38 SD Leavitt et al
Kekkonen M Hebert PD (2014) DNA barcode-baseddelineation of putative species efficient start fortaxonomic workflows Mol Ecol Res doi1011111755-099812233
Kelly LJ Hollingsworth PM Coppins BJ Ellis CJHarrold P Tosh J Yahr R (2011) DNA barcoding oflichenized fungi demonstrates high identification suc-cess in a floristic context New Phytol 191(1)288ndash300doi101111j1469-8137201103677x
Kiss L (2012) Limits of nuclear ribosomal DNA internaltranscribed spacer (ITS) sequences as species barcodesfor fungi Proc Natl Acad Sci 109(27)E1811ndashE1811doi101073pnas1207143109
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Knowles LL Carstens BC (2007) Delimiting specieswithout monophyletic gene trees Syst Biol 56(6)887ndash895 doi10108010635150701701091
Kroken S Taylor JW (2001) A gene genealogical approachto recognize phylogenetic species boundaries in thelichenized fungus Letharia Mycologia 93(1)38ndash53
Kubatko LS Degnan JH (2007) Inconsistency of phylo-genetic estimates from concatenated data under coa-lescence Syst Biol 56(1)17ndash24 doi10108010635150601146041
Latch EK Dharmarajan G Glaubitz JC Rhodes OE Jr(2006) Relative performance of Bayesian clusteringsoftware for inferring population substructure andindividual assignments at low levels of populationdifferentiation Conserv Genet 7(2)295ndash302 doi101007s10592-005-9098-1
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Le Gac M Hood ME Fournier E Giraud T (2007)Phylogenetic evidence of host-specific cryptic speciesin the anther smut fungus Evolution 61(1)15ndash26doi101111j1558-5646200700002x
Leacheacute AD (2009) Species tree discordance traces tophylogeographic clade boundaries in North Americanfence lizards (Sceloporus) Syst Biol 58(6)547ndash559doi101093sysbiosyp057
Leacheacute AD Fujita MK (2010) Bayesian species delim-itation in West African forest geckos (Hemidactylusfasciatus) Proc R Soc B Biol Sci 2773071ndash3077doi101098rspb20100662
Leacheacute AD Koo M Spencer C Papenfuss T Fisher RMcGuire J (2009) Quantifying ecological morpho-logical and genetic variation to delimit species in thecoast horned lizard species complex (Phrynosoma)Proc Natl Acad Sci USA 10612418ndash12423 doi101073pnas0906380106
Leacheacute AD Fujita MK Minin VN Bouckaert RR (2014)Species delimitation using genome-wide SNP dataSystematic Biology doi101093sysbiosyu018
Leavitt SD Fankhauser JD Leavitt DH Porter LDJohnson LA St Clair LL (2011a) Complex patterns ofspeciation in cosmopolitan ldquorock posyrdquo lichensmdashdiscovering and delimiting cryptic fungal species in
the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
Leavitt SD Esslinger TL Lumbsch HT (2012c) Neogene-dominated diversification in neotropical montanelichens dating divergence events in the lichen-form-ing fungal genus Oropogon (Parmeliaceae) Am J Bot99(11)1764ndash1777 doi103732ajb1200146
Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Lumbsch HT St Clair LL(2013c) DNA barcode identification of lichen-formingfungal species in the Rhizoplaca melanophthalmaspecies-complex (Lecanorales Lecanoraceae) includ-ing five new species MycoKeys 71ndash22 doi103897mycokeys74508
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Vondraacutek J Thorsten Lum-bsch H Clair LLS (2013d) Local representation ofglobal diversity in a cosmopolitan lichen-formingfungal species complex (Rhizoplaca Ascomycota)J Biogeogr 40(9)1792ndash1806 doi101111jbi12118
Leavitt SD Lumbsch HT Stenroos S St Clair LL(2013e) Pleistocene speciation in North American
2 The Dynamic Discipline of Species Delimitation hellip 39
lichenized fungi and the impact of alternative speciescircumscriptions and rates of molecular evolution ondivergence estimates PLoS ONE 8(12)e85240doi101371journalpone0085240
Leavitt SD Esslinger TL Hansen ES Divakar PK CrespoA Loomis BF Lumbsch HT (2014) DNA barcoding ofbrown Parmeliae (Parmeliaceae) species a molecularapproach for accurate specimen identification empha-sizing species in Greenland Organ Divers Evol 14(1)11ndash20 doi101007s13127-013-0147-1
Leliaert F Verbruggen H Vanormelingen P Steen FLopez-Bautista JM Zuccarello GC De Clerck O(2014) DNA-based species delimitation in algae Eur JPhycol 49(2) doi101080096702622014904524
Leuckert C (1985) Probleme der Flechten-Chemotaxon-omiemdashStoffkombinationen und ihre taxonomischeWertung Ber Deut Bot Ges 98401ndash408
Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
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Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
Lumbsch HT (1994) Die Lecanora subfusca-Gruppe inAustralasien J Hattori Bot Lab 771ndash175
Lumbsch HT (1998a) Taxonomic use of metabolic data inlichen-forming fungi In Frisvad JC Bridge PDArora DK (eds) Chemical Fungal Taxonomy MarcelDekker New York pp 345ndash387
Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
Mayden RL (1997) A hierarchy of species concepts thedenouement in the saga of the species problem InClaridge MF Dawah HA Wilson MR (eds) Speciesthe units of biodiversity Chapman amp Hall Londonpp 381ndash424
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McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
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40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
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Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
Mishler BD Brandon RN (1987) Individuality pluralismand the phylogenetic species concept Biol Philos 2397
Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
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Nylander W (1866a) Circa novum in studio Lichenumcriterium chemicum Flora 49198ndash201
Nylander W (1866b) Hypochlorite of lime and hydrate ofpotash Two new criteria for the study of lichens(Tanslated and communicated by the Rev W ALeighton) Bot J Linn Soc 9358ndash365
OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
OrsquoNeill EM Schwartz R Bullock CT Williams JSShaffer HB Aguilar-Miguel X Parra-Olea G Weis-rock DW (2013) Parallel tagged amplicon sequencingreveals major lineages and phylogenetic structure inthe North American tiger salamander (Ambystomatigrinum) species complex Mol Ecol 22(1)111ndash129doi101111mec12049
Orock EA Leavitt SD Fonge BA St Clair LL LumbschHT (2012) DNA-based identification of lichen-form-ing fungi Can publicly available sequence databasesaid in lichen diversity inventories of Mount Cameroon(West Africa) The Lichenologist 44(6)833ndash839doi101017S0024282912000424
Otaacutelora MAG Martiacutenez I Aragoacuten G Molina MC (2010)Phylogeography and divergence date estimates of alichen species complex with a disjunct distributionpattern Am J Bot 97(2)216ndash223 doi103732ajb0900064
Padial J Castroviejo-Fisher S Kohler J Vila C ChaparroJ De la Riva I (2009) Deciphering the products ofevolution at the species level the need for anintegrative taxonomy Zoolog Scr 38(4)431ndash447doi101111j1463-6409200800381x
Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
Papong K Luumlcking R Thammathaworn A BoonpragobK (2009) Four new taxa of Chroodiscus (thelotremoidGraphidaceae) from Southeast Asia The Bryologist112(1)152ndash163 doi1016390007-2745-1121152
Park S-Y Choi J Kim JA Jeong M-H Kim S Lee Y-HHur J-S (2013a) Draft genome sequence of Cladoniamacilenta KoLRI003786 a lichen-forming fungusproducing biruloquinone Genome Announcements 1(5)e00695-13 doi101128genomeA00695-13
Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
Peacuterez-Ortega S Fernaacutendez-Mendoza F Raggio J VivasM Ascaso C Sancho LG Printzen C de los Riacuteos A(2012) Extreme phenotypic variation in Cetrariaaculeata (lichenized Ascomycota) adaptation or inci-dental modification Ann Bot 109(6)1133ndash1148doi101093aobmcs042
Pino-Bodas R Burgaz A Martiacuten M Lumbsch HT (2011)Phenotypical plasticity and homoplasy complicatespecies delimitation in the Cladonia gracilis group(Cladoniaceae Ascomycota) Organ Divers Evol 11(5)343ndash355 doi101007s13127-011-0062-2
Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
Pino-Bodas R Martiacuten M Burgaz A (2012b) Cladoniasubturgida and C iberica (Cladoniaceae) form asingle morphologically and chemically polymorphicspecies Mycol Prog 11(1)269ndash278 doi101007s11557-011-0746-1
Pino-Bodas R Ahti T Stenroos S Martiacuten MP BurgazAR (2013a) Multilocus approach to species recogni-tion in the Cladonia humilis complex (CladoniaceaeAscomycota) Am J Bot 100(4)664ndash678 doi103732ajb1200162
Pino-Bodas R Martiacuten AP Burgaz AR Lumbsch HT(2013b) Species delimitation in Cladonia (Ascomy-cota) a challenge to the DNA barcoding philosophyMol Ecol Resour 13(6)1058ndash1068 doi1011111755-099812086
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Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
Pringle A Baker DM Platt JL Wares JP Latgeacute JPTaylor JW (2005) Cryptic speciation in the cosmo-politan and clonal human pathogenic fungus Asper-gillus fumigatus Evolution 59(9)1886ndash1899 doi10155404-2411
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Pritchard JK Stephens M Donnelly P (2000) Inference ofpopulation structure using multilocus genotype dataGenetics 155(2)945ndash959
Puillandre N Lambert A Brouillet S Achaz G (2012)ABGD automatic barcode gap discovery for primaryspecies delimitation Mol Ecol 21(8)1864ndash1877doi101111j1365-294X201105239x
Rannala B Yang Z (2003) Bayes estimation of speciesdivergence times and ancestral population sizes usingDNA sequences from multiple loci Genetics1641645ndash1656
Raxworthy CJ Ingram CM Rabibisoa N Pearson RG(2007) Applications of ecological niche modelingfor species delimitation a review and empiricalevaluation using day geckos (Phelsuma) fromMadagascar Syst Biol 56(6)907ndash923 doi10108010635150701775111
Ray J (1686) Historia planarum vol 1 Clark LondonReese Naeligsborg R Ekman S Tibell L (2007) Molecular
phylogeny of the genus Lecania (Ramalinaceaelichenized Ascomycota) Mycol Res 111(5)581ndash591doi101016jmycres200703001
Reid N Carstens B (2012) Phylogenetic estimation errorcan decrease the accuracy of species delimitation aBayesian implementation of the general mixed Yule-coalescent model BMC Evol Biol 12(1)196 doi1011861471-2148-12-196
Rissler L Apodaca J (2007) Adding more ecology intospecies delimitation ecological niche models andphylogeography help define cryptic species in theBlack Salamander (Aneides flavipunctatus) Syst Biol56924ndash942 doi10108010635150701703063
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Rivas Plata E Lumbsch HT (2011) Parallel evolution andphenotypic divergence in lichenized fungi a casestudy in the lichen-forming fungal family Graphida-ceae (Ascomycota Lecanoromycetes Ostropales)Mol Phylogenet Evol 61(1)45ndash63 doi101016jympev201104025
Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
Ross KG Gotzek D Ascunce MS Shoemaker DD (2010)Species delimitation a case study in a problematic anttaxon Syst Biol 59(2)162ndash184 doi101093sysbiosyp089
Rubin BER Ree RH Moreau CS (2012) Inferringphylogenies from RAD sequence data PLoS ONE 7(4)e33394 doi101371journalpone0033394
Rubinoff D (2006) Utility of mitochondrial DNA barcodesin species conservation Conserv Biol 20(4)1026ndash1033 doi101111j1523-1739200600372x
Ruiz-Sanchez E Sosa V (2010) Delimiting speciesboundaries within the Neotropical bamboo Otatea(Poaceae Bambusoideae) using molecular morpho-logical and ecological data Mol Phylogenet Evol 54(2)344ndash356 doi101016jympev200910035
Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
morphological characters Antarctic Sci 22 (SpecialIssue 06)727ndash741 doi101017S0954102010000477
Salicini I Ibaacutentildeez C Juste J (2011) Multilocus phylogenyand species delimitation within the Nattererrsquos batspecies complex in the Western Palearctic MolPhylogenet Evol 61(3)888ndash898 doi101016jympev201108010
Satler JD Carstens BC Hedin M (2013) Multilocusspecies delimitation in a complex of morphologicallyconserved trapdoor spiders (Mygalomorphae Antro-diaetidae Aliatypus) Syst Biol 62(6)805ndash823 doi101093sysbiosyt041
Schlick-Steiner BC Steiner FM Seifert B Stauffer CChristian E Crozier RH (2010) Integrative taxonomya multisource approach to exploring biodiversityAnnu Rev Entomol 55(1)421ndash438 doi101146annurev-ento-112408-085432
Schoch CL Seifert KA Huhndorf S Robert V SpougeJL Levesque CA Chen W Fungal Barcoding Con-sortium (2012) Nuclear ribosomal internal transcribedspacer (ITS) region as a universal DNA barcodemarker for Fungi Proc Natl Acad Sci 109(16)6241ndash6246 doi101073pnas1117018109
Seacuterusiaux E Villarreal AJC Wheeler T Goffinet B(2011) Recent origin active speciation and dispersalfor the lichen genus Nephroma (Peltigerales) inMacaronesia J Biogeogr 38(6)1138ndash1151 doi101111j1365-2699201002469x
Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
Simpson GG (1951) The species concept Evolution5285ndash298
Sites JW Marshall JC (2003) Delimiting species a renais-sance issue in systematic biology Trends Ecol Evol18462ndash470 doi101016S0169-5347(03)00184-8
Sites JW Marshall JC (2004) Operational criteria fordelimiting species Annu Rev Ecol Evol Syst 35(1)199ndash227 doi101146annurevecolsys35112202130128
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Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
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Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
Tewksbury J Anderson JGT Bakker JD Billo TJDunwiddie PW Groom MJ Hampton SE HermanSG Levey DJ Machnicki NJ del Rio CM PowerME Rowell K Salomon AK Stacey L TrombulakSC Wheeler TA (2014) Natural historyrsquos place inscience and society BioScience doi101093bioscibiu032
Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
Werth S Cornejo C Scheidegger C (2013) Characteriza-tion of microsatellite loci in the lichen fungus Lobariapulmonaria (Lobariaceae) Appl Plant Sci 1(2)1200290 doi103732apps1200290
Westberg M Arup U Kaumlrnefelt I (2007) Phylogeneticstudies in the Candelariaceae (lichenized Ascomy-cota) based on nuclear ITS DNA sequence dataMycol Res 111(11)1277ndash1284 doi101017S0953756204002102
Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
2 The Dynamic Discipline of Species Delimitation hellip 43
(Lepidoptera Lycaenidae) Front Zoo 4(1)8 doi1011861742-9994-4-8
Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
Structuram
amdashpo
pulatio
nassignmentusing
Bayesianclustering
Implem
entstheclustering
algorithm
used
inST
RUCTURE(see
above)
that
clusters
samples
into
populatio
nsby
minim
izingHardyndashWeinb
ergdisequ
ilibrium
foragiven
partitioninglevelHow
everStructuram
aalso
includes
theadditio
nof
reversible-jum
pMCMCto
identifytheop
timalpartition
inglevelNearlyanytype
ofgenetic
datacanbe
inpu
tinto
Structuram
aandtheprog
ram
canassign
individu
alsto
populatio
nwith
orwith
outtheadmixture
Limita
tions
Tem
poraldivergence
andrelatio
nships
amongputativ
egroups
isnot
explicitlyestim
atedE
quivalence
togenetic
clusters
tospecies-levelg
roupsisuncertain
andvalid
ationapproaches
canbe
used
toassess
evolutionary
independence
ofclusters
Source
httpctegberkeleyedu
structuram
aindexhtmldescribedin
Huelsenbeck
etal(201
1)
Genotyp
icdata
ABGDmdashbarcodegapusinggenetic
distances
ldquoAutom
aticBarcode
Gap
Discoveryrdquosortssequencesinto
hypotheticalspeciesbasedon
thebarcodegap
The
methoduses
arecursiveapproach
topartition
thedata
andtestfor
sign
ificant
gapsA
BGDisfastsim
plemethodto
split
asequence
alignm
entd
atasetinto
candidatespeciesthat
should
becomplem
entedwith
otherevidence
inan
integrative
taxono
mic
approach
Limita
tions
Single-locus
data
aloneshould
only
beused
toprovideaprelim
inary
perspectiveof
speciesboundaries
andnotas
thesole
evidence
inspecies
circum
scriptions
Source
httpwwwabisnv
jussieufrpublicabg
ddescribedin
Puillandreet
al(201
2)
Single-locus
sequence
alignm
ent
GMYCampbG
MYCmdashgene
tree
The
GMYC
approach
combinesacoalescent
model
ofintraspecificbranchingwith
aYulemod
elforinterspecificbranching
which
isthen
fitto
aninferred
sing
le-gene
topology
toestim
atespeciesboundaries
andastatistical
measure
ofconfi
denceforthe
inferred
boundariesA
san
inputtheGMYCapproach
requires
anultram
etricgene
tree
andrecent
refinementscanaccountforuncertaintyin
phylogenetic
relatio
nships
and
parametersof
theGMYCmod
elT
heGMYCisgenerally
stable
across
awiderangeof
circum
stancesincludingvariousmethods
ofphylogeneticreconstructio
nthepresence
ofahigh
numbersingletonshigh
numbers
ofsampled
speciesandgaps
inintraspecific
sampling
theaccuracy
oftheGMYCismostsign
ificantly
affected
bythemean
populatio
nsize
relativ
eto
divergence
times
betweenthem
Limita
tions
The
GMYCmay
delim
itwell-supportedclades
ofhaplotypes
asindependent
lineagesandas
such
may
beproneto
over-delim
itatio
nSingle-locus
data
aloneshould
only
beused
toprov
ideaprelim
inaryperspectiveof
speciesbo
undaries
andno
tas
the
sole
evidence
inspeciescircum
scriptions
Source
(http
r-forger-projecto
rgprojectssplitshttpssitesgooglecom
site
noahmreidhom
esoftware)describedin
Monaghanet
al(200
9)Fu
jisaw
aandBarrac-
loug
h(201
3)andPo
nset
al(200
6)
Sing
le-locus
ultram
etricgene
tree (con
tinued)
20 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
PTPamp
bPTPmdash
gene
tree
The
Poissontree
processes(PTP)
modelcanbe
used
toinferputativ
especiesboundaries
onagivenphylogenetic
inputtreePT
Pcaninferputativ
especiesboundaries
that
are
consistent
with
thePS
CAnim
portantadvantageof
thismethodisthat
itmod
els
speciatio
nin
term
sof
thenumberof
substitutionsthereby
circum
ventingthepotentially
error-proneandcompute-intensive
processof
generatin
gultram
etrictreeswhich
are
required
asan
inpu
tfor
GMYCmod
el(see
abov
e)F
urthermoreitappearsthatthePT
Pmodel
may
outperform
theGMYCandotherOTU-picking
methods
whenevolutionary
distancesaresm
all
Limita
tions
Single-locus
data
aloneshould
only
beused
toprovideaprelim
inary
perspectiveof
speciesboundaries
andnotas
thesole
evidence
inspecies
circum
scriptions
Source
httpscoh-itsorgexelix
iswebsoftwarePT
Pindexhtmldescribedin
Zhang
etal(201
3)
Sing
le-locus
gene
tree
GeneiousSp
eciesDelim
itatio
nplug
-inmdash
gene
tree
Aplug-into
theGeneioussoftwareprovides
anexploratorytool
allowingtheuser
toassess
phylog
enetic
supportanddiagno
sabilityof
speciesdefinedas
user-selected
collections
oftaxa
onuser-sup
pliedtreesThe
plug
-incompu
tesstatisticsrelatin
gto
the
probability
oftheob
served
mon
ophyly
orexclusivity
having
occurred
bychance
ina
coalescent
processandassesses
thewith
in-andbetween-speciesgenetic
distancesto
infertheprobability
with
which
mem
bers
ofaputativ
especiesmight
beidentifi
edsuccessfully
with
tree-based
methods
Limita
tions
The
plug-insummarizes
measuresof
phylogenetic
supportand
diagnosabilityof
speciesdefinedas
user-selectedcollections
oftaxabutitdoes
not
providedefinitiv
esupportforspeciesgroupsItassumes
speciesaremonophyletic
Source
Implem
entedas
aplug-into
Geneious(geneiouscom)describedin
Mastersetal
(201
1)
Sing
le-locus
gene
tree
BPamp
Pmdashmultispecies
coalescent
model
for
speciesvalid
ation
Thisapproach
tospeciesdelim
itatio
nuses
aBayesianmodelingapproach
togeneratethe
posteriorprobabilitiesof
speciesassignmentstaking
accountof
uncertaintiesdueto
unknow
ngene
treesandtheancestralcoalescent
processThe
methodrelieson
auser-
specified
guidetreeimplem
entin
gareversible-jum
pMarkovchainMonte
Carlo
search
ofparameter
spacethatincludes
θpopulatio
ndivergenceand
estim
ated
distributio
nsof
gene
treesfrom
multip
leloci
Limita
tions
Misspecificatio
nsof
priorsandthegu
idetree
canresultin
inflatedspeciatio
nprobabilitiesitassumes
norecombinatio
nandcomputatio
nallim
itatio
nsrestrict
itsutility
with
next-generationdatasetswith
100s
ofloci
Source
httpabacusgeneuclacuksoftwarehtmldescribedin
YangandRannala
(201
0)andRannala
andYang(200
3)
Multilocus
sequence
alignm
entsandgroup
mem
bership
(con
tinued)
2 The Dynamic Discipline of Species Delimitation hellip 21
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
DISSE
CTmdashmultispecies
coalescent
model
forspeciesdelim
itatio
nDISSE
CTexplores
thefullspaceof
possible
clusteringsof
individualsandspeciestree
topologies
inaBayesianfram
ework
Toavoidtheneed
forreversible-jum
pMCMCit
uses
anapproxim
ationin
theform
ofapriorthatisamodificatio
nof
thebirthndashdeathprior
forthespeciestreeItisim
plem
entedas
partof
BEAST
andrequires
only
afewchanges
from
astandard
BEAST
analysisA
nalysesof
simulated
andem
piricald
atasuggestthat
themethodisshow
nto
beinsensitive
tothedegree
ofapproxim
ation
butq
uitesensitive
tootherparameters
Limita
tions
Recently
describedmethodlackingathorough
theoretical
andem
pirical
evaluatio
nItappearsthat
alargenu
mberof
sequencesarerequ
ired
todraw
firm
conclusion
sSo
urce
httpcodegooglecompbeast-m
cmcamphttpwwwin
driid
com
dissectinbeast
htmldescribedin
JonesandOxelm
an(201
4)
Multilocus
sequence
data
SpeD
eSTEMmdashmultispecies
coalescent
mod
elfordiscov
ery
valid
ation
andjoint
estim
ation
Thismaxim
umlik
elihoo
dandorinform
ationtheory-based
methodwas
developedto
test
speciesboundaries
inasystem
with
existin
gsubspecies
taxonomy(CarstensandDew
ey20
10)andcomputestheprobability
ofthegene
treesgiventhespeciestree
forall
hierarchical
perm
utations
oflin
eage
grou
pingSp
eciesbo
undaries
arecomparedusing
Akaikeinform
ationcriteria
andphylogenetic
uncertaintyin
thespeciestree
topologies
does
notaffect
speciesdelim
itatio
nsLimita
tions
Accuracyisdependenton
quality
ofthegene
tree
estim
ates
Source
(http
carstenslaborgohio-stateedusoftwarehtml)criteria
describedin
Ence
andCarstens(201
1)
Multilocus
sequence
alignm
entsandgroup
mem
bership
Browniemdash
multispecies
coalescent
model
forspeciesdelim
itatio
nThe
nonp
aram
etricheuristic
speciesdelim
itatio
napproach
implem
entedin
theprog
ram
Brownie(O
rsquoMeara
2010)jointly
sortsanonym
oussamples
into
speciesandinfers
aspeciestree
from
inputg
enetreesfrom
differentlociassumingthatforaspeciatio
neven
thecorrespondingnodeson
gene
treeswill
bemoreconsistent
with
each
than
the
divergenceswith
inspecies
Limita
tions
Findingboth
theoptim
umspeciestree
andspeciesboundaries
remains
compu
tatio
nally
challenging
andBrowniehasbeen
show
nto
frequently
yieldincorrect
resultsT
heaccuracy
ofthemethodislik
elycorrelated
with
nodalsupportvalues
inthe
individu
algene
topo
logies
Source
httpwwwbrianom
earain
fobrownie
describedin
OrsquoM
eara
(201
0)
Individual
gene
trees
(con
tinued)
22 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
BFD
mdashmultispecies
coalescent
model
for
speciesdelim
itatio
nThe
recently
developedmethod
Bayes
factor
delim
itatio
n(with
genomicdataB
FD)
combinesady
namic
programmingalgorithm
forestim
atingspeciestreesthat
bypasses
thecompu
tatio
nally
intensiveMCMCintegrationov
ergene
treesto
prov
idearigorous
techniqueforspeciesdelim
itatio
nstudiesusinggenome-wideSN
PdataCom
petin
gspeciesdelim
itatio
nmodelsarecomparedusingBayes
factorsanditappearsthat
this
approach
isrobustto
samplesizes(iefew
loci
andlim
itedsamples
perspecies)
and
misspecificatio
nof
thepriorforpo
pulatio
nsize
(θ)
Limita
tions
Recently
describedmethodlackingathorough
theoretical
andem
pirical
evaluatio
nSo
urce
httpwwwbeast2orgwikiindexphpBFD
describedin
Leacheacuteet
al(201
4)
Genom
e-wideSN
Pdata
2 The Dynamic Discipline of Species Delimitation hellip 23
distances among organisms belonging to thesame species are smaller than distances amongorganisms from different species (Puillandreet al 2012 Hebert et al 2003) Genetic distanceapproaches hold particular promise as an identi-fication tool shortcutting the difficulties of mor-phology-based identification (Hebert et al 2004)although in practice a barcode gap may not existfor many groups (Wiemers and Fiedler 2007)Furthermore the role of distance-based approa-ches using a single genetic marker for speciesdiscovery remains more controversial (Rubinoff2006) and without other corroborating evidenceOTUs inferred from single-locus dataset shouldonly be considered ldquocandidaterdquo species Tree-based methods aim to detect monophyletic cladescorresponding to species-level diversity bydetecting discontinuities associated within inter-and intraspecific branching patterns (Fujisawaand Barraclough 2013 Zhang et al 2013Monaghan et al 2009 Pons et al 2006) Tree-based species delimitation methods can also beused on the basis of other properties related tophylogenetic tree topologies (monophyly con-cordance with geography etc (Sites and Marshall2003 2004) Both distance- and tree-basedmethods have been applied for assessing speciesboundaries in lichen-forming fungi (Leavitt et al2012c 2014 Parnmen et al 2012 Del-Pradoet al 2010 2011 Molina et al 2011)
A number of tree-based methods partiallyautomate the species delimitation process withspecific bioinformatical analyses (Table 21)including the general mixed Yule coalescent(GMYC) approach (Fujisawa and Barraclough2013 Monaghan et al 2009 Pons et al 2006)and the Poisson tree processes (PTP) model(Zhang et al 2013) These methods provide rel-atively straightforward and objective pipelinesfor delimiting putative species-level lineagesfrom inferred gene trees by fitting within- andbetween-species branching models to an inferredsingle-locus topology A number of other tree-based methods for species delimitation areeffectively summarized in Sites and Marshall(2004)
When only single-locus data are available theGMYC has been shown to be a relatively robust
tool for species delimitation The GMYCapproach combines a coalescent model of intra-specific branching with a Yule model for inter-specific branching which is then fit to an inferredsingle-gene topology to estimate species bound-aries and a statistical measure of confidence for theinferred boundaries As an input the GMYCapproach requires an ultrametric gene tree andrecent refinements can account for uncertainty inphylogenetic relationships and parameters of theGMYC model (Fujisawa and Barraclough 2013Reid and Carstens 2012) Regardless of theseimprovements it may be difficult to accuratelyinfer an adequate ultrametric tree for large data-sets Although it appears that the GMYC isgenerally stable across a wide range of circum-stances including various methods of phyloge-netic reconstruction the presence of a highnumber singletons high numbers of sampledspecies and gaps in intraspecific sampling theaccuracy of the GMYC is most significantlyaffected by the mean population size relative todivergence times between them (Fujisawa andBarraclough 2013 Talavera et al 2013) Fur-thermore research suggests that the so-calledsingle-threshold version of the GMYC methodlikely outperforms the multiple-thresholdapproach (Fujisawa and Barraclough 2013Monaghan et al 2009) However other studiessuggest that theGMYCmethodmay often providehigher estimates for the total number ofOTUs thanother molecular species delimitation methods(Hamilton et al 2014 Miralles and Vences 2013Talavera et al 2013) warranting a cautiousinterpretation of results from GMYC analyses
Compared to the GMYC approach therecently introduced PTP method for speciesdelimitation has been suggested to be moreaccurate for preliminary species delimitation(Zhang et al 2013) Relative to the GMYC PTPoffers a more straightforward implementationrequiring a simple phylogenetic tree rather thanan ultrametric chronogram However moreresearch is required to assess the general perfor-mance of PTP across wide range of empirical andsimulated species delimitation studies At thispoint data support the use of the GMYC andPTP methods as objective and reasonable starting
24 SD Leavitt et al
points for species delimitation using single-genetopologies
The ldquoSpecies Delimitationrdquo plug-in to theGeneious software provides statistical approachesfor exploring species boundaries in single-genetopologies (Masters et al 2011) Using a priorispecimen assignments to putative species theldquoSpecies Delimitationrdquo plug-in computes statis-tics relating to the probability of the observedmonophyly or exclusivity having occurred bychance in a coalescent process and assesses thewithin- and between-species genetic distances inorder to infer the probability with which membersof a putative species might be identified suc-cessfully with tree-based methods An importantcontribution of the ldquoSpecies Delimitationrdquo plug-in is that it provides an objective means for usersto assess putative species within an empiricalstatistic-based framework rather than qualitativeevaluations of what level of hierarchy constitutesa species in a phylogeny
In contrast to simple sequence similaritythresholds (OTU-picking) for delimiting putativeevolutionarily significant units the AutomaticBarcode Gap Discovery (ABGD) method is anautomated procedure that sorts sequences intohypothetical species based on the existence of abarcode gap observed whenever intraspecificgenetic distances are less than those amongorganisms from different species (Puillandreet al 2012) Ultimately ABGD is a fast simplemethod that can be used to group individualsrepresented in a single-locus sequence alignmentinto candidate species that should be comple-mented with other lines of evidence in an inte-grative taxonomic approach (Kekkonen andHebert 2014 Puillandre et al 2012)
In general the ABGD GMYC and PTP ana-lytical protocols using single-locus data arerepeatable and computationally relatively fastproviding a valuable starting point for a pre-liminary perspective into species boundaries inunderstudied groups that can be validated withsubsequent studies (Kekkonen and Hebert 2014)Similarly analyses of single-locus data can beused to corroborate traditional phenotype-basedspecies boundaries and identify candidate speciesthat have previously been hidden within nominal
species However single-locus species delimita-tionmethodsmaybeparticularly prone to failure inrecognizing significant proportions of species-level biodiversity due to the strict criterion forreciprocal monophyly or alternatively providespurious inflations of estimated species diversitybased on genetic differences that donot correspondto species-level lineages For example recentestimates suggest that the incidence of species-level gene tree paraphyly is approximately 20 (Ross 2014) suggesting that analyses of single-locus datasets would likely fail to accurately deli-mit species in one in every five cases In contrastrecent empirical studies suggest that in some casesthe GMYC provides a striking overestimate ofspecies diversity (Miralles and Vences 2013)
The internal transcribed spacer region (ITS)has played a central role in molecular phyloge-netic studies of lichenized fungi and has recentlybeen adopted as the official barcoding marker forfungi (Schoch et al 2012) In many cases the ITSis sufficiently variable to resolve species bound-aries for lichenized fungi (Schoch et al 2012Kelly et al 2011) although accurate specimenidentification using sequence-based identificationapproaches requires a thoroughly curated refer-ence database (Leavitt et al 2014 Orock et al2012 Kelly et al 2011) In spite of the generalutility of the ITS marker a number of issues maypotentially limit its effective use in speciesdelimitation studies including the potential forintragenomic variation of the nuclear ribosomaltandem repeat and difficulties in aligning ITSsequences from divergent taxa (Kiss 2012)Because the ITS has been formally adopted as theofficial barcoding marker for fungi we recom-mend that species delimitation studies attempt toinclude this region for consistency across studiesHowever we recognize that in some taxonomicgroups the inclusion of the ITS may be prob-lematic and therefore suggest a careful screeningof other markers to identify appropriate loci forresolving species-level relationships
Ultimately the success of any single-locusspecies delimitation method largely depends onthe evolutionary history of the species groupunder study and the variability of the selectedmarker (Fig 22) Species delimitation will be
2 The Dynamic Discipline of Species Delimitation hellip 25
more difficult in more recently diverged lineagesand in cases with some level of interspecific geneflow relative to older well-diverged lineages(eg Leavitt et al 2012c 2013e) In the end wereemphasize that single-locus data alone shouldonly be used to provide a preliminary perspectiveof species boundaries and not as the sole evi-dence in species circumscriptions
222 Sampling Across the GenomeMultilocus Sequence Dataand Genome-Wide Markers
Although single-locus methods can provide anefficient approach for preliminary large-scaleassessments of species diversity there are
significant limitations particularly in recentlydiverged species groups where retention ofancestral polymorphisms and incomplete lineagesorting will likely result in different neutral locihaving unique gene topologies that do not mirrorthe speciation process (Knowles and Carstens2007 Heled and Drummond 2010 Taylor et al2000) In contrast to single-locus and strictlyphenotype-based approaches for species delimi-tation analyses of genetic data collected fromindependent genomic regions can provide robusthypotheses of species boundaries with increasingconfidence (Satler et al 2013 Zhang et al 2011Yang and Rannala 2010) Sequences from mul-tiple independent loci provide an importantsource of data for species delimitation studiesincluding recently developed models that
Fig 22 A simplified diagram illustrating the process ofspeciation through time in a single gene history andresulting gene topologies sampled at two points in thespeciation history (modified from Leliaert et al 2014)aEach dot represents a distinct gene copy and each row onenon-overlapping generation with lines connecting genecopies to their ancestors in the previous generation (onerow below) dashed diagonal lines represent reproductivebarriers two hypothetical sampling intervals at differentpoints in the speciation history are shown Tx and Ty
species delimitation methods using single-locus data areeffective only when species are reciprocally monophyleticin the sampled gene tree genetic clustering and coalescent-based species delimitation methods can circumscribespecies when species may not be monophyletic in sampledgenetic loci b Gene topology representing sampledhaplotypes at time Tx (shown in panel a) hypotheticalspecies are reciprocally monophyletic c Gene topologyrepresenting sampled haplotypes at time Ty (shown in panela) hypothetical species are para- and polyphyletic
26 SD Leavitt et al
combine individual gene genealogies and speciesphylogenies via modeling the coalescent historyof markers (Yang and Rannala 2010 Edwards2009 Knowles and Carstens 2007) As aresponse to advances in sequencing technologiesbioinformatical approaches for multimarker spe-cies delimitation analyses continue to be devel-oped (Table 21 Camargo and Sites 2013)
Long-term reproductive isolation of candidatespecies can be assessed with multilocus sequencedata by evaluating genealogical concordance ofunlinked markers (Baum and Shaw 1995 Aviseand Ball 1990) Within species the mixingeffects of recombination cause unlinked loci tohave distinct genealogical histories but geneticdrift and long-term divergence leads to concor-dant genealogical histories at loci across thegenome Relationships of individuals belongingto distinct candidate species can be evaluatedusing gene genealogies (eg haplotype networksor single-gene topologies) to identify lineagesthat exhibit genealogical exclusivity acrossunlinked neutral loci (Hudson and Coyne 2002Dettman et al 2003b Avise and Ball 1990) Thepresence of the same clades in the majority ofsingle-locus genealogies is taken as evidence thatthe clades represent reproductively isolated lin-eages (Dettman et al 2003a Pringle et al 2005)In practice the criteria of reciprocal monophylyand genealogical concordance of unlinked lociprovide a conservative approach for assessingspecies boundaries due to the fact that a sub-stantial amount of time is required after the initialdivergence of species until ancestral polymor-phisms have fully sorted (Knowles and Carstens2007 Hudson and Coyne 2002) Consequentlygroups with recent divergence histories willlikely go undiscovered under a genealogicalconcordance criterion due to the fact that speciesboundaries likely are not reflected in genegenealogies For example it would take morethan 1 million years after speciation before spe-cies would be delimited if 15 loci were sampledin species with an effective population size (Ne)of 100000 assuming one generation a yearunder a strict reciprocal monophyly criterion(Knowles and Carstens 2007) The amount oftime required for a species to be recognized using
a criterion of reciprocal monophyly increasesproportionally with increasing population sizes(Hudson and Coyne 2002) In a number ofstudies of lichen-forming fungi a genealogicalconcordance criterion has been used to bothdelimit previously unrecognized species-levellineages and validate some conventional pheno-type-based species (Leavitt et al 2012a c2013b Molina et al 2011 Kroken and Taylor2001)
Although analyses of molecular sequence datarelying on reciprocal monophyly or fixed char-acter differences for species delimitation canserve as important criteria for identifying speciesthese criteria are not commonly met acrossmultiple loci particularly in recent speciationhistories (Fig 22) To accommodate theobserved conflict among genealogies from mul-tiple loci with the underlying speciation historythe recent merge of coalescent theory with phy-logenetics has driven a major paradigm shift inspecies delimitation and molecular systematics ingeneral (Fujita et al 2012 Edwards 2009) Themultispecies coalescent model can be applied togenealogical histories from multiple independentloci to assemble separate coalescent processesoccurring in populations into a species tree(Degnan and Rosenberg 2009 Rannala andYang 2003) Within this coalescent-basedframework multiple individuals can be assignedto a single speciespopulation and the speciationhistory of ancestral and descendant species-levellineages can be inferred as a ldquospecies treerdquo incontrast to estimating gene genealogies fromindividual samples (Degnan and Rosenberg2006 2009 Rannala and Yang 2003)
A number of multispecies coalescent-basedspecies delimitation methods have recently beenintroduced offering an exciting framework forempirically assessing species boundaries byselecting the best species tree model from a set ofalternative models representing differenthypotheses of species boundaries (Table 21)For example SpeDeSTEM (Ence and Carstens2011) finds the maximum likelihood values for aspecies tree assuming all putative species areseparate lineages and for alternative species treeswhere two or more species are collapsed into a
2 The Dynamic Discipline of Species Delimitation hellip 27
single lineage The best fitting model is thenselected using the Akaike information criterionunder the assumption of constant populationsizes and fixed gene topologies across the speciestree The species delimitation program BayesianPhylogenetic and Phylogeography (BPampPYangand Rannala 2010) accommodates gene treeuncertainty and variable population sizes andsamples from the Bayesian posterior distributionof species delimitation models using reversible-jump Markov chain Monte Carlo (rjMCMC)sampling BPampP requires a user-provided guidetree with resolution finer than the species leveland evaluates alternative modes derived from allpossible subtrees that are generated by collapsingor splitting nodes on the guide tree (Yang andRannala 2010 Rannala and Yang 2003) Theproportion of time spent on each model is pro-portional to the posterior probability of themodel ie ldquospeciation probabilitiesrdquo WhileBPampP ranks among the most popular coalescent-based species delimitation programs (Fujita et al2012) misspecifications of the guide tree andorthe prior distribution for population size (θ) canresult in strong support for models containing aninflated number of species (Leacheacute and Fujita2010 Zhang et al 2011) The nonparametricheuristic species delimitation approach imple-mented in the program Brownie (OrsquoMeara 2010)jointly sorts anonymous samples into species andinfers a species tree from input gene trees fromdifferent loci assuming that for a speciationevent the corresponding nodes on gene trees willbe more consistent with each than the diver-gences within species However finding both theoptimum species tree and species boundariesremains computationally challenging andBrownie has been shown to frequently yieldincorrect results (OrsquoMeara 2010)
Most recently available coalescent-basedspecies delimitation methods require individualassignments to a species or population a priori Ina number of scenarios correct assignments ofsamples to species may be difficult includingpresence of cryptic species incongruencebetween conventional species and moleculardata or simply the fact that accurate specimenidentification in complex groups with subtle or
difficult to discern diagnostic characters is anontrivial task (Leavitt et al 2013e) Statisticalmethods for assessing individual assignment andspecies detection prior to coalescent-based spe-cies delimitation and species tree reconstructionand species provide a more objective approachfor understanding species boundaries
Population assignment tests using a variety ofclustering algorithms using genetic data offer auseful approach for grouping individuals intoputative reproductively isolated groups (Table 21Hausdorf and Hennig 2010 Corander et al2004 2008 Falush et al 2003 Pritchard et al2000) particularly for species delimitation prob-lems that exist at the interface of traditionalpopulation genetic and phylogenetic analyses(Carstens et al 2013 Weisrock et al 2010Knowles and Carstens 2007)
The programs STRUCTURE (Falush et al2003 Pritchard et al 2000) and STRUCTURA-MA (Huelsenbeck et al 2011) cluster samplesinto populations by minimizing HardyndashWeinbergdisequilibrium for a given number of populationclusters using unlinked genetic markers In gen-eral unlinked markers are not available for mostgroups of lichen-forming fungi and a numberof studies have used information from multilocussequence data for STRUCTURE analyses(Leavitt et al 2011a b c 2013e Fernaacutendez-Mendoza and Printzen 2013 Fernaacutendez-Mendoza et al 2011) Varying approaches havebeen implemented to convert DNA sequencedata to allelic data for Bayesian clustering (seeOrsquoNeill et al 2013) STRUCTURE is expectedto perform well when there is sufficient inde-pendence across regions such that linkage dis-equilibrium within regions does not dominate thedata (STRUCTURE manual) but can also beeffective using multilocus sequence data andtreating all SNPs as independent loci regardlessof physical linkage within each locus (OrsquoNeillet al 2013 Falush et al 2003) A recent study ofthe lichen-forming genus Letharia showed thatBayesian clustering implemented in the programSTRUCTURE was generally able to recover thesame putative Letharia lineages circumscribedemploying a gene genealogical approach inKroken and Taylorrsquos iconic species delimitation
28 SD Leavitt et al
study (Altermann et al 2014 Kroken and Taylor2001) Altermann et al (2014) show that popu-lation assignments were largely consistent acrossa range of scenarios including extensiveamounts of missing data the exclusion of SNPsfrom variable markers and inferences based onSNPs from as few as three gene regions Incontrast to STRUCTURE and STRUCTURA-MA the program BAPS (Corander et al 2008)can explicitly infer genetic structure using hap-loid sequence data or other linked geneticmarkers in the ldquoclustering with linked locirdquomodel Another advantage of BAPS is that theprogram is much more computationally efficientthan STRUCTURE or STRUCTURAMA Sim-ulation studies indicate that both BAPS andSTRUCTURE perform well at low levels ofpopulation differentiation and when clusters arenot well differentiated (Latch et al 2006)
In practice inferring the most appropriatelevel of population structure using Bayesianclustering algorithms remains challenging (Latchet al 2006 Evanno et al 2005) BAPS andSTRUCTURAMA can infer both individualassignments and the most likely number ofgenetic clusters (Corander et al 2008 Huelsen-beck et al 2011) and the ad hoc ΔK statisticproposed by Evanno et al (2005) provides anobjective approach for identifying the uppermosthierarchical level of structure However carefulconsideration of other levels of populationstructure may ultimately reveal more biologicallymeaning results and researchers should examineindividual assignments across a range of geneticgroupings Some markers generated fromindependent genomic regions such as SNPs andfast-evolving microsatellites can be used to dis-tinguish fine-scale population structuring on thebasis of allele frequencies and species delimita-tion analyses based on these markers may implythe risk of severe taxonomic oversplitting Inthese cases validation approaches such asBPampP and SpeDeSTEM and corroborationamong different species delimitation approachescan provide perspective between intraspecificpopulation structure and species-level clusters
Another limitation of clustering approaches isthat they do not assess or take into account
evolutionarily divergences and relationshipsamong population clusters Coalescent-basedspecies tree methods (discussed below) provide ameans to assess the diversification histories ofpopulations inferred from clustering analysesTherefore a potential working protocol for aninformed species delimitation study that takesinto account population structure could consist offirst applying a genetic clustering analysis under apopulation genetics criterion (eg BAPSSTRUCTURE STRUCTURAMA) to identifygenetically distinct population clusters that can beconsidered ldquocandidate speciesrdquo From these can-didate species a species tree can be inferred forfocal group using coalescent-based species treereconstruction methods (eg BEAST) Subse-quently a coalescent-based validation methodcan be applied to assess whether the distinctpopulation clusters represent independent evolu-tionary lineages (eg BPampP) This multistepapproach would provide a consistent and standardcriterion for distinguishing between population-and species-level lineages (Camargo and Sites2013) and has been applied to a number of casesincluding lichen-forming fungi (Leavitt et al2011a b c 2013e Leacheacute and Fujita 2010)
High-throughput sequencing methods providethe means to effectively sample hundreds tothousands of loci from across a species genome fora large number of species and continue to revolu-tionize studies that can be performed even in non-model organisms Targeted high-throughputsequencing approaches such as anchored phy-logenomics transcriptome sequencing reduced-representation genomic library sequencing(restriction-site-associated DNA sequencingRAD-Seq and genotype-by-sequencing GBS)and high-throughput amplicon sequencing pro-vide important insight into species boundaries fora number groups although none of these approa-ches has yet been applied to studies of lichenizedfungi
For example restriction-site-associated(RAD-Seq) DNA can simultaneously detect andgenotype thousands of genome-wide SNPs byfocusing the sequencing effort on a reducedrepresentation of the entire genome (Baird et al2008) and has been successfully applied to
2 The Dynamic Discipline of Species Delimitation hellip 29
intraspecies (Lewis et al 2007 Miller et al 2007Emerson et al 2010) and interspecies studies(Rubin et al 2012 Eaton and Ree 2013 Wagneret al 2013) This approach has provided strikinginsight into the recent adaption radiation of LakeVictoria cichlids (Wagner et al 2013) Analternative approach targeting the sequencing ofspecific loci using next-generation sequencingplatforms provides an efficient means of gener-ating data for loci of known genomic locationorthology size and expected level of variation(OrsquoNeill et al 2013) Markers can be targetedusing well-established PCR techniques or newerhybridization techniques and subsequentlypooled for high-throughput sequencing usingparallel-tagged sequencing of multiple individualsamples within a single sequencing run (OrsquoNeillet al 2013) A recent study of North Americantiger salamanders (Ambystoma tigrinum)demonstrated the potential for amplicon-basedparallel-tagged sequencing to rapidly generatelarge-scale genomic data for species delimitationand species tree research (OrsquoNeill et al 2013)
Although these methods provide an enormousamount of data and insight into diversification atan unprecedented scale computational limita-tions restrict the applicable analytical methodsfor large datasets although computational andanalytical advances are happening rapidly Mostrecent coalescent-based species delimitation andspecies tree models using gene trees have beenlimited to handle tens of loci for multiple indi-viduals and combining hundreds or thousands ofgene trees into a single species delimitationframework presents considerable computationalchallenges (Camargo and Sites 2013) Therecently developed method Bayes factor delim-itation (with genomic data BFD) combines adynamic programming algorithm for estimatingspecies trees that bypasses the computationallyintensive MCMC integration over gene trees toprovide a rigorous technique for species delimi-tation studies using genome-wide SNP data(Leacheacute et al 2014) Competing species delimi-tation models are compared using Bayes factorsand it appears that this approach is robust tosample sizes (ie few loci and limited samples
per species) and misspecification of the prior forpopulation size (θ) (Leacheacute et al 2014)
Fungi are an ideal model for assessing diver-gence in eukaryotes due to their simple mor-phologies small genomes broad ecological rolesand diverse lifestyles (Gladieux et al 2014)However the use of genomic data from high-throughput sequencing methods have not yet beenincluded in species delimitation studies of lichen-forming fungi This is due in part to challenges indealing with metagenomic data containing geno-mic information from a plethora of symbiontsassociated with a lichen thallus scant genomicresources and discipline-specific inertia A num-ber of lichen-forming fungal genomes are cur-rently available including Endocarponpusillum(Wang et al 2014) Cladonia spp (Park et al2013a 2014) Caloplaca flavorubescens (Parket al 2013b) and Cladonia greyi and Xanthoriaparientina through the Fungal Genomics Programat Joint Genome Institute of the United StatesDepartment of Energy (httpwwwjgidoegov)The foreseeable ongoing expansion of genomicresources for lichen-forming fungi will be centralto developing approaches for delimiting speciesusing high-throughput sequencing Specificallygenomic resources will be crucial in identifyingnovel markers (variable genes regions SNPsmicrosatellites etc) identifying conserved fungalmarkers for targeted high-throughput sequencingapproaches and references for entire genomecomparisons (Devkota et al 2014 Werth et al2013)
23 Can We Make SpeciesDelimitation in Lichen-FormingFungi Truly Integrative
The widespread availability of genetic data hascreated a biased viewpoint that only genetic datashould be used for statistical species delimitationHowever an ongoing appeal to researchers toassess species boundaries from multiple andcomplementary perspectives (phylogeneticspopulation genetics comparative morphologydevelopment ecology etc) has resulted in an
30 SD Leavitt et al
integrative taxonomic framework bringing theseconceptual and methodological perspectivestogether (Hamilton et al 2014 Fujita et al 2012Salicini et al 2011 Yeates et al 2011 Padialet al 2010 Dayrat 2005 Will et al 2005 Wiensand Penkrot 2002) In practice any study linkingdifferent kinds of data to support hypotheses ofspecies boundaries including mapping morpho-logical characters onto a molecular phylogenycan be considered integrative Integrative meth-ods for species delimitation fall across a broadspectrum ranging from verbal and qualitativeassessments of data classes to quantitative meth-ods that allow different data types to contribute tostatistical species delimitation (Yeates et al2011) In most taxonomic studies utilizing bothmolecular and morphological data expert opinionis eventually used to some degree to evaluate thefinal status of a candidate species (eg Bond andStockman 2008) In this sense many studiesusing evidence for independent data sources fordelimiting species boundaries use an iterativeapproach (Yeates et al 2011) where speciesboundaries can be tested within a hypothetico-deductive framework with diverse datasets
From a practical perspective we advocate aprocess of iterative taxonomy (sensu Yeates et al2011) to circumscribe and refine species limitsusing multiple lines of evidence This iterativeprocess involves comparisons of morphologicaldata with a phylogenetic hypothesis (ie single-locus gene tree or concatenated multilocusphylogeny) to identify the least inclusive mono-phyletic clade in the topology characterized by atleast one unambiguously diagnostic morphologi-cal character (Miralles and Vences 2013) Thisphylogenetic tree-informed approach to assessingspecies boundaries represents a common practicein studies of lichen-forming fungi Previouslyunrecognized species-level clades with corre-sponding subtle or overlooked phenotypiccharacters have been commonly observed in bothcrustose lichens [eg Acarospraceae (Wedin et al2009) Graphidaceae (Rivas Plata and Lumbsch2011 Papong et al 2009) Lecanoraceae (Leavittet al 2011a) Lecideaceae (Ruprecht et al 2010)Mycoblastaceae (Spribille et al 2011) andTeloschistaceae (Vondraacutek et al 2009 Muggia
et al 2008)] and foliose and fruticose lichens [egLobariaceae (Moncada et al 2014 McDonaldet al 2003) Parmeliaceae (Leavitt et al 2013aWirtz et al 2012 Divakar et al 2005 Molinaet al 2004 Kroken and Taylor 2001) Peltigera-ceae (OrsquoBrien et al 2009 Goffinet et al 2003)Physciaceae (Elix et al 2009 Divakar et al2007) and Sphaerophoraceae (Houmlgnabba andWedin 2003)] As a specific example Luumlckinget al (2008) used a combination of medullarychemistry and underside pigmentation in speci-mens from the Heterodermia obscurata group inCosta Rica to corroborate monophyletic clades inan ITS gene tree as species-level lineages
While this iterative approach to speciesdelimitation and taxonomy has proven valuablefor understanding species boundaries anddescribing new taxa in some groups of lichenizedfungi a posteriori examination of morphologicaland chemical features has failed to reveal diag-nostic phenotypic characters in a number ofstudies (Muggia et al 2014 Leavitt et al 2011aOtaacutelora et al 2010 OrsquoBrien et al 2009) Fur-thermore a study of widespread species in thegenus Melanelixia (Parmeliaceae) indicated thatphenotypically cryptic lichen-forming fungalspecies-level lineages may be relatively ancientand diagnosable phenotypic differences may beabsent even millions of years after the initialdivergence (Leavitt et al 2012b) The latter studyhighlights the fact that species-level lineages maycommonly exist without any observable diag-nostic phenotypic characters calling into ques-tion the impetus for a universal application ofintegrative taxonomy
In keeping with the principle that as manylines of evidence as available should be com-bined to delimit species (Dayrat 2005) the for-malized integrative taxonomic approach (ITAXMiralles and Vences 2013) uses a mtDNA guidetree and observations from different types of datathat might provide conclusive evidence for theindependence of lineages and thus their identityas different species Miralles and Vences (2013)provide a non-exhaustive list of species delimi-tation criteria to be integrated in the ITAXapproach including (a) sympatric occurrencewithout admixture as revealed by consistent
2 The Dynamic Discipline of Species Delimitation hellip 31
differences in morphological or molecular char-acters at the same geographic location (b) strongdifferences in a behavioral morphological orgenetic character known to mediate prematingisolation (c) unviability or infertility of hybrids(d) lack of gene flow across a geographicalhybrid zone (e) congruent diagnostic differencesbetween sister lineages in various unlinkedmorphological character (f) absence of haplo-type sharing in several unlinked nuclear loci and(g) a combination of criteria endashf Speciesboundaries are based on seeking the least inclu-sive monophyletic group in the mtDNA treewhich fulfills at least one of the criteria listedabove Clearly the ITAX approach is sensitive tosample size and in order to reliably support thedistinctiveness of a given species it has beenrecommended that the sampling strategy includesat least five individuals per species (Miralles andVences 2013) A similar approach could beadopted for lichenized fungi using an ITS genetopology as guide tree rather than mtDNA
A total evidence approach including concat-enation has been a common approach for inte-grating information from different sourcesincluding independent genetic markers in phy-logenetic reconstructions (Kluge 1989 Wiens1998 de Queiroz et al 1995) Proponents forconcatenation of independent data in phyloge-netic analyses argue that when combining alldata the underlying signal of speciation mayemerge even if weakly contradictory signalis contained in the individual data partitions(Gatesy et al 1999) For example establishing apreliminary perspective of species boundariesfrom multilocus sequence data using concatena-tion may provide a reasonable starting point forscreening for cryptic species and species treeinference (Šlapeta et al 2006 Leavitt et al2011a Le Gac et al 2007 Leacheacute 2009)However concatenation and consensus methodsimply a risk of obtaining inflated support forincorrect relationships and information aboutvariance in gene coalescence is lost (Degnan andRosenberg 2006 2009 Kubatko and Degnan2007 Edwards 2009) In spite of the impetus forintegrating evidence from independent data
sources into empirical species delimitation stud-ies and taxonomy (Fujita et al 2012 Padial et al2009) most currently available species delimi-tation methods are unable to accommodate non-genetic data sources in a statistical framework
231 Selecting the Appropriate Data
In the face of increasing availability of geneticdata and associated bioinformatical approachesfor delimiting species researchers should care-fully consider what information is being sacri-ficed by the failure to consider non-genetic datain species delimitation studies and whetheraccuracy could be improved by the addition ofmultiple data types Morphological data havehistorically served as a proxy to identify repro-ductively isolated groups (ie ldquospeciesrdquo) (Ray1686 Fujita et al 2012) Current methods fordelimiting species using non-genetic data (egchemistry morphology and ecology) remainwoefully understudied For example morphol-ogy-based species circumscriptions are generallybased on one or more qualitative (or quantitative)morphological characters that do not appear tooverlap with other species However ascertain-ing that a given trait is truly fixed within apopulation with statistical confidence requiresunrealistic sample sizes even when allowing forsome level of polymorphism in the diagnosticcharacter (Wiens and Servedio 2000) Now var-ious combinations of datamdashfrom morphologygenetics geography and ecologymdashare acceptedas standard information for species delimitationstudies (Ruiz-Sanchez and Sosa 2010 Ross et al2010 Edwards and Knowles 2014)
Below we briefly discuss appropriate datasources for species delimitation studies of lichen-forming fungi However homoplastic characters(similar traits that are not derived from a com-mon ancestor) are common among many traitscommonly used to circumscribe fungal taxa thebiological significance of secondary metabolitevariation remains largely unknown (Lawrey1986) and ecological niches may be difficultto adequately characterize and model due to
32 SD Leavitt et al
microhabitat requirements and data resolutionTherefore we advocate a cautious approach toselecting appropriate and relevant data forassessing species boundaries
In spite of the potential challenges and limi-tations of using phenotypic data these traits haveprovided a plethora of valuable information forunderstanding species boundaries For examplein the Melanelixia fuliginosa group (Parmelia-ceae) a morphometric analysis using color isi-dia and marginal zone free of isidia ascharacters revealed a general pattern of differ-entiation between material formerly recognizedas subspecies fuliginosa and glabratula in spiteof the fact that considerable overlap betweengroups occurs in some characters individually(Arup and Berlin 2011) Furthermore the dis-tinction of the two groups was supported by aphylogenetic analysis of the ITS marker andecological differences with M fuliginosaoccurring predominantly on rock and M glab-ratula on bark (Arup and Berlin 2011) Thisstudy of the M fuliginosa group provides a fit-ting example of using multiple independentsuites of data including ecology morphologyand genetic information to establish a robusthypothesis of species boundaries
In lichen systematics phenotypic dataincluding thallus organization secondary metab-olites mode of reproduction ascoma-type andontogeny ascus and ascospore characters havehistorically played a prominent role (Printzen2009) Ascomatal characters have traditionallyheld a major role in higher-level classification(Printzen 2009) in contrast to species-levelclassification which also tends to include a widearray of vegetative and chemical charactersCommonly assessed morphological charactersinclude thallus form and size cortical features(eg maculae and pseudocyphellae) presenceformcolor of attachment structure (eg rhizines)and reproductive mode (ascomata vs vegetativediaspores) (Printzen 2009) Different morpho-logical types of vegetative diasporesmdashcorticatedisidia and ecorticated sorediamdashand their locationare commonly used to distinguish speciesAscomatal characters including morphologylocation (laminal vs marginal) position (sessile
vs immersed) presence of thalline margins colorof an apothecial disc and presence or color ofpruina are also commonly used in speciesdelimitations of lichen-forming fungi (Printzen2009) Other important characters may includethalline characters form color size and septationof ascospores size and form and structure of ascithe hamathecium type of epihymenium andhypothecium and the type of excipulum orperidium conidiomatal characters etc (Printzen2009)
Assessments of secondary metabolites hasplayed an important role in lichen taxonomybeginning with the introduction of simple spottests by Nylander (1866a b) The use of chem-istry in lichen taxonomy has been discussed indetail in numerous reviews (Lumbsch 1998a bRogers 1989 Brodo 1986 Egan 1986 Leuckert1985 Brodo 1978 Hawksworth 1976 Culber-son 1969 1970) and we refer readers to thesevaluable sources for a more comprehensive per-spective on lichen chemistry In short extrolites(secondary metabolites) belong to various clas-ses the most common and diverse include dep-sides depsidones chlorinated xanthones andanthraquinones (Lumbsch 2002 Culberson1969) The presence or absence of specificextrolites or their replacements by another sub-stance is widely used to distinguish speciesparticularly when correlated with differences ingeographic distributions However if morpho-logical or geographical differences betweenpopulations containing different extrolites are notapparent the taxonomic significance has beendisputed with some authors distinguishing themas species and others preferring to regard them aschemical races within a species In addition tosimply using the presence or absence of extro-lites Culberson and Culberson (1976) proposedto arrange lichen substances into chemosyn-dromes of closely related substances The pres-ence or absence of these chemosyndromes maypotentially be used as characters to delimit spe-cies regarding differences involving the samechemosyndromes as intraspecific variation anddistinct chemosyndromes as evidence for inter-specific populations (Lumbsch 1994 Gowan1986)
2 The Dynamic Discipline of Species Delimitation hellip 33
Due to the fact that species delimitationstudies often incorporate a substantial biogeo-graphical or ecological component ecologicalniche modeling plays an increasing role in phy-logenetic and taxonomic research Niche mod-eling can provide evidence for ecologicalisolation between populations based on eitherconserved or divergent ecological niches andtherefore can provide additional evidence sup-porting lineage independence between putativespecies The application of ecological nichemodeling has been applied to species delimita-tion studies in a number of cases (Ruiz-Sanchezand Sosa 2010 Leacheacute et al 2009 Raxworthyet al 2007 Rissler and Apodaca 2007) althoughit has not been explicitly applied to assess speciesboundaries in lichen-forming fungi By mappingthe spatial distribution of environmental suit-ability of climatic variables for candidate speciesthe application of ecological niche modeling canbe particularly important in cases where specieshave allopatric distributions (Raxworthy et al2007)
Ecological niche modeling utilizes knownassociations between a speciesrsquo occurrencelocalities and environmental variables to defineabiotic conditions within which populations canbe maintained (Guisan and Thuiller 2005) Themethodological approach for modeling is basedon four general properties (i) The current knownspeciesrsquo localities is the dependent variable (ii)the distribution is modeled as a map composed ofgrid cells at a specified resolution (iii) a range ofenvironmental variables (eg temperature pre-cipitation and solar exposure) are collected todescribe the characteristics of each cell and (iv)classifying the degree to which each cell is eithersuitable or unsuitable for each species under arange of models (Guisan and Thuiller 2005)
Ecological data also have the potential to playan important role in understanding speciesboundaries in lichen-forming fungi For exampleNash and Zavada (1977) demonstrated that Xant-hoparmelia populations with distinct chemistriesoccurring in the northern portion of the SonoranDesert exhibit habitat selection among differentrock substrates within a region with relativelyuniform climate and topography In another case
the parmelioid species Parmelia mayi is mor-phologically indistinguishable from P saxatilisbut can be separated by bioclimatic features andgenetic and chemical characters (Molina et al2011) McCune and Printzen(2011) assess distri-butions and climatic niches of species in theLecanora varia group inwesternUSAandprovidea model that uses continental influence and annualtemperature as themajor factors predicting speciesdistributions The distribution of Usnea hirta alichen commonly used in air quality biomonitor-ing research was modeled for a section of theWhite River National Forest in central Coloradobased on the presence of U hirta at 72 biomoni-toring reference sites distributed in the inter-mountain western United States (Shrestha et al2012) The best model for predicting U hirtadistribution included four variablesmdashsolar radia-tion average monthly precipitation and averagemonthly minimum and maximum temperatures(Shrestha et al 2012) These studies supportthe potential use of ecological niche modelingmethods in species delimitation studies of lichen-forming fungi
24 Conclusions What AboutTaxonomy
In most cases species circumscription and tax-onomy requires some degree of qualitativejudgment and individual interpretation Integrat-ing multiple types of data into an empiricalframework for delimiting species boundarieswhere species boundaries can be tested within ahypothetico-deductive framework with diversedatasets can provide robust hypotheses of speciesboundaries and taxonomic stability (Yeates et al2011) However it has long been known toevolutionary biologists that distinct species donot need to have diagnosable morphologicaldifferences (Mayr 1963) and increasing avail-ability of genetic data has allowed researchers toidentify species and to rigorously test speciesboundaries with a level of precision that wasunimaginable a decade ago While analyticaladvances in statistical species delimitation havebeen largely based on genetic data the utility of
34 SD Leavitt et al
these approaches to formal taxonomy remainselusive Due to these challenges an eclecticapproach to delimiting species and cautionagainst the reliance on any single dataset ormethod is required when delimiting species
While results from many recent studies maycontradict traditional species boundaries acrossmany groups of lichen-forming fungi we areoptimistic that this research represents substantialprogress toward a more accurate perspective onspecies boundaries and diversity in fungi As aresult of ongoing research of species boundariesin lichen-forming fungi the taxonomic value ofmany phenotypes is now better understood ourunderstanding of ecological evolutionary andbiogeographic patterns has improved and we canbegin to better understand patterns of symbiontinteractions in lichens Integrating new data(including novel morphological characters andgenetic data) will be essential to accurately rep-resent species-level diversity across all groups oflichenized fungi Hopefully an improved per-spective on lichen diversity also increases ourappreciation of these incredible symbiotic sys-tems While we are strong advocates for theapplication of independent data types in devel-oping an integrative taxonomy there is anincreasing need to formally recognize the exis-tence of phenotypically cryptic species-levellineages in lichen-forming fungi (see Hibbettet al 2011) In some cases a molecular taxon-omy may provide the most practical approach toconsistent treatment of mycobiont species withinlichen groups where diagnostic morphologicalcharacters are unidentifiable or practically notfeasible (Leavitt et al 2013c d)
These are exciting times for taxonomists andphylogeneticists A closer look at lichen taxon-omy with the inclusion of new data will help usto better understand the diversity of these fasci-nating organisms accurately interpret distribu-tion patterns and play a more important role inmeaningful conservation practices Howeversome level of uncertainty will accompany pro-gress In many taxonomic groups our traditionalapproach for species identification will likelyneed to be substantially modified The search forcorroborating morphological support for cryptic
species identified using molecular data willrequire meticulous and creative approaches toassess phenotypic variation in potentially unor-thodox ways We are hopeful that lichenologistswho traditionally have been eager to include newmethods such as chromatography in their rou-tine identifications will be amenable to includemolecular techniques to their routine examina-tion of specimens for identification and classifi-cation Although this may prove difficult toachieve by single individuals especially citizenscientists that traditionally play an important rolein lichen taxonomy the increasing numberof collaborative projects in the discipline (egLumbsch et al 2011 Crespo et al 2010Gueidan et al 2009) make us optimistic thatbroad-scale collaborative approaches will facili-tate the inclusion of molecular data in lichenresearch at all levels
Acknowledgments We are indebted to various col-leagues for valuable thought-provoking discussionnotably Matthew Nelsen (University of Chicago) AnaCrespo (Universidad Complutense de Madrid) PradeepDivakar (Universidad Complutense de Madrid) BeckettSterner (The Field Museum) and Joyce Havstad (TheField Museum) We also thank anonymous reviewers whoprovided valuable comments that improved this chapterSupport by the US National Science Foundation isgratefully acknowledged (ldquoHidden diversity in parmelioidlichensrdquo DEB-0949147)
References
Ahti T Hawksworth DL (2005) Xanthoparmelia steno-phylla the correct name for X somloeumlnsis one of themost widespread usnic acid containing species of thegenus The Lichenologist 37(4)363ndash366 doi101017S0024282905015197
Altermann S Leavitt SD Goward T Nelsen MPLumbsch HT (2014) How do you solve a problemlike Letharia A new look at cryptic species in lichen-forming fungi using Bayesian clustering and SNPSsfrom multilocus sequence data PLoS ONE 9(5)e97556 doi101371journalpone0097556
Amo de Paz G Cubas P Crespo A Elix JA Lumbsch HT(2012) Transoceanic dispersal and subsequent diver-sification on separate continents shaped diversity ofthe Xanthoparmelia pulla group (Ascomycota) PLoSONE 7(6)e39683 doi101371journalpone0039683
Arguumlello A Del Prado R Cubas P Crespo A (2007)Parmelina quercina (Parmeliaceae Lecanorales)
2 The Dynamic Discipline of Species Delimitation hellip 35
includes four phylogenetically supported morphospe-cies Biol J Linn Soc 91(3)455ndash467 doi101111j1095-8312200700810x
Arup U Berlin ES (2011) A taxonomic study ofMelanelixia fuliginosa in Europe The Lichenologist43(02)89ndash97 doi101017S0024282910000678
Arup U Grube M (2000) Is Rhizoplaca (Lecanoraleslichenized Ascomycota) a monophyletic genus Can JBot 78(3)318ndash327 doi101139b00-006
Avise J Ball R (1990) Principles of genealogicalconcordance in species concepts and biological tax-onomy Oxf Surv Evol Biol 745ndash67
Baird NA Etter PD Atwood TS Currey MC Shiver ALLewis ZA Selker EU Cresko WA Johnson EA(2008) Rapid SNP discovery and genetic mappingusing sequenced RAD markers PLoS ONE 3(10)e3376 doi101371journalpone0003376
Baum DA Shaw KL (1995) Genealogical perspectives onthe species problem In Hoch PC Stephenson AG(eds) Experimental and molecular approaches to plantbiosystematics Missouri Botanical Garden St Louispp 289ndash303
Beaumont MA Nielsen R Robert C Hey J Gaggiotti OKnowles L Estoup A Panchal M Corander JHickerson M Sisson SA Fagundes N Chikhi LBeerli P Vitalis R Cornuet J-M Huelsenbeck J FollM Yang Z Rousset F Balding D Excoffier L (2010)In defence of model-based inference in phylogeogra-phy Mol Ecol 19(3)436ndash446 doi101111j1365-294X200904515x
Bickford D Lohman DJ Sodhi NS Ng PKL Meier RWinker K Ingram KK Das I (2007) Cryptic speciesas a window on diversity and conservation TrendsEcol Evol 22(3)148ndash155 doi101016jtree200611004
Bonan GB Shugart HH (1989) Environmental factors andecological processes in boreal forests Annu Rev EcolSyst 20(1989)1ndash28
Bond J Stockman A (2008) An integrative method fordelimiting cohesion species finding the population-species interface in a group of Californian trapdoorspiders with extreme genetic divergence and geo-graphic structuring Syst Biol 57(4)628ndash646 doi10108010635150802302443
Brodo IM (1978) Changing concepts regarding chemicaldiversity in lichens The Lichenologist 10(1)1ndash11doi101017S0024282978000031
Brodo IM (1986) Interpreting chemical variation inlichens for systematic purposes The Bryologist 89(2)132ndash138
Caley MJ Fisher R Mengersen K (2014) Global speciesrichness estimates have not converged Trends EcolEvol 29(4)187ndash188 doi101016jtree201402002
Camargo A Sites JW (2013) Species delimitation adecade after the Renaissance In Pavlinov I (ed) Thespecies problemmdashongoing issues InTech doi10577252664
Camargo A Morando M Avila LJ Sites JW (2012)Species delimitation with ABC and other coalescent-based methods a test of accuracy with simulations and
an empirical example with lizards of the Liolaemusdarwinii complex (Squamata Liolaemidae) Evolution66(9)2834ndash2849 doi101111j1558-5646201201640x
Carstens BC Dewey TA (2010) Species delimitationusing a combined coalescent and information-theoreticapproach an example from North American Myotisbats Syst Biol 59(4)400ndash414 doi101093sysbiosyq024
Carstens BC Pelletier TA Reid NM Satler JD (2013)How to fail at species delimitation Mol Ecol 22(17)4369ndash4383 doi101111mec12413
Corander J Marttinen P (2006) Bayesian identification ofadmixture events using multi-locus molecular mark-ers Mol Ecol 15(10)2833ndash2843 doi101111j1365-294X200602994x
Corander J Waldmann P Marttinen P Sillanpaa M(2004) BAPS 2 enhanced possibilities for the analysisof genetic population structure Bioinformatics 20(15)2363ndash2369 doi101093bioinformaticsbth250
Corander J Marttinen P Mantyniemi S (2006) Bayesianidentification of stock mixtures from molecular markerdata Fish Bull 104550ndash558
Corander J Marttinen P Siren J Tang J (2008) EnhancedBayesian modelling in BAPS software for learninggenetic structures of populations BMC Bioinf 9(1)539 doi1011861471-2105-9-539
Coyne JA Orr HA (2004) Speciation Sinauer AssociatesSunderland
Cracraft J (1983) Species concepts and speciation anal-ysis Curr Ornithol 1159ndash187
Crespo A Lumbsch HT (2010) Cryptic species in lichen-forming fungi IMA Fungus 1167ndash170 doi105598imafungus2010010209
Crespo A Peacuterez-Ortega S (2009) Cryptic species andspecies pairs in lichens a discussion on the relation-ship between molecular phylogenies and morpholog-ical characters Anales del Jardin Botanico de Madrid66(S1)71ndash81 doi103989ajbm2225
Crespo A Kauff F Divakar PK del Prado R Perez-Ortega S Amo de Paz G Ferencova Z Blanco ORoca-Valiente B Nunez-Zapata J Cubas P ArguelloA Elix JA Esslinger TL Hawksworth DL MillanesA Molina MC Wedin M Ahti T Aptroot A et al(2010) Phylogenetic generic classification of parmeli-oid lichens (Parmeliaceae Ascomycota) based onmolecular morphological and chemical evidenceTaxon 59(6)1735ndash1753
Culberson WL (1969) The use of chemistry in thesystematics of the lichens Taxon 18498ndash505
Culberson WL (1970) Chemosystematics and ecology oflichen-forming fungi Annu Rev Ecol Syst 1153ndash170
Culberson CF Culberson WL (1976) Chemosyndromicvariation in lichens Syst Bot 1325ndash339
Darwin C (1859) On the origin of species by means ofnatural selection or the preservation of favoured racesin the struggle for life J Murray London
Dayrat B (2005) Towards integrative taxonomy Biol JLinn Soc 85(3)407ndash415 doi101111j1095-8312200500503x
36 SD Leavitt et al
de Queiroz K (1998) The general lineage concept ofspecies species criteria and the process of speciationa conceptual unification and terminological recom-mendations In Howard DJ Berlocher SH (eds)Endless forms species and speciation Oxford Uni-versity Press Oxford pp 57ndash75
de Queiroz K (1999) The general lineage concept ofspecies and the defining properties of the speciescategory In Wilson RA (ed) Species new interdis-ciplinary essays MIT Press Cambridge pp 49ndash89
de Queiroz K (2007) Species concepts and speciesdelimitation Syst Biol 56(6)879ndash886 doi10108010635150701701083
de Queiroz A Donoghue MJ Kim J (1995) Separateversus combined analysis of phylogenetic evidenceAnnu Rev Ecol Syst 26(1)657ndash681 doi101146annureves26110195003301
Degnan J Rosenberg N (2006) Discordance of speciestrees with their most likely gene trees PLoS Genet 2(5)e68 doi101371journalpgen0020068
Degnan JH Rosenberg NA (2009) Gene tree discordancephylogenetic inference and the multispecies coales-cent Trends Ecol Evol 24(6)332ndash340 doihttpdxdoiorg101016jtree200901009
Del-Prado R Cubas P Lumbsch HT Divakar PK BlancoO de Paz GA Molina MC Crespo A (2010) Geneticdistances within and among species in monophyleticlineages of Parmeliaceae (Ascomycota) as a tool fortaxon delimitation Mol Phylogenet Evol 56(1)125ndash133 doi101016jympev201004014
Del-Prado R Divakar PK Crespo A (2011) Using geneticdistances in addition to ITS molecular phylogeny toidentify potential species in the Parmotrema reticul-atum complex a case study The Lichenologist 43(06)569ndash583 doi101017S0024282911000582
Del-Prado R Blanco O Lumbsch HT Divakar PK ElixJA Molina MC Crespo A (2013) Molecular phylog-eny and historical biogeography of the lichen-formingfungal genus Flavoparmelia (Ascomycota Parmelia-ceae) Taxon 62(5)928ndash939 doi101270562522
Dettman J Jacobson D Taylor J (2003a) A multilocusgenealogical approach to phylogenetic species recog-nition in the model eukaryote Neurospora Evolution57(12)2703ndash2720 doi101111j0014-38202003tb01514x
Dettman JR Jacobson DJ Turner E Pringle A TaylorJW (2003b) Reproductive isolation and phylogeneticdivergence in Neurospora comparing methods ofspecies recognition in a model eukaryote Evolution57(12)2721 doi10155403-074
Devkota S Cornejo C Werth S Chaudhary RP Sche-idegger C (2014) Characterization of microsatelliteloci in the Himalayan lichen fungus Lobaria pindar-ensis (Lobariaceae) Appl Plant Sci 2(5)1300101doi103732apps1300101
Divakar PK Molina MC Lumbsch HT Crespo A (2005)Parmelia barrenoae a new lichen species related toParmelia sulcata (Parmeliaceae) based on molecularand morphological data The Lichenologist 37(01)37ndash46 doi101017S0024282904014641
Divakar PK Amo De paz G del Prado R Esslinger TLCrespo A (2007) Upper cortex anatomy corroboratesphylogenetic hypothesis in species of Physconia(Ascomycota Lecanoromycetes) Mycol Res 111(11)1311ndash1320 doi101016jmycres200708009
Divakar PK Figueras G Hladun N Crespo A (2010)Molecular phylogenetic studies reveal an undescribedspecies within the North American concept of Mela-nelixia glabra (Parmeliaceae) Fungal Divers 42(1)47ndash55 doi101007s13225-010-0027-3
Donoghue MJ Gauthier A (2004) Implementing thephylocode Trends Ecol Evol 19(6)281ndash282 doi101016jtree200404004
Eaton DAR Ree RH (2013) Inferring phylogeny andintrogression using RADseq data an example fromflowering plants (Pedicularis Orobanchaceae) SystBiol 62(5)689ndash706 doi101093sysbiosyt032
Edwards SV (2009) Is a new and general theory ofmolecular systematics emerging Evolution 63(1)1ndash19 doi101111j1558-5646200800549x
Edwards DL Knowles LL (2014) Species detection andindividual assignment in species delimitation canintegrative data increase efficacy Proc R Soc B BiolSci 281(1777) doi101098rspb20132765
Egan RS (1986) Correlations and non-correlations ofchemical variation patterns with lichen morphologyand geography The Bryologist 8999ndash110
Elix JA Corush J Lumbsch HT (2009) Triterpenechemosyndromes and subtle morphological characterscharacterise lineages in the Physcia aipolia group inAustralia (Ascomycota) Syst Biodivers 7(04)479ndash487 doi101017S1477200009990223
Emerson KJ Merz CR Catchen JM Hohenlohe PACresko WA Bradshaw WE Holzapfel CM (2010)Resolving postglacial phylogeography using high-throughput sequencing Proc Natl Acad Sci 107(37)16196ndash16200 doi101073pnas1006538107
Ence DD Carstens BC (2011) SpedeSTEM a rapid andaccurate method for species delimitation Mol EcolResour 11(3)473ndash480 doi101111j1755-0998201002947x
EvannoGRegnaut SGoudet J (2005)Detecting thenumberof clusters of individuals using the software STRUC-TURE a simulation study Mol Ecol 14(8)2611ndash2620doi101111j1365-294X200502553x
Falush D Stephens M Pritchard JK (2003) Inference ofpopulation structure using multilocus genotype datalinked loci and correlated allele frequencies Genetics164(4)1567ndash1587 doi101111j1471-8286200701758x
Fan HH Kubatko LS (2011) Estimating species treesusing approximate Bayesian computation Mol Phylo-genet Evol 59(2)354ndash363 doi101016jympev201102019
Fernaacutendez-Mendoza F Printzen C (2013) Pleistoceneexpansion of the bipolar lichen Cetraria aculeata intothe Southern hemisphere Mol Ecol 22(7)1961ndash1983doi101111mec12210
Fernaacutendez-Mendoza F Domaschke S Garciacutea MA JordanP Martin MP Printzen C (2011) Population structure
2 The Dynamic Discipline of Species Delimitation hellip 37
of mycobionts and photobionts of the widespreadlichen Cetraria aculeata Mol Ecol 20(6)1208ndash1232doi101111j1365-294X201004993x
Fraley C Raftery A (2007) Model-based methods ofclassification Using the mclust software in chemo-metrics J Stat Softw 18i06 [Available online at httpwwwdoajorgdoajfunc=abstractampid=218544]
Fujisawa T Barraclough TG (2013) Delimiting speciesusing single-locus data and the generalized mixed yulecoalescent (GMYC) approach a revised method andevaluation on simulated datasets Syst Biol 62(5)707ndash724 doi101093sysbiosyt033
Fujita MK Leacheacute AD Burbrink FT McGuire JA MoritzC (2012) Coalescent-based species delimitation in anintegrative taxonomy Trends Ecol Evol 27(9)480ndash488 doi101016jtree201204012
Gatesy J OrsquoGrady P Baker RH (1999) Corroborationamong data sets in simultaneous analysis hiddensupport for phylogenetic relationships among higherlevel Artiodactyl taxa Cladistics 15(3)271ndash313doi101111j1096-00311999tb00268x
Gladieux P Ropars J Badouin H Branca A Aguileta Gde Vienne DM Rodriacuteguez de la Vega RC Branco SGiraud T (2014) Fungal evolutionary genomicsprovides insight into the mechanisms of adaptivedivergence in eukaryotes Mol Ecol 23(4)753ndash773doi101111mec12631
Goffinet B Miadlikowska J Goward T (2003) Phyloge-netic inferences based on nrDNA sequences supportfive morphospecies within the Peltigera didactylacomplex (Lichenized Ascomycota) The Bryologist106(3)349ndash364 doi10163901
Gowan SP (1986) Evolution of secondary natural productsin the genus Porpidia (Ascomycata Porpidiaceae) AmJ Bot 73606
Griffin PC Hoffmann AA (2014) Limited genetic diver-gence among Australian alpine Poa tussock grassescoupled with regional structuring points to ongoinggene flow and taxonomic challenges Ann Bot doi101093aobmcu017
Grube M Hawksworth DL (2007) Trouble with lichenthe re-evaluation and re-interpretation of thallus formand fruit body types in the molecular era Mycol Res111(9)1116ndash1132 doi101016jmycres200704008
Gueidan C Savi S Thues H Roux C Keller C Tibell LPrieto M Heimarsson S Breuss O Orange A FrobergL Wynns AA Navarro-Rosines P Krzewicka BPykaelae J Grube M Lutzoni F (2009) Genericclassification of the Verrucariaceae (Ascomycota)based on molecular and morphological evidencerecent progress and remaining challenges Taxon 58(1)184ndash208
Guillot G Mortier F Estoup A (2005) Geneland acomputer package for landscape genetics Mol EcolNotes 5(3)712ndash715 doi101111j1471-8286200501031x
Guillot G Renaud S Ledevin R Michaux J Claude J(2012) A unifying model for the analysis of pheno-typic genetic and geographic data Syst Biol 61(6)897ndash911 doi101093sysbiosys038
Guisan A Thuiller W (2005) Predicting species distribu-tion offering more than simple habitat models EcolLett 8(9)993ndash1009 doi101111j1461-0248200500792x
Hale ME (1990) A synopsis of the lichen genusXanthoparmelia (Vainio) Hale (AscomycotinaParmeliaceae) Smithsonian Institution Press Wash-ington DC
Hamilton CA Hendrixson BE Brewer MS Bond JE(2014) An evaluation of sampling effects on multipleDNA barcoding methods leads to an integrativeapproach for delimiting species a case study ofthe North American tarantula genus Aphonopelma(Araneae Mygalomorphae Theraphosidae) MolPhylogenet Evol 7179ndash93 doi101016jympev201311007
Hausdorf B (2011) Progress toward a general speciesconcept Evolution 65(4)923ndash931 doi101111j1558-5646201101231x
Hausdorf B Hennig C (2010) Species delimitation usingdominant and codominant multilocus markers SystBiol 59(5)491ndash503 doi101093sysbiosyq039
Hawksworth DL (1976) Lichen chemotaxonomy InBailey RH (ed) Lichenology progress and problemsAcademic London pp 139ndash184
Hebert PDN Cywinska A Ball SL deWaard JR (2003)Biological identifications through DNA barcodesProc R Soc Lond B Biol Sci 270(1512)313ndash321doi101098rspb20022218
Hebert PDN Stoeckle MY Zemlak TS Francis CM(2004) Identification of birds through DNA barcodesPLoS Biol 2(10)e312 doi101371journalpbio0020312
Heled J Drummond AJ (2010) Bayesian inference ofspecies trees from multilocus data Mol Biol Evol 27(3)570ndash580 doi101093molbevmsp274
Hey J (2006) On the failure of modern species conceptsTrends Ecol Evol 21(8)447ndash450 doi101016jtree200605011
Hibbett DS OhmanA Glotzer D NuhnMKirk P NilssonRH (2011) Progress in molecular and morphologicaltaxon discovery in Fungi and options for formalclassification on environmental sequences Fungal BiolRev 2538ndash47 doi101016jfbr201101001
Houmlgnabba F Wedin M (2003) Molecular phylogeny ofthe Sphaerophorus globosus species complex Cladis-tics 19(3)224ndash232 doi101111j1096-00312003tb00365x
Hudson RR Coyne JA (2002) Mathematical conse-quences of the genealogical species concept Evolu-tion 56(8)1557 doi101111j0014-38202002tb01467x
Huelsenbeck JP Andolfatto P Huelsenbeck ET (2011)Structurama Bayesian inference of population struc-ture Evol Bioinf Online 7(2011)55ndash59 doi104137EBOS6761
Jones G Oxelman B (2014) DISSECT an assignment-free Bayesian discovery method for species delimita-tion under the multispecies coalescent Bioinformaticsdoi101101003178
38 SD Leavitt et al
Kekkonen M Hebert PD (2014) DNA barcode-baseddelineation of putative species efficient start fortaxonomic workflows Mol Ecol Res doi1011111755-099812233
Kelly LJ Hollingsworth PM Coppins BJ Ellis CJHarrold P Tosh J Yahr R (2011) DNA barcoding oflichenized fungi demonstrates high identification suc-cess in a floristic context New Phytol 191(1)288ndash300doi101111j1469-8137201103677x
Kiss L (2012) Limits of nuclear ribosomal DNA internaltranscribed spacer (ITS) sequences as species barcodesfor fungi Proc Natl Acad Sci 109(27)E1811ndashE1811doi101073pnas1207143109
Kluge AG (1989) A concern for evidence and aphylogenetic hypothesis for relationships among Epi-crates (Boidae Serpentes) Syst Zool 38(1)7ndash25doi101093sysbio3817
Knowles LL Carstens BC (2007) Delimiting specieswithout monophyletic gene trees Syst Biol 56(6)887ndash895 doi10108010635150701701091
Kroken S Taylor JW (2001) A gene genealogical approachto recognize phylogenetic species boundaries in thelichenized fungus Letharia Mycologia 93(1)38ndash53
Kubatko LS Degnan JH (2007) Inconsistency of phylo-genetic estimates from concatenated data under coa-lescence Syst Biol 56(1)17ndash24 doi10108010635150601146041
Latch EK Dharmarajan G Glaubitz JC Rhodes OE Jr(2006) Relative performance of Bayesian clusteringsoftware for inferring population substructure andindividual assignments at low levels of populationdifferentiation Conserv Genet 7(2)295ndash302 doi101007s10592-005-9098-1
Lawrey JD (1986) Biological role of lichen substancesThe Bryologist 89(2)111ndash122
Le Gac M Hood ME Fournier E Giraud T (2007)Phylogenetic evidence of host-specific cryptic speciesin the anther smut fungus Evolution 61(1)15ndash26doi101111j1558-5646200700002x
Leacheacute AD (2009) Species tree discordance traces tophylogeographic clade boundaries in North Americanfence lizards (Sceloporus) Syst Biol 58(6)547ndash559doi101093sysbiosyp057
Leacheacute AD Fujita MK (2010) Bayesian species delim-itation in West African forest geckos (Hemidactylusfasciatus) Proc R Soc B Biol Sci 2773071ndash3077doi101098rspb20100662
Leacheacute AD Koo M Spencer C Papenfuss T Fisher RMcGuire J (2009) Quantifying ecological morpho-logical and genetic variation to delimit species in thecoast horned lizard species complex (Phrynosoma)Proc Natl Acad Sci USA 10612418ndash12423 doi101073pnas0906380106
Leacheacute AD Fujita MK Minin VN Bouckaert RR (2014)Species delimitation using genome-wide SNP dataSystematic Biology doi101093sysbiosyu018
Leavitt SD Fankhauser JD Leavitt DH Porter LDJohnson LA St Clair LL (2011a) Complex patterns ofspeciation in cosmopolitan ldquorock posyrdquo lichensmdashdiscovering and delimiting cryptic fungal species in
the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
Leavitt SD Esslinger TL Lumbsch HT (2012c) Neogene-dominated diversification in neotropical montanelichens dating divergence events in the lichen-form-ing fungal genus Oropogon (Parmeliaceae) Am J Bot99(11)1764ndash1777 doi103732ajb1200146
Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Lumbsch HT St Clair LL(2013c) DNA barcode identification of lichen-formingfungal species in the Rhizoplaca melanophthalmaspecies-complex (Lecanorales Lecanoraceae) includ-ing five new species MycoKeys 71ndash22 doi103897mycokeys74508
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Vondraacutek J Thorsten Lum-bsch H Clair LLS (2013d) Local representation ofglobal diversity in a cosmopolitan lichen-formingfungal species complex (Rhizoplaca Ascomycota)J Biogeogr 40(9)1792ndash1806 doi101111jbi12118
Leavitt SD Lumbsch HT Stenroos S St Clair LL(2013e) Pleistocene speciation in North American
2 The Dynamic Discipline of Species Delimitation hellip 39
lichenized fungi and the impact of alternative speciescircumscriptions and rates of molecular evolution ondivergence estimates PLoS ONE 8(12)e85240doi101371journalpone0085240
Leavitt SD Esslinger TL Hansen ES Divakar PK CrespoA Loomis BF Lumbsch HT (2014) DNA barcoding ofbrown Parmeliae (Parmeliaceae) species a molecularapproach for accurate specimen identification empha-sizing species in Greenland Organ Divers Evol 14(1)11ndash20 doi101007s13127-013-0147-1
Leliaert F Verbruggen H Vanormelingen P Steen FLopez-Bautista JM Zuccarello GC De Clerck O(2014) DNA-based species delimitation in algae Eur JPhycol 49(2) doi101080096702622014904524
Leuckert C (1985) Probleme der Flechten-Chemotaxon-omiemdashStoffkombinationen und ihre taxonomischeWertung Ber Deut Bot Ges 98401ndash408
Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
Lohtander K Kaumlllersjouml M Roland M Tehler A (2000)The family physciaceae in fennoscandia phylogenyinferred from its sequences Mycologia 92(4)728ndash735
Longton RE (1997) The role of bryophytes and lichens inpolar ecosystems In Woodin SJ Marquiss M (eds)Ecology of Arctic Environments Blackwell ScienceOxford pp 69ndash96 (Special publication No 13)
Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
Lumbsch HT (1994) Die Lecanora subfusca-Gruppe inAustralasien J Hattori Bot Lab 771ndash175
Lumbsch HT (1998a) Taxonomic use of metabolic data inlichen-forming fungi In Frisvad JC Bridge PDArora DK (eds) Chemical Fungal Taxonomy MarcelDekker New York pp 345ndash387
Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
Mayden RL (1997) A hierarchy of species concepts thedenouement in the saga of the species problem InClaridge MF Dawah HA Wilson MR (eds) Speciesthe units of biodiversity Chapman amp Hall Londonpp 381ndash424
Mayr E (1963) Animal species and evolution HarvardUniversity Press Cambridge
Mayr E (1970) Populations species and evolutionBelknap Press of Harvard University Press Cambridge
McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
McDonald T Miadlikowska J Lutzoni F (2003) Thelichen genus Sticta in the Great Smoky Mountains a
40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
McKay BD Mays HL Wu Y Li H Yao C-T Nishiumi IZou F (2013) An empirical comparison of character-based and coalescent-based approaches to speciesdelimitation in a young avian complex Mol Ecol 22(19)4943ndash4957 doi101111mec12446
Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
Mishler BD Brandon RN (1987) Individuality pluralismand the phylogenetic species concept Biol Philos 2397
Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
Nash TH Zavada M (1977) Population studies amongSonoran Desert species of Parmelia subg Xanthop-armelia (Parmeliaceae) Am J Bot 64(6)664ndash669
Nylander W (1866a) Circa novum in studio Lichenumcriterium chemicum Flora 49198ndash201
Nylander W (1866b) Hypochlorite of lime and hydrate ofpotash Two new criteria for the study of lichens(Tanslated and communicated by the Rev W ALeighton) Bot J Linn Soc 9358ndash365
OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
OrsquoNeill EM Schwartz R Bullock CT Williams JSShaffer HB Aguilar-Miguel X Parra-Olea G Weis-rock DW (2013) Parallel tagged amplicon sequencingreveals major lineages and phylogenetic structure inthe North American tiger salamander (Ambystomatigrinum) species complex Mol Ecol 22(1)111ndash129doi101111mec12049
Orock EA Leavitt SD Fonge BA St Clair LL LumbschHT (2012) DNA-based identification of lichen-form-ing fungi Can publicly available sequence databasesaid in lichen diversity inventories of Mount Cameroon(West Africa) The Lichenologist 44(6)833ndash839doi101017S0024282912000424
Otaacutelora MAG Martiacutenez I Aragoacuten G Molina MC (2010)Phylogeography and divergence date estimates of alichen species complex with a disjunct distributionpattern Am J Bot 97(2)216ndash223 doi103732ajb0900064
Padial J Castroviejo-Fisher S Kohler J Vila C ChaparroJ De la Riva I (2009) Deciphering the products ofevolution at the species level the need for anintegrative taxonomy Zoolog Scr 38(4)431ndash447doi101111j1463-6409200800381x
Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
Papong K Luumlcking R Thammathaworn A BoonpragobK (2009) Four new taxa of Chroodiscus (thelotremoidGraphidaceae) from Southeast Asia The Bryologist112(1)152ndash163 doi1016390007-2745-1121152
Park S-Y Choi J Kim JA Jeong M-H Kim S Lee Y-HHur J-S (2013a) Draft genome sequence of Cladoniamacilenta KoLRI003786 a lichen-forming fungusproducing biruloquinone Genome Announcements 1(5)e00695-13 doi101128genomeA00695-13
Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
Peacuterez-Ortega S Fernaacutendez-Mendoza F Raggio J VivasM Ascaso C Sancho LG Printzen C de los Riacuteos A(2012) Extreme phenotypic variation in Cetrariaaculeata (lichenized Ascomycota) adaptation or inci-dental modification Ann Bot 109(6)1133ndash1148doi101093aobmcs042
Pino-Bodas R Burgaz A Martiacuten M Lumbsch HT (2011)Phenotypical plasticity and homoplasy complicatespecies delimitation in the Cladonia gracilis group(Cladoniaceae Ascomycota) Organ Divers Evol 11(5)343ndash355 doi101007s13127-011-0062-2
Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
Pino-Bodas R Martiacuten M Burgaz A (2012b) Cladoniasubturgida and C iberica (Cladoniaceae) form asingle morphologically and chemically polymorphicspecies Mycol Prog 11(1)269ndash278 doi101007s11557-011-0746-1
Pino-Bodas R Ahti T Stenroos S Martiacuten MP BurgazAR (2013a) Multilocus approach to species recogni-tion in the Cladonia humilis complex (CladoniaceaeAscomycota) Am J Bot 100(4)664ndash678 doi103732ajb1200162
Pino-Bodas R Martiacuten AP Burgaz AR Lumbsch HT(2013b) Species delimitation in Cladonia (Ascomy-cota) a challenge to the DNA barcoding philosophyMol Ecol Resour 13(6)1058ndash1068 doi1011111755-099812086
Pons J Barraclough TG Gomez-Zurita J Cardoso ADuran DP Hazell S Kamoun S Sumlin WD VoglerAP (2006) Sequence-based species delimitation for theDNA taxonomy of undescribed insects Syst Biol 55(4)595ndash609 doi10108010635150600852011
Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
Pringle A Baker DM Platt JL Wares JP Latgeacute JPTaylor JW (2005) Cryptic speciation in the cosmo-politan and clonal human pathogenic fungus Asper-gillus fumigatus Evolution 59(9)1886ndash1899 doi10155404-2411
Printzen C (2009) Lichen systematics the role ofmorphological and molecular data to reconstructphylogenetic relationships Progress in botany vol71 Springer Berlin pp 233ndash275
Pritchard JK Stephens M Donnelly P (2000) Inference ofpopulation structure using multilocus genotype dataGenetics 155(2)945ndash959
Puillandre N Lambert A Brouillet S Achaz G (2012)ABGD automatic barcode gap discovery for primaryspecies delimitation Mol Ecol 21(8)1864ndash1877doi101111j1365-294X201105239x
Rannala B Yang Z (2003) Bayes estimation of speciesdivergence times and ancestral population sizes usingDNA sequences from multiple loci Genetics1641645ndash1656
Raxworthy CJ Ingram CM Rabibisoa N Pearson RG(2007) Applications of ecological niche modelingfor species delimitation a review and empiricalevaluation using day geckos (Phelsuma) fromMadagascar Syst Biol 56(6)907ndash923 doi10108010635150701775111
Ray J (1686) Historia planarum vol 1 Clark LondonReese Naeligsborg R Ekman S Tibell L (2007) Molecular
phylogeny of the genus Lecania (Ramalinaceaelichenized Ascomycota) Mycol Res 111(5)581ndash591doi101016jmycres200703001
Reid N Carstens B (2012) Phylogenetic estimation errorcan decrease the accuracy of species delimitation aBayesian implementation of the general mixed Yule-coalescent model BMC Evol Biol 12(1)196 doi1011861471-2148-12-196
Rissler L Apodaca J (2007) Adding more ecology intospecies delimitation ecological niche models andphylogeography help define cryptic species in theBlack Salamander (Aneides flavipunctatus) Syst Biol56924ndash942 doi10108010635150701703063
Rivas Plata E Luumlcking R (2013) High diversity ofGraphidaceae (lichenized Ascomycota Ostropales) inAmazonian Peruacute Fungal Divers 58(1)13ndash32 doi101007s13225-012-0172-y
Rivas Plata E Lumbsch HT (2011) Parallel evolution andphenotypic divergence in lichenized fungi a casestudy in the lichen-forming fungal family Graphida-ceae (Ascomycota Lecanoromycetes Ostropales)Mol Phylogenet Evol 61(1)45ndash63 doi101016jympev201104025
Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
Ross KG Gotzek D Ascunce MS Shoemaker DD (2010)Species delimitation a case study in a problematic anttaxon Syst Biol 59(2)162ndash184 doi101093sysbiosyp089
Rubin BER Ree RH Moreau CS (2012) Inferringphylogenies from RAD sequence data PLoS ONE 7(4)e33394 doi101371journalpone0033394
Rubinoff D (2006) Utility of mitochondrial DNA barcodesin species conservation Conserv Biol 20(4)1026ndash1033 doi101111j1523-1739200600372x
Ruiz-Sanchez E Sosa V (2010) Delimiting speciesboundaries within the Neotropical bamboo Otatea(Poaceae Bambusoideae) using molecular morpho-logical and ecological data Mol Phylogenet Evol 54(2)344ndash356 doi101016jympev200910035
Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
morphological characters Antarctic Sci 22 (SpecialIssue 06)727ndash741 doi101017S0954102010000477
Salicini I Ibaacutentildeez C Juste J (2011) Multilocus phylogenyand species delimitation within the Nattererrsquos batspecies complex in the Western Palearctic MolPhylogenet Evol 61(3)888ndash898 doi101016jympev201108010
Satler JD Carstens BC Hedin M (2013) Multilocusspecies delimitation in a complex of morphologicallyconserved trapdoor spiders (Mygalomorphae Antro-diaetidae Aliatypus) Syst Biol 62(6)805ndash823 doi101093sysbiosyt041
Schlick-Steiner BC Steiner FM Seifert B Stauffer CChristian E Crozier RH (2010) Integrative taxonomya multisource approach to exploring biodiversityAnnu Rev Entomol 55(1)421ndash438 doi101146annurev-ento-112408-085432
Schoch CL Seifert KA Huhndorf S Robert V SpougeJL Levesque CA Chen W Fungal Barcoding Con-sortium (2012) Nuclear ribosomal internal transcribedspacer (ITS) region as a universal DNA barcodemarker for Fungi Proc Natl Acad Sci 109(16)6241ndash6246 doi101073pnas1117018109
Seacuterusiaux E Villarreal AJC Wheeler T Goffinet B(2011) Recent origin active speciation and dispersalfor the lichen genus Nephroma (Peltigerales) inMacaronesia J Biogeogr 38(6)1138ndash1151 doi101111j1365-2699201002469x
Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
Simpson GG (1951) The species concept Evolution5285ndash298
Sites JW Marshall JC (2003) Delimiting species a renais-sance issue in systematic biology Trends Ecol Evol18462ndash470 doi101016S0169-5347(03)00184-8
Sites JW Marshall JC (2004) Operational criteria fordelimiting species Annu Rev Ecol Evol Syst 35(1)199ndash227 doi101146annurevecolsys35112202130128
Šlapeta J Loacutepez-Garciacutea P Moreira D (2006) Globaldispersal and ancient cryptic species in the smallestmarine eukaryotes Mol Biol Evol 23(1)23ndash29doi101093molbevmsj001
Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
Talavera G Dincă V Vila R (2013) Factors affectingspecies delimitations with the GMYC model insightsfrom a butterfly survey Methods Ecol Evol 4(12)1101ndash1110 doi1011112041-210X12107
Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
Tewksbury J Anderson JGT Bakker JD Billo TJDunwiddie PW Groom MJ Hampton SE HermanSG Levey DJ Machnicki NJ del Rio CM PowerME Rowell K Salomon AK Stacey L TrombulakSC Wheeler TA (2014) Natural historyrsquos place inscience and society BioScience doi101093bioscibiu032
Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
Werth S Cornejo C Scheidegger C (2013) Characteriza-tion of microsatellite loci in the lichen fungus Lobariapulmonaria (Lobariaceae) Appl Plant Sci 1(2)1200290 doi103732apps1200290
Westberg M Arup U Kaumlrnefelt I (2007) Phylogeneticstudies in the Candelariaceae (lichenized Ascomy-cota) based on nuclear ITS DNA sequence dataMycol Res 111(11)1277ndash1284 doi101017S0953756204002102
Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
2 The Dynamic Discipline of Species Delimitation hellip 43
(Lepidoptera Lycaenidae) Front Zoo 4(1)8 doi1011861742-9994-4-8
Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
PTPamp
bPTPmdash
gene
tree
The
Poissontree
processes(PTP)
modelcanbe
used
toinferputativ
especiesboundaries
onagivenphylogenetic
inputtreePT
Pcaninferputativ
especiesboundaries
that
are
consistent
with
thePS
CAnim
portantadvantageof
thismethodisthat
itmod
els
speciatio
nin
term
sof
thenumberof
substitutionsthereby
circum
ventingthepotentially
error-proneandcompute-intensive
processof
generatin
gultram
etrictreeswhich
are
required
asan
inpu
tfor
GMYCmod
el(see
abov
e)F
urthermoreitappearsthatthePT
Pmodel
may
outperform
theGMYCandotherOTU-picking
methods
whenevolutionary
distancesaresm
all
Limita
tions
Single-locus
data
aloneshould
only
beused
toprovideaprelim
inary
perspectiveof
speciesboundaries
andnotas
thesole
evidence
inspecies
circum
scriptions
Source
httpscoh-itsorgexelix
iswebsoftwarePT
Pindexhtmldescribedin
Zhang
etal(201
3)
Sing
le-locus
gene
tree
GeneiousSp
eciesDelim
itatio
nplug
-inmdash
gene
tree
Aplug-into
theGeneioussoftwareprovides
anexploratorytool
allowingtheuser
toassess
phylog
enetic
supportanddiagno
sabilityof
speciesdefinedas
user-selected
collections
oftaxa
onuser-sup
pliedtreesThe
plug
-incompu
tesstatisticsrelatin
gto
the
probability
oftheob
served
mon
ophyly
orexclusivity
having
occurred
bychance
ina
coalescent
processandassesses
thewith
in-andbetween-speciesgenetic
distancesto
infertheprobability
with
which
mem
bers
ofaputativ
especiesmight
beidentifi
edsuccessfully
with
tree-based
methods
Limita
tions
The
plug-insummarizes
measuresof
phylogenetic
supportand
diagnosabilityof
speciesdefinedas
user-selectedcollections
oftaxabutitdoes
not
providedefinitiv
esupportforspeciesgroupsItassumes
speciesaremonophyletic
Source
Implem
entedas
aplug-into
Geneious(geneiouscom)describedin
Mastersetal
(201
1)
Sing
le-locus
gene
tree
BPamp
Pmdashmultispecies
coalescent
model
for
speciesvalid
ation
Thisapproach
tospeciesdelim
itatio
nuses
aBayesianmodelingapproach
togeneratethe
posteriorprobabilitiesof
speciesassignmentstaking
accountof
uncertaintiesdueto
unknow
ngene
treesandtheancestralcoalescent
processThe
methodrelieson
auser-
specified
guidetreeimplem
entin
gareversible-jum
pMarkovchainMonte
Carlo
search
ofparameter
spacethatincludes
θpopulatio
ndivergenceand
estim
ated
distributio
nsof
gene
treesfrom
multip
leloci
Limita
tions
Misspecificatio
nsof
priorsandthegu
idetree
canresultin
inflatedspeciatio
nprobabilitiesitassumes
norecombinatio
nandcomputatio
nallim
itatio
nsrestrict
itsutility
with
next-generationdatasetswith
100s
ofloci
Source
httpabacusgeneuclacuksoftwarehtmldescribedin
YangandRannala
(201
0)andRannala
andYang(200
3)
Multilocus
sequence
alignm
entsandgroup
mem
bership
(con
tinued)
2 The Dynamic Discipline of Species Delimitation hellip 21
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
DISSE
CTmdashmultispecies
coalescent
model
forspeciesdelim
itatio
nDISSE
CTexplores
thefullspaceof
possible
clusteringsof
individualsandspeciestree
topologies
inaBayesianfram
ework
Toavoidtheneed
forreversible-jum
pMCMCit
uses
anapproxim
ationin
theform
ofapriorthatisamodificatio
nof
thebirthndashdeathprior
forthespeciestreeItisim
plem
entedas
partof
BEAST
andrequires
only
afewchanges
from
astandard
BEAST
analysisA
nalysesof
simulated
andem
piricald
atasuggestthat
themethodisshow
nto
beinsensitive
tothedegree
ofapproxim
ation
butq
uitesensitive
tootherparameters
Limita
tions
Recently
describedmethodlackingathorough
theoretical
andem
pirical
evaluatio
nItappearsthat
alargenu
mberof
sequencesarerequ
ired
todraw
firm
conclusion
sSo
urce
httpcodegooglecompbeast-m
cmcamphttpwwwin
driid
com
dissectinbeast
htmldescribedin
JonesandOxelm
an(201
4)
Multilocus
sequence
data
SpeD
eSTEMmdashmultispecies
coalescent
mod
elfordiscov
ery
valid
ation
andjoint
estim
ation
Thismaxim
umlik
elihoo
dandorinform
ationtheory-based
methodwas
developedto
test
speciesboundaries
inasystem
with
existin
gsubspecies
taxonomy(CarstensandDew
ey20
10)andcomputestheprobability
ofthegene
treesgiventhespeciestree
forall
hierarchical
perm
utations
oflin
eage
grou
pingSp
eciesbo
undaries
arecomparedusing
Akaikeinform
ationcriteria
andphylogenetic
uncertaintyin
thespeciestree
topologies
does
notaffect
speciesdelim
itatio
nsLimita
tions
Accuracyisdependenton
quality
ofthegene
tree
estim
ates
Source
(http
carstenslaborgohio-stateedusoftwarehtml)criteria
describedin
Ence
andCarstens(201
1)
Multilocus
sequence
alignm
entsandgroup
mem
bership
Browniemdash
multispecies
coalescent
model
forspeciesdelim
itatio
nThe
nonp
aram
etricheuristic
speciesdelim
itatio
napproach
implem
entedin
theprog
ram
Brownie(O
rsquoMeara
2010)jointly
sortsanonym
oussamples
into
speciesandinfers
aspeciestree
from
inputg
enetreesfrom
differentlociassumingthatforaspeciatio
neven
thecorrespondingnodeson
gene
treeswill
bemoreconsistent
with
each
than
the
divergenceswith
inspecies
Limita
tions
Findingboth
theoptim
umspeciestree
andspeciesboundaries
remains
compu
tatio
nally
challenging
andBrowniehasbeen
show
nto
frequently
yieldincorrect
resultsT
heaccuracy
ofthemethodislik
elycorrelated
with
nodalsupportvalues
inthe
individu
algene
topo
logies
Source
httpwwwbrianom
earain
fobrownie
describedin
OrsquoM
eara
(201
0)
Individual
gene
trees
(con
tinued)
22 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
BFD
mdashmultispecies
coalescent
model
for
speciesdelim
itatio
nThe
recently
developedmethod
Bayes
factor
delim
itatio
n(with
genomicdataB
FD)
combinesady
namic
programmingalgorithm
forestim
atingspeciestreesthat
bypasses
thecompu
tatio
nally
intensiveMCMCintegrationov
ergene
treesto
prov
idearigorous
techniqueforspeciesdelim
itatio
nstudiesusinggenome-wideSN
PdataCom
petin
gspeciesdelim
itatio
nmodelsarecomparedusingBayes
factorsanditappearsthat
this
approach
isrobustto
samplesizes(iefew
loci
andlim
itedsamples
perspecies)
and
misspecificatio
nof
thepriorforpo
pulatio
nsize
(θ)
Limita
tions
Recently
describedmethodlackingathorough
theoretical
andem
pirical
evaluatio
nSo
urce
httpwwwbeast2orgwikiindexphpBFD
describedin
Leacheacuteet
al(201
4)
Genom
e-wideSN
Pdata
2 The Dynamic Discipline of Species Delimitation hellip 23
distances among organisms belonging to thesame species are smaller than distances amongorganisms from different species (Puillandreet al 2012 Hebert et al 2003) Genetic distanceapproaches hold particular promise as an identi-fication tool shortcutting the difficulties of mor-phology-based identification (Hebert et al 2004)although in practice a barcode gap may not existfor many groups (Wiemers and Fiedler 2007)Furthermore the role of distance-based approa-ches using a single genetic marker for speciesdiscovery remains more controversial (Rubinoff2006) and without other corroborating evidenceOTUs inferred from single-locus dataset shouldonly be considered ldquocandidaterdquo species Tree-based methods aim to detect monophyletic cladescorresponding to species-level diversity bydetecting discontinuities associated within inter-and intraspecific branching patterns (Fujisawaand Barraclough 2013 Zhang et al 2013Monaghan et al 2009 Pons et al 2006) Tree-based species delimitation methods can also beused on the basis of other properties related tophylogenetic tree topologies (monophyly con-cordance with geography etc (Sites and Marshall2003 2004) Both distance- and tree-basedmethods have been applied for assessing speciesboundaries in lichen-forming fungi (Leavitt et al2012c 2014 Parnmen et al 2012 Del-Pradoet al 2010 2011 Molina et al 2011)
A number of tree-based methods partiallyautomate the species delimitation process withspecific bioinformatical analyses (Table 21)including the general mixed Yule coalescent(GMYC) approach (Fujisawa and Barraclough2013 Monaghan et al 2009 Pons et al 2006)and the Poisson tree processes (PTP) model(Zhang et al 2013) These methods provide rel-atively straightforward and objective pipelinesfor delimiting putative species-level lineagesfrom inferred gene trees by fitting within- andbetween-species branching models to an inferredsingle-locus topology A number of other tree-based methods for species delimitation areeffectively summarized in Sites and Marshall(2004)
When only single-locus data are available theGMYC has been shown to be a relatively robust
tool for species delimitation The GMYCapproach combines a coalescent model of intra-specific branching with a Yule model for inter-specific branching which is then fit to an inferredsingle-gene topology to estimate species bound-aries and a statistical measure of confidence for theinferred boundaries As an input the GMYCapproach requires an ultrametric gene tree andrecent refinements can account for uncertainty inphylogenetic relationships and parameters of theGMYC model (Fujisawa and Barraclough 2013Reid and Carstens 2012) Regardless of theseimprovements it may be difficult to accuratelyinfer an adequate ultrametric tree for large data-sets Although it appears that the GMYC isgenerally stable across a wide range of circum-stances including various methods of phyloge-netic reconstruction the presence of a highnumber singletons high numbers of sampledspecies and gaps in intraspecific sampling theaccuracy of the GMYC is most significantlyaffected by the mean population size relative todivergence times between them (Fujisawa andBarraclough 2013 Talavera et al 2013) Fur-thermore research suggests that the so-calledsingle-threshold version of the GMYC methodlikely outperforms the multiple-thresholdapproach (Fujisawa and Barraclough 2013Monaghan et al 2009) However other studiessuggest that theGMYCmethodmay often providehigher estimates for the total number ofOTUs thanother molecular species delimitation methods(Hamilton et al 2014 Miralles and Vences 2013Talavera et al 2013) warranting a cautiousinterpretation of results from GMYC analyses
Compared to the GMYC approach therecently introduced PTP method for speciesdelimitation has been suggested to be moreaccurate for preliminary species delimitation(Zhang et al 2013) Relative to the GMYC PTPoffers a more straightforward implementationrequiring a simple phylogenetic tree rather thanan ultrametric chronogram However moreresearch is required to assess the general perfor-mance of PTP across wide range of empirical andsimulated species delimitation studies At thispoint data support the use of the GMYC andPTP methods as objective and reasonable starting
24 SD Leavitt et al
points for species delimitation using single-genetopologies
The ldquoSpecies Delimitationrdquo plug-in to theGeneious software provides statistical approachesfor exploring species boundaries in single-genetopologies (Masters et al 2011) Using a priorispecimen assignments to putative species theldquoSpecies Delimitationrdquo plug-in computes statis-tics relating to the probability of the observedmonophyly or exclusivity having occurred bychance in a coalescent process and assesses thewithin- and between-species genetic distances inorder to infer the probability with which membersof a putative species might be identified suc-cessfully with tree-based methods An importantcontribution of the ldquoSpecies Delimitationrdquo plug-in is that it provides an objective means for usersto assess putative species within an empiricalstatistic-based framework rather than qualitativeevaluations of what level of hierarchy constitutesa species in a phylogeny
In contrast to simple sequence similaritythresholds (OTU-picking) for delimiting putativeevolutionarily significant units the AutomaticBarcode Gap Discovery (ABGD) method is anautomated procedure that sorts sequences intohypothetical species based on the existence of abarcode gap observed whenever intraspecificgenetic distances are less than those amongorganisms from different species (Puillandreet al 2012) Ultimately ABGD is a fast simplemethod that can be used to group individualsrepresented in a single-locus sequence alignmentinto candidate species that should be comple-mented with other lines of evidence in an inte-grative taxonomic approach (Kekkonen andHebert 2014 Puillandre et al 2012)
In general the ABGD GMYC and PTP ana-lytical protocols using single-locus data arerepeatable and computationally relatively fastproviding a valuable starting point for a pre-liminary perspective into species boundaries inunderstudied groups that can be validated withsubsequent studies (Kekkonen and Hebert 2014)Similarly analyses of single-locus data can beused to corroborate traditional phenotype-basedspecies boundaries and identify candidate speciesthat have previously been hidden within nominal
species However single-locus species delimita-tionmethodsmaybeparticularly prone to failure inrecognizing significant proportions of species-level biodiversity due to the strict criterion forreciprocal monophyly or alternatively providespurious inflations of estimated species diversitybased on genetic differences that donot correspondto species-level lineages For example recentestimates suggest that the incidence of species-level gene tree paraphyly is approximately 20 (Ross 2014) suggesting that analyses of single-locus datasets would likely fail to accurately deli-mit species in one in every five cases In contrastrecent empirical studies suggest that in some casesthe GMYC provides a striking overestimate ofspecies diversity (Miralles and Vences 2013)
The internal transcribed spacer region (ITS)has played a central role in molecular phyloge-netic studies of lichenized fungi and has recentlybeen adopted as the official barcoding marker forfungi (Schoch et al 2012) In many cases the ITSis sufficiently variable to resolve species bound-aries for lichenized fungi (Schoch et al 2012Kelly et al 2011) although accurate specimenidentification using sequence-based identificationapproaches requires a thoroughly curated refer-ence database (Leavitt et al 2014 Orock et al2012 Kelly et al 2011) In spite of the generalutility of the ITS marker a number of issues maypotentially limit its effective use in speciesdelimitation studies including the potential forintragenomic variation of the nuclear ribosomaltandem repeat and difficulties in aligning ITSsequences from divergent taxa (Kiss 2012)Because the ITS has been formally adopted as theofficial barcoding marker for fungi we recom-mend that species delimitation studies attempt toinclude this region for consistency across studiesHowever we recognize that in some taxonomicgroups the inclusion of the ITS may be prob-lematic and therefore suggest a careful screeningof other markers to identify appropriate loci forresolving species-level relationships
Ultimately the success of any single-locusspecies delimitation method largely depends onthe evolutionary history of the species groupunder study and the variability of the selectedmarker (Fig 22) Species delimitation will be
2 The Dynamic Discipline of Species Delimitation hellip 25
more difficult in more recently diverged lineagesand in cases with some level of interspecific geneflow relative to older well-diverged lineages(eg Leavitt et al 2012c 2013e) In the end wereemphasize that single-locus data alone shouldonly be used to provide a preliminary perspectiveof species boundaries and not as the sole evi-dence in species circumscriptions
222 Sampling Across the GenomeMultilocus Sequence Dataand Genome-Wide Markers
Although single-locus methods can provide anefficient approach for preliminary large-scaleassessments of species diversity there are
significant limitations particularly in recentlydiverged species groups where retention ofancestral polymorphisms and incomplete lineagesorting will likely result in different neutral locihaving unique gene topologies that do not mirrorthe speciation process (Knowles and Carstens2007 Heled and Drummond 2010 Taylor et al2000) In contrast to single-locus and strictlyphenotype-based approaches for species delimi-tation analyses of genetic data collected fromindependent genomic regions can provide robusthypotheses of species boundaries with increasingconfidence (Satler et al 2013 Zhang et al 2011Yang and Rannala 2010) Sequences from mul-tiple independent loci provide an importantsource of data for species delimitation studiesincluding recently developed models that
Fig 22 A simplified diagram illustrating the process ofspeciation through time in a single gene history andresulting gene topologies sampled at two points in thespeciation history (modified from Leliaert et al 2014)aEach dot represents a distinct gene copy and each row onenon-overlapping generation with lines connecting genecopies to their ancestors in the previous generation (onerow below) dashed diagonal lines represent reproductivebarriers two hypothetical sampling intervals at differentpoints in the speciation history are shown Tx and Ty
species delimitation methods using single-locus data areeffective only when species are reciprocally monophyleticin the sampled gene tree genetic clustering and coalescent-based species delimitation methods can circumscribespecies when species may not be monophyletic in sampledgenetic loci b Gene topology representing sampledhaplotypes at time Tx (shown in panel a) hypotheticalspecies are reciprocally monophyletic c Gene topologyrepresenting sampled haplotypes at time Ty (shown in panela) hypothetical species are para- and polyphyletic
26 SD Leavitt et al
combine individual gene genealogies and speciesphylogenies via modeling the coalescent historyof markers (Yang and Rannala 2010 Edwards2009 Knowles and Carstens 2007) As aresponse to advances in sequencing technologiesbioinformatical approaches for multimarker spe-cies delimitation analyses continue to be devel-oped (Table 21 Camargo and Sites 2013)
Long-term reproductive isolation of candidatespecies can be assessed with multilocus sequencedata by evaluating genealogical concordance ofunlinked markers (Baum and Shaw 1995 Aviseand Ball 1990) Within species the mixingeffects of recombination cause unlinked loci tohave distinct genealogical histories but geneticdrift and long-term divergence leads to concor-dant genealogical histories at loci across thegenome Relationships of individuals belongingto distinct candidate species can be evaluatedusing gene genealogies (eg haplotype networksor single-gene topologies) to identify lineagesthat exhibit genealogical exclusivity acrossunlinked neutral loci (Hudson and Coyne 2002Dettman et al 2003b Avise and Ball 1990) Thepresence of the same clades in the majority ofsingle-locus genealogies is taken as evidence thatthe clades represent reproductively isolated lin-eages (Dettman et al 2003a Pringle et al 2005)In practice the criteria of reciprocal monophylyand genealogical concordance of unlinked lociprovide a conservative approach for assessingspecies boundaries due to the fact that a sub-stantial amount of time is required after the initialdivergence of species until ancestral polymor-phisms have fully sorted (Knowles and Carstens2007 Hudson and Coyne 2002) Consequentlygroups with recent divergence histories willlikely go undiscovered under a genealogicalconcordance criterion due to the fact that speciesboundaries likely are not reflected in genegenealogies For example it would take morethan 1 million years after speciation before spe-cies would be delimited if 15 loci were sampledin species with an effective population size (Ne)of 100000 assuming one generation a yearunder a strict reciprocal monophyly criterion(Knowles and Carstens 2007) The amount oftime required for a species to be recognized using
a criterion of reciprocal monophyly increasesproportionally with increasing population sizes(Hudson and Coyne 2002) In a number ofstudies of lichen-forming fungi a genealogicalconcordance criterion has been used to bothdelimit previously unrecognized species-levellineages and validate some conventional pheno-type-based species (Leavitt et al 2012a c2013b Molina et al 2011 Kroken and Taylor2001)
Although analyses of molecular sequence datarelying on reciprocal monophyly or fixed char-acter differences for species delimitation canserve as important criteria for identifying speciesthese criteria are not commonly met acrossmultiple loci particularly in recent speciationhistories (Fig 22) To accommodate theobserved conflict among genealogies from mul-tiple loci with the underlying speciation historythe recent merge of coalescent theory with phy-logenetics has driven a major paradigm shift inspecies delimitation and molecular systematics ingeneral (Fujita et al 2012 Edwards 2009) Themultispecies coalescent model can be applied togenealogical histories from multiple independentloci to assemble separate coalescent processesoccurring in populations into a species tree(Degnan and Rosenberg 2009 Rannala andYang 2003) Within this coalescent-basedframework multiple individuals can be assignedto a single speciespopulation and the speciationhistory of ancestral and descendant species-levellineages can be inferred as a ldquospecies treerdquo incontrast to estimating gene genealogies fromindividual samples (Degnan and Rosenberg2006 2009 Rannala and Yang 2003)
A number of multispecies coalescent-basedspecies delimitation methods have recently beenintroduced offering an exciting framework forempirically assessing species boundaries byselecting the best species tree model from a set ofalternative models representing differenthypotheses of species boundaries (Table 21)For example SpeDeSTEM (Ence and Carstens2011) finds the maximum likelihood values for aspecies tree assuming all putative species areseparate lineages and for alternative species treeswhere two or more species are collapsed into a
2 The Dynamic Discipline of Species Delimitation hellip 27
single lineage The best fitting model is thenselected using the Akaike information criterionunder the assumption of constant populationsizes and fixed gene topologies across the speciestree The species delimitation program BayesianPhylogenetic and Phylogeography (BPampPYangand Rannala 2010) accommodates gene treeuncertainty and variable population sizes andsamples from the Bayesian posterior distributionof species delimitation models using reversible-jump Markov chain Monte Carlo (rjMCMC)sampling BPampP requires a user-provided guidetree with resolution finer than the species leveland evaluates alternative modes derived from allpossible subtrees that are generated by collapsingor splitting nodes on the guide tree (Yang andRannala 2010 Rannala and Yang 2003) Theproportion of time spent on each model is pro-portional to the posterior probability of themodel ie ldquospeciation probabilitiesrdquo WhileBPampP ranks among the most popular coalescent-based species delimitation programs (Fujita et al2012) misspecifications of the guide tree andorthe prior distribution for population size (θ) canresult in strong support for models containing aninflated number of species (Leacheacute and Fujita2010 Zhang et al 2011) The nonparametricheuristic species delimitation approach imple-mented in the program Brownie (OrsquoMeara 2010)jointly sorts anonymous samples into species andinfers a species tree from input gene trees fromdifferent loci assuming that for a speciationevent the corresponding nodes on gene trees willbe more consistent with each than the diver-gences within species However finding both theoptimum species tree and species boundariesremains computationally challenging andBrownie has been shown to frequently yieldincorrect results (OrsquoMeara 2010)
Most recently available coalescent-basedspecies delimitation methods require individualassignments to a species or population a priori Ina number of scenarios correct assignments ofsamples to species may be difficult includingpresence of cryptic species incongruencebetween conventional species and moleculardata or simply the fact that accurate specimenidentification in complex groups with subtle or
difficult to discern diagnostic characters is anontrivial task (Leavitt et al 2013e) Statisticalmethods for assessing individual assignment andspecies detection prior to coalescent-based spe-cies delimitation and species tree reconstructionand species provide a more objective approachfor understanding species boundaries
Population assignment tests using a variety ofclustering algorithms using genetic data offer auseful approach for grouping individuals intoputative reproductively isolated groups (Table 21Hausdorf and Hennig 2010 Corander et al2004 2008 Falush et al 2003 Pritchard et al2000) particularly for species delimitation prob-lems that exist at the interface of traditionalpopulation genetic and phylogenetic analyses(Carstens et al 2013 Weisrock et al 2010Knowles and Carstens 2007)
The programs STRUCTURE (Falush et al2003 Pritchard et al 2000) and STRUCTURA-MA (Huelsenbeck et al 2011) cluster samplesinto populations by minimizing HardyndashWeinbergdisequilibrium for a given number of populationclusters using unlinked genetic markers In gen-eral unlinked markers are not available for mostgroups of lichen-forming fungi and a numberof studies have used information from multilocussequence data for STRUCTURE analyses(Leavitt et al 2011a b c 2013e Fernaacutendez-Mendoza and Printzen 2013 Fernaacutendez-Mendoza et al 2011) Varying approaches havebeen implemented to convert DNA sequencedata to allelic data for Bayesian clustering (seeOrsquoNeill et al 2013) STRUCTURE is expectedto perform well when there is sufficient inde-pendence across regions such that linkage dis-equilibrium within regions does not dominate thedata (STRUCTURE manual) but can also beeffective using multilocus sequence data andtreating all SNPs as independent loci regardlessof physical linkage within each locus (OrsquoNeillet al 2013 Falush et al 2003) A recent study ofthe lichen-forming genus Letharia showed thatBayesian clustering implemented in the programSTRUCTURE was generally able to recover thesame putative Letharia lineages circumscribedemploying a gene genealogical approach inKroken and Taylorrsquos iconic species delimitation
28 SD Leavitt et al
study (Altermann et al 2014 Kroken and Taylor2001) Altermann et al (2014) show that popu-lation assignments were largely consistent acrossa range of scenarios including extensiveamounts of missing data the exclusion of SNPsfrom variable markers and inferences based onSNPs from as few as three gene regions Incontrast to STRUCTURE and STRUCTURA-MA the program BAPS (Corander et al 2008)can explicitly infer genetic structure using hap-loid sequence data or other linked geneticmarkers in the ldquoclustering with linked locirdquomodel Another advantage of BAPS is that theprogram is much more computationally efficientthan STRUCTURE or STRUCTURAMA Sim-ulation studies indicate that both BAPS andSTRUCTURE perform well at low levels ofpopulation differentiation and when clusters arenot well differentiated (Latch et al 2006)
In practice inferring the most appropriatelevel of population structure using Bayesianclustering algorithms remains challenging (Latchet al 2006 Evanno et al 2005) BAPS andSTRUCTURAMA can infer both individualassignments and the most likely number ofgenetic clusters (Corander et al 2008 Huelsen-beck et al 2011) and the ad hoc ΔK statisticproposed by Evanno et al (2005) provides anobjective approach for identifying the uppermosthierarchical level of structure However carefulconsideration of other levels of populationstructure may ultimately reveal more biologicallymeaning results and researchers should examineindividual assignments across a range of geneticgroupings Some markers generated fromindependent genomic regions such as SNPs andfast-evolving microsatellites can be used to dis-tinguish fine-scale population structuring on thebasis of allele frequencies and species delimita-tion analyses based on these markers may implythe risk of severe taxonomic oversplitting Inthese cases validation approaches such asBPampP and SpeDeSTEM and corroborationamong different species delimitation approachescan provide perspective between intraspecificpopulation structure and species-level clusters
Another limitation of clustering approaches isthat they do not assess or take into account
evolutionarily divergences and relationshipsamong population clusters Coalescent-basedspecies tree methods (discussed below) provide ameans to assess the diversification histories ofpopulations inferred from clustering analysesTherefore a potential working protocol for aninformed species delimitation study that takesinto account population structure could consist offirst applying a genetic clustering analysis under apopulation genetics criterion (eg BAPSSTRUCTURE STRUCTURAMA) to identifygenetically distinct population clusters that can beconsidered ldquocandidate speciesrdquo From these can-didate species a species tree can be inferred forfocal group using coalescent-based species treereconstruction methods (eg BEAST) Subse-quently a coalescent-based validation methodcan be applied to assess whether the distinctpopulation clusters represent independent evolu-tionary lineages (eg BPampP) This multistepapproach would provide a consistent and standardcriterion for distinguishing between population-and species-level lineages (Camargo and Sites2013) and has been applied to a number of casesincluding lichen-forming fungi (Leavitt et al2011a b c 2013e Leacheacute and Fujita 2010)
High-throughput sequencing methods providethe means to effectively sample hundreds tothousands of loci from across a species genome fora large number of species and continue to revolu-tionize studies that can be performed even in non-model organisms Targeted high-throughputsequencing approaches such as anchored phy-logenomics transcriptome sequencing reduced-representation genomic library sequencing(restriction-site-associated DNA sequencingRAD-Seq and genotype-by-sequencing GBS)and high-throughput amplicon sequencing pro-vide important insight into species boundaries fora number groups although none of these approa-ches has yet been applied to studies of lichenizedfungi
For example restriction-site-associated(RAD-Seq) DNA can simultaneously detect andgenotype thousands of genome-wide SNPs byfocusing the sequencing effort on a reducedrepresentation of the entire genome (Baird et al2008) and has been successfully applied to
2 The Dynamic Discipline of Species Delimitation hellip 29
intraspecies (Lewis et al 2007 Miller et al 2007Emerson et al 2010) and interspecies studies(Rubin et al 2012 Eaton and Ree 2013 Wagneret al 2013) This approach has provided strikinginsight into the recent adaption radiation of LakeVictoria cichlids (Wagner et al 2013) Analternative approach targeting the sequencing ofspecific loci using next-generation sequencingplatforms provides an efficient means of gener-ating data for loci of known genomic locationorthology size and expected level of variation(OrsquoNeill et al 2013) Markers can be targetedusing well-established PCR techniques or newerhybridization techniques and subsequentlypooled for high-throughput sequencing usingparallel-tagged sequencing of multiple individualsamples within a single sequencing run (OrsquoNeillet al 2013) A recent study of North Americantiger salamanders (Ambystoma tigrinum)demonstrated the potential for amplicon-basedparallel-tagged sequencing to rapidly generatelarge-scale genomic data for species delimitationand species tree research (OrsquoNeill et al 2013)
Although these methods provide an enormousamount of data and insight into diversification atan unprecedented scale computational limita-tions restrict the applicable analytical methodsfor large datasets although computational andanalytical advances are happening rapidly Mostrecent coalescent-based species delimitation andspecies tree models using gene trees have beenlimited to handle tens of loci for multiple indi-viduals and combining hundreds or thousands ofgene trees into a single species delimitationframework presents considerable computationalchallenges (Camargo and Sites 2013) Therecently developed method Bayes factor delim-itation (with genomic data BFD) combines adynamic programming algorithm for estimatingspecies trees that bypasses the computationallyintensive MCMC integration over gene trees toprovide a rigorous technique for species delimi-tation studies using genome-wide SNP data(Leacheacute et al 2014) Competing species delimi-tation models are compared using Bayes factorsand it appears that this approach is robust tosample sizes (ie few loci and limited samples
per species) and misspecification of the prior forpopulation size (θ) (Leacheacute et al 2014)
Fungi are an ideal model for assessing diver-gence in eukaryotes due to their simple mor-phologies small genomes broad ecological rolesand diverse lifestyles (Gladieux et al 2014)However the use of genomic data from high-throughput sequencing methods have not yet beenincluded in species delimitation studies of lichen-forming fungi This is due in part to challenges indealing with metagenomic data containing geno-mic information from a plethora of symbiontsassociated with a lichen thallus scant genomicresources and discipline-specific inertia A num-ber of lichen-forming fungal genomes are cur-rently available including Endocarponpusillum(Wang et al 2014) Cladonia spp (Park et al2013a 2014) Caloplaca flavorubescens (Parket al 2013b) and Cladonia greyi and Xanthoriaparientina through the Fungal Genomics Programat Joint Genome Institute of the United StatesDepartment of Energy (httpwwwjgidoegov)The foreseeable ongoing expansion of genomicresources for lichen-forming fungi will be centralto developing approaches for delimiting speciesusing high-throughput sequencing Specificallygenomic resources will be crucial in identifyingnovel markers (variable genes regions SNPsmicrosatellites etc) identifying conserved fungalmarkers for targeted high-throughput sequencingapproaches and references for entire genomecomparisons (Devkota et al 2014 Werth et al2013)
23 Can We Make SpeciesDelimitation in Lichen-FormingFungi Truly Integrative
The widespread availability of genetic data hascreated a biased viewpoint that only genetic datashould be used for statistical species delimitationHowever an ongoing appeal to researchers toassess species boundaries from multiple andcomplementary perspectives (phylogeneticspopulation genetics comparative morphologydevelopment ecology etc) has resulted in an
30 SD Leavitt et al
integrative taxonomic framework bringing theseconceptual and methodological perspectivestogether (Hamilton et al 2014 Fujita et al 2012Salicini et al 2011 Yeates et al 2011 Padialet al 2010 Dayrat 2005 Will et al 2005 Wiensand Penkrot 2002) In practice any study linkingdifferent kinds of data to support hypotheses ofspecies boundaries including mapping morpho-logical characters onto a molecular phylogenycan be considered integrative Integrative meth-ods for species delimitation fall across a broadspectrum ranging from verbal and qualitativeassessments of data classes to quantitative meth-ods that allow different data types to contribute tostatistical species delimitation (Yeates et al2011) In most taxonomic studies utilizing bothmolecular and morphological data expert opinionis eventually used to some degree to evaluate thefinal status of a candidate species (eg Bond andStockman 2008) In this sense many studiesusing evidence for independent data sources fordelimiting species boundaries use an iterativeapproach (Yeates et al 2011) where speciesboundaries can be tested within a hypothetico-deductive framework with diverse datasets
From a practical perspective we advocate aprocess of iterative taxonomy (sensu Yeates et al2011) to circumscribe and refine species limitsusing multiple lines of evidence This iterativeprocess involves comparisons of morphologicaldata with a phylogenetic hypothesis (ie single-locus gene tree or concatenated multilocusphylogeny) to identify the least inclusive mono-phyletic clade in the topology characterized by atleast one unambiguously diagnostic morphologi-cal character (Miralles and Vences 2013) Thisphylogenetic tree-informed approach to assessingspecies boundaries represents a common practicein studies of lichen-forming fungi Previouslyunrecognized species-level clades with corre-sponding subtle or overlooked phenotypiccharacters have been commonly observed in bothcrustose lichens [eg Acarospraceae (Wedin et al2009) Graphidaceae (Rivas Plata and Lumbsch2011 Papong et al 2009) Lecanoraceae (Leavittet al 2011a) Lecideaceae (Ruprecht et al 2010)Mycoblastaceae (Spribille et al 2011) andTeloschistaceae (Vondraacutek et al 2009 Muggia
et al 2008)] and foliose and fruticose lichens [egLobariaceae (Moncada et al 2014 McDonaldet al 2003) Parmeliaceae (Leavitt et al 2013aWirtz et al 2012 Divakar et al 2005 Molinaet al 2004 Kroken and Taylor 2001) Peltigera-ceae (OrsquoBrien et al 2009 Goffinet et al 2003)Physciaceae (Elix et al 2009 Divakar et al2007) and Sphaerophoraceae (Houmlgnabba andWedin 2003)] As a specific example Luumlckinget al (2008) used a combination of medullarychemistry and underside pigmentation in speci-mens from the Heterodermia obscurata group inCosta Rica to corroborate monophyletic clades inan ITS gene tree as species-level lineages
While this iterative approach to speciesdelimitation and taxonomy has proven valuablefor understanding species boundaries anddescribing new taxa in some groups of lichenizedfungi a posteriori examination of morphologicaland chemical features has failed to reveal diag-nostic phenotypic characters in a number ofstudies (Muggia et al 2014 Leavitt et al 2011aOtaacutelora et al 2010 OrsquoBrien et al 2009) Fur-thermore a study of widespread species in thegenus Melanelixia (Parmeliaceae) indicated thatphenotypically cryptic lichen-forming fungalspecies-level lineages may be relatively ancientand diagnosable phenotypic differences may beabsent even millions of years after the initialdivergence (Leavitt et al 2012b) The latter studyhighlights the fact that species-level lineages maycommonly exist without any observable diag-nostic phenotypic characters calling into ques-tion the impetus for a universal application ofintegrative taxonomy
In keeping with the principle that as manylines of evidence as available should be com-bined to delimit species (Dayrat 2005) the for-malized integrative taxonomic approach (ITAXMiralles and Vences 2013) uses a mtDNA guidetree and observations from different types of datathat might provide conclusive evidence for theindependence of lineages and thus their identityas different species Miralles and Vences (2013)provide a non-exhaustive list of species delimi-tation criteria to be integrated in the ITAXapproach including (a) sympatric occurrencewithout admixture as revealed by consistent
2 The Dynamic Discipline of Species Delimitation hellip 31
differences in morphological or molecular char-acters at the same geographic location (b) strongdifferences in a behavioral morphological orgenetic character known to mediate prematingisolation (c) unviability or infertility of hybrids(d) lack of gene flow across a geographicalhybrid zone (e) congruent diagnostic differencesbetween sister lineages in various unlinkedmorphological character (f) absence of haplo-type sharing in several unlinked nuclear loci and(g) a combination of criteria endashf Speciesboundaries are based on seeking the least inclu-sive monophyletic group in the mtDNA treewhich fulfills at least one of the criteria listedabove Clearly the ITAX approach is sensitive tosample size and in order to reliably support thedistinctiveness of a given species it has beenrecommended that the sampling strategy includesat least five individuals per species (Miralles andVences 2013) A similar approach could beadopted for lichenized fungi using an ITS genetopology as guide tree rather than mtDNA
A total evidence approach including concat-enation has been a common approach for inte-grating information from different sourcesincluding independent genetic markers in phy-logenetic reconstructions (Kluge 1989 Wiens1998 de Queiroz et al 1995) Proponents forconcatenation of independent data in phyloge-netic analyses argue that when combining alldata the underlying signal of speciation mayemerge even if weakly contradictory signalis contained in the individual data partitions(Gatesy et al 1999) For example establishing apreliminary perspective of species boundariesfrom multilocus sequence data using concatena-tion may provide a reasonable starting point forscreening for cryptic species and species treeinference (Šlapeta et al 2006 Leavitt et al2011a Le Gac et al 2007 Leacheacute 2009)However concatenation and consensus methodsimply a risk of obtaining inflated support forincorrect relationships and information aboutvariance in gene coalescence is lost (Degnan andRosenberg 2006 2009 Kubatko and Degnan2007 Edwards 2009) In spite of the impetus forintegrating evidence from independent data
sources into empirical species delimitation stud-ies and taxonomy (Fujita et al 2012 Padial et al2009) most currently available species delimi-tation methods are unable to accommodate non-genetic data sources in a statistical framework
231 Selecting the Appropriate Data
In the face of increasing availability of geneticdata and associated bioinformatical approachesfor delimiting species researchers should care-fully consider what information is being sacri-ficed by the failure to consider non-genetic datain species delimitation studies and whetheraccuracy could be improved by the addition ofmultiple data types Morphological data havehistorically served as a proxy to identify repro-ductively isolated groups (ie ldquospeciesrdquo) (Ray1686 Fujita et al 2012) Current methods fordelimiting species using non-genetic data (egchemistry morphology and ecology) remainwoefully understudied For example morphol-ogy-based species circumscriptions are generallybased on one or more qualitative (or quantitative)morphological characters that do not appear tooverlap with other species However ascertain-ing that a given trait is truly fixed within apopulation with statistical confidence requiresunrealistic sample sizes even when allowing forsome level of polymorphism in the diagnosticcharacter (Wiens and Servedio 2000) Now var-ious combinations of datamdashfrom morphologygenetics geography and ecologymdashare acceptedas standard information for species delimitationstudies (Ruiz-Sanchez and Sosa 2010 Ross et al2010 Edwards and Knowles 2014)
Below we briefly discuss appropriate datasources for species delimitation studies of lichen-forming fungi However homoplastic characters(similar traits that are not derived from a com-mon ancestor) are common among many traitscommonly used to circumscribe fungal taxa thebiological significance of secondary metabolitevariation remains largely unknown (Lawrey1986) and ecological niches may be difficultto adequately characterize and model due to
32 SD Leavitt et al
microhabitat requirements and data resolutionTherefore we advocate a cautious approach toselecting appropriate and relevant data forassessing species boundaries
In spite of the potential challenges and limi-tations of using phenotypic data these traits haveprovided a plethora of valuable information forunderstanding species boundaries For examplein the Melanelixia fuliginosa group (Parmelia-ceae) a morphometric analysis using color isi-dia and marginal zone free of isidia ascharacters revealed a general pattern of differ-entiation between material formerly recognizedas subspecies fuliginosa and glabratula in spiteof the fact that considerable overlap betweengroups occurs in some characters individually(Arup and Berlin 2011) Furthermore the dis-tinction of the two groups was supported by aphylogenetic analysis of the ITS marker andecological differences with M fuliginosaoccurring predominantly on rock and M glab-ratula on bark (Arup and Berlin 2011) Thisstudy of the M fuliginosa group provides a fit-ting example of using multiple independentsuites of data including ecology morphologyand genetic information to establish a robusthypothesis of species boundaries
In lichen systematics phenotypic dataincluding thallus organization secondary metab-olites mode of reproduction ascoma-type andontogeny ascus and ascospore characters havehistorically played a prominent role (Printzen2009) Ascomatal characters have traditionallyheld a major role in higher-level classification(Printzen 2009) in contrast to species-levelclassification which also tends to include a widearray of vegetative and chemical charactersCommonly assessed morphological charactersinclude thallus form and size cortical features(eg maculae and pseudocyphellae) presenceformcolor of attachment structure (eg rhizines)and reproductive mode (ascomata vs vegetativediaspores) (Printzen 2009) Different morpho-logical types of vegetative diasporesmdashcorticatedisidia and ecorticated sorediamdashand their locationare commonly used to distinguish speciesAscomatal characters including morphologylocation (laminal vs marginal) position (sessile
vs immersed) presence of thalline margins colorof an apothecial disc and presence or color ofpruina are also commonly used in speciesdelimitations of lichen-forming fungi (Printzen2009) Other important characters may includethalline characters form color size and septationof ascospores size and form and structure of ascithe hamathecium type of epihymenium andhypothecium and the type of excipulum orperidium conidiomatal characters etc (Printzen2009)
Assessments of secondary metabolites hasplayed an important role in lichen taxonomybeginning with the introduction of simple spottests by Nylander (1866a b) The use of chem-istry in lichen taxonomy has been discussed indetail in numerous reviews (Lumbsch 1998a bRogers 1989 Brodo 1986 Egan 1986 Leuckert1985 Brodo 1978 Hawksworth 1976 Culber-son 1969 1970) and we refer readers to thesevaluable sources for a more comprehensive per-spective on lichen chemistry In short extrolites(secondary metabolites) belong to various clas-ses the most common and diverse include dep-sides depsidones chlorinated xanthones andanthraquinones (Lumbsch 2002 Culberson1969) The presence or absence of specificextrolites or their replacements by another sub-stance is widely used to distinguish speciesparticularly when correlated with differences ingeographic distributions However if morpho-logical or geographical differences betweenpopulations containing different extrolites are notapparent the taxonomic significance has beendisputed with some authors distinguishing themas species and others preferring to regard them aschemical races within a species In addition tosimply using the presence or absence of extro-lites Culberson and Culberson (1976) proposedto arrange lichen substances into chemosyn-dromes of closely related substances The pres-ence or absence of these chemosyndromes maypotentially be used as characters to delimit spe-cies regarding differences involving the samechemosyndromes as intraspecific variation anddistinct chemosyndromes as evidence for inter-specific populations (Lumbsch 1994 Gowan1986)
2 The Dynamic Discipline of Species Delimitation hellip 33
Due to the fact that species delimitationstudies often incorporate a substantial biogeo-graphical or ecological component ecologicalniche modeling plays an increasing role in phy-logenetic and taxonomic research Niche mod-eling can provide evidence for ecologicalisolation between populations based on eitherconserved or divergent ecological niches andtherefore can provide additional evidence sup-porting lineage independence between putativespecies The application of ecological nichemodeling has been applied to species delimita-tion studies in a number of cases (Ruiz-Sanchezand Sosa 2010 Leacheacute et al 2009 Raxworthyet al 2007 Rissler and Apodaca 2007) althoughit has not been explicitly applied to assess speciesboundaries in lichen-forming fungi By mappingthe spatial distribution of environmental suit-ability of climatic variables for candidate speciesthe application of ecological niche modeling canbe particularly important in cases where specieshave allopatric distributions (Raxworthy et al2007)
Ecological niche modeling utilizes knownassociations between a speciesrsquo occurrencelocalities and environmental variables to defineabiotic conditions within which populations canbe maintained (Guisan and Thuiller 2005) Themethodological approach for modeling is basedon four general properties (i) The current knownspeciesrsquo localities is the dependent variable (ii)the distribution is modeled as a map composed ofgrid cells at a specified resolution (iii) a range ofenvironmental variables (eg temperature pre-cipitation and solar exposure) are collected todescribe the characteristics of each cell and (iv)classifying the degree to which each cell is eithersuitable or unsuitable for each species under arange of models (Guisan and Thuiller 2005)
Ecological data also have the potential to playan important role in understanding speciesboundaries in lichen-forming fungi For exampleNash and Zavada (1977) demonstrated that Xant-hoparmelia populations with distinct chemistriesoccurring in the northern portion of the SonoranDesert exhibit habitat selection among differentrock substrates within a region with relativelyuniform climate and topography In another case
the parmelioid species Parmelia mayi is mor-phologically indistinguishable from P saxatilisbut can be separated by bioclimatic features andgenetic and chemical characters (Molina et al2011) McCune and Printzen(2011) assess distri-butions and climatic niches of species in theLecanora varia group inwesternUSAandprovidea model that uses continental influence and annualtemperature as themajor factors predicting speciesdistributions The distribution of Usnea hirta alichen commonly used in air quality biomonitor-ing research was modeled for a section of theWhite River National Forest in central Coloradobased on the presence of U hirta at 72 biomoni-toring reference sites distributed in the inter-mountain western United States (Shrestha et al2012) The best model for predicting U hirtadistribution included four variablesmdashsolar radia-tion average monthly precipitation and averagemonthly minimum and maximum temperatures(Shrestha et al 2012) These studies supportthe potential use of ecological niche modelingmethods in species delimitation studies of lichen-forming fungi
24 Conclusions What AboutTaxonomy
In most cases species circumscription and tax-onomy requires some degree of qualitativejudgment and individual interpretation Integrat-ing multiple types of data into an empiricalframework for delimiting species boundarieswhere species boundaries can be tested within ahypothetico-deductive framework with diversedatasets can provide robust hypotheses of speciesboundaries and taxonomic stability (Yeates et al2011) However it has long been known toevolutionary biologists that distinct species donot need to have diagnosable morphologicaldifferences (Mayr 1963) and increasing avail-ability of genetic data has allowed researchers toidentify species and to rigorously test speciesboundaries with a level of precision that wasunimaginable a decade ago While analyticaladvances in statistical species delimitation havebeen largely based on genetic data the utility of
34 SD Leavitt et al
these approaches to formal taxonomy remainselusive Due to these challenges an eclecticapproach to delimiting species and cautionagainst the reliance on any single dataset ormethod is required when delimiting species
While results from many recent studies maycontradict traditional species boundaries acrossmany groups of lichen-forming fungi we areoptimistic that this research represents substantialprogress toward a more accurate perspective onspecies boundaries and diversity in fungi As aresult of ongoing research of species boundariesin lichen-forming fungi the taxonomic value ofmany phenotypes is now better understood ourunderstanding of ecological evolutionary andbiogeographic patterns has improved and we canbegin to better understand patterns of symbiontinteractions in lichens Integrating new data(including novel morphological characters andgenetic data) will be essential to accurately rep-resent species-level diversity across all groups oflichenized fungi Hopefully an improved per-spective on lichen diversity also increases ourappreciation of these incredible symbiotic sys-tems While we are strong advocates for theapplication of independent data types in devel-oping an integrative taxonomy there is anincreasing need to formally recognize the exis-tence of phenotypically cryptic species-levellineages in lichen-forming fungi (see Hibbettet al 2011) In some cases a molecular taxon-omy may provide the most practical approach toconsistent treatment of mycobiont species withinlichen groups where diagnostic morphologicalcharacters are unidentifiable or practically notfeasible (Leavitt et al 2013c d)
These are exciting times for taxonomists andphylogeneticists A closer look at lichen taxon-omy with the inclusion of new data will help usto better understand the diversity of these fasci-nating organisms accurately interpret distribu-tion patterns and play a more important role inmeaningful conservation practices Howeversome level of uncertainty will accompany pro-gress In many taxonomic groups our traditionalapproach for species identification will likelyneed to be substantially modified The search forcorroborating morphological support for cryptic
species identified using molecular data willrequire meticulous and creative approaches toassess phenotypic variation in potentially unor-thodox ways We are hopeful that lichenologistswho traditionally have been eager to include newmethods such as chromatography in their rou-tine identifications will be amenable to includemolecular techniques to their routine examina-tion of specimens for identification and classifi-cation Although this may prove difficult toachieve by single individuals especially citizenscientists that traditionally play an important rolein lichen taxonomy the increasing numberof collaborative projects in the discipline (egLumbsch et al 2011 Crespo et al 2010Gueidan et al 2009) make us optimistic thatbroad-scale collaborative approaches will facili-tate the inclusion of molecular data in lichenresearch at all levels
Acknowledgments We are indebted to various col-leagues for valuable thought-provoking discussionnotably Matthew Nelsen (University of Chicago) AnaCrespo (Universidad Complutense de Madrid) PradeepDivakar (Universidad Complutense de Madrid) BeckettSterner (The Field Museum) and Joyce Havstad (TheField Museum) We also thank anonymous reviewers whoprovided valuable comments that improved this chapterSupport by the US National Science Foundation isgratefully acknowledged (ldquoHidden diversity in parmelioidlichensrdquo DEB-0949147)
References
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Altermann S Leavitt SD Goward T Nelsen MPLumbsch HT (2014) How do you solve a problemlike Letharia A new look at cryptic species in lichen-forming fungi using Bayesian clustering and SNPSsfrom multilocus sequence data PLoS ONE 9(5)e97556 doi101371journalpone0097556
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2 The Dynamic Discipline of Species Delimitation hellip 35
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Culberson WL (1969) The use of chemistry in thesystematics of the lichens Taxon 18498ndash505
Culberson WL (1970) Chemosystematics and ecology oflichen-forming fungi Annu Rev Ecol Syst 1153ndash170
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Darwin C (1859) On the origin of species by means ofnatural selection or the preservation of favoured racesin the struggle for life J Murray London
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de Queiroz K (1998) The general lineage concept ofspecies species criteria and the process of speciationa conceptual unification and terminological recom-mendations In Howard DJ Berlocher SH (eds)Endless forms species and speciation Oxford Uni-versity Press Oxford pp 57ndash75
de Queiroz K (1999) The general lineage concept ofspecies and the defining properties of the speciescategory In Wilson RA (ed) Species new interdis-ciplinary essays MIT Press Cambridge pp 49ndash89
de Queiroz K (2007) Species concepts and speciesdelimitation Syst Biol 56(6)879ndash886 doi10108010635150701701083
de Queiroz A Donoghue MJ Kim J (1995) Separateversus combined analysis of phylogenetic evidenceAnnu Rev Ecol Syst 26(1)657ndash681 doi101146annureves26110195003301
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Degnan JH Rosenberg NA (2009) Gene tree discordancephylogenetic inference and the multispecies coales-cent Trends Ecol Evol 24(6)332ndash340 doihttpdxdoiorg101016jtree200901009
Del-Prado R Cubas P Lumbsch HT Divakar PK BlancoO de Paz GA Molina MC Crespo A (2010) Geneticdistances within and among species in monophyleticlineages of Parmeliaceae (Ascomycota) as a tool fortaxon delimitation Mol Phylogenet Evol 56(1)125ndash133 doi101016jympev201004014
Del-Prado R Divakar PK Crespo A (2011) Using geneticdistances in addition to ITS molecular phylogeny toidentify potential species in the Parmotrema reticul-atum complex a case study The Lichenologist 43(06)569ndash583 doi101017S0024282911000582
Del-Prado R Blanco O Lumbsch HT Divakar PK ElixJA Molina MC Crespo A (2013) Molecular phylog-eny and historical biogeography of the lichen-formingfungal genus Flavoparmelia (Ascomycota Parmelia-ceae) Taxon 62(5)928ndash939 doi101270562522
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Dettman JR Jacobson DJ Turner E Pringle A TaylorJW (2003b) Reproductive isolation and phylogeneticdivergence in Neurospora comparing methods ofspecies recognition in a model eukaryote Evolution57(12)2721 doi10155403-074
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Divakar PK Molina MC Lumbsch HT Crespo A (2005)Parmelia barrenoae a new lichen species related toParmelia sulcata (Parmeliaceae) based on molecularand morphological data The Lichenologist 37(01)37ndash46 doi101017S0024282904014641
Divakar PK Amo De paz G del Prado R Esslinger TLCrespo A (2007) Upper cortex anatomy corroboratesphylogenetic hypothesis in species of Physconia(Ascomycota Lecanoromycetes) Mycol Res 111(11)1311ndash1320 doi101016jmycres200708009
Divakar PK Figueras G Hladun N Crespo A (2010)Molecular phylogenetic studies reveal an undescribedspecies within the North American concept of Mela-nelixia glabra (Parmeliaceae) Fungal Divers 42(1)47ndash55 doi101007s13225-010-0027-3
Donoghue MJ Gauthier A (2004) Implementing thephylocode Trends Ecol Evol 19(6)281ndash282 doi101016jtree200404004
Eaton DAR Ree RH (2013) Inferring phylogeny andintrogression using RADseq data an example fromflowering plants (Pedicularis Orobanchaceae) SystBiol 62(5)689ndash706 doi101093sysbiosyt032
Edwards SV (2009) Is a new and general theory ofmolecular systematics emerging Evolution 63(1)1ndash19 doi101111j1558-5646200800549x
Edwards DL Knowles LL (2014) Species detection andindividual assignment in species delimitation canintegrative data increase efficacy Proc R Soc B BiolSci 281(1777) doi101098rspb20132765
Egan RS (1986) Correlations and non-correlations ofchemical variation patterns with lichen morphologyand geography The Bryologist 8999ndash110
Elix JA Corush J Lumbsch HT (2009) Triterpenechemosyndromes and subtle morphological characterscharacterise lineages in the Physcia aipolia group inAustralia (Ascomycota) Syst Biodivers 7(04)479ndash487 doi101017S1477200009990223
Emerson KJ Merz CR Catchen JM Hohenlohe PACresko WA Bradshaw WE Holzapfel CM (2010)Resolving postglacial phylogeography using high-throughput sequencing Proc Natl Acad Sci 107(37)16196ndash16200 doi101073pnas1006538107
Ence DD Carstens BC (2011) SpedeSTEM a rapid andaccurate method for species delimitation Mol EcolResour 11(3)473ndash480 doi101111j1755-0998201002947x
EvannoGRegnaut SGoudet J (2005)Detecting thenumberof clusters of individuals using the software STRUC-TURE a simulation study Mol Ecol 14(8)2611ndash2620doi101111j1365-294X200502553x
Falush D Stephens M Pritchard JK (2003) Inference ofpopulation structure using multilocus genotype datalinked loci and correlated allele frequencies Genetics164(4)1567ndash1587 doi101111j1471-8286200701758x
Fan HH Kubatko LS (2011) Estimating species treesusing approximate Bayesian computation Mol Phylo-genet Evol 59(2)354ndash363 doi101016jympev201102019
Fernaacutendez-Mendoza F Printzen C (2013) Pleistoceneexpansion of the bipolar lichen Cetraria aculeata intothe Southern hemisphere Mol Ecol 22(7)1961ndash1983doi101111mec12210
Fernaacutendez-Mendoza F Domaschke S Garciacutea MA JordanP Martin MP Printzen C (2011) Population structure
2 The Dynamic Discipline of Species Delimitation hellip 37
of mycobionts and photobionts of the widespreadlichen Cetraria aculeata Mol Ecol 20(6)1208ndash1232doi101111j1365-294X201004993x
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Fujisawa T Barraclough TG (2013) Delimiting speciesusing single-locus data and the generalized mixed yulecoalescent (GMYC) approach a revised method andevaluation on simulated datasets Syst Biol 62(5)707ndash724 doi101093sysbiosyt033
Fujita MK Leacheacute AD Burbrink FT McGuire JA MoritzC (2012) Coalescent-based species delimitation in anintegrative taxonomy Trends Ecol Evol 27(9)480ndash488 doi101016jtree201204012
Gatesy J OrsquoGrady P Baker RH (1999) Corroborationamong data sets in simultaneous analysis hiddensupport for phylogenetic relationships among higherlevel Artiodactyl taxa Cladistics 15(3)271ndash313doi101111j1096-00311999tb00268x
Gladieux P Ropars J Badouin H Branca A Aguileta Gde Vienne DM Rodriacuteguez de la Vega RC Branco SGiraud T (2014) Fungal evolutionary genomicsprovides insight into the mechanisms of adaptivedivergence in eukaryotes Mol Ecol 23(4)753ndash773doi101111mec12631
Goffinet B Miadlikowska J Goward T (2003) Phyloge-netic inferences based on nrDNA sequences supportfive morphospecies within the Peltigera didactylacomplex (Lichenized Ascomycota) The Bryologist106(3)349ndash364 doi10163901
Gowan SP (1986) Evolution of secondary natural productsin the genus Porpidia (Ascomycata Porpidiaceae) AmJ Bot 73606
Griffin PC Hoffmann AA (2014) Limited genetic diver-gence among Australian alpine Poa tussock grassescoupled with regional structuring points to ongoinggene flow and taxonomic challenges Ann Bot doi101093aobmcu017
Grube M Hawksworth DL (2007) Trouble with lichenthe re-evaluation and re-interpretation of thallus formand fruit body types in the molecular era Mycol Res111(9)1116ndash1132 doi101016jmycres200704008
Gueidan C Savi S Thues H Roux C Keller C Tibell LPrieto M Heimarsson S Breuss O Orange A FrobergL Wynns AA Navarro-Rosines P Krzewicka BPykaelae J Grube M Lutzoni F (2009) Genericclassification of the Verrucariaceae (Ascomycota)based on molecular and morphological evidencerecent progress and remaining challenges Taxon 58(1)184ndash208
Guillot G Mortier F Estoup A (2005) Geneland acomputer package for landscape genetics Mol EcolNotes 5(3)712ndash715 doi101111j1471-8286200501031x
Guillot G Renaud S Ledevin R Michaux J Claude J(2012) A unifying model for the analysis of pheno-typic genetic and geographic data Syst Biol 61(6)897ndash911 doi101093sysbiosys038
Guisan A Thuiller W (2005) Predicting species distribu-tion offering more than simple habitat models EcolLett 8(9)993ndash1009 doi101111j1461-0248200500792x
Hale ME (1990) A synopsis of the lichen genusXanthoparmelia (Vainio) Hale (AscomycotinaParmeliaceae) Smithsonian Institution Press Wash-ington DC
Hamilton CA Hendrixson BE Brewer MS Bond JE(2014) An evaluation of sampling effects on multipleDNA barcoding methods leads to an integrativeapproach for delimiting species a case study ofthe North American tarantula genus Aphonopelma(Araneae Mygalomorphae Theraphosidae) MolPhylogenet Evol 7179ndash93 doi101016jympev201311007
Hausdorf B (2011) Progress toward a general speciesconcept Evolution 65(4)923ndash931 doi101111j1558-5646201101231x
Hausdorf B Hennig C (2010) Species delimitation usingdominant and codominant multilocus markers SystBiol 59(5)491ndash503 doi101093sysbiosyq039
Hawksworth DL (1976) Lichen chemotaxonomy InBailey RH (ed) Lichenology progress and problemsAcademic London pp 139ndash184
Hebert PDN Cywinska A Ball SL deWaard JR (2003)Biological identifications through DNA barcodesProc R Soc Lond B Biol Sci 270(1512)313ndash321doi101098rspb20022218
Hebert PDN Stoeckle MY Zemlak TS Francis CM(2004) Identification of birds through DNA barcodesPLoS Biol 2(10)e312 doi101371journalpbio0020312
Heled J Drummond AJ (2010) Bayesian inference ofspecies trees from multilocus data Mol Biol Evol 27(3)570ndash580 doi101093molbevmsp274
Hey J (2006) On the failure of modern species conceptsTrends Ecol Evol 21(8)447ndash450 doi101016jtree200605011
Hibbett DS OhmanA Glotzer D NuhnMKirk P NilssonRH (2011) Progress in molecular and morphologicaltaxon discovery in Fungi and options for formalclassification on environmental sequences Fungal BiolRev 2538ndash47 doi101016jfbr201101001
Houmlgnabba F Wedin M (2003) Molecular phylogeny ofthe Sphaerophorus globosus species complex Cladis-tics 19(3)224ndash232 doi101111j1096-00312003tb00365x
Hudson RR Coyne JA (2002) Mathematical conse-quences of the genealogical species concept Evolu-tion 56(8)1557 doi101111j0014-38202002tb01467x
Huelsenbeck JP Andolfatto P Huelsenbeck ET (2011)Structurama Bayesian inference of population struc-ture Evol Bioinf Online 7(2011)55ndash59 doi104137EBOS6761
Jones G Oxelman B (2014) DISSECT an assignment-free Bayesian discovery method for species delimita-tion under the multispecies coalescent Bioinformaticsdoi101101003178
38 SD Leavitt et al
Kekkonen M Hebert PD (2014) DNA barcode-baseddelineation of putative species efficient start fortaxonomic workflows Mol Ecol Res doi1011111755-099812233
Kelly LJ Hollingsworth PM Coppins BJ Ellis CJHarrold P Tosh J Yahr R (2011) DNA barcoding oflichenized fungi demonstrates high identification suc-cess in a floristic context New Phytol 191(1)288ndash300doi101111j1469-8137201103677x
Kiss L (2012) Limits of nuclear ribosomal DNA internaltranscribed spacer (ITS) sequences as species barcodesfor fungi Proc Natl Acad Sci 109(27)E1811ndashE1811doi101073pnas1207143109
Kluge AG (1989) A concern for evidence and aphylogenetic hypothesis for relationships among Epi-crates (Boidae Serpentes) Syst Zool 38(1)7ndash25doi101093sysbio3817
Knowles LL Carstens BC (2007) Delimiting specieswithout monophyletic gene trees Syst Biol 56(6)887ndash895 doi10108010635150701701091
Kroken S Taylor JW (2001) A gene genealogical approachto recognize phylogenetic species boundaries in thelichenized fungus Letharia Mycologia 93(1)38ndash53
Kubatko LS Degnan JH (2007) Inconsistency of phylo-genetic estimates from concatenated data under coa-lescence Syst Biol 56(1)17ndash24 doi10108010635150601146041
Latch EK Dharmarajan G Glaubitz JC Rhodes OE Jr(2006) Relative performance of Bayesian clusteringsoftware for inferring population substructure andindividual assignments at low levels of populationdifferentiation Conserv Genet 7(2)295ndash302 doi101007s10592-005-9098-1
Lawrey JD (1986) Biological role of lichen substancesThe Bryologist 89(2)111ndash122
Le Gac M Hood ME Fournier E Giraud T (2007)Phylogenetic evidence of host-specific cryptic speciesin the anther smut fungus Evolution 61(1)15ndash26doi101111j1558-5646200700002x
Leacheacute AD (2009) Species tree discordance traces tophylogeographic clade boundaries in North Americanfence lizards (Sceloporus) Syst Biol 58(6)547ndash559doi101093sysbiosyp057
Leacheacute AD Fujita MK (2010) Bayesian species delim-itation in West African forest geckos (Hemidactylusfasciatus) Proc R Soc B Biol Sci 2773071ndash3077doi101098rspb20100662
Leacheacute AD Koo M Spencer C Papenfuss T Fisher RMcGuire J (2009) Quantifying ecological morpho-logical and genetic variation to delimit species in thecoast horned lizard species complex (Phrynosoma)Proc Natl Acad Sci USA 10612418ndash12423 doi101073pnas0906380106
Leacheacute AD Fujita MK Minin VN Bouckaert RR (2014)Species delimitation using genome-wide SNP dataSystematic Biology doi101093sysbiosyu018
Leavitt SD Fankhauser JD Leavitt DH Porter LDJohnson LA St Clair LL (2011a) Complex patterns ofspeciation in cosmopolitan ldquorock posyrdquo lichensmdashdiscovering and delimiting cryptic fungal species in
the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
Leavitt SD Esslinger TL Lumbsch HT (2012c) Neogene-dominated diversification in neotropical montanelichens dating divergence events in the lichen-form-ing fungal genus Oropogon (Parmeliaceae) Am J Bot99(11)1764ndash1777 doi103732ajb1200146
Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Lumbsch HT St Clair LL(2013c) DNA barcode identification of lichen-formingfungal species in the Rhizoplaca melanophthalmaspecies-complex (Lecanorales Lecanoraceae) includ-ing five new species MycoKeys 71ndash22 doi103897mycokeys74508
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Vondraacutek J Thorsten Lum-bsch H Clair LLS (2013d) Local representation ofglobal diversity in a cosmopolitan lichen-formingfungal species complex (Rhizoplaca Ascomycota)J Biogeogr 40(9)1792ndash1806 doi101111jbi12118
Leavitt SD Lumbsch HT Stenroos S St Clair LL(2013e) Pleistocene speciation in North American
2 The Dynamic Discipline of Species Delimitation hellip 39
lichenized fungi and the impact of alternative speciescircumscriptions and rates of molecular evolution ondivergence estimates PLoS ONE 8(12)e85240doi101371journalpone0085240
Leavitt SD Esslinger TL Hansen ES Divakar PK CrespoA Loomis BF Lumbsch HT (2014) DNA barcoding ofbrown Parmeliae (Parmeliaceae) species a molecularapproach for accurate specimen identification empha-sizing species in Greenland Organ Divers Evol 14(1)11ndash20 doi101007s13127-013-0147-1
Leliaert F Verbruggen H Vanormelingen P Steen FLopez-Bautista JM Zuccarello GC De Clerck O(2014) DNA-based species delimitation in algae Eur JPhycol 49(2) doi101080096702622014904524
Leuckert C (1985) Probleme der Flechten-Chemotaxon-omiemdashStoffkombinationen und ihre taxonomischeWertung Ber Deut Bot Ges 98401ndash408
Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
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Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
Lumbsch HT (1994) Die Lecanora subfusca-Gruppe inAustralasien J Hattori Bot Lab 771ndash175
Lumbsch HT (1998a) Taxonomic use of metabolic data inlichen-forming fungi In Frisvad JC Bridge PDArora DK (eds) Chemical Fungal Taxonomy MarcelDekker New York pp 345ndash387
Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
Mayden RL (1997) A hierarchy of species concepts thedenouement in the saga of the species problem InClaridge MF Dawah HA Wilson MR (eds) Speciesthe units of biodiversity Chapman amp Hall Londonpp 381ndash424
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Mayr E (1970) Populations species and evolutionBelknap Press of Harvard University Press Cambridge
McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
McDonald T Miadlikowska J Lutzoni F (2003) Thelichen genus Sticta in the Great Smoky Mountains a
40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
McKay BD Mays HL Wu Y Li H Yao C-T Nishiumi IZou F (2013) An empirical comparison of character-based and coalescent-based approaches to speciesdelimitation in a young avian complex Mol Ecol 22(19)4943ndash4957 doi101111mec12446
Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
Mishler BD Brandon RN (1987) Individuality pluralismand the phylogenetic species concept Biol Philos 2397
Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
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Nylander W (1866a) Circa novum in studio Lichenumcriterium chemicum Flora 49198ndash201
Nylander W (1866b) Hypochlorite of lime and hydrate ofpotash Two new criteria for the study of lichens(Tanslated and communicated by the Rev W ALeighton) Bot J Linn Soc 9358ndash365
OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
OrsquoNeill EM Schwartz R Bullock CT Williams JSShaffer HB Aguilar-Miguel X Parra-Olea G Weis-rock DW (2013) Parallel tagged amplicon sequencingreveals major lineages and phylogenetic structure inthe North American tiger salamander (Ambystomatigrinum) species complex Mol Ecol 22(1)111ndash129doi101111mec12049
Orock EA Leavitt SD Fonge BA St Clair LL LumbschHT (2012) DNA-based identification of lichen-form-ing fungi Can publicly available sequence databasesaid in lichen diversity inventories of Mount Cameroon(West Africa) The Lichenologist 44(6)833ndash839doi101017S0024282912000424
Otaacutelora MAG Martiacutenez I Aragoacuten G Molina MC (2010)Phylogeography and divergence date estimates of alichen species complex with a disjunct distributionpattern Am J Bot 97(2)216ndash223 doi103732ajb0900064
Padial J Castroviejo-Fisher S Kohler J Vila C ChaparroJ De la Riva I (2009) Deciphering the products ofevolution at the species level the need for anintegrative taxonomy Zoolog Scr 38(4)431ndash447doi101111j1463-6409200800381x
Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
Papong K Luumlcking R Thammathaworn A BoonpragobK (2009) Four new taxa of Chroodiscus (thelotremoidGraphidaceae) from Southeast Asia The Bryologist112(1)152ndash163 doi1016390007-2745-1121152
Park S-Y Choi J Kim JA Jeong M-H Kim S Lee Y-HHur J-S (2013a) Draft genome sequence of Cladoniamacilenta KoLRI003786 a lichen-forming fungusproducing biruloquinone Genome Announcements 1(5)e00695-13 doi101128genomeA00695-13
Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
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Pino-Bodas R Burgaz A Martiacuten M Lumbsch HT (2011)Phenotypical plasticity and homoplasy complicatespecies delimitation in the Cladonia gracilis group(Cladoniaceae Ascomycota) Organ Divers Evol 11(5)343ndash355 doi101007s13127-011-0062-2
Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
Pino-Bodas R Martiacuten M Burgaz A (2012b) Cladoniasubturgida and C iberica (Cladoniaceae) form asingle morphologically and chemically polymorphicspecies Mycol Prog 11(1)269ndash278 doi101007s11557-011-0746-1
Pino-Bodas R Ahti T Stenroos S Martiacuten MP BurgazAR (2013a) Multilocus approach to species recogni-tion in the Cladonia humilis complex (CladoniaceaeAscomycota) Am J Bot 100(4)664ndash678 doi103732ajb1200162
Pino-Bodas R Martiacuten AP Burgaz AR Lumbsch HT(2013b) Species delimitation in Cladonia (Ascomy-cota) a challenge to the DNA barcoding philosophyMol Ecol Resour 13(6)1058ndash1068 doi1011111755-099812086
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Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
Pringle A Baker DM Platt JL Wares JP Latgeacute JPTaylor JW (2005) Cryptic speciation in the cosmo-politan and clonal human pathogenic fungus Asper-gillus fumigatus Evolution 59(9)1886ndash1899 doi10155404-2411
Printzen C (2009) Lichen systematics the role ofmorphological and molecular data to reconstructphylogenetic relationships Progress in botany vol71 Springer Berlin pp 233ndash275
Pritchard JK Stephens M Donnelly P (2000) Inference ofpopulation structure using multilocus genotype dataGenetics 155(2)945ndash959
Puillandre N Lambert A Brouillet S Achaz G (2012)ABGD automatic barcode gap discovery for primaryspecies delimitation Mol Ecol 21(8)1864ndash1877doi101111j1365-294X201105239x
Rannala B Yang Z (2003) Bayes estimation of speciesdivergence times and ancestral population sizes usingDNA sequences from multiple loci Genetics1641645ndash1656
Raxworthy CJ Ingram CM Rabibisoa N Pearson RG(2007) Applications of ecological niche modelingfor species delimitation a review and empiricalevaluation using day geckos (Phelsuma) fromMadagascar Syst Biol 56(6)907ndash923 doi10108010635150701775111
Ray J (1686) Historia planarum vol 1 Clark LondonReese Naeligsborg R Ekman S Tibell L (2007) Molecular
phylogeny of the genus Lecania (Ramalinaceaelichenized Ascomycota) Mycol Res 111(5)581ndash591doi101016jmycres200703001
Reid N Carstens B (2012) Phylogenetic estimation errorcan decrease the accuracy of species delimitation aBayesian implementation of the general mixed Yule-coalescent model BMC Evol Biol 12(1)196 doi1011861471-2148-12-196
Rissler L Apodaca J (2007) Adding more ecology intospecies delimitation ecological niche models andphylogeography help define cryptic species in theBlack Salamander (Aneides flavipunctatus) Syst Biol56924ndash942 doi10108010635150701703063
Rivas Plata E Luumlcking R (2013) High diversity ofGraphidaceae (lichenized Ascomycota Ostropales) inAmazonian Peruacute Fungal Divers 58(1)13ndash32 doi101007s13225-012-0172-y
Rivas Plata E Lumbsch HT (2011) Parallel evolution andphenotypic divergence in lichenized fungi a casestudy in the lichen-forming fungal family Graphida-ceae (Ascomycota Lecanoromycetes Ostropales)Mol Phylogenet Evol 61(1)45ndash63 doi101016jympev201104025
Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
Ross KG Gotzek D Ascunce MS Shoemaker DD (2010)Species delimitation a case study in a problematic anttaxon Syst Biol 59(2)162ndash184 doi101093sysbiosyp089
Rubin BER Ree RH Moreau CS (2012) Inferringphylogenies from RAD sequence data PLoS ONE 7(4)e33394 doi101371journalpone0033394
Rubinoff D (2006) Utility of mitochondrial DNA barcodesin species conservation Conserv Biol 20(4)1026ndash1033 doi101111j1523-1739200600372x
Ruiz-Sanchez E Sosa V (2010) Delimiting speciesboundaries within the Neotropical bamboo Otatea(Poaceae Bambusoideae) using molecular morpho-logical and ecological data Mol Phylogenet Evol 54(2)344ndash356 doi101016jympev200910035
Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
morphological characters Antarctic Sci 22 (SpecialIssue 06)727ndash741 doi101017S0954102010000477
Salicini I Ibaacutentildeez C Juste J (2011) Multilocus phylogenyand species delimitation within the Nattererrsquos batspecies complex in the Western Palearctic MolPhylogenet Evol 61(3)888ndash898 doi101016jympev201108010
Satler JD Carstens BC Hedin M (2013) Multilocusspecies delimitation in a complex of morphologicallyconserved trapdoor spiders (Mygalomorphae Antro-diaetidae Aliatypus) Syst Biol 62(6)805ndash823 doi101093sysbiosyt041
Schlick-Steiner BC Steiner FM Seifert B Stauffer CChristian E Crozier RH (2010) Integrative taxonomya multisource approach to exploring biodiversityAnnu Rev Entomol 55(1)421ndash438 doi101146annurev-ento-112408-085432
Schoch CL Seifert KA Huhndorf S Robert V SpougeJL Levesque CA Chen W Fungal Barcoding Con-sortium (2012) Nuclear ribosomal internal transcribedspacer (ITS) region as a universal DNA barcodemarker for Fungi Proc Natl Acad Sci 109(16)6241ndash6246 doi101073pnas1117018109
Seacuterusiaux E Villarreal AJC Wheeler T Goffinet B(2011) Recent origin active speciation and dispersalfor the lichen genus Nephroma (Peltigerales) inMacaronesia J Biogeogr 38(6)1138ndash1151 doi101111j1365-2699201002469x
Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
Simpson GG (1951) The species concept Evolution5285ndash298
Sites JW Marshall JC (2003) Delimiting species a renais-sance issue in systematic biology Trends Ecol Evol18462ndash470 doi101016S0169-5347(03)00184-8
Sites JW Marshall JC (2004) Operational criteria fordelimiting species Annu Rev Ecol Evol Syst 35(1)199ndash227 doi101146annurevecolsys35112202130128
Šlapeta J Loacutepez-Garciacutea P Moreira D (2006) Globaldispersal and ancient cryptic species in the smallestmarine eukaryotes Mol Biol Evol 23(1)23ndash29doi101093molbevmsj001
Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
Talavera G Dincă V Vila R (2013) Factors affectingspecies delimitations with the GMYC model insightsfrom a butterfly survey Methods Ecol Evol 4(12)1101ndash1110 doi1011112041-210X12107
Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
Tewksbury J Anderson JGT Bakker JD Billo TJDunwiddie PW Groom MJ Hampton SE HermanSG Levey DJ Machnicki NJ del Rio CM PowerME Rowell K Salomon AK Stacey L TrombulakSC Wheeler TA (2014) Natural historyrsquos place inscience and society BioScience doi101093bioscibiu032
Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
Werth S Cornejo C Scheidegger C (2013) Characteriza-tion of microsatellite loci in the lichen fungus Lobariapulmonaria (Lobariaceae) Appl Plant Sci 1(2)1200290 doi103732apps1200290
Westberg M Arup U Kaumlrnefelt I (2007) Phylogeneticstudies in the Candelariaceae (lichenized Ascomy-cota) based on nuclear ITS DNA sequence dataMycol Res 111(11)1277ndash1284 doi101017S0953756204002102
Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
2 The Dynamic Discipline of Species Delimitation hellip 43
(Lepidoptera Lycaenidae) Front Zoo 4(1)8 doi1011861742-9994-4-8
Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
DISSE
CTmdashmultispecies
coalescent
model
forspeciesdelim
itatio
nDISSE
CTexplores
thefullspaceof
possible
clusteringsof
individualsandspeciestree
topologies
inaBayesianfram
ework
Toavoidtheneed
forreversible-jum
pMCMCit
uses
anapproxim
ationin
theform
ofapriorthatisamodificatio
nof
thebirthndashdeathprior
forthespeciestreeItisim
plem
entedas
partof
BEAST
andrequires
only
afewchanges
from
astandard
BEAST
analysisA
nalysesof
simulated
andem
piricald
atasuggestthat
themethodisshow
nto
beinsensitive
tothedegree
ofapproxim
ation
butq
uitesensitive
tootherparameters
Limita
tions
Recently
describedmethodlackingathorough
theoretical
andem
pirical
evaluatio
nItappearsthat
alargenu
mberof
sequencesarerequ
ired
todraw
firm
conclusion
sSo
urce
httpcodegooglecompbeast-m
cmcamphttpwwwin
driid
com
dissectinbeast
htmldescribedin
JonesandOxelm
an(201
4)
Multilocus
sequence
data
SpeD
eSTEMmdashmultispecies
coalescent
mod
elfordiscov
ery
valid
ation
andjoint
estim
ation
Thismaxim
umlik
elihoo
dandorinform
ationtheory-based
methodwas
developedto
test
speciesboundaries
inasystem
with
existin
gsubspecies
taxonomy(CarstensandDew
ey20
10)andcomputestheprobability
ofthegene
treesgiventhespeciestree
forall
hierarchical
perm
utations
oflin
eage
grou
pingSp
eciesbo
undaries
arecomparedusing
Akaikeinform
ationcriteria
andphylogenetic
uncertaintyin
thespeciestree
topologies
does
notaffect
speciesdelim
itatio
nsLimita
tions
Accuracyisdependenton
quality
ofthegene
tree
estim
ates
Source
(http
carstenslaborgohio-stateedusoftwarehtml)criteria
describedin
Ence
andCarstens(201
1)
Multilocus
sequence
alignm
entsandgroup
mem
bership
Browniemdash
multispecies
coalescent
model
forspeciesdelim
itatio
nThe
nonp
aram
etricheuristic
speciesdelim
itatio
napproach
implem
entedin
theprog
ram
Brownie(O
rsquoMeara
2010)jointly
sortsanonym
oussamples
into
speciesandinfers
aspeciestree
from
inputg
enetreesfrom
differentlociassumingthatforaspeciatio
neven
thecorrespondingnodeson
gene
treeswill
bemoreconsistent
with
each
than
the
divergenceswith
inspecies
Limita
tions
Findingboth
theoptim
umspeciestree
andspeciesboundaries
remains
compu
tatio
nally
challenging
andBrowniehasbeen
show
nto
frequently
yieldincorrect
resultsT
heaccuracy
ofthemethodislik
elycorrelated
with
nodalsupportvalues
inthe
individu
algene
topo
logies
Source
httpwwwbrianom
earain
fobrownie
describedin
OrsquoM
eara
(201
0)
Individual
gene
trees
(con
tinued)
22 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
BFD
mdashmultispecies
coalescent
model
for
speciesdelim
itatio
nThe
recently
developedmethod
Bayes
factor
delim
itatio
n(with
genomicdataB
FD)
combinesady
namic
programmingalgorithm
forestim
atingspeciestreesthat
bypasses
thecompu
tatio
nally
intensiveMCMCintegrationov
ergene
treesto
prov
idearigorous
techniqueforspeciesdelim
itatio
nstudiesusinggenome-wideSN
PdataCom
petin
gspeciesdelim
itatio
nmodelsarecomparedusingBayes
factorsanditappearsthat
this
approach
isrobustto
samplesizes(iefew
loci
andlim
itedsamples
perspecies)
and
misspecificatio
nof
thepriorforpo
pulatio
nsize
(θ)
Limita
tions
Recently
describedmethodlackingathorough
theoretical
andem
pirical
evaluatio
nSo
urce
httpwwwbeast2orgwikiindexphpBFD
describedin
Leacheacuteet
al(201
4)
Genom
e-wideSN
Pdata
2 The Dynamic Discipline of Species Delimitation hellip 23
distances among organisms belonging to thesame species are smaller than distances amongorganisms from different species (Puillandreet al 2012 Hebert et al 2003) Genetic distanceapproaches hold particular promise as an identi-fication tool shortcutting the difficulties of mor-phology-based identification (Hebert et al 2004)although in practice a barcode gap may not existfor many groups (Wiemers and Fiedler 2007)Furthermore the role of distance-based approa-ches using a single genetic marker for speciesdiscovery remains more controversial (Rubinoff2006) and without other corroborating evidenceOTUs inferred from single-locus dataset shouldonly be considered ldquocandidaterdquo species Tree-based methods aim to detect monophyletic cladescorresponding to species-level diversity bydetecting discontinuities associated within inter-and intraspecific branching patterns (Fujisawaand Barraclough 2013 Zhang et al 2013Monaghan et al 2009 Pons et al 2006) Tree-based species delimitation methods can also beused on the basis of other properties related tophylogenetic tree topologies (monophyly con-cordance with geography etc (Sites and Marshall2003 2004) Both distance- and tree-basedmethods have been applied for assessing speciesboundaries in lichen-forming fungi (Leavitt et al2012c 2014 Parnmen et al 2012 Del-Pradoet al 2010 2011 Molina et al 2011)
A number of tree-based methods partiallyautomate the species delimitation process withspecific bioinformatical analyses (Table 21)including the general mixed Yule coalescent(GMYC) approach (Fujisawa and Barraclough2013 Monaghan et al 2009 Pons et al 2006)and the Poisson tree processes (PTP) model(Zhang et al 2013) These methods provide rel-atively straightforward and objective pipelinesfor delimiting putative species-level lineagesfrom inferred gene trees by fitting within- andbetween-species branching models to an inferredsingle-locus topology A number of other tree-based methods for species delimitation areeffectively summarized in Sites and Marshall(2004)
When only single-locus data are available theGMYC has been shown to be a relatively robust
tool for species delimitation The GMYCapproach combines a coalescent model of intra-specific branching with a Yule model for inter-specific branching which is then fit to an inferredsingle-gene topology to estimate species bound-aries and a statistical measure of confidence for theinferred boundaries As an input the GMYCapproach requires an ultrametric gene tree andrecent refinements can account for uncertainty inphylogenetic relationships and parameters of theGMYC model (Fujisawa and Barraclough 2013Reid and Carstens 2012) Regardless of theseimprovements it may be difficult to accuratelyinfer an adequate ultrametric tree for large data-sets Although it appears that the GMYC isgenerally stable across a wide range of circum-stances including various methods of phyloge-netic reconstruction the presence of a highnumber singletons high numbers of sampledspecies and gaps in intraspecific sampling theaccuracy of the GMYC is most significantlyaffected by the mean population size relative todivergence times between them (Fujisawa andBarraclough 2013 Talavera et al 2013) Fur-thermore research suggests that the so-calledsingle-threshold version of the GMYC methodlikely outperforms the multiple-thresholdapproach (Fujisawa and Barraclough 2013Monaghan et al 2009) However other studiessuggest that theGMYCmethodmay often providehigher estimates for the total number ofOTUs thanother molecular species delimitation methods(Hamilton et al 2014 Miralles and Vences 2013Talavera et al 2013) warranting a cautiousinterpretation of results from GMYC analyses
Compared to the GMYC approach therecently introduced PTP method for speciesdelimitation has been suggested to be moreaccurate for preliminary species delimitation(Zhang et al 2013) Relative to the GMYC PTPoffers a more straightforward implementationrequiring a simple phylogenetic tree rather thanan ultrametric chronogram However moreresearch is required to assess the general perfor-mance of PTP across wide range of empirical andsimulated species delimitation studies At thispoint data support the use of the GMYC andPTP methods as objective and reasonable starting
24 SD Leavitt et al
points for species delimitation using single-genetopologies
The ldquoSpecies Delimitationrdquo plug-in to theGeneious software provides statistical approachesfor exploring species boundaries in single-genetopologies (Masters et al 2011) Using a priorispecimen assignments to putative species theldquoSpecies Delimitationrdquo plug-in computes statis-tics relating to the probability of the observedmonophyly or exclusivity having occurred bychance in a coalescent process and assesses thewithin- and between-species genetic distances inorder to infer the probability with which membersof a putative species might be identified suc-cessfully with tree-based methods An importantcontribution of the ldquoSpecies Delimitationrdquo plug-in is that it provides an objective means for usersto assess putative species within an empiricalstatistic-based framework rather than qualitativeevaluations of what level of hierarchy constitutesa species in a phylogeny
In contrast to simple sequence similaritythresholds (OTU-picking) for delimiting putativeevolutionarily significant units the AutomaticBarcode Gap Discovery (ABGD) method is anautomated procedure that sorts sequences intohypothetical species based on the existence of abarcode gap observed whenever intraspecificgenetic distances are less than those amongorganisms from different species (Puillandreet al 2012) Ultimately ABGD is a fast simplemethod that can be used to group individualsrepresented in a single-locus sequence alignmentinto candidate species that should be comple-mented with other lines of evidence in an inte-grative taxonomic approach (Kekkonen andHebert 2014 Puillandre et al 2012)
In general the ABGD GMYC and PTP ana-lytical protocols using single-locus data arerepeatable and computationally relatively fastproviding a valuable starting point for a pre-liminary perspective into species boundaries inunderstudied groups that can be validated withsubsequent studies (Kekkonen and Hebert 2014)Similarly analyses of single-locus data can beused to corroborate traditional phenotype-basedspecies boundaries and identify candidate speciesthat have previously been hidden within nominal
species However single-locus species delimita-tionmethodsmaybeparticularly prone to failure inrecognizing significant proportions of species-level biodiversity due to the strict criterion forreciprocal monophyly or alternatively providespurious inflations of estimated species diversitybased on genetic differences that donot correspondto species-level lineages For example recentestimates suggest that the incidence of species-level gene tree paraphyly is approximately 20 (Ross 2014) suggesting that analyses of single-locus datasets would likely fail to accurately deli-mit species in one in every five cases In contrastrecent empirical studies suggest that in some casesthe GMYC provides a striking overestimate ofspecies diversity (Miralles and Vences 2013)
The internal transcribed spacer region (ITS)has played a central role in molecular phyloge-netic studies of lichenized fungi and has recentlybeen adopted as the official barcoding marker forfungi (Schoch et al 2012) In many cases the ITSis sufficiently variable to resolve species bound-aries for lichenized fungi (Schoch et al 2012Kelly et al 2011) although accurate specimenidentification using sequence-based identificationapproaches requires a thoroughly curated refer-ence database (Leavitt et al 2014 Orock et al2012 Kelly et al 2011) In spite of the generalutility of the ITS marker a number of issues maypotentially limit its effective use in speciesdelimitation studies including the potential forintragenomic variation of the nuclear ribosomaltandem repeat and difficulties in aligning ITSsequences from divergent taxa (Kiss 2012)Because the ITS has been formally adopted as theofficial barcoding marker for fungi we recom-mend that species delimitation studies attempt toinclude this region for consistency across studiesHowever we recognize that in some taxonomicgroups the inclusion of the ITS may be prob-lematic and therefore suggest a careful screeningof other markers to identify appropriate loci forresolving species-level relationships
Ultimately the success of any single-locusspecies delimitation method largely depends onthe evolutionary history of the species groupunder study and the variability of the selectedmarker (Fig 22) Species delimitation will be
2 The Dynamic Discipline of Species Delimitation hellip 25
more difficult in more recently diverged lineagesand in cases with some level of interspecific geneflow relative to older well-diverged lineages(eg Leavitt et al 2012c 2013e) In the end wereemphasize that single-locus data alone shouldonly be used to provide a preliminary perspectiveof species boundaries and not as the sole evi-dence in species circumscriptions
222 Sampling Across the GenomeMultilocus Sequence Dataand Genome-Wide Markers
Although single-locus methods can provide anefficient approach for preliminary large-scaleassessments of species diversity there are
significant limitations particularly in recentlydiverged species groups where retention ofancestral polymorphisms and incomplete lineagesorting will likely result in different neutral locihaving unique gene topologies that do not mirrorthe speciation process (Knowles and Carstens2007 Heled and Drummond 2010 Taylor et al2000) In contrast to single-locus and strictlyphenotype-based approaches for species delimi-tation analyses of genetic data collected fromindependent genomic regions can provide robusthypotheses of species boundaries with increasingconfidence (Satler et al 2013 Zhang et al 2011Yang and Rannala 2010) Sequences from mul-tiple independent loci provide an importantsource of data for species delimitation studiesincluding recently developed models that
Fig 22 A simplified diagram illustrating the process ofspeciation through time in a single gene history andresulting gene topologies sampled at two points in thespeciation history (modified from Leliaert et al 2014)aEach dot represents a distinct gene copy and each row onenon-overlapping generation with lines connecting genecopies to their ancestors in the previous generation (onerow below) dashed diagonal lines represent reproductivebarriers two hypothetical sampling intervals at differentpoints in the speciation history are shown Tx and Ty
species delimitation methods using single-locus data areeffective only when species are reciprocally monophyleticin the sampled gene tree genetic clustering and coalescent-based species delimitation methods can circumscribespecies when species may not be monophyletic in sampledgenetic loci b Gene topology representing sampledhaplotypes at time Tx (shown in panel a) hypotheticalspecies are reciprocally monophyletic c Gene topologyrepresenting sampled haplotypes at time Ty (shown in panela) hypothetical species are para- and polyphyletic
26 SD Leavitt et al
combine individual gene genealogies and speciesphylogenies via modeling the coalescent historyof markers (Yang and Rannala 2010 Edwards2009 Knowles and Carstens 2007) As aresponse to advances in sequencing technologiesbioinformatical approaches for multimarker spe-cies delimitation analyses continue to be devel-oped (Table 21 Camargo and Sites 2013)
Long-term reproductive isolation of candidatespecies can be assessed with multilocus sequencedata by evaluating genealogical concordance ofunlinked markers (Baum and Shaw 1995 Aviseand Ball 1990) Within species the mixingeffects of recombination cause unlinked loci tohave distinct genealogical histories but geneticdrift and long-term divergence leads to concor-dant genealogical histories at loci across thegenome Relationships of individuals belongingto distinct candidate species can be evaluatedusing gene genealogies (eg haplotype networksor single-gene topologies) to identify lineagesthat exhibit genealogical exclusivity acrossunlinked neutral loci (Hudson and Coyne 2002Dettman et al 2003b Avise and Ball 1990) Thepresence of the same clades in the majority ofsingle-locus genealogies is taken as evidence thatthe clades represent reproductively isolated lin-eages (Dettman et al 2003a Pringle et al 2005)In practice the criteria of reciprocal monophylyand genealogical concordance of unlinked lociprovide a conservative approach for assessingspecies boundaries due to the fact that a sub-stantial amount of time is required after the initialdivergence of species until ancestral polymor-phisms have fully sorted (Knowles and Carstens2007 Hudson and Coyne 2002) Consequentlygroups with recent divergence histories willlikely go undiscovered under a genealogicalconcordance criterion due to the fact that speciesboundaries likely are not reflected in genegenealogies For example it would take morethan 1 million years after speciation before spe-cies would be delimited if 15 loci were sampledin species with an effective population size (Ne)of 100000 assuming one generation a yearunder a strict reciprocal monophyly criterion(Knowles and Carstens 2007) The amount oftime required for a species to be recognized using
a criterion of reciprocal monophyly increasesproportionally with increasing population sizes(Hudson and Coyne 2002) In a number ofstudies of lichen-forming fungi a genealogicalconcordance criterion has been used to bothdelimit previously unrecognized species-levellineages and validate some conventional pheno-type-based species (Leavitt et al 2012a c2013b Molina et al 2011 Kroken and Taylor2001)
Although analyses of molecular sequence datarelying on reciprocal monophyly or fixed char-acter differences for species delimitation canserve as important criteria for identifying speciesthese criteria are not commonly met acrossmultiple loci particularly in recent speciationhistories (Fig 22) To accommodate theobserved conflict among genealogies from mul-tiple loci with the underlying speciation historythe recent merge of coalescent theory with phy-logenetics has driven a major paradigm shift inspecies delimitation and molecular systematics ingeneral (Fujita et al 2012 Edwards 2009) Themultispecies coalescent model can be applied togenealogical histories from multiple independentloci to assemble separate coalescent processesoccurring in populations into a species tree(Degnan and Rosenberg 2009 Rannala andYang 2003) Within this coalescent-basedframework multiple individuals can be assignedto a single speciespopulation and the speciationhistory of ancestral and descendant species-levellineages can be inferred as a ldquospecies treerdquo incontrast to estimating gene genealogies fromindividual samples (Degnan and Rosenberg2006 2009 Rannala and Yang 2003)
A number of multispecies coalescent-basedspecies delimitation methods have recently beenintroduced offering an exciting framework forempirically assessing species boundaries byselecting the best species tree model from a set ofalternative models representing differenthypotheses of species boundaries (Table 21)For example SpeDeSTEM (Ence and Carstens2011) finds the maximum likelihood values for aspecies tree assuming all putative species areseparate lineages and for alternative species treeswhere two or more species are collapsed into a
2 The Dynamic Discipline of Species Delimitation hellip 27
single lineage The best fitting model is thenselected using the Akaike information criterionunder the assumption of constant populationsizes and fixed gene topologies across the speciestree The species delimitation program BayesianPhylogenetic and Phylogeography (BPampPYangand Rannala 2010) accommodates gene treeuncertainty and variable population sizes andsamples from the Bayesian posterior distributionof species delimitation models using reversible-jump Markov chain Monte Carlo (rjMCMC)sampling BPampP requires a user-provided guidetree with resolution finer than the species leveland evaluates alternative modes derived from allpossible subtrees that are generated by collapsingor splitting nodes on the guide tree (Yang andRannala 2010 Rannala and Yang 2003) Theproportion of time spent on each model is pro-portional to the posterior probability of themodel ie ldquospeciation probabilitiesrdquo WhileBPampP ranks among the most popular coalescent-based species delimitation programs (Fujita et al2012) misspecifications of the guide tree andorthe prior distribution for population size (θ) canresult in strong support for models containing aninflated number of species (Leacheacute and Fujita2010 Zhang et al 2011) The nonparametricheuristic species delimitation approach imple-mented in the program Brownie (OrsquoMeara 2010)jointly sorts anonymous samples into species andinfers a species tree from input gene trees fromdifferent loci assuming that for a speciationevent the corresponding nodes on gene trees willbe more consistent with each than the diver-gences within species However finding both theoptimum species tree and species boundariesremains computationally challenging andBrownie has been shown to frequently yieldincorrect results (OrsquoMeara 2010)
Most recently available coalescent-basedspecies delimitation methods require individualassignments to a species or population a priori Ina number of scenarios correct assignments ofsamples to species may be difficult includingpresence of cryptic species incongruencebetween conventional species and moleculardata or simply the fact that accurate specimenidentification in complex groups with subtle or
difficult to discern diagnostic characters is anontrivial task (Leavitt et al 2013e) Statisticalmethods for assessing individual assignment andspecies detection prior to coalescent-based spe-cies delimitation and species tree reconstructionand species provide a more objective approachfor understanding species boundaries
Population assignment tests using a variety ofclustering algorithms using genetic data offer auseful approach for grouping individuals intoputative reproductively isolated groups (Table 21Hausdorf and Hennig 2010 Corander et al2004 2008 Falush et al 2003 Pritchard et al2000) particularly for species delimitation prob-lems that exist at the interface of traditionalpopulation genetic and phylogenetic analyses(Carstens et al 2013 Weisrock et al 2010Knowles and Carstens 2007)
The programs STRUCTURE (Falush et al2003 Pritchard et al 2000) and STRUCTURA-MA (Huelsenbeck et al 2011) cluster samplesinto populations by minimizing HardyndashWeinbergdisequilibrium for a given number of populationclusters using unlinked genetic markers In gen-eral unlinked markers are not available for mostgroups of lichen-forming fungi and a numberof studies have used information from multilocussequence data for STRUCTURE analyses(Leavitt et al 2011a b c 2013e Fernaacutendez-Mendoza and Printzen 2013 Fernaacutendez-Mendoza et al 2011) Varying approaches havebeen implemented to convert DNA sequencedata to allelic data for Bayesian clustering (seeOrsquoNeill et al 2013) STRUCTURE is expectedto perform well when there is sufficient inde-pendence across regions such that linkage dis-equilibrium within regions does not dominate thedata (STRUCTURE manual) but can also beeffective using multilocus sequence data andtreating all SNPs as independent loci regardlessof physical linkage within each locus (OrsquoNeillet al 2013 Falush et al 2003) A recent study ofthe lichen-forming genus Letharia showed thatBayesian clustering implemented in the programSTRUCTURE was generally able to recover thesame putative Letharia lineages circumscribedemploying a gene genealogical approach inKroken and Taylorrsquos iconic species delimitation
28 SD Leavitt et al
study (Altermann et al 2014 Kroken and Taylor2001) Altermann et al (2014) show that popu-lation assignments were largely consistent acrossa range of scenarios including extensiveamounts of missing data the exclusion of SNPsfrom variable markers and inferences based onSNPs from as few as three gene regions Incontrast to STRUCTURE and STRUCTURA-MA the program BAPS (Corander et al 2008)can explicitly infer genetic structure using hap-loid sequence data or other linked geneticmarkers in the ldquoclustering with linked locirdquomodel Another advantage of BAPS is that theprogram is much more computationally efficientthan STRUCTURE or STRUCTURAMA Sim-ulation studies indicate that both BAPS andSTRUCTURE perform well at low levels ofpopulation differentiation and when clusters arenot well differentiated (Latch et al 2006)
In practice inferring the most appropriatelevel of population structure using Bayesianclustering algorithms remains challenging (Latchet al 2006 Evanno et al 2005) BAPS andSTRUCTURAMA can infer both individualassignments and the most likely number ofgenetic clusters (Corander et al 2008 Huelsen-beck et al 2011) and the ad hoc ΔK statisticproposed by Evanno et al (2005) provides anobjective approach for identifying the uppermosthierarchical level of structure However carefulconsideration of other levels of populationstructure may ultimately reveal more biologicallymeaning results and researchers should examineindividual assignments across a range of geneticgroupings Some markers generated fromindependent genomic regions such as SNPs andfast-evolving microsatellites can be used to dis-tinguish fine-scale population structuring on thebasis of allele frequencies and species delimita-tion analyses based on these markers may implythe risk of severe taxonomic oversplitting Inthese cases validation approaches such asBPampP and SpeDeSTEM and corroborationamong different species delimitation approachescan provide perspective between intraspecificpopulation structure and species-level clusters
Another limitation of clustering approaches isthat they do not assess or take into account
evolutionarily divergences and relationshipsamong population clusters Coalescent-basedspecies tree methods (discussed below) provide ameans to assess the diversification histories ofpopulations inferred from clustering analysesTherefore a potential working protocol for aninformed species delimitation study that takesinto account population structure could consist offirst applying a genetic clustering analysis under apopulation genetics criterion (eg BAPSSTRUCTURE STRUCTURAMA) to identifygenetically distinct population clusters that can beconsidered ldquocandidate speciesrdquo From these can-didate species a species tree can be inferred forfocal group using coalescent-based species treereconstruction methods (eg BEAST) Subse-quently a coalescent-based validation methodcan be applied to assess whether the distinctpopulation clusters represent independent evolu-tionary lineages (eg BPampP) This multistepapproach would provide a consistent and standardcriterion for distinguishing between population-and species-level lineages (Camargo and Sites2013) and has been applied to a number of casesincluding lichen-forming fungi (Leavitt et al2011a b c 2013e Leacheacute and Fujita 2010)
High-throughput sequencing methods providethe means to effectively sample hundreds tothousands of loci from across a species genome fora large number of species and continue to revolu-tionize studies that can be performed even in non-model organisms Targeted high-throughputsequencing approaches such as anchored phy-logenomics transcriptome sequencing reduced-representation genomic library sequencing(restriction-site-associated DNA sequencingRAD-Seq and genotype-by-sequencing GBS)and high-throughput amplicon sequencing pro-vide important insight into species boundaries fora number groups although none of these approa-ches has yet been applied to studies of lichenizedfungi
For example restriction-site-associated(RAD-Seq) DNA can simultaneously detect andgenotype thousands of genome-wide SNPs byfocusing the sequencing effort on a reducedrepresentation of the entire genome (Baird et al2008) and has been successfully applied to
2 The Dynamic Discipline of Species Delimitation hellip 29
intraspecies (Lewis et al 2007 Miller et al 2007Emerson et al 2010) and interspecies studies(Rubin et al 2012 Eaton and Ree 2013 Wagneret al 2013) This approach has provided strikinginsight into the recent adaption radiation of LakeVictoria cichlids (Wagner et al 2013) Analternative approach targeting the sequencing ofspecific loci using next-generation sequencingplatforms provides an efficient means of gener-ating data for loci of known genomic locationorthology size and expected level of variation(OrsquoNeill et al 2013) Markers can be targetedusing well-established PCR techniques or newerhybridization techniques and subsequentlypooled for high-throughput sequencing usingparallel-tagged sequencing of multiple individualsamples within a single sequencing run (OrsquoNeillet al 2013) A recent study of North Americantiger salamanders (Ambystoma tigrinum)demonstrated the potential for amplicon-basedparallel-tagged sequencing to rapidly generatelarge-scale genomic data for species delimitationand species tree research (OrsquoNeill et al 2013)
Although these methods provide an enormousamount of data and insight into diversification atan unprecedented scale computational limita-tions restrict the applicable analytical methodsfor large datasets although computational andanalytical advances are happening rapidly Mostrecent coalescent-based species delimitation andspecies tree models using gene trees have beenlimited to handle tens of loci for multiple indi-viduals and combining hundreds or thousands ofgene trees into a single species delimitationframework presents considerable computationalchallenges (Camargo and Sites 2013) Therecently developed method Bayes factor delim-itation (with genomic data BFD) combines adynamic programming algorithm for estimatingspecies trees that bypasses the computationallyintensive MCMC integration over gene trees toprovide a rigorous technique for species delimi-tation studies using genome-wide SNP data(Leacheacute et al 2014) Competing species delimi-tation models are compared using Bayes factorsand it appears that this approach is robust tosample sizes (ie few loci and limited samples
per species) and misspecification of the prior forpopulation size (θ) (Leacheacute et al 2014)
Fungi are an ideal model for assessing diver-gence in eukaryotes due to their simple mor-phologies small genomes broad ecological rolesand diverse lifestyles (Gladieux et al 2014)However the use of genomic data from high-throughput sequencing methods have not yet beenincluded in species delimitation studies of lichen-forming fungi This is due in part to challenges indealing with metagenomic data containing geno-mic information from a plethora of symbiontsassociated with a lichen thallus scant genomicresources and discipline-specific inertia A num-ber of lichen-forming fungal genomes are cur-rently available including Endocarponpusillum(Wang et al 2014) Cladonia spp (Park et al2013a 2014) Caloplaca flavorubescens (Parket al 2013b) and Cladonia greyi and Xanthoriaparientina through the Fungal Genomics Programat Joint Genome Institute of the United StatesDepartment of Energy (httpwwwjgidoegov)The foreseeable ongoing expansion of genomicresources for lichen-forming fungi will be centralto developing approaches for delimiting speciesusing high-throughput sequencing Specificallygenomic resources will be crucial in identifyingnovel markers (variable genes regions SNPsmicrosatellites etc) identifying conserved fungalmarkers for targeted high-throughput sequencingapproaches and references for entire genomecomparisons (Devkota et al 2014 Werth et al2013)
23 Can We Make SpeciesDelimitation in Lichen-FormingFungi Truly Integrative
The widespread availability of genetic data hascreated a biased viewpoint that only genetic datashould be used for statistical species delimitationHowever an ongoing appeal to researchers toassess species boundaries from multiple andcomplementary perspectives (phylogeneticspopulation genetics comparative morphologydevelopment ecology etc) has resulted in an
30 SD Leavitt et al
integrative taxonomic framework bringing theseconceptual and methodological perspectivestogether (Hamilton et al 2014 Fujita et al 2012Salicini et al 2011 Yeates et al 2011 Padialet al 2010 Dayrat 2005 Will et al 2005 Wiensand Penkrot 2002) In practice any study linkingdifferent kinds of data to support hypotheses ofspecies boundaries including mapping morpho-logical characters onto a molecular phylogenycan be considered integrative Integrative meth-ods for species delimitation fall across a broadspectrum ranging from verbal and qualitativeassessments of data classes to quantitative meth-ods that allow different data types to contribute tostatistical species delimitation (Yeates et al2011) In most taxonomic studies utilizing bothmolecular and morphological data expert opinionis eventually used to some degree to evaluate thefinal status of a candidate species (eg Bond andStockman 2008) In this sense many studiesusing evidence for independent data sources fordelimiting species boundaries use an iterativeapproach (Yeates et al 2011) where speciesboundaries can be tested within a hypothetico-deductive framework with diverse datasets
From a practical perspective we advocate aprocess of iterative taxonomy (sensu Yeates et al2011) to circumscribe and refine species limitsusing multiple lines of evidence This iterativeprocess involves comparisons of morphologicaldata with a phylogenetic hypothesis (ie single-locus gene tree or concatenated multilocusphylogeny) to identify the least inclusive mono-phyletic clade in the topology characterized by atleast one unambiguously diagnostic morphologi-cal character (Miralles and Vences 2013) Thisphylogenetic tree-informed approach to assessingspecies boundaries represents a common practicein studies of lichen-forming fungi Previouslyunrecognized species-level clades with corre-sponding subtle or overlooked phenotypiccharacters have been commonly observed in bothcrustose lichens [eg Acarospraceae (Wedin et al2009) Graphidaceae (Rivas Plata and Lumbsch2011 Papong et al 2009) Lecanoraceae (Leavittet al 2011a) Lecideaceae (Ruprecht et al 2010)Mycoblastaceae (Spribille et al 2011) andTeloschistaceae (Vondraacutek et al 2009 Muggia
et al 2008)] and foliose and fruticose lichens [egLobariaceae (Moncada et al 2014 McDonaldet al 2003) Parmeliaceae (Leavitt et al 2013aWirtz et al 2012 Divakar et al 2005 Molinaet al 2004 Kroken and Taylor 2001) Peltigera-ceae (OrsquoBrien et al 2009 Goffinet et al 2003)Physciaceae (Elix et al 2009 Divakar et al2007) and Sphaerophoraceae (Houmlgnabba andWedin 2003)] As a specific example Luumlckinget al (2008) used a combination of medullarychemistry and underside pigmentation in speci-mens from the Heterodermia obscurata group inCosta Rica to corroborate monophyletic clades inan ITS gene tree as species-level lineages
While this iterative approach to speciesdelimitation and taxonomy has proven valuablefor understanding species boundaries anddescribing new taxa in some groups of lichenizedfungi a posteriori examination of morphologicaland chemical features has failed to reveal diag-nostic phenotypic characters in a number ofstudies (Muggia et al 2014 Leavitt et al 2011aOtaacutelora et al 2010 OrsquoBrien et al 2009) Fur-thermore a study of widespread species in thegenus Melanelixia (Parmeliaceae) indicated thatphenotypically cryptic lichen-forming fungalspecies-level lineages may be relatively ancientand diagnosable phenotypic differences may beabsent even millions of years after the initialdivergence (Leavitt et al 2012b) The latter studyhighlights the fact that species-level lineages maycommonly exist without any observable diag-nostic phenotypic characters calling into ques-tion the impetus for a universal application ofintegrative taxonomy
In keeping with the principle that as manylines of evidence as available should be com-bined to delimit species (Dayrat 2005) the for-malized integrative taxonomic approach (ITAXMiralles and Vences 2013) uses a mtDNA guidetree and observations from different types of datathat might provide conclusive evidence for theindependence of lineages and thus their identityas different species Miralles and Vences (2013)provide a non-exhaustive list of species delimi-tation criteria to be integrated in the ITAXapproach including (a) sympatric occurrencewithout admixture as revealed by consistent
2 The Dynamic Discipline of Species Delimitation hellip 31
differences in morphological or molecular char-acters at the same geographic location (b) strongdifferences in a behavioral morphological orgenetic character known to mediate prematingisolation (c) unviability or infertility of hybrids(d) lack of gene flow across a geographicalhybrid zone (e) congruent diagnostic differencesbetween sister lineages in various unlinkedmorphological character (f) absence of haplo-type sharing in several unlinked nuclear loci and(g) a combination of criteria endashf Speciesboundaries are based on seeking the least inclu-sive monophyletic group in the mtDNA treewhich fulfills at least one of the criteria listedabove Clearly the ITAX approach is sensitive tosample size and in order to reliably support thedistinctiveness of a given species it has beenrecommended that the sampling strategy includesat least five individuals per species (Miralles andVences 2013) A similar approach could beadopted for lichenized fungi using an ITS genetopology as guide tree rather than mtDNA
A total evidence approach including concat-enation has been a common approach for inte-grating information from different sourcesincluding independent genetic markers in phy-logenetic reconstructions (Kluge 1989 Wiens1998 de Queiroz et al 1995) Proponents forconcatenation of independent data in phyloge-netic analyses argue that when combining alldata the underlying signal of speciation mayemerge even if weakly contradictory signalis contained in the individual data partitions(Gatesy et al 1999) For example establishing apreliminary perspective of species boundariesfrom multilocus sequence data using concatena-tion may provide a reasonable starting point forscreening for cryptic species and species treeinference (Šlapeta et al 2006 Leavitt et al2011a Le Gac et al 2007 Leacheacute 2009)However concatenation and consensus methodsimply a risk of obtaining inflated support forincorrect relationships and information aboutvariance in gene coalescence is lost (Degnan andRosenberg 2006 2009 Kubatko and Degnan2007 Edwards 2009) In spite of the impetus forintegrating evidence from independent data
sources into empirical species delimitation stud-ies and taxonomy (Fujita et al 2012 Padial et al2009) most currently available species delimi-tation methods are unable to accommodate non-genetic data sources in a statistical framework
231 Selecting the Appropriate Data
In the face of increasing availability of geneticdata and associated bioinformatical approachesfor delimiting species researchers should care-fully consider what information is being sacri-ficed by the failure to consider non-genetic datain species delimitation studies and whetheraccuracy could be improved by the addition ofmultiple data types Morphological data havehistorically served as a proxy to identify repro-ductively isolated groups (ie ldquospeciesrdquo) (Ray1686 Fujita et al 2012) Current methods fordelimiting species using non-genetic data (egchemistry morphology and ecology) remainwoefully understudied For example morphol-ogy-based species circumscriptions are generallybased on one or more qualitative (or quantitative)morphological characters that do not appear tooverlap with other species However ascertain-ing that a given trait is truly fixed within apopulation with statistical confidence requiresunrealistic sample sizes even when allowing forsome level of polymorphism in the diagnosticcharacter (Wiens and Servedio 2000) Now var-ious combinations of datamdashfrom morphologygenetics geography and ecologymdashare acceptedas standard information for species delimitationstudies (Ruiz-Sanchez and Sosa 2010 Ross et al2010 Edwards and Knowles 2014)
Below we briefly discuss appropriate datasources for species delimitation studies of lichen-forming fungi However homoplastic characters(similar traits that are not derived from a com-mon ancestor) are common among many traitscommonly used to circumscribe fungal taxa thebiological significance of secondary metabolitevariation remains largely unknown (Lawrey1986) and ecological niches may be difficultto adequately characterize and model due to
32 SD Leavitt et al
microhabitat requirements and data resolutionTherefore we advocate a cautious approach toselecting appropriate and relevant data forassessing species boundaries
In spite of the potential challenges and limi-tations of using phenotypic data these traits haveprovided a plethora of valuable information forunderstanding species boundaries For examplein the Melanelixia fuliginosa group (Parmelia-ceae) a morphometric analysis using color isi-dia and marginal zone free of isidia ascharacters revealed a general pattern of differ-entiation between material formerly recognizedas subspecies fuliginosa and glabratula in spiteof the fact that considerable overlap betweengroups occurs in some characters individually(Arup and Berlin 2011) Furthermore the dis-tinction of the two groups was supported by aphylogenetic analysis of the ITS marker andecological differences with M fuliginosaoccurring predominantly on rock and M glab-ratula on bark (Arup and Berlin 2011) Thisstudy of the M fuliginosa group provides a fit-ting example of using multiple independentsuites of data including ecology morphologyand genetic information to establish a robusthypothesis of species boundaries
In lichen systematics phenotypic dataincluding thallus organization secondary metab-olites mode of reproduction ascoma-type andontogeny ascus and ascospore characters havehistorically played a prominent role (Printzen2009) Ascomatal characters have traditionallyheld a major role in higher-level classification(Printzen 2009) in contrast to species-levelclassification which also tends to include a widearray of vegetative and chemical charactersCommonly assessed morphological charactersinclude thallus form and size cortical features(eg maculae and pseudocyphellae) presenceformcolor of attachment structure (eg rhizines)and reproductive mode (ascomata vs vegetativediaspores) (Printzen 2009) Different morpho-logical types of vegetative diasporesmdashcorticatedisidia and ecorticated sorediamdashand their locationare commonly used to distinguish speciesAscomatal characters including morphologylocation (laminal vs marginal) position (sessile
vs immersed) presence of thalline margins colorof an apothecial disc and presence or color ofpruina are also commonly used in speciesdelimitations of lichen-forming fungi (Printzen2009) Other important characters may includethalline characters form color size and septationof ascospores size and form and structure of ascithe hamathecium type of epihymenium andhypothecium and the type of excipulum orperidium conidiomatal characters etc (Printzen2009)
Assessments of secondary metabolites hasplayed an important role in lichen taxonomybeginning with the introduction of simple spottests by Nylander (1866a b) The use of chem-istry in lichen taxonomy has been discussed indetail in numerous reviews (Lumbsch 1998a bRogers 1989 Brodo 1986 Egan 1986 Leuckert1985 Brodo 1978 Hawksworth 1976 Culber-son 1969 1970) and we refer readers to thesevaluable sources for a more comprehensive per-spective on lichen chemistry In short extrolites(secondary metabolites) belong to various clas-ses the most common and diverse include dep-sides depsidones chlorinated xanthones andanthraquinones (Lumbsch 2002 Culberson1969) The presence or absence of specificextrolites or their replacements by another sub-stance is widely used to distinguish speciesparticularly when correlated with differences ingeographic distributions However if morpho-logical or geographical differences betweenpopulations containing different extrolites are notapparent the taxonomic significance has beendisputed with some authors distinguishing themas species and others preferring to regard them aschemical races within a species In addition tosimply using the presence or absence of extro-lites Culberson and Culberson (1976) proposedto arrange lichen substances into chemosyn-dromes of closely related substances The pres-ence or absence of these chemosyndromes maypotentially be used as characters to delimit spe-cies regarding differences involving the samechemosyndromes as intraspecific variation anddistinct chemosyndromes as evidence for inter-specific populations (Lumbsch 1994 Gowan1986)
2 The Dynamic Discipline of Species Delimitation hellip 33
Due to the fact that species delimitationstudies often incorporate a substantial biogeo-graphical or ecological component ecologicalniche modeling plays an increasing role in phy-logenetic and taxonomic research Niche mod-eling can provide evidence for ecologicalisolation between populations based on eitherconserved or divergent ecological niches andtherefore can provide additional evidence sup-porting lineage independence between putativespecies The application of ecological nichemodeling has been applied to species delimita-tion studies in a number of cases (Ruiz-Sanchezand Sosa 2010 Leacheacute et al 2009 Raxworthyet al 2007 Rissler and Apodaca 2007) althoughit has not been explicitly applied to assess speciesboundaries in lichen-forming fungi By mappingthe spatial distribution of environmental suit-ability of climatic variables for candidate speciesthe application of ecological niche modeling canbe particularly important in cases where specieshave allopatric distributions (Raxworthy et al2007)
Ecological niche modeling utilizes knownassociations between a speciesrsquo occurrencelocalities and environmental variables to defineabiotic conditions within which populations canbe maintained (Guisan and Thuiller 2005) Themethodological approach for modeling is basedon four general properties (i) The current knownspeciesrsquo localities is the dependent variable (ii)the distribution is modeled as a map composed ofgrid cells at a specified resolution (iii) a range ofenvironmental variables (eg temperature pre-cipitation and solar exposure) are collected todescribe the characteristics of each cell and (iv)classifying the degree to which each cell is eithersuitable or unsuitable for each species under arange of models (Guisan and Thuiller 2005)
Ecological data also have the potential to playan important role in understanding speciesboundaries in lichen-forming fungi For exampleNash and Zavada (1977) demonstrated that Xant-hoparmelia populations with distinct chemistriesoccurring in the northern portion of the SonoranDesert exhibit habitat selection among differentrock substrates within a region with relativelyuniform climate and topography In another case
the parmelioid species Parmelia mayi is mor-phologically indistinguishable from P saxatilisbut can be separated by bioclimatic features andgenetic and chemical characters (Molina et al2011) McCune and Printzen(2011) assess distri-butions and climatic niches of species in theLecanora varia group inwesternUSAandprovidea model that uses continental influence and annualtemperature as themajor factors predicting speciesdistributions The distribution of Usnea hirta alichen commonly used in air quality biomonitor-ing research was modeled for a section of theWhite River National Forest in central Coloradobased on the presence of U hirta at 72 biomoni-toring reference sites distributed in the inter-mountain western United States (Shrestha et al2012) The best model for predicting U hirtadistribution included four variablesmdashsolar radia-tion average monthly precipitation and averagemonthly minimum and maximum temperatures(Shrestha et al 2012) These studies supportthe potential use of ecological niche modelingmethods in species delimitation studies of lichen-forming fungi
24 Conclusions What AboutTaxonomy
In most cases species circumscription and tax-onomy requires some degree of qualitativejudgment and individual interpretation Integrat-ing multiple types of data into an empiricalframework for delimiting species boundarieswhere species boundaries can be tested within ahypothetico-deductive framework with diversedatasets can provide robust hypotheses of speciesboundaries and taxonomic stability (Yeates et al2011) However it has long been known toevolutionary biologists that distinct species donot need to have diagnosable morphologicaldifferences (Mayr 1963) and increasing avail-ability of genetic data has allowed researchers toidentify species and to rigorously test speciesboundaries with a level of precision that wasunimaginable a decade ago While analyticaladvances in statistical species delimitation havebeen largely based on genetic data the utility of
34 SD Leavitt et al
these approaches to formal taxonomy remainselusive Due to these challenges an eclecticapproach to delimiting species and cautionagainst the reliance on any single dataset ormethod is required when delimiting species
While results from many recent studies maycontradict traditional species boundaries acrossmany groups of lichen-forming fungi we areoptimistic that this research represents substantialprogress toward a more accurate perspective onspecies boundaries and diversity in fungi As aresult of ongoing research of species boundariesin lichen-forming fungi the taxonomic value ofmany phenotypes is now better understood ourunderstanding of ecological evolutionary andbiogeographic patterns has improved and we canbegin to better understand patterns of symbiontinteractions in lichens Integrating new data(including novel morphological characters andgenetic data) will be essential to accurately rep-resent species-level diversity across all groups oflichenized fungi Hopefully an improved per-spective on lichen diversity also increases ourappreciation of these incredible symbiotic sys-tems While we are strong advocates for theapplication of independent data types in devel-oping an integrative taxonomy there is anincreasing need to formally recognize the exis-tence of phenotypically cryptic species-levellineages in lichen-forming fungi (see Hibbettet al 2011) In some cases a molecular taxon-omy may provide the most practical approach toconsistent treatment of mycobiont species withinlichen groups where diagnostic morphologicalcharacters are unidentifiable or practically notfeasible (Leavitt et al 2013c d)
These are exciting times for taxonomists andphylogeneticists A closer look at lichen taxon-omy with the inclusion of new data will help usto better understand the diversity of these fasci-nating organisms accurately interpret distribu-tion patterns and play a more important role inmeaningful conservation practices Howeversome level of uncertainty will accompany pro-gress In many taxonomic groups our traditionalapproach for species identification will likelyneed to be substantially modified The search forcorroborating morphological support for cryptic
species identified using molecular data willrequire meticulous and creative approaches toassess phenotypic variation in potentially unor-thodox ways We are hopeful that lichenologistswho traditionally have been eager to include newmethods such as chromatography in their rou-tine identifications will be amenable to includemolecular techniques to their routine examina-tion of specimens for identification and classifi-cation Although this may prove difficult toachieve by single individuals especially citizenscientists that traditionally play an important rolein lichen taxonomy the increasing numberof collaborative projects in the discipline (egLumbsch et al 2011 Crespo et al 2010Gueidan et al 2009) make us optimistic thatbroad-scale collaborative approaches will facili-tate the inclusion of molecular data in lichenresearch at all levels
Acknowledgments We are indebted to various col-leagues for valuable thought-provoking discussionnotably Matthew Nelsen (University of Chicago) AnaCrespo (Universidad Complutense de Madrid) PradeepDivakar (Universidad Complutense de Madrid) BeckettSterner (The Field Museum) and Joyce Havstad (TheField Museum) We also thank anonymous reviewers whoprovided valuable comments that improved this chapterSupport by the US National Science Foundation isgratefully acknowledged (ldquoHidden diversity in parmelioidlichensrdquo DEB-0949147)
References
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Altermann S Leavitt SD Goward T Nelsen MPLumbsch HT (2014) How do you solve a problemlike Letharia A new look at cryptic species in lichen-forming fungi using Bayesian clustering and SNPSsfrom multilocus sequence data PLoS ONE 9(5)e97556 doi101371journalpone0097556
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Culberson WL (1970) Chemosystematics and ecology oflichen-forming fungi Annu Rev Ecol Syst 1153ndash170
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de Queiroz K (1999) The general lineage concept ofspecies and the defining properties of the speciescategory In Wilson RA (ed) Species new interdis-ciplinary essays MIT Press Cambridge pp 49ndash89
de Queiroz K (2007) Species concepts and speciesdelimitation Syst Biol 56(6)879ndash886 doi10108010635150701701083
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Degnan JH Rosenberg NA (2009) Gene tree discordancephylogenetic inference and the multispecies coales-cent Trends Ecol Evol 24(6)332ndash340 doihttpdxdoiorg101016jtree200901009
Del-Prado R Cubas P Lumbsch HT Divakar PK BlancoO de Paz GA Molina MC Crespo A (2010) Geneticdistances within and among species in monophyleticlineages of Parmeliaceae (Ascomycota) as a tool fortaxon delimitation Mol Phylogenet Evol 56(1)125ndash133 doi101016jympev201004014
Del-Prado R Divakar PK Crespo A (2011) Using geneticdistances in addition to ITS molecular phylogeny toidentify potential species in the Parmotrema reticul-atum complex a case study The Lichenologist 43(06)569ndash583 doi101017S0024282911000582
Del-Prado R Blanco O Lumbsch HT Divakar PK ElixJA Molina MC Crespo A (2013) Molecular phylog-eny and historical biogeography of the lichen-formingfungal genus Flavoparmelia (Ascomycota Parmelia-ceae) Taxon 62(5)928ndash939 doi101270562522
Dettman J Jacobson D Taylor J (2003a) A multilocusgenealogical approach to phylogenetic species recog-nition in the model eukaryote Neurospora Evolution57(12)2703ndash2720 doi101111j0014-38202003tb01514x
Dettman JR Jacobson DJ Turner E Pringle A TaylorJW (2003b) Reproductive isolation and phylogeneticdivergence in Neurospora comparing methods ofspecies recognition in a model eukaryote Evolution57(12)2721 doi10155403-074
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Divakar PK Molina MC Lumbsch HT Crespo A (2005)Parmelia barrenoae a new lichen species related toParmelia sulcata (Parmeliaceae) based on molecularand morphological data The Lichenologist 37(01)37ndash46 doi101017S0024282904014641
Divakar PK Amo De paz G del Prado R Esslinger TLCrespo A (2007) Upper cortex anatomy corroboratesphylogenetic hypothesis in species of Physconia(Ascomycota Lecanoromycetes) Mycol Res 111(11)1311ndash1320 doi101016jmycres200708009
Divakar PK Figueras G Hladun N Crespo A (2010)Molecular phylogenetic studies reveal an undescribedspecies within the North American concept of Mela-nelixia glabra (Parmeliaceae) Fungal Divers 42(1)47ndash55 doi101007s13225-010-0027-3
Donoghue MJ Gauthier A (2004) Implementing thephylocode Trends Ecol Evol 19(6)281ndash282 doi101016jtree200404004
Eaton DAR Ree RH (2013) Inferring phylogeny andintrogression using RADseq data an example fromflowering plants (Pedicularis Orobanchaceae) SystBiol 62(5)689ndash706 doi101093sysbiosyt032
Edwards SV (2009) Is a new and general theory ofmolecular systematics emerging Evolution 63(1)1ndash19 doi101111j1558-5646200800549x
Edwards DL Knowles LL (2014) Species detection andindividual assignment in species delimitation canintegrative data increase efficacy Proc R Soc B BiolSci 281(1777) doi101098rspb20132765
Egan RS (1986) Correlations and non-correlations ofchemical variation patterns with lichen morphologyand geography The Bryologist 8999ndash110
Elix JA Corush J Lumbsch HT (2009) Triterpenechemosyndromes and subtle morphological characterscharacterise lineages in the Physcia aipolia group inAustralia (Ascomycota) Syst Biodivers 7(04)479ndash487 doi101017S1477200009990223
Emerson KJ Merz CR Catchen JM Hohenlohe PACresko WA Bradshaw WE Holzapfel CM (2010)Resolving postglacial phylogeography using high-throughput sequencing Proc Natl Acad Sci 107(37)16196ndash16200 doi101073pnas1006538107
Ence DD Carstens BC (2011) SpedeSTEM a rapid andaccurate method for species delimitation Mol EcolResour 11(3)473ndash480 doi101111j1755-0998201002947x
EvannoGRegnaut SGoudet J (2005)Detecting thenumberof clusters of individuals using the software STRUC-TURE a simulation study Mol Ecol 14(8)2611ndash2620doi101111j1365-294X200502553x
Falush D Stephens M Pritchard JK (2003) Inference ofpopulation structure using multilocus genotype datalinked loci and correlated allele frequencies Genetics164(4)1567ndash1587 doi101111j1471-8286200701758x
Fan HH Kubatko LS (2011) Estimating species treesusing approximate Bayesian computation Mol Phylo-genet Evol 59(2)354ndash363 doi101016jympev201102019
Fernaacutendez-Mendoza F Printzen C (2013) Pleistoceneexpansion of the bipolar lichen Cetraria aculeata intothe Southern hemisphere Mol Ecol 22(7)1961ndash1983doi101111mec12210
Fernaacutendez-Mendoza F Domaschke S Garciacutea MA JordanP Martin MP Printzen C (2011) Population structure
2 The Dynamic Discipline of Species Delimitation hellip 37
of mycobionts and photobionts of the widespreadlichen Cetraria aculeata Mol Ecol 20(6)1208ndash1232doi101111j1365-294X201004993x
Fraley C Raftery A (2007) Model-based methods ofclassification Using the mclust software in chemo-metrics J Stat Softw 18i06 [Available online at httpwwwdoajorgdoajfunc=abstractampid=218544]
Fujisawa T Barraclough TG (2013) Delimiting speciesusing single-locus data and the generalized mixed yulecoalescent (GMYC) approach a revised method andevaluation on simulated datasets Syst Biol 62(5)707ndash724 doi101093sysbiosyt033
Fujita MK Leacheacute AD Burbrink FT McGuire JA MoritzC (2012) Coalescent-based species delimitation in anintegrative taxonomy Trends Ecol Evol 27(9)480ndash488 doi101016jtree201204012
Gatesy J OrsquoGrady P Baker RH (1999) Corroborationamong data sets in simultaneous analysis hiddensupport for phylogenetic relationships among higherlevel Artiodactyl taxa Cladistics 15(3)271ndash313doi101111j1096-00311999tb00268x
Gladieux P Ropars J Badouin H Branca A Aguileta Gde Vienne DM Rodriacuteguez de la Vega RC Branco SGiraud T (2014) Fungal evolutionary genomicsprovides insight into the mechanisms of adaptivedivergence in eukaryotes Mol Ecol 23(4)753ndash773doi101111mec12631
Goffinet B Miadlikowska J Goward T (2003) Phyloge-netic inferences based on nrDNA sequences supportfive morphospecies within the Peltigera didactylacomplex (Lichenized Ascomycota) The Bryologist106(3)349ndash364 doi10163901
Gowan SP (1986) Evolution of secondary natural productsin the genus Porpidia (Ascomycata Porpidiaceae) AmJ Bot 73606
Griffin PC Hoffmann AA (2014) Limited genetic diver-gence among Australian alpine Poa tussock grassescoupled with regional structuring points to ongoinggene flow and taxonomic challenges Ann Bot doi101093aobmcu017
Grube M Hawksworth DL (2007) Trouble with lichenthe re-evaluation and re-interpretation of thallus formand fruit body types in the molecular era Mycol Res111(9)1116ndash1132 doi101016jmycres200704008
Gueidan C Savi S Thues H Roux C Keller C Tibell LPrieto M Heimarsson S Breuss O Orange A FrobergL Wynns AA Navarro-Rosines P Krzewicka BPykaelae J Grube M Lutzoni F (2009) Genericclassification of the Verrucariaceae (Ascomycota)based on molecular and morphological evidencerecent progress and remaining challenges Taxon 58(1)184ndash208
Guillot G Mortier F Estoup A (2005) Geneland acomputer package for landscape genetics Mol EcolNotes 5(3)712ndash715 doi101111j1471-8286200501031x
Guillot G Renaud S Ledevin R Michaux J Claude J(2012) A unifying model for the analysis of pheno-typic genetic and geographic data Syst Biol 61(6)897ndash911 doi101093sysbiosys038
Guisan A Thuiller W (2005) Predicting species distribu-tion offering more than simple habitat models EcolLett 8(9)993ndash1009 doi101111j1461-0248200500792x
Hale ME (1990) A synopsis of the lichen genusXanthoparmelia (Vainio) Hale (AscomycotinaParmeliaceae) Smithsonian Institution Press Wash-ington DC
Hamilton CA Hendrixson BE Brewer MS Bond JE(2014) An evaluation of sampling effects on multipleDNA barcoding methods leads to an integrativeapproach for delimiting species a case study ofthe North American tarantula genus Aphonopelma(Araneae Mygalomorphae Theraphosidae) MolPhylogenet Evol 7179ndash93 doi101016jympev201311007
Hausdorf B (2011) Progress toward a general speciesconcept Evolution 65(4)923ndash931 doi101111j1558-5646201101231x
Hausdorf B Hennig C (2010) Species delimitation usingdominant and codominant multilocus markers SystBiol 59(5)491ndash503 doi101093sysbiosyq039
Hawksworth DL (1976) Lichen chemotaxonomy InBailey RH (ed) Lichenology progress and problemsAcademic London pp 139ndash184
Hebert PDN Cywinska A Ball SL deWaard JR (2003)Biological identifications through DNA barcodesProc R Soc Lond B Biol Sci 270(1512)313ndash321doi101098rspb20022218
Hebert PDN Stoeckle MY Zemlak TS Francis CM(2004) Identification of birds through DNA barcodesPLoS Biol 2(10)e312 doi101371journalpbio0020312
Heled J Drummond AJ (2010) Bayesian inference ofspecies trees from multilocus data Mol Biol Evol 27(3)570ndash580 doi101093molbevmsp274
Hey J (2006) On the failure of modern species conceptsTrends Ecol Evol 21(8)447ndash450 doi101016jtree200605011
Hibbett DS OhmanA Glotzer D NuhnMKirk P NilssonRH (2011) Progress in molecular and morphologicaltaxon discovery in Fungi and options for formalclassification on environmental sequences Fungal BiolRev 2538ndash47 doi101016jfbr201101001
Houmlgnabba F Wedin M (2003) Molecular phylogeny ofthe Sphaerophorus globosus species complex Cladis-tics 19(3)224ndash232 doi101111j1096-00312003tb00365x
Hudson RR Coyne JA (2002) Mathematical conse-quences of the genealogical species concept Evolu-tion 56(8)1557 doi101111j0014-38202002tb01467x
Huelsenbeck JP Andolfatto P Huelsenbeck ET (2011)Structurama Bayesian inference of population struc-ture Evol Bioinf Online 7(2011)55ndash59 doi104137EBOS6761
Jones G Oxelman B (2014) DISSECT an assignment-free Bayesian discovery method for species delimita-tion under the multispecies coalescent Bioinformaticsdoi101101003178
38 SD Leavitt et al
Kekkonen M Hebert PD (2014) DNA barcode-baseddelineation of putative species efficient start fortaxonomic workflows Mol Ecol Res doi1011111755-099812233
Kelly LJ Hollingsworth PM Coppins BJ Ellis CJHarrold P Tosh J Yahr R (2011) DNA barcoding oflichenized fungi demonstrates high identification suc-cess in a floristic context New Phytol 191(1)288ndash300doi101111j1469-8137201103677x
Kiss L (2012) Limits of nuclear ribosomal DNA internaltranscribed spacer (ITS) sequences as species barcodesfor fungi Proc Natl Acad Sci 109(27)E1811ndashE1811doi101073pnas1207143109
Kluge AG (1989) A concern for evidence and aphylogenetic hypothesis for relationships among Epi-crates (Boidae Serpentes) Syst Zool 38(1)7ndash25doi101093sysbio3817
Knowles LL Carstens BC (2007) Delimiting specieswithout monophyletic gene trees Syst Biol 56(6)887ndash895 doi10108010635150701701091
Kroken S Taylor JW (2001) A gene genealogical approachto recognize phylogenetic species boundaries in thelichenized fungus Letharia Mycologia 93(1)38ndash53
Kubatko LS Degnan JH (2007) Inconsistency of phylo-genetic estimates from concatenated data under coa-lescence Syst Biol 56(1)17ndash24 doi10108010635150601146041
Latch EK Dharmarajan G Glaubitz JC Rhodes OE Jr(2006) Relative performance of Bayesian clusteringsoftware for inferring population substructure andindividual assignments at low levels of populationdifferentiation Conserv Genet 7(2)295ndash302 doi101007s10592-005-9098-1
Lawrey JD (1986) Biological role of lichen substancesThe Bryologist 89(2)111ndash122
Le Gac M Hood ME Fournier E Giraud T (2007)Phylogenetic evidence of host-specific cryptic speciesin the anther smut fungus Evolution 61(1)15ndash26doi101111j1558-5646200700002x
Leacheacute AD (2009) Species tree discordance traces tophylogeographic clade boundaries in North Americanfence lizards (Sceloporus) Syst Biol 58(6)547ndash559doi101093sysbiosyp057
Leacheacute AD Fujita MK (2010) Bayesian species delim-itation in West African forest geckos (Hemidactylusfasciatus) Proc R Soc B Biol Sci 2773071ndash3077doi101098rspb20100662
Leacheacute AD Koo M Spencer C Papenfuss T Fisher RMcGuire J (2009) Quantifying ecological morpho-logical and genetic variation to delimit species in thecoast horned lizard species complex (Phrynosoma)Proc Natl Acad Sci USA 10612418ndash12423 doi101073pnas0906380106
Leacheacute AD Fujita MK Minin VN Bouckaert RR (2014)Species delimitation using genome-wide SNP dataSystematic Biology doi101093sysbiosyu018
Leavitt SD Fankhauser JD Leavitt DH Porter LDJohnson LA St Clair LL (2011a) Complex patterns ofspeciation in cosmopolitan ldquorock posyrdquo lichensmdashdiscovering and delimiting cryptic fungal species in
the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
Leavitt SD Esslinger TL Lumbsch HT (2012c) Neogene-dominated diversification in neotropical montanelichens dating divergence events in the lichen-form-ing fungal genus Oropogon (Parmeliaceae) Am J Bot99(11)1764ndash1777 doi103732ajb1200146
Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Lumbsch HT St Clair LL(2013c) DNA barcode identification of lichen-formingfungal species in the Rhizoplaca melanophthalmaspecies-complex (Lecanorales Lecanoraceae) includ-ing five new species MycoKeys 71ndash22 doi103897mycokeys74508
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Vondraacutek J Thorsten Lum-bsch H Clair LLS (2013d) Local representation ofglobal diversity in a cosmopolitan lichen-formingfungal species complex (Rhizoplaca Ascomycota)J Biogeogr 40(9)1792ndash1806 doi101111jbi12118
Leavitt SD Lumbsch HT Stenroos S St Clair LL(2013e) Pleistocene speciation in North American
2 The Dynamic Discipline of Species Delimitation hellip 39
lichenized fungi and the impact of alternative speciescircumscriptions and rates of molecular evolution ondivergence estimates PLoS ONE 8(12)e85240doi101371journalpone0085240
Leavitt SD Esslinger TL Hansen ES Divakar PK CrespoA Loomis BF Lumbsch HT (2014) DNA barcoding ofbrown Parmeliae (Parmeliaceae) species a molecularapproach for accurate specimen identification empha-sizing species in Greenland Organ Divers Evol 14(1)11ndash20 doi101007s13127-013-0147-1
Leliaert F Verbruggen H Vanormelingen P Steen FLopez-Bautista JM Zuccarello GC De Clerck O(2014) DNA-based species delimitation in algae Eur JPhycol 49(2) doi101080096702622014904524
Leuckert C (1985) Probleme der Flechten-Chemotaxon-omiemdashStoffkombinationen und ihre taxonomischeWertung Ber Deut Bot Ges 98401ndash408
Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
Lohtander K Kaumlllersjouml M Roland M Tehler A (2000)The family physciaceae in fennoscandia phylogenyinferred from its sequences Mycologia 92(4)728ndash735
Longton RE (1997) The role of bryophytes and lichens inpolar ecosystems In Woodin SJ Marquiss M (eds)Ecology of Arctic Environments Blackwell ScienceOxford pp 69ndash96 (Special publication No 13)
Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
Lumbsch HT (1994) Die Lecanora subfusca-Gruppe inAustralasien J Hattori Bot Lab 771ndash175
Lumbsch HT (1998a) Taxonomic use of metabolic data inlichen-forming fungi In Frisvad JC Bridge PDArora DK (eds) Chemical Fungal Taxonomy MarcelDekker New York pp 345ndash387
Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
Mayden RL (1997) A hierarchy of species concepts thedenouement in the saga of the species problem InClaridge MF Dawah HA Wilson MR (eds) Speciesthe units of biodiversity Chapman amp Hall Londonpp 381ndash424
Mayr E (1963) Animal species and evolution HarvardUniversity Press Cambridge
Mayr E (1970) Populations species and evolutionBelknap Press of Harvard University Press Cambridge
McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
McDonald T Miadlikowska J Lutzoni F (2003) Thelichen genus Sticta in the Great Smoky Mountains a
40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
McKay BD Mays HL Wu Y Li H Yao C-T Nishiumi IZou F (2013) An empirical comparison of character-based and coalescent-based approaches to speciesdelimitation in a young avian complex Mol Ecol 22(19)4943ndash4957 doi101111mec12446
Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
Mishler BD Brandon RN (1987) Individuality pluralismand the phylogenetic species concept Biol Philos 2397
Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
Nash TH Zavada M (1977) Population studies amongSonoran Desert species of Parmelia subg Xanthop-armelia (Parmeliaceae) Am J Bot 64(6)664ndash669
Nylander W (1866a) Circa novum in studio Lichenumcriterium chemicum Flora 49198ndash201
Nylander W (1866b) Hypochlorite of lime and hydrate ofpotash Two new criteria for the study of lichens(Tanslated and communicated by the Rev W ALeighton) Bot J Linn Soc 9358ndash365
OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
OrsquoNeill EM Schwartz R Bullock CT Williams JSShaffer HB Aguilar-Miguel X Parra-Olea G Weis-rock DW (2013) Parallel tagged amplicon sequencingreveals major lineages and phylogenetic structure inthe North American tiger salamander (Ambystomatigrinum) species complex Mol Ecol 22(1)111ndash129doi101111mec12049
Orock EA Leavitt SD Fonge BA St Clair LL LumbschHT (2012) DNA-based identification of lichen-form-ing fungi Can publicly available sequence databasesaid in lichen diversity inventories of Mount Cameroon(West Africa) The Lichenologist 44(6)833ndash839doi101017S0024282912000424
Otaacutelora MAG Martiacutenez I Aragoacuten G Molina MC (2010)Phylogeography and divergence date estimates of alichen species complex with a disjunct distributionpattern Am J Bot 97(2)216ndash223 doi103732ajb0900064
Padial J Castroviejo-Fisher S Kohler J Vila C ChaparroJ De la Riva I (2009) Deciphering the products ofevolution at the species level the need for anintegrative taxonomy Zoolog Scr 38(4)431ndash447doi101111j1463-6409200800381x
Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
Papong K Luumlcking R Thammathaworn A BoonpragobK (2009) Four new taxa of Chroodiscus (thelotremoidGraphidaceae) from Southeast Asia The Bryologist112(1)152ndash163 doi1016390007-2745-1121152
Park S-Y Choi J Kim JA Jeong M-H Kim S Lee Y-HHur J-S (2013a) Draft genome sequence of Cladoniamacilenta KoLRI003786 a lichen-forming fungusproducing biruloquinone Genome Announcements 1(5)e00695-13 doi101128genomeA00695-13
Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
Peacuterez-Ortega S Fernaacutendez-Mendoza F Raggio J VivasM Ascaso C Sancho LG Printzen C de los Riacuteos A(2012) Extreme phenotypic variation in Cetrariaaculeata (lichenized Ascomycota) adaptation or inci-dental modification Ann Bot 109(6)1133ndash1148doi101093aobmcs042
Pino-Bodas R Burgaz A Martiacuten M Lumbsch HT (2011)Phenotypical plasticity and homoplasy complicatespecies delimitation in the Cladonia gracilis group(Cladoniaceae Ascomycota) Organ Divers Evol 11(5)343ndash355 doi101007s13127-011-0062-2
Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
Pino-Bodas R Martiacuten M Burgaz A (2012b) Cladoniasubturgida and C iberica (Cladoniaceae) form asingle morphologically and chemically polymorphicspecies Mycol Prog 11(1)269ndash278 doi101007s11557-011-0746-1
Pino-Bodas R Ahti T Stenroos S Martiacuten MP BurgazAR (2013a) Multilocus approach to species recogni-tion in the Cladonia humilis complex (CladoniaceaeAscomycota) Am J Bot 100(4)664ndash678 doi103732ajb1200162
Pino-Bodas R Martiacuten AP Burgaz AR Lumbsch HT(2013b) Species delimitation in Cladonia (Ascomy-cota) a challenge to the DNA barcoding philosophyMol Ecol Resour 13(6)1058ndash1068 doi1011111755-099812086
Pons J Barraclough TG Gomez-Zurita J Cardoso ADuran DP Hazell S Kamoun S Sumlin WD VoglerAP (2006) Sequence-based species delimitation for theDNA taxonomy of undescribed insects Syst Biol 55(4)595ndash609 doi10108010635150600852011
Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
Pringle A Baker DM Platt JL Wares JP Latgeacute JPTaylor JW (2005) Cryptic speciation in the cosmo-politan and clonal human pathogenic fungus Asper-gillus fumigatus Evolution 59(9)1886ndash1899 doi10155404-2411
Printzen C (2009) Lichen systematics the role ofmorphological and molecular data to reconstructphylogenetic relationships Progress in botany vol71 Springer Berlin pp 233ndash275
Pritchard JK Stephens M Donnelly P (2000) Inference ofpopulation structure using multilocus genotype dataGenetics 155(2)945ndash959
Puillandre N Lambert A Brouillet S Achaz G (2012)ABGD automatic barcode gap discovery for primaryspecies delimitation Mol Ecol 21(8)1864ndash1877doi101111j1365-294X201105239x
Rannala B Yang Z (2003) Bayes estimation of speciesdivergence times and ancestral population sizes usingDNA sequences from multiple loci Genetics1641645ndash1656
Raxworthy CJ Ingram CM Rabibisoa N Pearson RG(2007) Applications of ecological niche modelingfor species delimitation a review and empiricalevaluation using day geckos (Phelsuma) fromMadagascar Syst Biol 56(6)907ndash923 doi10108010635150701775111
Ray J (1686) Historia planarum vol 1 Clark LondonReese Naeligsborg R Ekman S Tibell L (2007) Molecular
phylogeny of the genus Lecania (Ramalinaceaelichenized Ascomycota) Mycol Res 111(5)581ndash591doi101016jmycres200703001
Reid N Carstens B (2012) Phylogenetic estimation errorcan decrease the accuracy of species delimitation aBayesian implementation of the general mixed Yule-coalescent model BMC Evol Biol 12(1)196 doi1011861471-2148-12-196
Rissler L Apodaca J (2007) Adding more ecology intospecies delimitation ecological niche models andphylogeography help define cryptic species in theBlack Salamander (Aneides flavipunctatus) Syst Biol56924ndash942 doi10108010635150701703063
Rivas Plata E Luumlcking R (2013) High diversity ofGraphidaceae (lichenized Ascomycota Ostropales) inAmazonian Peruacute Fungal Divers 58(1)13ndash32 doi101007s13225-012-0172-y
Rivas Plata E Lumbsch HT (2011) Parallel evolution andphenotypic divergence in lichenized fungi a casestudy in the lichen-forming fungal family Graphida-ceae (Ascomycota Lecanoromycetes Ostropales)Mol Phylogenet Evol 61(1)45ndash63 doi101016jympev201104025
Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
Ross KG Gotzek D Ascunce MS Shoemaker DD (2010)Species delimitation a case study in a problematic anttaxon Syst Biol 59(2)162ndash184 doi101093sysbiosyp089
Rubin BER Ree RH Moreau CS (2012) Inferringphylogenies from RAD sequence data PLoS ONE 7(4)e33394 doi101371journalpone0033394
Rubinoff D (2006) Utility of mitochondrial DNA barcodesin species conservation Conserv Biol 20(4)1026ndash1033 doi101111j1523-1739200600372x
Ruiz-Sanchez E Sosa V (2010) Delimiting speciesboundaries within the Neotropical bamboo Otatea(Poaceae Bambusoideae) using molecular morpho-logical and ecological data Mol Phylogenet Evol 54(2)344ndash356 doi101016jympev200910035
Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
morphological characters Antarctic Sci 22 (SpecialIssue 06)727ndash741 doi101017S0954102010000477
Salicini I Ibaacutentildeez C Juste J (2011) Multilocus phylogenyand species delimitation within the Nattererrsquos batspecies complex in the Western Palearctic MolPhylogenet Evol 61(3)888ndash898 doi101016jympev201108010
Satler JD Carstens BC Hedin M (2013) Multilocusspecies delimitation in a complex of morphologicallyconserved trapdoor spiders (Mygalomorphae Antro-diaetidae Aliatypus) Syst Biol 62(6)805ndash823 doi101093sysbiosyt041
Schlick-Steiner BC Steiner FM Seifert B Stauffer CChristian E Crozier RH (2010) Integrative taxonomya multisource approach to exploring biodiversityAnnu Rev Entomol 55(1)421ndash438 doi101146annurev-ento-112408-085432
Schoch CL Seifert KA Huhndorf S Robert V SpougeJL Levesque CA Chen W Fungal Barcoding Con-sortium (2012) Nuclear ribosomal internal transcribedspacer (ITS) region as a universal DNA barcodemarker for Fungi Proc Natl Acad Sci 109(16)6241ndash6246 doi101073pnas1117018109
Seacuterusiaux E Villarreal AJC Wheeler T Goffinet B(2011) Recent origin active speciation and dispersalfor the lichen genus Nephroma (Peltigerales) inMacaronesia J Biogeogr 38(6)1138ndash1151 doi101111j1365-2699201002469x
Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
Simpson GG (1951) The species concept Evolution5285ndash298
Sites JW Marshall JC (2003) Delimiting species a renais-sance issue in systematic biology Trends Ecol Evol18462ndash470 doi101016S0169-5347(03)00184-8
Sites JW Marshall JC (2004) Operational criteria fordelimiting species Annu Rev Ecol Evol Syst 35(1)199ndash227 doi101146annurevecolsys35112202130128
Šlapeta J Loacutepez-Garciacutea P Moreira D (2006) Globaldispersal and ancient cryptic species in the smallestmarine eukaryotes Mol Biol Evol 23(1)23ndash29doi101093molbevmsj001
Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
Talavera G Dincă V Vila R (2013) Factors affectingspecies delimitations with the GMYC model insightsfrom a butterfly survey Methods Ecol Evol 4(12)1101ndash1110 doi1011112041-210X12107
Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
Tewksbury J Anderson JGT Bakker JD Billo TJDunwiddie PW Groom MJ Hampton SE HermanSG Levey DJ Machnicki NJ del Rio CM PowerME Rowell K Salomon AK Stacey L TrombulakSC Wheeler TA (2014) Natural historyrsquos place inscience and society BioScience doi101093bioscibiu032
Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
Werth S Cornejo C Scheidegger C (2013) Characteriza-tion of microsatellite loci in the lichen fungus Lobariapulmonaria (Lobariaceae) Appl Plant Sci 1(2)1200290 doi103732apps1200290
Westberg M Arup U Kaumlrnefelt I (2007) Phylogeneticstudies in the Candelariaceae (lichenized Ascomy-cota) based on nuclear ITS DNA sequence dataMycol Res 111(11)1277ndash1284 doi101017S0953756204002102
Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
2 The Dynamic Discipline of Species Delimitation hellip 43
(Lepidoptera Lycaenidae) Front Zoo 4(1)8 doi1011861742-9994-4-8
Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
Table
21
(con
tinued)
Metho
dDescriptio
nInpu
tdata
BFD
mdashmultispecies
coalescent
model
for
speciesdelim
itatio
nThe
recently
developedmethod
Bayes
factor
delim
itatio
n(with
genomicdataB
FD)
combinesady
namic
programmingalgorithm
forestim
atingspeciestreesthat
bypasses
thecompu
tatio
nally
intensiveMCMCintegrationov
ergene
treesto
prov
idearigorous
techniqueforspeciesdelim
itatio
nstudiesusinggenome-wideSN
PdataCom
petin
gspeciesdelim
itatio
nmodelsarecomparedusingBayes
factorsanditappearsthat
this
approach
isrobustto
samplesizes(iefew
loci
andlim
itedsamples
perspecies)
and
misspecificatio
nof
thepriorforpo
pulatio
nsize
(θ)
Limita
tions
Recently
describedmethodlackingathorough
theoretical
andem
pirical
evaluatio
nSo
urce
httpwwwbeast2orgwikiindexphpBFD
describedin
Leacheacuteet
al(201
4)
Genom
e-wideSN
Pdata
2 The Dynamic Discipline of Species Delimitation hellip 23
distances among organisms belonging to thesame species are smaller than distances amongorganisms from different species (Puillandreet al 2012 Hebert et al 2003) Genetic distanceapproaches hold particular promise as an identi-fication tool shortcutting the difficulties of mor-phology-based identification (Hebert et al 2004)although in practice a barcode gap may not existfor many groups (Wiemers and Fiedler 2007)Furthermore the role of distance-based approa-ches using a single genetic marker for speciesdiscovery remains more controversial (Rubinoff2006) and without other corroborating evidenceOTUs inferred from single-locus dataset shouldonly be considered ldquocandidaterdquo species Tree-based methods aim to detect monophyletic cladescorresponding to species-level diversity bydetecting discontinuities associated within inter-and intraspecific branching patterns (Fujisawaand Barraclough 2013 Zhang et al 2013Monaghan et al 2009 Pons et al 2006) Tree-based species delimitation methods can also beused on the basis of other properties related tophylogenetic tree topologies (monophyly con-cordance with geography etc (Sites and Marshall2003 2004) Both distance- and tree-basedmethods have been applied for assessing speciesboundaries in lichen-forming fungi (Leavitt et al2012c 2014 Parnmen et al 2012 Del-Pradoet al 2010 2011 Molina et al 2011)
A number of tree-based methods partiallyautomate the species delimitation process withspecific bioinformatical analyses (Table 21)including the general mixed Yule coalescent(GMYC) approach (Fujisawa and Barraclough2013 Monaghan et al 2009 Pons et al 2006)and the Poisson tree processes (PTP) model(Zhang et al 2013) These methods provide rel-atively straightforward and objective pipelinesfor delimiting putative species-level lineagesfrom inferred gene trees by fitting within- andbetween-species branching models to an inferredsingle-locus topology A number of other tree-based methods for species delimitation areeffectively summarized in Sites and Marshall(2004)
When only single-locus data are available theGMYC has been shown to be a relatively robust
tool for species delimitation The GMYCapproach combines a coalescent model of intra-specific branching with a Yule model for inter-specific branching which is then fit to an inferredsingle-gene topology to estimate species bound-aries and a statistical measure of confidence for theinferred boundaries As an input the GMYCapproach requires an ultrametric gene tree andrecent refinements can account for uncertainty inphylogenetic relationships and parameters of theGMYC model (Fujisawa and Barraclough 2013Reid and Carstens 2012) Regardless of theseimprovements it may be difficult to accuratelyinfer an adequate ultrametric tree for large data-sets Although it appears that the GMYC isgenerally stable across a wide range of circum-stances including various methods of phyloge-netic reconstruction the presence of a highnumber singletons high numbers of sampledspecies and gaps in intraspecific sampling theaccuracy of the GMYC is most significantlyaffected by the mean population size relative todivergence times between them (Fujisawa andBarraclough 2013 Talavera et al 2013) Fur-thermore research suggests that the so-calledsingle-threshold version of the GMYC methodlikely outperforms the multiple-thresholdapproach (Fujisawa and Barraclough 2013Monaghan et al 2009) However other studiessuggest that theGMYCmethodmay often providehigher estimates for the total number ofOTUs thanother molecular species delimitation methods(Hamilton et al 2014 Miralles and Vences 2013Talavera et al 2013) warranting a cautiousinterpretation of results from GMYC analyses
Compared to the GMYC approach therecently introduced PTP method for speciesdelimitation has been suggested to be moreaccurate for preliminary species delimitation(Zhang et al 2013) Relative to the GMYC PTPoffers a more straightforward implementationrequiring a simple phylogenetic tree rather thanan ultrametric chronogram However moreresearch is required to assess the general perfor-mance of PTP across wide range of empirical andsimulated species delimitation studies At thispoint data support the use of the GMYC andPTP methods as objective and reasonable starting
24 SD Leavitt et al
points for species delimitation using single-genetopologies
The ldquoSpecies Delimitationrdquo plug-in to theGeneious software provides statistical approachesfor exploring species boundaries in single-genetopologies (Masters et al 2011) Using a priorispecimen assignments to putative species theldquoSpecies Delimitationrdquo plug-in computes statis-tics relating to the probability of the observedmonophyly or exclusivity having occurred bychance in a coalescent process and assesses thewithin- and between-species genetic distances inorder to infer the probability with which membersof a putative species might be identified suc-cessfully with tree-based methods An importantcontribution of the ldquoSpecies Delimitationrdquo plug-in is that it provides an objective means for usersto assess putative species within an empiricalstatistic-based framework rather than qualitativeevaluations of what level of hierarchy constitutesa species in a phylogeny
In contrast to simple sequence similaritythresholds (OTU-picking) for delimiting putativeevolutionarily significant units the AutomaticBarcode Gap Discovery (ABGD) method is anautomated procedure that sorts sequences intohypothetical species based on the existence of abarcode gap observed whenever intraspecificgenetic distances are less than those amongorganisms from different species (Puillandreet al 2012) Ultimately ABGD is a fast simplemethod that can be used to group individualsrepresented in a single-locus sequence alignmentinto candidate species that should be comple-mented with other lines of evidence in an inte-grative taxonomic approach (Kekkonen andHebert 2014 Puillandre et al 2012)
In general the ABGD GMYC and PTP ana-lytical protocols using single-locus data arerepeatable and computationally relatively fastproviding a valuable starting point for a pre-liminary perspective into species boundaries inunderstudied groups that can be validated withsubsequent studies (Kekkonen and Hebert 2014)Similarly analyses of single-locus data can beused to corroborate traditional phenotype-basedspecies boundaries and identify candidate speciesthat have previously been hidden within nominal
species However single-locus species delimita-tionmethodsmaybeparticularly prone to failure inrecognizing significant proportions of species-level biodiversity due to the strict criterion forreciprocal monophyly or alternatively providespurious inflations of estimated species diversitybased on genetic differences that donot correspondto species-level lineages For example recentestimates suggest that the incidence of species-level gene tree paraphyly is approximately 20 (Ross 2014) suggesting that analyses of single-locus datasets would likely fail to accurately deli-mit species in one in every five cases In contrastrecent empirical studies suggest that in some casesthe GMYC provides a striking overestimate ofspecies diversity (Miralles and Vences 2013)
The internal transcribed spacer region (ITS)has played a central role in molecular phyloge-netic studies of lichenized fungi and has recentlybeen adopted as the official barcoding marker forfungi (Schoch et al 2012) In many cases the ITSis sufficiently variable to resolve species bound-aries for lichenized fungi (Schoch et al 2012Kelly et al 2011) although accurate specimenidentification using sequence-based identificationapproaches requires a thoroughly curated refer-ence database (Leavitt et al 2014 Orock et al2012 Kelly et al 2011) In spite of the generalutility of the ITS marker a number of issues maypotentially limit its effective use in speciesdelimitation studies including the potential forintragenomic variation of the nuclear ribosomaltandem repeat and difficulties in aligning ITSsequences from divergent taxa (Kiss 2012)Because the ITS has been formally adopted as theofficial barcoding marker for fungi we recom-mend that species delimitation studies attempt toinclude this region for consistency across studiesHowever we recognize that in some taxonomicgroups the inclusion of the ITS may be prob-lematic and therefore suggest a careful screeningof other markers to identify appropriate loci forresolving species-level relationships
Ultimately the success of any single-locusspecies delimitation method largely depends onthe evolutionary history of the species groupunder study and the variability of the selectedmarker (Fig 22) Species delimitation will be
2 The Dynamic Discipline of Species Delimitation hellip 25
more difficult in more recently diverged lineagesand in cases with some level of interspecific geneflow relative to older well-diverged lineages(eg Leavitt et al 2012c 2013e) In the end wereemphasize that single-locus data alone shouldonly be used to provide a preliminary perspectiveof species boundaries and not as the sole evi-dence in species circumscriptions
222 Sampling Across the GenomeMultilocus Sequence Dataand Genome-Wide Markers
Although single-locus methods can provide anefficient approach for preliminary large-scaleassessments of species diversity there are
significant limitations particularly in recentlydiverged species groups where retention ofancestral polymorphisms and incomplete lineagesorting will likely result in different neutral locihaving unique gene topologies that do not mirrorthe speciation process (Knowles and Carstens2007 Heled and Drummond 2010 Taylor et al2000) In contrast to single-locus and strictlyphenotype-based approaches for species delimi-tation analyses of genetic data collected fromindependent genomic regions can provide robusthypotheses of species boundaries with increasingconfidence (Satler et al 2013 Zhang et al 2011Yang and Rannala 2010) Sequences from mul-tiple independent loci provide an importantsource of data for species delimitation studiesincluding recently developed models that
Fig 22 A simplified diagram illustrating the process ofspeciation through time in a single gene history andresulting gene topologies sampled at two points in thespeciation history (modified from Leliaert et al 2014)aEach dot represents a distinct gene copy and each row onenon-overlapping generation with lines connecting genecopies to their ancestors in the previous generation (onerow below) dashed diagonal lines represent reproductivebarriers two hypothetical sampling intervals at differentpoints in the speciation history are shown Tx and Ty
species delimitation methods using single-locus data areeffective only when species are reciprocally monophyleticin the sampled gene tree genetic clustering and coalescent-based species delimitation methods can circumscribespecies when species may not be monophyletic in sampledgenetic loci b Gene topology representing sampledhaplotypes at time Tx (shown in panel a) hypotheticalspecies are reciprocally monophyletic c Gene topologyrepresenting sampled haplotypes at time Ty (shown in panela) hypothetical species are para- and polyphyletic
26 SD Leavitt et al
combine individual gene genealogies and speciesphylogenies via modeling the coalescent historyof markers (Yang and Rannala 2010 Edwards2009 Knowles and Carstens 2007) As aresponse to advances in sequencing technologiesbioinformatical approaches for multimarker spe-cies delimitation analyses continue to be devel-oped (Table 21 Camargo and Sites 2013)
Long-term reproductive isolation of candidatespecies can be assessed with multilocus sequencedata by evaluating genealogical concordance ofunlinked markers (Baum and Shaw 1995 Aviseand Ball 1990) Within species the mixingeffects of recombination cause unlinked loci tohave distinct genealogical histories but geneticdrift and long-term divergence leads to concor-dant genealogical histories at loci across thegenome Relationships of individuals belongingto distinct candidate species can be evaluatedusing gene genealogies (eg haplotype networksor single-gene topologies) to identify lineagesthat exhibit genealogical exclusivity acrossunlinked neutral loci (Hudson and Coyne 2002Dettman et al 2003b Avise and Ball 1990) Thepresence of the same clades in the majority ofsingle-locus genealogies is taken as evidence thatthe clades represent reproductively isolated lin-eages (Dettman et al 2003a Pringle et al 2005)In practice the criteria of reciprocal monophylyand genealogical concordance of unlinked lociprovide a conservative approach for assessingspecies boundaries due to the fact that a sub-stantial amount of time is required after the initialdivergence of species until ancestral polymor-phisms have fully sorted (Knowles and Carstens2007 Hudson and Coyne 2002) Consequentlygroups with recent divergence histories willlikely go undiscovered under a genealogicalconcordance criterion due to the fact that speciesboundaries likely are not reflected in genegenealogies For example it would take morethan 1 million years after speciation before spe-cies would be delimited if 15 loci were sampledin species with an effective population size (Ne)of 100000 assuming one generation a yearunder a strict reciprocal monophyly criterion(Knowles and Carstens 2007) The amount oftime required for a species to be recognized using
a criterion of reciprocal monophyly increasesproportionally with increasing population sizes(Hudson and Coyne 2002) In a number ofstudies of lichen-forming fungi a genealogicalconcordance criterion has been used to bothdelimit previously unrecognized species-levellineages and validate some conventional pheno-type-based species (Leavitt et al 2012a c2013b Molina et al 2011 Kroken and Taylor2001)
Although analyses of molecular sequence datarelying on reciprocal monophyly or fixed char-acter differences for species delimitation canserve as important criteria for identifying speciesthese criteria are not commonly met acrossmultiple loci particularly in recent speciationhistories (Fig 22) To accommodate theobserved conflict among genealogies from mul-tiple loci with the underlying speciation historythe recent merge of coalescent theory with phy-logenetics has driven a major paradigm shift inspecies delimitation and molecular systematics ingeneral (Fujita et al 2012 Edwards 2009) Themultispecies coalescent model can be applied togenealogical histories from multiple independentloci to assemble separate coalescent processesoccurring in populations into a species tree(Degnan and Rosenberg 2009 Rannala andYang 2003) Within this coalescent-basedframework multiple individuals can be assignedto a single speciespopulation and the speciationhistory of ancestral and descendant species-levellineages can be inferred as a ldquospecies treerdquo incontrast to estimating gene genealogies fromindividual samples (Degnan and Rosenberg2006 2009 Rannala and Yang 2003)
A number of multispecies coalescent-basedspecies delimitation methods have recently beenintroduced offering an exciting framework forempirically assessing species boundaries byselecting the best species tree model from a set ofalternative models representing differenthypotheses of species boundaries (Table 21)For example SpeDeSTEM (Ence and Carstens2011) finds the maximum likelihood values for aspecies tree assuming all putative species areseparate lineages and for alternative species treeswhere two or more species are collapsed into a
2 The Dynamic Discipline of Species Delimitation hellip 27
single lineage The best fitting model is thenselected using the Akaike information criterionunder the assumption of constant populationsizes and fixed gene topologies across the speciestree The species delimitation program BayesianPhylogenetic and Phylogeography (BPampPYangand Rannala 2010) accommodates gene treeuncertainty and variable population sizes andsamples from the Bayesian posterior distributionof species delimitation models using reversible-jump Markov chain Monte Carlo (rjMCMC)sampling BPampP requires a user-provided guidetree with resolution finer than the species leveland evaluates alternative modes derived from allpossible subtrees that are generated by collapsingor splitting nodes on the guide tree (Yang andRannala 2010 Rannala and Yang 2003) Theproportion of time spent on each model is pro-portional to the posterior probability of themodel ie ldquospeciation probabilitiesrdquo WhileBPampP ranks among the most popular coalescent-based species delimitation programs (Fujita et al2012) misspecifications of the guide tree andorthe prior distribution for population size (θ) canresult in strong support for models containing aninflated number of species (Leacheacute and Fujita2010 Zhang et al 2011) The nonparametricheuristic species delimitation approach imple-mented in the program Brownie (OrsquoMeara 2010)jointly sorts anonymous samples into species andinfers a species tree from input gene trees fromdifferent loci assuming that for a speciationevent the corresponding nodes on gene trees willbe more consistent with each than the diver-gences within species However finding both theoptimum species tree and species boundariesremains computationally challenging andBrownie has been shown to frequently yieldincorrect results (OrsquoMeara 2010)
Most recently available coalescent-basedspecies delimitation methods require individualassignments to a species or population a priori Ina number of scenarios correct assignments ofsamples to species may be difficult includingpresence of cryptic species incongruencebetween conventional species and moleculardata or simply the fact that accurate specimenidentification in complex groups with subtle or
difficult to discern diagnostic characters is anontrivial task (Leavitt et al 2013e) Statisticalmethods for assessing individual assignment andspecies detection prior to coalescent-based spe-cies delimitation and species tree reconstructionand species provide a more objective approachfor understanding species boundaries
Population assignment tests using a variety ofclustering algorithms using genetic data offer auseful approach for grouping individuals intoputative reproductively isolated groups (Table 21Hausdorf and Hennig 2010 Corander et al2004 2008 Falush et al 2003 Pritchard et al2000) particularly for species delimitation prob-lems that exist at the interface of traditionalpopulation genetic and phylogenetic analyses(Carstens et al 2013 Weisrock et al 2010Knowles and Carstens 2007)
The programs STRUCTURE (Falush et al2003 Pritchard et al 2000) and STRUCTURA-MA (Huelsenbeck et al 2011) cluster samplesinto populations by minimizing HardyndashWeinbergdisequilibrium for a given number of populationclusters using unlinked genetic markers In gen-eral unlinked markers are not available for mostgroups of lichen-forming fungi and a numberof studies have used information from multilocussequence data for STRUCTURE analyses(Leavitt et al 2011a b c 2013e Fernaacutendez-Mendoza and Printzen 2013 Fernaacutendez-Mendoza et al 2011) Varying approaches havebeen implemented to convert DNA sequencedata to allelic data for Bayesian clustering (seeOrsquoNeill et al 2013) STRUCTURE is expectedto perform well when there is sufficient inde-pendence across regions such that linkage dis-equilibrium within regions does not dominate thedata (STRUCTURE manual) but can also beeffective using multilocus sequence data andtreating all SNPs as independent loci regardlessof physical linkage within each locus (OrsquoNeillet al 2013 Falush et al 2003) A recent study ofthe lichen-forming genus Letharia showed thatBayesian clustering implemented in the programSTRUCTURE was generally able to recover thesame putative Letharia lineages circumscribedemploying a gene genealogical approach inKroken and Taylorrsquos iconic species delimitation
28 SD Leavitt et al
study (Altermann et al 2014 Kroken and Taylor2001) Altermann et al (2014) show that popu-lation assignments were largely consistent acrossa range of scenarios including extensiveamounts of missing data the exclusion of SNPsfrom variable markers and inferences based onSNPs from as few as three gene regions Incontrast to STRUCTURE and STRUCTURA-MA the program BAPS (Corander et al 2008)can explicitly infer genetic structure using hap-loid sequence data or other linked geneticmarkers in the ldquoclustering with linked locirdquomodel Another advantage of BAPS is that theprogram is much more computationally efficientthan STRUCTURE or STRUCTURAMA Sim-ulation studies indicate that both BAPS andSTRUCTURE perform well at low levels ofpopulation differentiation and when clusters arenot well differentiated (Latch et al 2006)
In practice inferring the most appropriatelevel of population structure using Bayesianclustering algorithms remains challenging (Latchet al 2006 Evanno et al 2005) BAPS andSTRUCTURAMA can infer both individualassignments and the most likely number ofgenetic clusters (Corander et al 2008 Huelsen-beck et al 2011) and the ad hoc ΔK statisticproposed by Evanno et al (2005) provides anobjective approach for identifying the uppermosthierarchical level of structure However carefulconsideration of other levels of populationstructure may ultimately reveal more biologicallymeaning results and researchers should examineindividual assignments across a range of geneticgroupings Some markers generated fromindependent genomic regions such as SNPs andfast-evolving microsatellites can be used to dis-tinguish fine-scale population structuring on thebasis of allele frequencies and species delimita-tion analyses based on these markers may implythe risk of severe taxonomic oversplitting Inthese cases validation approaches such asBPampP and SpeDeSTEM and corroborationamong different species delimitation approachescan provide perspective between intraspecificpopulation structure and species-level clusters
Another limitation of clustering approaches isthat they do not assess or take into account
evolutionarily divergences and relationshipsamong population clusters Coalescent-basedspecies tree methods (discussed below) provide ameans to assess the diversification histories ofpopulations inferred from clustering analysesTherefore a potential working protocol for aninformed species delimitation study that takesinto account population structure could consist offirst applying a genetic clustering analysis under apopulation genetics criterion (eg BAPSSTRUCTURE STRUCTURAMA) to identifygenetically distinct population clusters that can beconsidered ldquocandidate speciesrdquo From these can-didate species a species tree can be inferred forfocal group using coalescent-based species treereconstruction methods (eg BEAST) Subse-quently a coalescent-based validation methodcan be applied to assess whether the distinctpopulation clusters represent independent evolu-tionary lineages (eg BPampP) This multistepapproach would provide a consistent and standardcriterion for distinguishing between population-and species-level lineages (Camargo and Sites2013) and has been applied to a number of casesincluding lichen-forming fungi (Leavitt et al2011a b c 2013e Leacheacute and Fujita 2010)
High-throughput sequencing methods providethe means to effectively sample hundreds tothousands of loci from across a species genome fora large number of species and continue to revolu-tionize studies that can be performed even in non-model organisms Targeted high-throughputsequencing approaches such as anchored phy-logenomics transcriptome sequencing reduced-representation genomic library sequencing(restriction-site-associated DNA sequencingRAD-Seq and genotype-by-sequencing GBS)and high-throughput amplicon sequencing pro-vide important insight into species boundaries fora number groups although none of these approa-ches has yet been applied to studies of lichenizedfungi
For example restriction-site-associated(RAD-Seq) DNA can simultaneously detect andgenotype thousands of genome-wide SNPs byfocusing the sequencing effort on a reducedrepresentation of the entire genome (Baird et al2008) and has been successfully applied to
2 The Dynamic Discipline of Species Delimitation hellip 29
intraspecies (Lewis et al 2007 Miller et al 2007Emerson et al 2010) and interspecies studies(Rubin et al 2012 Eaton and Ree 2013 Wagneret al 2013) This approach has provided strikinginsight into the recent adaption radiation of LakeVictoria cichlids (Wagner et al 2013) Analternative approach targeting the sequencing ofspecific loci using next-generation sequencingplatforms provides an efficient means of gener-ating data for loci of known genomic locationorthology size and expected level of variation(OrsquoNeill et al 2013) Markers can be targetedusing well-established PCR techniques or newerhybridization techniques and subsequentlypooled for high-throughput sequencing usingparallel-tagged sequencing of multiple individualsamples within a single sequencing run (OrsquoNeillet al 2013) A recent study of North Americantiger salamanders (Ambystoma tigrinum)demonstrated the potential for amplicon-basedparallel-tagged sequencing to rapidly generatelarge-scale genomic data for species delimitationand species tree research (OrsquoNeill et al 2013)
Although these methods provide an enormousamount of data and insight into diversification atan unprecedented scale computational limita-tions restrict the applicable analytical methodsfor large datasets although computational andanalytical advances are happening rapidly Mostrecent coalescent-based species delimitation andspecies tree models using gene trees have beenlimited to handle tens of loci for multiple indi-viduals and combining hundreds or thousands ofgene trees into a single species delimitationframework presents considerable computationalchallenges (Camargo and Sites 2013) Therecently developed method Bayes factor delim-itation (with genomic data BFD) combines adynamic programming algorithm for estimatingspecies trees that bypasses the computationallyintensive MCMC integration over gene trees toprovide a rigorous technique for species delimi-tation studies using genome-wide SNP data(Leacheacute et al 2014) Competing species delimi-tation models are compared using Bayes factorsand it appears that this approach is robust tosample sizes (ie few loci and limited samples
per species) and misspecification of the prior forpopulation size (θ) (Leacheacute et al 2014)
Fungi are an ideal model for assessing diver-gence in eukaryotes due to their simple mor-phologies small genomes broad ecological rolesand diverse lifestyles (Gladieux et al 2014)However the use of genomic data from high-throughput sequencing methods have not yet beenincluded in species delimitation studies of lichen-forming fungi This is due in part to challenges indealing with metagenomic data containing geno-mic information from a plethora of symbiontsassociated with a lichen thallus scant genomicresources and discipline-specific inertia A num-ber of lichen-forming fungal genomes are cur-rently available including Endocarponpusillum(Wang et al 2014) Cladonia spp (Park et al2013a 2014) Caloplaca flavorubescens (Parket al 2013b) and Cladonia greyi and Xanthoriaparientina through the Fungal Genomics Programat Joint Genome Institute of the United StatesDepartment of Energy (httpwwwjgidoegov)The foreseeable ongoing expansion of genomicresources for lichen-forming fungi will be centralto developing approaches for delimiting speciesusing high-throughput sequencing Specificallygenomic resources will be crucial in identifyingnovel markers (variable genes regions SNPsmicrosatellites etc) identifying conserved fungalmarkers for targeted high-throughput sequencingapproaches and references for entire genomecomparisons (Devkota et al 2014 Werth et al2013)
23 Can We Make SpeciesDelimitation in Lichen-FormingFungi Truly Integrative
The widespread availability of genetic data hascreated a biased viewpoint that only genetic datashould be used for statistical species delimitationHowever an ongoing appeal to researchers toassess species boundaries from multiple andcomplementary perspectives (phylogeneticspopulation genetics comparative morphologydevelopment ecology etc) has resulted in an
30 SD Leavitt et al
integrative taxonomic framework bringing theseconceptual and methodological perspectivestogether (Hamilton et al 2014 Fujita et al 2012Salicini et al 2011 Yeates et al 2011 Padialet al 2010 Dayrat 2005 Will et al 2005 Wiensand Penkrot 2002) In practice any study linkingdifferent kinds of data to support hypotheses ofspecies boundaries including mapping morpho-logical characters onto a molecular phylogenycan be considered integrative Integrative meth-ods for species delimitation fall across a broadspectrum ranging from verbal and qualitativeassessments of data classes to quantitative meth-ods that allow different data types to contribute tostatistical species delimitation (Yeates et al2011) In most taxonomic studies utilizing bothmolecular and morphological data expert opinionis eventually used to some degree to evaluate thefinal status of a candidate species (eg Bond andStockman 2008) In this sense many studiesusing evidence for independent data sources fordelimiting species boundaries use an iterativeapproach (Yeates et al 2011) where speciesboundaries can be tested within a hypothetico-deductive framework with diverse datasets
From a practical perspective we advocate aprocess of iterative taxonomy (sensu Yeates et al2011) to circumscribe and refine species limitsusing multiple lines of evidence This iterativeprocess involves comparisons of morphologicaldata with a phylogenetic hypothesis (ie single-locus gene tree or concatenated multilocusphylogeny) to identify the least inclusive mono-phyletic clade in the topology characterized by atleast one unambiguously diagnostic morphologi-cal character (Miralles and Vences 2013) Thisphylogenetic tree-informed approach to assessingspecies boundaries represents a common practicein studies of lichen-forming fungi Previouslyunrecognized species-level clades with corre-sponding subtle or overlooked phenotypiccharacters have been commonly observed in bothcrustose lichens [eg Acarospraceae (Wedin et al2009) Graphidaceae (Rivas Plata and Lumbsch2011 Papong et al 2009) Lecanoraceae (Leavittet al 2011a) Lecideaceae (Ruprecht et al 2010)Mycoblastaceae (Spribille et al 2011) andTeloschistaceae (Vondraacutek et al 2009 Muggia
et al 2008)] and foliose and fruticose lichens [egLobariaceae (Moncada et al 2014 McDonaldet al 2003) Parmeliaceae (Leavitt et al 2013aWirtz et al 2012 Divakar et al 2005 Molinaet al 2004 Kroken and Taylor 2001) Peltigera-ceae (OrsquoBrien et al 2009 Goffinet et al 2003)Physciaceae (Elix et al 2009 Divakar et al2007) and Sphaerophoraceae (Houmlgnabba andWedin 2003)] As a specific example Luumlckinget al (2008) used a combination of medullarychemistry and underside pigmentation in speci-mens from the Heterodermia obscurata group inCosta Rica to corroborate monophyletic clades inan ITS gene tree as species-level lineages
While this iterative approach to speciesdelimitation and taxonomy has proven valuablefor understanding species boundaries anddescribing new taxa in some groups of lichenizedfungi a posteriori examination of morphologicaland chemical features has failed to reveal diag-nostic phenotypic characters in a number ofstudies (Muggia et al 2014 Leavitt et al 2011aOtaacutelora et al 2010 OrsquoBrien et al 2009) Fur-thermore a study of widespread species in thegenus Melanelixia (Parmeliaceae) indicated thatphenotypically cryptic lichen-forming fungalspecies-level lineages may be relatively ancientand diagnosable phenotypic differences may beabsent even millions of years after the initialdivergence (Leavitt et al 2012b) The latter studyhighlights the fact that species-level lineages maycommonly exist without any observable diag-nostic phenotypic characters calling into ques-tion the impetus for a universal application ofintegrative taxonomy
In keeping with the principle that as manylines of evidence as available should be com-bined to delimit species (Dayrat 2005) the for-malized integrative taxonomic approach (ITAXMiralles and Vences 2013) uses a mtDNA guidetree and observations from different types of datathat might provide conclusive evidence for theindependence of lineages and thus their identityas different species Miralles and Vences (2013)provide a non-exhaustive list of species delimi-tation criteria to be integrated in the ITAXapproach including (a) sympatric occurrencewithout admixture as revealed by consistent
2 The Dynamic Discipline of Species Delimitation hellip 31
differences in morphological or molecular char-acters at the same geographic location (b) strongdifferences in a behavioral morphological orgenetic character known to mediate prematingisolation (c) unviability or infertility of hybrids(d) lack of gene flow across a geographicalhybrid zone (e) congruent diagnostic differencesbetween sister lineages in various unlinkedmorphological character (f) absence of haplo-type sharing in several unlinked nuclear loci and(g) a combination of criteria endashf Speciesboundaries are based on seeking the least inclu-sive monophyletic group in the mtDNA treewhich fulfills at least one of the criteria listedabove Clearly the ITAX approach is sensitive tosample size and in order to reliably support thedistinctiveness of a given species it has beenrecommended that the sampling strategy includesat least five individuals per species (Miralles andVences 2013) A similar approach could beadopted for lichenized fungi using an ITS genetopology as guide tree rather than mtDNA
A total evidence approach including concat-enation has been a common approach for inte-grating information from different sourcesincluding independent genetic markers in phy-logenetic reconstructions (Kluge 1989 Wiens1998 de Queiroz et al 1995) Proponents forconcatenation of independent data in phyloge-netic analyses argue that when combining alldata the underlying signal of speciation mayemerge even if weakly contradictory signalis contained in the individual data partitions(Gatesy et al 1999) For example establishing apreliminary perspective of species boundariesfrom multilocus sequence data using concatena-tion may provide a reasonable starting point forscreening for cryptic species and species treeinference (Šlapeta et al 2006 Leavitt et al2011a Le Gac et al 2007 Leacheacute 2009)However concatenation and consensus methodsimply a risk of obtaining inflated support forincorrect relationships and information aboutvariance in gene coalescence is lost (Degnan andRosenberg 2006 2009 Kubatko and Degnan2007 Edwards 2009) In spite of the impetus forintegrating evidence from independent data
sources into empirical species delimitation stud-ies and taxonomy (Fujita et al 2012 Padial et al2009) most currently available species delimi-tation methods are unable to accommodate non-genetic data sources in a statistical framework
231 Selecting the Appropriate Data
In the face of increasing availability of geneticdata and associated bioinformatical approachesfor delimiting species researchers should care-fully consider what information is being sacri-ficed by the failure to consider non-genetic datain species delimitation studies and whetheraccuracy could be improved by the addition ofmultiple data types Morphological data havehistorically served as a proxy to identify repro-ductively isolated groups (ie ldquospeciesrdquo) (Ray1686 Fujita et al 2012) Current methods fordelimiting species using non-genetic data (egchemistry morphology and ecology) remainwoefully understudied For example morphol-ogy-based species circumscriptions are generallybased on one or more qualitative (or quantitative)morphological characters that do not appear tooverlap with other species However ascertain-ing that a given trait is truly fixed within apopulation with statistical confidence requiresunrealistic sample sizes even when allowing forsome level of polymorphism in the diagnosticcharacter (Wiens and Servedio 2000) Now var-ious combinations of datamdashfrom morphologygenetics geography and ecologymdashare acceptedas standard information for species delimitationstudies (Ruiz-Sanchez and Sosa 2010 Ross et al2010 Edwards and Knowles 2014)
Below we briefly discuss appropriate datasources for species delimitation studies of lichen-forming fungi However homoplastic characters(similar traits that are not derived from a com-mon ancestor) are common among many traitscommonly used to circumscribe fungal taxa thebiological significance of secondary metabolitevariation remains largely unknown (Lawrey1986) and ecological niches may be difficultto adequately characterize and model due to
32 SD Leavitt et al
microhabitat requirements and data resolutionTherefore we advocate a cautious approach toselecting appropriate and relevant data forassessing species boundaries
In spite of the potential challenges and limi-tations of using phenotypic data these traits haveprovided a plethora of valuable information forunderstanding species boundaries For examplein the Melanelixia fuliginosa group (Parmelia-ceae) a morphometric analysis using color isi-dia and marginal zone free of isidia ascharacters revealed a general pattern of differ-entiation between material formerly recognizedas subspecies fuliginosa and glabratula in spiteof the fact that considerable overlap betweengroups occurs in some characters individually(Arup and Berlin 2011) Furthermore the dis-tinction of the two groups was supported by aphylogenetic analysis of the ITS marker andecological differences with M fuliginosaoccurring predominantly on rock and M glab-ratula on bark (Arup and Berlin 2011) Thisstudy of the M fuliginosa group provides a fit-ting example of using multiple independentsuites of data including ecology morphologyand genetic information to establish a robusthypothesis of species boundaries
In lichen systematics phenotypic dataincluding thallus organization secondary metab-olites mode of reproduction ascoma-type andontogeny ascus and ascospore characters havehistorically played a prominent role (Printzen2009) Ascomatal characters have traditionallyheld a major role in higher-level classification(Printzen 2009) in contrast to species-levelclassification which also tends to include a widearray of vegetative and chemical charactersCommonly assessed morphological charactersinclude thallus form and size cortical features(eg maculae and pseudocyphellae) presenceformcolor of attachment structure (eg rhizines)and reproductive mode (ascomata vs vegetativediaspores) (Printzen 2009) Different morpho-logical types of vegetative diasporesmdashcorticatedisidia and ecorticated sorediamdashand their locationare commonly used to distinguish speciesAscomatal characters including morphologylocation (laminal vs marginal) position (sessile
vs immersed) presence of thalline margins colorof an apothecial disc and presence or color ofpruina are also commonly used in speciesdelimitations of lichen-forming fungi (Printzen2009) Other important characters may includethalline characters form color size and septationof ascospores size and form and structure of ascithe hamathecium type of epihymenium andhypothecium and the type of excipulum orperidium conidiomatal characters etc (Printzen2009)
Assessments of secondary metabolites hasplayed an important role in lichen taxonomybeginning with the introduction of simple spottests by Nylander (1866a b) The use of chem-istry in lichen taxonomy has been discussed indetail in numerous reviews (Lumbsch 1998a bRogers 1989 Brodo 1986 Egan 1986 Leuckert1985 Brodo 1978 Hawksworth 1976 Culber-son 1969 1970) and we refer readers to thesevaluable sources for a more comprehensive per-spective on lichen chemistry In short extrolites(secondary metabolites) belong to various clas-ses the most common and diverse include dep-sides depsidones chlorinated xanthones andanthraquinones (Lumbsch 2002 Culberson1969) The presence or absence of specificextrolites or their replacements by another sub-stance is widely used to distinguish speciesparticularly when correlated with differences ingeographic distributions However if morpho-logical or geographical differences betweenpopulations containing different extrolites are notapparent the taxonomic significance has beendisputed with some authors distinguishing themas species and others preferring to regard them aschemical races within a species In addition tosimply using the presence or absence of extro-lites Culberson and Culberson (1976) proposedto arrange lichen substances into chemosyn-dromes of closely related substances The pres-ence or absence of these chemosyndromes maypotentially be used as characters to delimit spe-cies regarding differences involving the samechemosyndromes as intraspecific variation anddistinct chemosyndromes as evidence for inter-specific populations (Lumbsch 1994 Gowan1986)
2 The Dynamic Discipline of Species Delimitation hellip 33
Due to the fact that species delimitationstudies often incorporate a substantial biogeo-graphical or ecological component ecologicalniche modeling plays an increasing role in phy-logenetic and taxonomic research Niche mod-eling can provide evidence for ecologicalisolation between populations based on eitherconserved or divergent ecological niches andtherefore can provide additional evidence sup-porting lineage independence between putativespecies The application of ecological nichemodeling has been applied to species delimita-tion studies in a number of cases (Ruiz-Sanchezand Sosa 2010 Leacheacute et al 2009 Raxworthyet al 2007 Rissler and Apodaca 2007) althoughit has not been explicitly applied to assess speciesboundaries in lichen-forming fungi By mappingthe spatial distribution of environmental suit-ability of climatic variables for candidate speciesthe application of ecological niche modeling canbe particularly important in cases where specieshave allopatric distributions (Raxworthy et al2007)
Ecological niche modeling utilizes knownassociations between a speciesrsquo occurrencelocalities and environmental variables to defineabiotic conditions within which populations canbe maintained (Guisan and Thuiller 2005) Themethodological approach for modeling is basedon four general properties (i) The current knownspeciesrsquo localities is the dependent variable (ii)the distribution is modeled as a map composed ofgrid cells at a specified resolution (iii) a range ofenvironmental variables (eg temperature pre-cipitation and solar exposure) are collected todescribe the characteristics of each cell and (iv)classifying the degree to which each cell is eithersuitable or unsuitable for each species under arange of models (Guisan and Thuiller 2005)
Ecological data also have the potential to playan important role in understanding speciesboundaries in lichen-forming fungi For exampleNash and Zavada (1977) demonstrated that Xant-hoparmelia populations with distinct chemistriesoccurring in the northern portion of the SonoranDesert exhibit habitat selection among differentrock substrates within a region with relativelyuniform climate and topography In another case
the parmelioid species Parmelia mayi is mor-phologically indistinguishable from P saxatilisbut can be separated by bioclimatic features andgenetic and chemical characters (Molina et al2011) McCune and Printzen(2011) assess distri-butions and climatic niches of species in theLecanora varia group inwesternUSAandprovidea model that uses continental influence and annualtemperature as themajor factors predicting speciesdistributions The distribution of Usnea hirta alichen commonly used in air quality biomonitor-ing research was modeled for a section of theWhite River National Forest in central Coloradobased on the presence of U hirta at 72 biomoni-toring reference sites distributed in the inter-mountain western United States (Shrestha et al2012) The best model for predicting U hirtadistribution included four variablesmdashsolar radia-tion average monthly precipitation and averagemonthly minimum and maximum temperatures(Shrestha et al 2012) These studies supportthe potential use of ecological niche modelingmethods in species delimitation studies of lichen-forming fungi
24 Conclusions What AboutTaxonomy
In most cases species circumscription and tax-onomy requires some degree of qualitativejudgment and individual interpretation Integrat-ing multiple types of data into an empiricalframework for delimiting species boundarieswhere species boundaries can be tested within ahypothetico-deductive framework with diversedatasets can provide robust hypotheses of speciesboundaries and taxonomic stability (Yeates et al2011) However it has long been known toevolutionary biologists that distinct species donot need to have diagnosable morphologicaldifferences (Mayr 1963) and increasing avail-ability of genetic data has allowed researchers toidentify species and to rigorously test speciesboundaries with a level of precision that wasunimaginable a decade ago While analyticaladvances in statistical species delimitation havebeen largely based on genetic data the utility of
34 SD Leavitt et al
these approaches to formal taxonomy remainselusive Due to these challenges an eclecticapproach to delimiting species and cautionagainst the reliance on any single dataset ormethod is required when delimiting species
While results from many recent studies maycontradict traditional species boundaries acrossmany groups of lichen-forming fungi we areoptimistic that this research represents substantialprogress toward a more accurate perspective onspecies boundaries and diversity in fungi As aresult of ongoing research of species boundariesin lichen-forming fungi the taxonomic value ofmany phenotypes is now better understood ourunderstanding of ecological evolutionary andbiogeographic patterns has improved and we canbegin to better understand patterns of symbiontinteractions in lichens Integrating new data(including novel morphological characters andgenetic data) will be essential to accurately rep-resent species-level diversity across all groups oflichenized fungi Hopefully an improved per-spective on lichen diversity also increases ourappreciation of these incredible symbiotic sys-tems While we are strong advocates for theapplication of independent data types in devel-oping an integrative taxonomy there is anincreasing need to formally recognize the exis-tence of phenotypically cryptic species-levellineages in lichen-forming fungi (see Hibbettet al 2011) In some cases a molecular taxon-omy may provide the most practical approach toconsistent treatment of mycobiont species withinlichen groups where diagnostic morphologicalcharacters are unidentifiable or practically notfeasible (Leavitt et al 2013c d)
These are exciting times for taxonomists andphylogeneticists A closer look at lichen taxon-omy with the inclusion of new data will help usto better understand the diversity of these fasci-nating organisms accurately interpret distribu-tion patterns and play a more important role inmeaningful conservation practices Howeversome level of uncertainty will accompany pro-gress In many taxonomic groups our traditionalapproach for species identification will likelyneed to be substantially modified The search forcorroborating morphological support for cryptic
species identified using molecular data willrequire meticulous and creative approaches toassess phenotypic variation in potentially unor-thodox ways We are hopeful that lichenologistswho traditionally have been eager to include newmethods such as chromatography in their rou-tine identifications will be amenable to includemolecular techniques to their routine examina-tion of specimens for identification and classifi-cation Although this may prove difficult toachieve by single individuals especially citizenscientists that traditionally play an important rolein lichen taxonomy the increasing numberof collaborative projects in the discipline (egLumbsch et al 2011 Crespo et al 2010Gueidan et al 2009) make us optimistic thatbroad-scale collaborative approaches will facili-tate the inclusion of molecular data in lichenresearch at all levels
Acknowledgments We are indebted to various col-leagues for valuable thought-provoking discussionnotably Matthew Nelsen (University of Chicago) AnaCrespo (Universidad Complutense de Madrid) PradeepDivakar (Universidad Complutense de Madrid) BeckettSterner (The Field Museum) and Joyce Havstad (TheField Museum) We also thank anonymous reviewers whoprovided valuable comments that improved this chapterSupport by the US National Science Foundation isgratefully acknowledged (ldquoHidden diversity in parmelioidlichensrdquo DEB-0949147)
References
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Altermann S Leavitt SD Goward T Nelsen MPLumbsch HT (2014) How do you solve a problemlike Letharia A new look at cryptic species in lichen-forming fungi using Bayesian clustering and SNPSsfrom multilocus sequence data PLoS ONE 9(5)e97556 doi101371journalpone0097556
Amo de Paz G Cubas P Crespo A Elix JA Lumbsch HT(2012) Transoceanic dispersal and subsequent diver-sification on separate continents shaped diversity ofthe Xanthoparmelia pulla group (Ascomycota) PLoSONE 7(6)e39683 doi101371journalpone0039683
Arguumlello A Del Prado R Cubas P Crespo A (2007)Parmelina quercina (Parmeliaceae Lecanorales)
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Arup U Berlin ES (2011) A taxonomic study ofMelanelixia fuliginosa in Europe The Lichenologist43(02)89ndash97 doi101017S0024282910000678
Arup U Grube M (2000) Is Rhizoplaca (Lecanoraleslichenized Ascomycota) a monophyletic genus Can JBot 78(3)318ndash327 doi101139b00-006
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Bond J Stockman A (2008) An integrative method fordelimiting cohesion species finding the population-species interface in a group of Californian trapdoorspiders with extreme genetic divergence and geo-graphic structuring Syst Biol 57(4)628ndash646 doi10108010635150802302443
Brodo IM (1978) Changing concepts regarding chemicaldiversity in lichens The Lichenologist 10(1)1ndash11doi101017S0024282978000031
Brodo IM (1986) Interpreting chemical variation inlichens for systematic purposes The Bryologist 89(2)132ndash138
Caley MJ Fisher R Mengersen K (2014) Global speciesrichness estimates have not converged Trends EcolEvol 29(4)187ndash188 doi101016jtree201402002
Camargo A Sites JW (2013) Species delimitation adecade after the Renaissance In Pavlinov I (ed) Thespecies problemmdashongoing issues InTech doi10577252664
Camargo A Morando M Avila LJ Sites JW (2012)Species delimitation with ABC and other coalescent-based methods a test of accuracy with simulations and
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Carstens BC Dewey TA (2010) Species delimitationusing a combined coalescent and information-theoreticapproach an example from North American Myotisbats Syst Biol 59(4)400ndash414 doi101093sysbiosyq024
Carstens BC Pelletier TA Reid NM Satler JD (2013)How to fail at species delimitation Mol Ecol 22(17)4369ndash4383 doi101111mec12413
Corander J Marttinen P (2006) Bayesian identification ofadmixture events using multi-locus molecular mark-ers Mol Ecol 15(10)2833ndash2843 doi101111j1365-294X200602994x
Corander J Waldmann P Marttinen P Sillanpaa M(2004) BAPS 2 enhanced possibilities for the analysisof genetic population structure Bioinformatics 20(15)2363ndash2369 doi101093bioinformaticsbth250
Corander J Marttinen P Mantyniemi S (2006) Bayesianidentification of stock mixtures from molecular markerdata Fish Bull 104550ndash558
Corander J Marttinen P Siren J Tang J (2008) EnhancedBayesian modelling in BAPS software for learninggenetic structures of populations BMC Bioinf 9(1)539 doi1011861471-2105-9-539
Coyne JA Orr HA (2004) Speciation Sinauer AssociatesSunderland
Cracraft J (1983) Species concepts and speciation anal-ysis Curr Ornithol 1159ndash187
Crespo A Lumbsch HT (2010) Cryptic species in lichen-forming fungi IMA Fungus 1167ndash170 doi105598imafungus2010010209
Crespo A Peacuterez-Ortega S (2009) Cryptic species andspecies pairs in lichens a discussion on the relation-ship between molecular phylogenies and morpholog-ical characters Anales del Jardin Botanico de Madrid66(S1)71ndash81 doi103989ajbm2225
Crespo A Kauff F Divakar PK del Prado R Perez-Ortega S Amo de Paz G Ferencova Z Blanco ORoca-Valiente B Nunez-Zapata J Cubas P ArguelloA Elix JA Esslinger TL Hawksworth DL MillanesA Molina MC Wedin M Ahti T Aptroot A et al(2010) Phylogenetic generic classification of parmeli-oid lichens (Parmeliaceae Ascomycota) based onmolecular morphological and chemical evidenceTaxon 59(6)1735ndash1753
Culberson WL (1969) The use of chemistry in thesystematics of the lichens Taxon 18498ndash505
Culberson WL (1970) Chemosystematics and ecology oflichen-forming fungi Annu Rev Ecol Syst 1153ndash170
Culberson CF Culberson WL (1976) Chemosyndromicvariation in lichens Syst Bot 1325ndash339
Darwin C (1859) On the origin of species by means ofnatural selection or the preservation of favoured racesin the struggle for life J Murray London
Dayrat B (2005) Towards integrative taxonomy Biol JLinn Soc 85(3)407ndash415 doi101111j1095-8312200500503x
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de Queiroz K (1998) The general lineage concept ofspecies species criteria and the process of speciationa conceptual unification and terminological recom-mendations In Howard DJ Berlocher SH (eds)Endless forms species and speciation Oxford Uni-versity Press Oxford pp 57ndash75
de Queiroz K (1999) The general lineage concept ofspecies and the defining properties of the speciescategory In Wilson RA (ed) Species new interdis-ciplinary essays MIT Press Cambridge pp 49ndash89
de Queiroz K (2007) Species concepts and speciesdelimitation Syst Biol 56(6)879ndash886 doi10108010635150701701083
de Queiroz A Donoghue MJ Kim J (1995) Separateversus combined analysis of phylogenetic evidenceAnnu Rev Ecol Syst 26(1)657ndash681 doi101146annureves26110195003301
Degnan J Rosenberg N (2006) Discordance of speciestrees with their most likely gene trees PLoS Genet 2(5)e68 doi101371journalpgen0020068
Degnan JH Rosenberg NA (2009) Gene tree discordancephylogenetic inference and the multispecies coales-cent Trends Ecol Evol 24(6)332ndash340 doihttpdxdoiorg101016jtree200901009
Del-Prado R Cubas P Lumbsch HT Divakar PK BlancoO de Paz GA Molina MC Crespo A (2010) Geneticdistances within and among species in monophyleticlineages of Parmeliaceae (Ascomycota) as a tool fortaxon delimitation Mol Phylogenet Evol 56(1)125ndash133 doi101016jympev201004014
Del-Prado R Divakar PK Crespo A (2011) Using geneticdistances in addition to ITS molecular phylogeny toidentify potential species in the Parmotrema reticul-atum complex a case study The Lichenologist 43(06)569ndash583 doi101017S0024282911000582
Del-Prado R Blanco O Lumbsch HT Divakar PK ElixJA Molina MC Crespo A (2013) Molecular phylog-eny and historical biogeography of the lichen-formingfungal genus Flavoparmelia (Ascomycota Parmelia-ceae) Taxon 62(5)928ndash939 doi101270562522
Dettman J Jacobson D Taylor J (2003a) A multilocusgenealogical approach to phylogenetic species recog-nition in the model eukaryote Neurospora Evolution57(12)2703ndash2720 doi101111j0014-38202003tb01514x
Dettman JR Jacobson DJ Turner E Pringle A TaylorJW (2003b) Reproductive isolation and phylogeneticdivergence in Neurospora comparing methods ofspecies recognition in a model eukaryote Evolution57(12)2721 doi10155403-074
Devkota S Cornejo C Werth S Chaudhary RP Sche-idegger C (2014) Characterization of microsatelliteloci in the Himalayan lichen fungus Lobaria pindar-ensis (Lobariaceae) Appl Plant Sci 2(5)1300101doi103732apps1300101
Divakar PK Molina MC Lumbsch HT Crespo A (2005)Parmelia barrenoae a new lichen species related toParmelia sulcata (Parmeliaceae) based on molecularand morphological data The Lichenologist 37(01)37ndash46 doi101017S0024282904014641
Divakar PK Amo De paz G del Prado R Esslinger TLCrespo A (2007) Upper cortex anatomy corroboratesphylogenetic hypothesis in species of Physconia(Ascomycota Lecanoromycetes) Mycol Res 111(11)1311ndash1320 doi101016jmycres200708009
Divakar PK Figueras G Hladun N Crespo A (2010)Molecular phylogenetic studies reveal an undescribedspecies within the North American concept of Mela-nelixia glabra (Parmeliaceae) Fungal Divers 42(1)47ndash55 doi101007s13225-010-0027-3
Donoghue MJ Gauthier A (2004) Implementing thephylocode Trends Ecol Evol 19(6)281ndash282 doi101016jtree200404004
Eaton DAR Ree RH (2013) Inferring phylogeny andintrogression using RADseq data an example fromflowering plants (Pedicularis Orobanchaceae) SystBiol 62(5)689ndash706 doi101093sysbiosyt032
Edwards SV (2009) Is a new and general theory ofmolecular systematics emerging Evolution 63(1)1ndash19 doi101111j1558-5646200800549x
Edwards DL Knowles LL (2014) Species detection andindividual assignment in species delimitation canintegrative data increase efficacy Proc R Soc B BiolSci 281(1777) doi101098rspb20132765
Egan RS (1986) Correlations and non-correlations ofchemical variation patterns with lichen morphologyand geography The Bryologist 8999ndash110
Elix JA Corush J Lumbsch HT (2009) Triterpenechemosyndromes and subtle morphological characterscharacterise lineages in the Physcia aipolia group inAustralia (Ascomycota) Syst Biodivers 7(04)479ndash487 doi101017S1477200009990223
Emerson KJ Merz CR Catchen JM Hohenlohe PACresko WA Bradshaw WE Holzapfel CM (2010)Resolving postglacial phylogeography using high-throughput sequencing Proc Natl Acad Sci 107(37)16196ndash16200 doi101073pnas1006538107
Ence DD Carstens BC (2011) SpedeSTEM a rapid andaccurate method for species delimitation Mol EcolResour 11(3)473ndash480 doi101111j1755-0998201002947x
EvannoGRegnaut SGoudet J (2005)Detecting thenumberof clusters of individuals using the software STRUC-TURE a simulation study Mol Ecol 14(8)2611ndash2620doi101111j1365-294X200502553x
Falush D Stephens M Pritchard JK (2003) Inference ofpopulation structure using multilocus genotype datalinked loci and correlated allele frequencies Genetics164(4)1567ndash1587 doi101111j1471-8286200701758x
Fan HH Kubatko LS (2011) Estimating species treesusing approximate Bayesian computation Mol Phylo-genet Evol 59(2)354ndash363 doi101016jympev201102019
Fernaacutendez-Mendoza F Printzen C (2013) Pleistoceneexpansion of the bipolar lichen Cetraria aculeata intothe Southern hemisphere Mol Ecol 22(7)1961ndash1983doi101111mec12210
Fernaacutendez-Mendoza F Domaschke S Garciacutea MA JordanP Martin MP Printzen C (2011) Population structure
2 The Dynamic Discipline of Species Delimitation hellip 37
of mycobionts and photobionts of the widespreadlichen Cetraria aculeata Mol Ecol 20(6)1208ndash1232doi101111j1365-294X201004993x
Fraley C Raftery A (2007) Model-based methods ofclassification Using the mclust software in chemo-metrics J Stat Softw 18i06 [Available online at httpwwwdoajorgdoajfunc=abstractampid=218544]
Fujisawa T Barraclough TG (2013) Delimiting speciesusing single-locus data and the generalized mixed yulecoalescent (GMYC) approach a revised method andevaluation on simulated datasets Syst Biol 62(5)707ndash724 doi101093sysbiosyt033
Fujita MK Leacheacute AD Burbrink FT McGuire JA MoritzC (2012) Coalescent-based species delimitation in anintegrative taxonomy Trends Ecol Evol 27(9)480ndash488 doi101016jtree201204012
Gatesy J OrsquoGrady P Baker RH (1999) Corroborationamong data sets in simultaneous analysis hiddensupport for phylogenetic relationships among higherlevel Artiodactyl taxa Cladistics 15(3)271ndash313doi101111j1096-00311999tb00268x
Gladieux P Ropars J Badouin H Branca A Aguileta Gde Vienne DM Rodriacuteguez de la Vega RC Branco SGiraud T (2014) Fungal evolutionary genomicsprovides insight into the mechanisms of adaptivedivergence in eukaryotes Mol Ecol 23(4)753ndash773doi101111mec12631
Goffinet B Miadlikowska J Goward T (2003) Phyloge-netic inferences based on nrDNA sequences supportfive morphospecies within the Peltigera didactylacomplex (Lichenized Ascomycota) The Bryologist106(3)349ndash364 doi10163901
Gowan SP (1986) Evolution of secondary natural productsin the genus Porpidia (Ascomycata Porpidiaceae) AmJ Bot 73606
Griffin PC Hoffmann AA (2014) Limited genetic diver-gence among Australian alpine Poa tussock grassescoupled with regional structuring points to ongoinggene flow and taxonomic challenges Ann Bot doi101093aobmcu017
Grube M Hawksworth DL (2007) Trouble with lichenthe re-evaluation and re-interpretation of thallus formand fruit body types in the molecular era Mycol Res111(9)1116ndash1132 doi101016jmycres200704008
Gueidan C Savi S Thues H Roux C Keller C Tibell LPrieto M Heimarsson S Breuss O Orange A FrobergL Wynns AA Navarro-Rosines P Krzewicka BPykaelae J Grube M Lutzoni F (2009) Genericclassification of the Verrucariaceae (Ascomycota)based on molecular and morphological evidencerecent progress and remaining challenges Taxon 58(1)184ndash208
Guillot G Mortier F Estoup A (2005) Geneland acomputer package for landscape genetics Mol EcolNotes 5(3)712ndash715 doi101111j1471-8286200501031x
Guillot G Renaud S Ledevin R Michaux J Claude J(2012) A unifying model for the analysis of pheno-typic genetic and geographic data Syst Biol 61(6)897ndash911 doi101093sysbiosys038
Guisan A Thuiller W (2005) Predicting species distribu-tion offering more than simple habitat models EcolLett 8(9)993ndash1009 doi101111j1461-0248200500792x
Hale ME (1990) A synopsis of the lichen genusXanthoparmelia (Vainio) Hale (AscomycotinaParmeliaceae) Smithsonian Institution Press Wash-ington DC
Hamilton CA Hendrixson BE Brewer MS Bond JE(2014) An evaluation of sampling effects on multipleDNA barcoding methods leads to an integrativeapproach for delimiting species a case study ofthe North American tarantula genus Aphonopelma(Araneae Mygalomorphae Theraphosidae) MolPhylogenet Evol 7179ndash93 doi101016jympev201311007
Hausdorf B (2011) Progress toward a general speciesconcept Evolution 65(4)923ndash931 doi101111j1558-5646201101231x
Hausdorf B Hennig C (2010) Species delimitation usingdominant and codominant multilocus markers SystBiol 59(5)491ndash503 doi101093sysbiosyq039
Hawksworth DL (1976) Lichen chemotaxonomy InBailey RH (ed) Lichenology progress and problemsAcademic London pp 139ndash184
Hebert PDN Cywinska A Ball SL deWaard JR (2003)Biological identifications through DNA barcodesProc R Soc Lond B Biol Sci 270(1512)313ndash321doi101098rspb20022218
Hebert PDN Stoeckle MY Zemlak TS Francis CM(2004) Identification of birds through DNA barcodesPLoS Biol 2(10)e312 doi101371journalpbio0020312
Heled J Drummond AJ (2010) Bayesian inference ofspecies trees from multilocus data Mol Biol Evol 27(3)570ndash580 doi101093molbevmsp274
Hey J (2006) On the failure of modern species conceptsTrends Ecol Evol 21(8)447ndash450 doi101016jtree200605011
Hibbett DS OhmanA Glotzer D NuhnMKirk P NilssonRH (2011) Progress in molecular and morphologicaltaxon discovery in Fungi and options for formalclassification on environmental sequences Fungal BiolRev 2538ndash47 doi101016jfbr201101001
Houmlgnabba F Wedin M (2003) Molecular phylogeny ofthe Sphaerophorus globosus species complex Cladis-tics 19(3)224ndash232 doi101111j1096-00312003tb00365x
Hudson RR Coyne JA (2002) Mathematical conse-quences of the genealogical species concept Evolu-tion 56(8)1557 doi101111j0014-38202002tb01467x
Huelsenbeck JP Andolfatto P Huelsenbeck ET (2011)Structurama Bayesian inference of population struc-ture Evol Bioinf Online 7(2011)55ndash59 doi104137EBOS6761
Jones G Oxelman B (2014) DISSECT an assignment-free Bayesian discovery method for species delimita-tion under the multispecies coalescent Bioinformaticsdoi101101003178
38 SD Leavitt et al
Kekkonen M Hebert PD (2014) DNA barcode-baseddelineation of putative species efficient start fortaxonomic workflows Mol Ecol Res doi1011111755-099812233
Kelly LJ Hollingsworth PM Coppins BJ Ellis CJHarrold P Tosh J Yahr R (2011) DNA barcoding oflichenized fungi demonstrates high identification suc-cess in a floristic context New Phytol 191(1)288ndash300doi101111j1469-8137201103677x
Kiss L (2012) Limits of nuclear ribosomal DNA internaltranscribed spacer (ITS) sequences as species barcodesfor fungi Proc Natl Acad Sci 109(27)E1811ndashE1811doi101073pnas1207143109
Kluge AG (1989) A concern for evidence and aphylogenetic hypothesis for relationships among Epi-crates (Boidae Serpentes) Syst Zool 38(1)7ndash25doi101093sysbio3817
Knowles LL Carstens BC (2007) Delimiting specieswithout monophyletic gene trees Syst Biol 56(6)887ndash895 doi10108010635150701701091
Kroken S Taylor JW (2001) A gene genealogical approachto recognize phylogenetic species boundaries in thelichenized fungus Letharia Mycologia 93(1)38ndash53
Kubatko LS Degnan JH (2007) Inconsistency of phylo-genetic estimates from concatenated data under coa-lescence Syst Biol 56(1)17ndash24 doi10108010635150601146041
Latch EK Dharmarajan G Glaubitz JC Rhodes OE Jr(2006) Relative performance of Bayesian clusteringsoftware for inferring population substructure andindividual assignments at low levels of populationdifferentiation Conserv Genet 7(2)295ndash302 doi101007s10592-005-9098-1
Lawrey JD (1986) Biological role of lichen substancesThe Bryologist 89(2)111ndash122
Le Gac M Hood ME Fournier E Giraud T (2007)Phylogenetic evidence of host-specific cryptic speciesin the anther smut fungus Evolution 61(1)15ndash26doi101111j1558-5646200700002x
Leacheacute AD (2009) Species tree discordance traces tophylogeographic clade boundaries in North Americanfence lizards (Sceloporus) Syst Biol 58(6)547ndash559doi101093sysbiosyp057
Leacheacute AD Fujita MK (2010) Bayesian species delim-itation in West African forest geckos (Hemidactylusfasciatus) Proc R Soc B Biol Sci 2773071ndash3077doi101098rspb20100662
Leacheacute AD Koo M Spencer C Papenfuss T Fisher RMcGuire J (2009) Quantifying ecological morpho-logical and genetic variation to delimit species in thecoast horned lizard species complex (Phrynosoma)Proc Natl Acad Sci USA 10612418ndash12423 doi101073pnas0906380106
Leacheacute AD Fujita MK Minin VN Bouckaert RR (2014)Species delimitation using genome-wide SNP dataSystematic Biology doi101093sysbiosyu018
Leavitt SD Fankhauser JD Leavitt DH Porter LDJohnson LA St Clair LL (2011a) Complex patterns ofspeciation in cosmopolitan ldquorock posyrdquo lichensmdashdiscovering and delimiting cryptic fungal species in
the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
Leavitt SD Esslinger TL Lumbsch HT (2012c) Neogene-dominated diversification in neotropical montanelichens dating divergence events in the lichen-form-ing fungal genus Oropogon (Parmeliaceae) Am J Bot99(11)1764ndash1777 doi103732ajb1200146
Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Lumbsch HT St Clair LL(2013c) DNA barcode identification of lichen-formingfungal species in the Rhizoplaca melanophthalmaspecies-complex (Lecanorales Lecanoraceae) includ-ing five new species MycoKeys 71ndash22 doi103897mycokeys74508
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Vondraacutek J Thorsten Lum-bsch H Clair LLS (2013d) Local representation ofglobal diversity in a cosmopolitan lichen-formingfungal species complex (Rhizoplaca Ascomycota)J Biogeogr 40(9)1792ndash1806 doi101111jbi12118
Leavitt SD Lumbsch HT Stenroos S St Clair LL(2013e) Pleistocene speciation in North American
2 The Dynamic Discipline of Species Delimitation hellip 39
lichenized fungi and the impact of alternative speciescircumscriptions and rates of molecular evolution ondivergence estimates PLoS ONE 8(12)e85240doi101371journalpone0085240
Leavitt SD Esslinger TL Hansen ES Divakar PK CrespoA Loomis BF Lumbsch HT (2014) DNA barcoding ofbrown Parmeliae (Parmeliaceae) species a molecularapproach for accurate specimen identification empha-sizing species in Greenland Organ Divers Evol 14(1)11ndash20 doi101007s13127-013-0147-1
Leliaert F Verbruggen H Vanormelingen P Steen FLopez-Bautista JM Zuccarello GC De Clerck O(2014) DNA-based species delimitation in algae Eur JPhycol 49(2) doi101080096702622014904524
Leuckert C (1985) Probleme der Flechten-Chemotaxon-omiemdashStoffkombinationen und ihre taxonomischeWertung Ber Deut Bot Ges 98401ndash408
Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
Lohtander K Kaumlllersjouml M Roland M Tehler A (2000)The family physciaceae in fennoscandia phylogenyinferred from its sequences Mycologia 92(4)728ndash735
Longton RE (1997) The role of bryophytes and lichens inpolar ecosystems In Woodin SJ Marquiss M (eds)Ecology of Arctic Environments Blackwell ScienceOxford pp 69ndash96 (Special publication No 13)
Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
Lumbsch HT (1994) Die Lecanora subfusca-Gruppe inAustralasien J Hattori Bot Lab 771ndash175
Lumbsch HT (1998a) Taxonomic use of metabolic data inlichen-forming fungi In Frisvad JC Bridge PDArora DK (eds) Chemical Fungal Taxonomy MarcelDekker New York pp 345ndash387
Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
Mayden RL (1997) A hierarchy of species concepts thedenouement in the saga of the species problem InClaridge MF Dawah HA Wilson MR (eds) Speciesthe units of biodiversity Chapman amp Hall Londonpp 381ndash424
Mayr E (1963) Animal species and evolution HarvardUniversity Press Cambridge
Mayr E (1970) Populations species and evolutionBelknap Press of Harvard University Press Cambridge
McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
McDonald T Miadlikowska J Lutzoni F (2003) Thelichen genus Sticta in the Great Smoky Mountains a
40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
McKay BD Mays HL Wu Y Li H Yao C-T Nishiumi IZou F (2013) An empirical comparison of character-based and coalescent-based approaches to speciesdelimitation in a young avian complex Mol Ecol 22(19)4943ndash4957 doi101111mec12446
Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
Mishler BD Brandon RN (1987) Individuality pluralismand the phylogenetic species concept Biol Philos 2397
Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
Nash TH Zavada M (1977) Population studies amongSonoran Desert species of Parmelia subg Xanthop-armelia (Parmeliaceae) Am J Bot 64(6)664ndash669
Nylander W (1866a) Circa novum in studio Lichenumcriterium chemicum Flora 49198ndash201
Nylander W (1866b) Hypochlorite of lime and hydrate ofpotash Two new criteria for the study of lichens(Tanslated and communicated by the Rev W ALeighton) Bot J Linn Soc 9358ndash365
OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
OrsquoNeill EM Schwartz R Bullock CT Williams JSShaffer HB Aguilar-Miguel X Parra-Olea G Weis-rock DW (2013) Parallel tagged amplicon sequencingreveals major lineages and phylogenetic structure inthe North American tiger salamander (Ambystomatigrinum) species complex Mol Ecol 22(1)111ndash129doi101111mec12049
Orock EA Leavitt SD Fonge BA St Clair LL LumbschHT (2012) DNA-based identification of lichen-form-ing fungi Can publicly available sequence databasesaid in lichen diversity inventories of Mount Cameroon(West Africa) The Lichenologist 44(6)833ndash839doi101017S0024282912000424
Otaacutelora MAG Martiacutenez I Aragoacuten G Molina MC (2010)Phylogeography and divergence date estimates of alichen species complex with a disjunct distributionpattern Am J Bot 97(2)216ndash223 doi103732ajb0900064
Padial J Castroviejo-Fisher S Kohler J Vila C ChaparroJ De la Riva I (2009) Deciphering the products ofevolution at the species level the need for anintegrative taxonomy Zoolog Scr 38(4)431ndash447doi101111j1463-6409200800381x
Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
Papong K Luumlcking R Thammathaworn A BoonpragobK (2009) Four new taxa of Chroodiscus (thelotremoidGraphidaceae) from Southeast Asia The Bryologist112(1)152ndash163 doi1016390007-2745-1121152
Park S-Y Choi J Kim JA Jeong M-H Kim S Lee Y-HHur J-S (2013a) Draft genome sequence of Cladoniamacilenta KoLRI003786 a lichen-forming fungusproducing biruloquinone Genome Announcements 1(5)e00695-13 doi101128genomeA00695-13
Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
Peacuterez-Ortega S Fernaacutendez-Mendoza F Raggio J VivasM Ascaso C Sancho LG Printzen C de los Riacuteos A(2012) Extreme phenotypic variation in Cetrariaaculeata (lichenized Ascomycota) adaptation or inci-dental modification Ann Bot 109(6)1133ndash1148doi101093aobmcs042
Pino-Bodas R Burgaz A Martiacuten M Lumbsch HT (2011)Phenotypical plasticity and homoplasy complicatespecies delimitation in the Cladonia gracilis group(Cladoniaceae Ascomycota) Organ Divers Evol 11(5)343ndash355 doi101007s13127-011-0062-2
Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
Pino-Bodas R Martiacuten M Burgaz A (2012b) Cladoniasubturgida and C iberica (Cladoniaceae) form asingle morphologically and chemically polymorphicspecies Mycol Prog 11(1)269ndash278 doi101007s11557-011-0746-1
Pino-Bodas R Ahti T Stenroos S Martiacuten MP BurgazAR (2013a) Multilocus approach to species recogni-tion in the Cladonia humilis complex (CladoniaceaeAscomycota) Am J Bot 100(4)664ndash678 doi103732ajb1200162
Pino-Bodas R Martiacuten AP Burgaz AR Lumbsch HT(2013b) Species delimitation in Cladonia (Ascomy-cota) a challenge to the DNA barcoding philosophyMol Ecol Resour 13(6)1058ndash1068 doi1011111755-099812086
Pons J Barraclough TG Gomez-Zurita J Cardoso ADuran DP Hazell S Kamoun S Sumlin WD VoglerAP (2006) Sequence-based species delimitation for theDNA taxonomy of undescribed insects Syst Biol 55(4)595ndash609 doi10108010635150600852011
Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
Pringle A Baker DM Platt JL Wares JP Latgeacute JPTaylor JW (2005) Cryptic speciation in the cosmo-politan and clonal human pathogenic fungus Asper-gillus fumigatus Evolution 59(9)1886ndash1899 doi10155404-2411
Printzen C (2009) Lichen systematics the role ofmorphological and molecular data to reconstructphylogenetic relationships Progress in botany vol71 Springer Berlin pp 233ndash275
Pritchard JK Stephens M Donnelly P (2000) Inference ofpopulation structure using multilocus genotype dataGenetics 155(2)945ndash959
Puillandre N Lambert A Brouillet S Achaz G (2012)ABGD automatic barcode gap discovery for primaryspecies delimitation Mol Ecol 21(8)1864ndash1877doi101111j1365-294X201105239x
Rannala B Yang Z (2003) Bayes estimation of speciesdivergence times and ancestral population sizes usingDNA sequences from multiple loci Genetics1641645ndash1656
Raxworthy CJ Ingram CM Rabibisoa N Pearson RG(2007) Applications of ecological niche modelingfor species delimitation a review and empiricalevaluation using day geckos (Phelsuma) fromMadagascar Syst Biol 56(6)907ndash923 doi10108010635150701775111
Ray J (1686) Historia planarum vol 1 Clark LondonReese Naeligsborg R Ekman S Tibell L (2007) Molecular
phylogeny of the genus Lecania (Ramalinaceaelichenized Ascomycota) Mycol Res 111(5)581ndash591doi101016jmycres200703001
Reid N Carstens B (2012) Phylogenetic estimation errorcan decrease the accuracy of species delimitation aBayesian implementation of the general mixed Yule-coalescent model BMC Evol Biol 12(1)196 doi1011861471-2148-12-196
Rissler L Apodaca J (2007) Adding more ecology intospecies delimitation ecological niche models andphylogeography help define cryptic species in theBlack Salamander (Aneides flavipunctatus) Syst Biol56924ndash942 doi10108010635150701703063
Rivas Plata E Luumlcking R (2013) High diversity ofGraphidaceae (lichenized Ascomycota Ostropales) inAmazonian Peruacute Fungal Divers 58(1)13ndash32 doi101007s13225-012-0172-y
Rivas Plata E Lumbsch HT (2011) Parallel evolution andphenotypic divergence in lichenized fungi a casestudy in the lichen-forming fungal family Graphida-ceae (Ascomycota Lecanoromycetes Ostropales)Mol Phylogenet Evol 61(1)45ndash63 doi101016jympev201104025
Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
Ross KG Gotzek D Ascunce MS Shoemaker DD (2010)Species delimitation a case study in a problematic anttaxon Syst Biol 59(2)162ndash184 doi101093sysbiosyp089
Rubin BER Ree RH Moreau CS (2012) Inferringphylogenies from RAD sequence data PLoS ONE 7(4)e33394 doi101371journalpone0033394
Rubinoff D (2006) Utility of mitochondrial DNA barcodesin species conservation Conserv Biol 20(4)1026ndash1033 doi101111j1523-1739200600372x
Ruiz-Sanchez E Sosa V (2010) Delimiting speciesboundaries within the Neotropical bamboo Otatea(Poaceae Bambusoideae) using molecular morpho-logical and ecological data Mol Phylogenet Evol 54(2)344ndash356 doi101016jympev200910035
Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
morphological characters Antarctic Sci 22 (SpecialIssue 06)727ndash741 doi101017S0954102010000477
Salicini I Ibaacutentildeez C Juste J (2011) Multilocus phylogenyand species delimitation within the Nattererrsquos batspecies complex in the Western Palearctic MolPhylogenet Evol 61(3)888ndash898 doi101016jympev201108010
Satler JD Carstens BC Hedin M (2013) Multilocusspecies delimitation in a complex of morphologicallyconserved trapdoor spiders (Mygalomorphae Antro-diaetidae Aliatypus) Syst Biol 62(6)805ndash823 doi101093sysbiosyt041
Schlick-Steiner BC Steiner FM Seifert B Stauffer CChristian E Crozier RH (2010) Integrative taxonomya multisource approach to exploring biodiversityAnnu Rev Entomol 55(1)421ndash438 doi101146annurev-ento-112408-085432
Schoch CL Seifert KA Huhndorf S Robert V SpougeJL Levesque CA Chen W Fungal Barcoding Con-sortium (2012) Nuclear ribosomal internal transcribedspacer (ITS) region as a universal DNA barcodemarker for Fungi Proc Natl Acad Sci 109(16)6241ndash6246 doi101073pnas1117018109
Seacuterusiaux E Villarreal AJC Wheeler T Goffinet B(2011) Recent origin active speciation and dispersalfor the lichen genus Nephroma (Peltigerales) inMacaronesia J Biogeogr 38(6)1138ndash1151 doi101111j1365-2699201002469x
Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
Simpson GG (1951) The species concept Evolution5285ndash298
Sites JW Marshall JC (2003) Delimiting species a renais-sance issue in systematic biology Trends Ecol Evol18462ndash470 doi101016S0169-5347(03)00184-8
Sites JW Marshall JC (2004) Operational criteria fordelimiting species Annu Rev Ecol Evol Syst 35(1)199ndash227 doi101146annurevecolsys35112202130128
Šlapeta J Loacutepez-Garciacutea P Moreira D (2006) Globaldispersal and ancient cryptic species in the smallestmarine eukaryotes Mol Biol Evol 23(1)23ndash29doi101093molbevmsj001
Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
Talavera G Dincă V Vila R (2013) Factors affectingspecies delimitations with the GMYC model insightsfrom a butterfly survey Methods Ecol Evol 4(12)1101ndash1110 doi1011112041-210X12107
Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
Tewksbury J Anderson JGT Bakker JD Billo TJDunwiddie PW Groom MJ Hampton SE HermanSG Levey DJ Machnicki NJ del Rio CM PowerME Rowell K Salomon AK Stacey L TrombulakSC Wheeler TA (2014) Natural historyrsquos place inscience and society BioScience doi101093bioscibiu032
Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
Werth S Cornejo C Scheidegger C (2013) Characteriza-tion of microsatellite loci in the lichen fungus Lobariapulmonaria (Lobariaceae) Appl Plant Sci 1(2)1200290 doi103732apps1200290
Westberg M Arup U Kaumlrnefelt I (2007) Phylogeneticstudies in the Candelariaceae (lichenized Ascomy-cota) based on nuclear ITS DNA sequence dataMycol Res 111(11)1277ndash1284 doi101017S0953756204002102
Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
2 The Dynamic Discipline of Species Delimitation hellip 43
(Lepidoptera Lycaenidae) Front Zoo 4(1)8 doi1011861742-9994-4-8
Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
distances among organisms belonging to thesame species are smaller than distances amongorganisms from different species (Puillandreet al 2012 Hebert et al 2003) Genetic distanceapproaches hold particular promise as an identi-fication tool shortcutting the difficulties of mor-phology-based identification (Hebert et al 2004)although in practice a barcode gap may not existfor many groups (Wiemers and Fiedler 2007)Furthermore the role of distance-based approa-ches using a single genetic marker for speciesdiscovery remains more controversial (Rubinoff2006) and without other corroborating evidenceOTUs inferred from single-locus dataset shouldonly be considered ldquocandidaterdquo species Tree-based methods aim to detect monophyletic cladescorresponding to species-level diversity bydetecting discontinuities associated within inter-and intraspecific branching patterns (Fujisawaand Barraclough 2013 Zhang et al 2013Monaghan et al 2009 Pons et al 2006) Tree-based species delimitation methods can also beused on the basis of other properties related tophylogenetic tree topologies (monophyly con-cordance with geography etc (Sites and Marshall2003 2004) Both distance- and tree-basedmethods have been applied for assessing speciesboundaries in lichen-forming fungi (Leavitt et al2012c 2014 Parnmen et al 2012 Del-Pradoet al 2010 2011 Molina et al 2011)
A number of tree-based methods partiallyautomate the species delimitation process withspecific bioinformatical analyses (Table 21)including the general mixed Yule coalescent(GMYC) approach (Fujisawa and Barraclough2013 Monaghan et al 2009 Pons et al 2006)and the Poisson tree processes (PTP) model(Zhang et al 2013) These methods provide rel-atively straightforward and objective pipelinesfor delimiting putative species-level lineagesfrom inferred gene trees by fitting within- andbetween-species branching models to an inferredsingle-locus topology A number of other tree-based methods for species delimitation areeffectively summarized in Sites and Marshall(2004)
When only single-locus data are available theGMYC has been shown to be a relatively robust
tool for species delimitation The GMYCapproach combines a coalescent model of intra-specific branching with a Yule model for inter-specific branching which is then fit to an inferredsingle-gene topology to estimate species bound-aries and a statistical measure of confidence for theinferred boundaries As an input the GMYCapproach requires an ultrametric gene tree andrecent refinements can account for uncertainty inphylogenetic relationships and parameters of theGMYC model (Fujisawa and Barraclough 2013Reid and Carstens 2012) Regardless of theseimprovements it may be difficult to accuratelyinfer an adequate ultrametric tree for large data-sets Although it appears that the GMYC isgenerally stable across a wide range of circum-stances including various methods of phyloge-netic reconstruction the presence of a highnumber singletons high numbers of sampledspecies and gaps in intraspecific sampling theaccuracy of the GMYC is most significantlyaffected by the mean population size relative todivergence times between them (Fujisawa andBarraclough 2013 Talavera et al 2013) Fur-thermore research suggests that the so-calledsingle-threshold version of the GMYC methodlikely outperforms the multiple-thresholdapproach (Fujisawa and Barraclough 2013Monaghan et al 2009) However other studiessuggest that theGMYCmethodmay often providehigher estimates for the total number ofOTUs thanother molecular species delimitation methods(Hamilton et al 2014 Miralles and Vences 2013Talavera et al 2013) warranting a cautiousinterpretation of results from GMYC analyses
Compared to the GMYC approach therecently introduced PTP method for speciesdelimitation has been suggested to be moreaccurate for preliminary species delimitation(Zhang et al 2013) Relative to the GMYC PTPoffers a more straightforward implementationrequiring a simple phylogenetic tree rather thanan ultrametric chronogram However moreresearch is required to assess the general perfor-mance of PTP across wide range of empirical andsimulated species delimitation studies At thispoint data support the use of the GMYC andPTP methods as objective and reasonable starting
24 SD Leavitt et al
points for species delimitation using single-genetopologies
The ldquoSpecies Delimitationrdquo plug-in to theGeneious software provides statistical approachesfor exploring species boundaries in single-genetopologies (Masters et al 2011) Using a priorispecimen assignments to putative species theldquoSpecies Delimitationrdquo plug-in computes statis-tics relating to the probability of the observedmonophyly or exclusivity having occurred bychance in a coalescent process and assesses thewithin- and between-species genetic distances inorder to infer the probability with which membersof a putative species might be identified suc-cessfully with tree-based methods An importantcontribution of the ldquoSpecies Delimitationrdquo plug-in is that it provides an objective means for usersto assess putative species within an empiricalstatistic-based framework rather than qualitativeevaluations of what level of hierarchy constitutesa species in a phylogeny
In contrast to simple sequence similaritythresholds (OTU-picking) for delimiting putativeevolutionarily significant units the AutomaticBarcode Gap Discovery (ABGD) method is anautomated procedure that sorts sequences intohypothetical species based on the existence of abarcode gap observed whenever intraspecificgenetic distances are less than those amongorganisms from different species (Puillandreet al 2012) Ultimately ABGD is a fast simplemethod that can be used to group individualsrepresented in a single-locus sequence alignmentinto candidate species that should be comple-mented with other lines of evidence in an inte-grative taxonomic approach (Kekkonen andHebert 2014 Puillandre et al 2012)
In general the ABGD GMYC and PTP ana-lytical protocols using single-locus data arerepeatable and computationally relatively fastproviding a valuable starting point for a pre-liminary perspective into species boundaries inunderstudied groups that can be validated withsubsequent studies (Kekkonen and Hebert 2014)Similarly analyses of single-locus data can beused to corroborate traditional phenotype-basedspecies boundaries and identify candidate speciesthat have previously been hidden within nominal
species However single-locus species delimita-tionmethodsmaybeparticularly prone to failure inrecognizing significant proportions of species-level biodiversity due to the strict criterion forreciprocal monophyly or alternatively providespurious inflations of estimated species diversitybased on genetic differences that donot correspondto species-level lineages For example recentestimates suggest that the incidence of species-level gene tree paraphyly is approximately 20 (Ross 2014) suggesting that analyses of single-locus datasets would likely fail to accurately deli-mit species in one in every five cases In contrastrecent empirical studies suggest that in some casesthe GMYC provides a striking overestimate ofspecies diversity (Miralles and Vences 2013)
The internal transcribed spacer region (ITS)has played a central role in molecular phyloge-netic studies of lichenized fungi and has recentlybeen adopted as the official barcoding marker forfungi (Schoch et al 2012) In many cases the ITSis sufficiently variable to resolve species bound-aries for lichenized fungi (Schoch et al 2012Kelly et al 2011) although accurate specimenidentification using sequence-based identificationapproaches requires a thoroughly curated refer-ence database (Leavitt et al 2014 Orock et al2012 Kelly et al 2011) In spite of the generalutility of the ITS marker a number of issues maypotentially limit its effective use in speciesdelimitation studies including the potential forintragenomic variation of the nuclear ribosomaltandem repeat and difficulties in aligning ITSsequences from divergent taxa (Kiss 2012)Because the ITS has been formally adopted as theofficial barcoding marker for fungi we recom-mend that species delimitation studies attempt toinclude this region for consistency across studiesHowever we recognize that in some taxonomicgroups the inclusion of the ITS may be prob-lematic and therefore suggest a careful screeningof other markers to identify appropriate loci forresolving species-level relationships
Ultimately the success of any single-locusspecies delimitation method largely depends onthe evolutionary history of the species groupunder study and the variability of the selectedmarker (Fig 22) Species delimitation will be
2 The Dynamic Discipline of Species Delimitation hellip 25
more difficult in more recently diverged lineagesand in cases with some level of interspecific geneflow relative to older well-diverged lineages(eg Leavitt et al 2012c 2013e) In the end wereemphasize that single-locus data alone shouldonly be used to provide a preliminary perspectiveof species boundaries and not as the sole evi-dence in species circumscriptions
222 Sampling Across the GenomeMultilocus Sequence Dataand Genome-Wide Markers
Although single-locus methods can provide anefficient approach for preliminary large-scaleassessments of species diversity there are
significant limitations particularly in recentlydiverged species groups where retention ofancestral polymorphisms and incomplete lineagesorting will likely result in different neutral locihaving unique gene topologies that do not mirrorthe speciation process (Knowles and Carstens2007 Heled and Drummond 2010 Taylor et al2000) In contrast to single-locus and strictlyphenotype-based approaches for species delimi-tation analyses of genetic data collected fromindependent genomic regions can provide robusthypotheses of species boundaries with increasingconfidence (Satler et al 2013 Zhang et al 2011Yang and Rannala 2010) Sequences from mul-tiple independent loci provide an importantsource of data for species delimitation studiesincluding recently developed models that
Fig 22 A simplified diagram illustrating the process ofspeciation through time in a single gene history andresulting gene topologies sampled at two points in thespeciation history (modified from Leliaert et al 2014)aEach dot represents a distinct gene copy and each row onenon-overlapping generation with lines connecting genecopies to their ancestors in the previous generation (onerow below) dashed diagonal lines represent reproductivebarriers two hypothetical sampling intervals at differentpoints in the speciation history are shown Tx and Ty
species delimitation methods using single-locus data areeffective only when species are reciprocally monophyleticin the sampled gene tree genetic clustering and coalescent-based species delimitation methods can circumscribespecies when species may not be monophyletic in sampledgenetic loci b Gene topology representing sampledhaplotypes at time Tx (shown in panel a) hypotheticalspecies are reciprocally monophyletic c Gene topologyrepresenting sampled haplotypes at time Ty (shown in panela) hypothetical species are para- and polyphyletic
26 SD Leavitt et al
combine individual gene genealogies and speciesphylogenies via modeling the coalescent historyof markers (Yang and Rannala 2010 Edwards2009 Knowles and Carstens 2007) As aresponse to advances in sequencing technologiesbioinformatical approaches for multimarker spe-cies delimitation analyses continue to be devel-oped (Table 21 Camargo and Sites 2013)
Long-term reproductive isolation of candidatespecies can be assessed with multilocus sequencedata by evaluating genealogical concordance ofunlinked markers (Baum and Shaw 1995 Aviseand Ball 1990) Within species the mixingeffects of recombination cause unlinked loci tohave distinct genealogical histories but geneticdrift and long-term divergence leads to concor-dant genealogical histories at loci across thegenome Relationships of individuals belongingto distinct candidate species can be evaluatedusing gene genealogies (eg haplotype networksor single-gene topologies) to identify lineagesthat exhibit genealogical exclusivity acrossunlinked neutral loci (Hudson and Coyne 2002Dettman et al 2003b Avise and Ball 1990) Thepresence of the same clades in the majority ofsingle-locus genealogies is taken as evidence thatthe clades represent reproductively isolated lin-eages (Dettman et al 2003a Pringle et al 2005)In practice the criteria of reciprocal monophylyand genealogical concordance of unlinked lociprovide a conservative approach for assessingspecies boundaries due to the fact that a sub-stantial amount of time is required after the initialdivergence of species until ancestral polymor-phisms have fully sorted (Knowles and Carstens2007 Hudson and Coyne 2002) Consequentlygroups with recent divergence histories willlikely go undiscovered under a genealogicalconcordance criterion due to the fact that speciesboundaries likely are not reflected in genegenealogies For example it would take morethan 1 million years after speciation before spe-cies would be delimited if 15 loci were sampledin species with an effective population size (Ne)of 100000 assuming one generation a yearunder a strict reciprocal monophyly criterion(Knowles and Carstens 2007) The amount oftime required for a species to be recognized using
a criterion of reciprocal monophyly increasesproportionally with increasing population sizes(Hudson and Coyne 2002) In a number ofstudies of lichen-forming fungi a genealogicalconcordance criterion has been used to bothdelimit previously unrecognized species-levellineages and validate some conventional pheno-type-based species (Leavitt et al 2012a c2013b Molina et al 2011 Kroken and Taylor2001)
Although analyses of molecular sequence datarelying on reciprocal monophyly or fixed char-acter differences for species delimitation canserve as important criteria for identifying speciesthese criteria are not commonly met acrossmultiple loci particularly in recent speciationhistories (Fig 22) To accommodate theobserved conflict among genealogies from mul-tiple loci with the underlying speciation historythe recent merge of coalescent theory with phy-logenetics has driven a major paradigm shift inspecies delimitation and molecular systematics ingeneral (Fujita et al 2012 Edwards 2009) Themultispecies coalescent model can be applied togenealogical histories from multiple independentloci to assemble separate coalescent processesoccurring in populations into a species tree(Degnan and Rosenberg 2009 Rannala andYang 2003) Within this coalescent-basedframework multiple individuals can be assignedto a single speciespopulation and the speciationhistory of ancestral and descendant species-levellineages can be inferred as a ldquospecies treerdquo incontrast to estimating gene genealogies fromindividual samples (Degnan and Rosenberg2006 2009 Rannala and Yang 2003)
A number of multispecies coalescent-basedspecies delimitation methods have recently beenintroduced offering an exciting framework forempirically assessing species boundaries byselecting the best species tree model from a set ofalternative models representing differenthypotheses of species boundaries (Table 21)For example SpeDeSTEM (Ence and Carstens2011) finds the maximum likelihood values for aspecies tree assuming all putative species areseparate lineages and for alternative species treeswhere two or more species are collapsed into a
2 The Dynamic Discipline of Species Delimitation hellip 27
single lineage The best fitting model is thenselected using the Akaike information criterionunder the assumption of constant populationsizes and fixed gene topologies across the speciestree The species delimitation program BayesianPhylogenetic and Phylogeography (BPampPYangand Rannala 2010) accommodates gene treeuncertainty and variable population sizes andsamples from the Bayesian posterior distributionof species delimitation models using reversible-jump Markov chain Monte Carlo (rjMCMC)sampling BPampP requires a user-provided guidetree with resolution finer than the species leveland evaluates alternative modes derived from allpossible subtrees that are generated by collapsingor splitting nodes on the guide tree (Yang andRannala 2010 Rannala and Yang 2003) Theproportion of time spent on each model is pro-portional to the posterior probability of themodel ie ldquospeciation probabilitiesrdquo WhileBPampP ranks among the most popular coalescent-based species delimitation programs (Fujita et al2012) misspecifications of the guide tree andorthe prior distribution for population size (θ) canresult in strong support for models containing aninflated number of species (Leacheacute and Fujita2010 Zhang et al 2011) The nonparametricheuristic species delimitation approach imple-mented in the program Brownie (OrsquoMeara 2010)jointly sorts anonymous samples into species andinfers a species tree from input gene trees fromdifferent loci assuming that for a speciationevent the corresponding nodes on gene trees willbe more consistent with each than the diver-gences within species However finding both theoptimum species tree and species boundariesremains computationally challenging andBrownie has been shown to frequently yieldincorrect results (OrsquoMeara 2010)
Most recently available coalescent-basedspecies delimitation methods require individualassignments to a species or population a priori Ina number of scenarios correct assignments ofsamples to species may be difficult includingpresence of cryptic species incongruencebetween conventional species and moleculardata or simply the fact that accurate specimenidentification in complex groups with subtle or
difficult to discern diagnostic characters is anontrivial task (Leavitt et al 2013e) Statisticalmethods for assessing individual assignment andspecies detection prior to coalescent-based spe-cies delimitation and species tree reconstructionand species provide a more objective approachfor understanding species boundaries
Population assignment tests using a variety ofclustering algorithms using genetic data offer auseful approach for grouping individuals intoputative reproductively isolated groups (Table 21Hausdorf and Hennig 2010 Corander et al2004 2008 Falush et al 2003 Pritchard et al2000) particularly for species delimitation prob-lems that exist at the interface of traditionalpopulation genetic and phylogenetic analyses(Carstens et al 2013 Weisrock et al 2010Knowles and Carstens 2007)
The programs STRUCTURE (Falush et al2003 Pritchard et al 2000) and STRUCTURA-MA (Huelsenbeck et al 2011) cluster samplesinto populations by minimizing HardyndashWeinbergdisequilibrium for a given number of populationclusters using unlinked genetic markers In gen-eral unlinked markers are not available for mostgroups of lichen-forming fungi and a numberof studies have used information from multilocussequence data for STRUCTURE analyses(Leavitt et al 2011a b c 2013e Fernaacutendez-Mendoza and Printzen 2013 Fernaacutendez-Mendoza et al 2011) Varying approaches havebeen implemented to convert DNA sequencedata to allelic data for Bayesian clustering (seeOrsquoNeill et al 2013) STRUCTURE is expectedto perform well when there is sufficient inde-pendence across regions such that linkage dis-equilibrium within regions does not dominate thedata (STRUCTURE manual) but can also beeffective using multilocus sequence data andtreating all SNPs as independent loci regardlessof physical linkage within each locus (OrsquoNeillet al 2013 Falush et al 2003) A recent study ofthe lichen-forming genus Letharia showed thatBayesian clustering implemented in the programSTRUCTURE was generally able to recover thesame putative Letharia lineages circumscribedemploying a gene genealogical approach inKroken and Taylorrsquos iconic species delimitation
28 SD Leavitt et al
study (Altermann et al 2014 Kroken and Taylor2001) Altermann et al (2014) show that popu-lation assignments were largely consistent acrossa range of scenarios including extensiveamounts of missing data the exclusion of SNPsfrom variable markers and inferences based onSNPs from as few as three gene regions Incontrast to STRUCTURE and STRUCTURA-MA the program BAPS (Corander et al 2008)can explicitly infer genetic structure using hap-loid sequence data or other linked geneticmarkers in the ldquoclustering with linked locirdquomodel Another advantage of BAPS is that theprogram is much more computationally efficientthan STRUCTURE or STRUCTURAMA Sim-ulation studies indicate that both BAPS andSTRUCTURE perform well at low levels ofpopulation differentiation and when clusters arenot well differentiated (Latch et al 2006)
In practice inferring the most appropriatelevel of population structure using Bayesianclustering algorithms remains challenging (Latchet al 2006 Evanno et al 2005) BAPS andSTRUCTURAMA can infer both individualassignments and the most likely number ofgenetic clusters (Corander et al 2008 Huelsen-beck et al 2011) and the ad hoc ΔK statisticproposed by Evanno et al (2005) provides anobjective approach for identifying the uppermosthierarchical level of structure However carefulconsideration of other levels of populationstructure may ultimately reveal more biologicallymeaning results and researchers should examineindividual assignments across a range of geneticgroupings Some markers generated fromindependent genomic regions such as SNPs andfast-evolving microsatellites can be used to dis-tinguish fine-scale population structuring on thebasis of allele frequencies and species delimita-tion analyses based on these markers may implythe risk of severe taxonomic oversplitting Inthese cases validation approaches such asBPampP and SpeDeSTEM and corroborationamong different species delimitation approachescan provide perspective between intraspecificpopulation structure and species-level clusters
Another limitation of clustering approaches isthat they do not assess or take into account
evolutionarily divergences and relationshipsamong population clusters Coalescent-basedspecies tree methods (discussed below) provide ameans to assess the diversification histories ofpopulations inferred from clustering analysesTherefore a potential working protocol for aninformed species delimitation study that takesinto account population structure could consist offirst applying a genetic clustering analysis under apopulation genetics criterion (eg BAPSSTRUCTURE STRUCTURAMA) to identifygenetically distinct population clusters that can beconsidered ldquocandidate speciesrdquo From these can-didate species a species tree can be inferred forfocal group using coalescent-based species treereconstruction methods (eg BEAST) Subse-quently a coalescent-based validation methodcan be applied to assess whether the distinctpopulation clusters represent independent evolu-tionary lineages (eg BPampP) This multistepapproach would provide a consistent and standardcriterion for distinguishing between population-and species-level lineages (Camargo and Sites2013) and has been applied to a number of casesincluding lichen-forming fungi (Leavitt et al2011a b c 2013e Leacheacute and Fujita 2010)
High-throughput sequencing methods providethe means to effectively sample hundreds tothousands of loci from across a species genome fora large number of species and continue to revolu-tionize studies that can be performed even in non-model organisms Targeted high-throughputsequencing approaches such as anchored phy-logenomics transcriptome sequencing reduced-representation genomic library sequencing(restriction-site-associated DNA sequencingRAD-Seq and genotype-by-sequencing GBS)and high-throughput amplicon sequencing pro-vide important insight into species boundaries fora number groups although none of these approa-ches has yet been applied to studies of lichenizedfungi
For example restriction-site-associated(RAD-Seq) DNA can simultaneously detect andgenotype thousands of genome-wide SNPs byfocusing the sequencing effort on a reducedrepresentation of the entire genome (Baird et al2008) and has been successfully applied to
2 The Dynamic Discipline of Species Delimitation hellip 29
intraspecies (Lewis et al 2007 Miller et al 2007Emerson et al 2010) and interspecies studies(Rubin et al 2012 Eaton and Ree 2013 Wagneret al 2013) This approach has provided strikinginsight into the recent adaption radiation of LakeVictoria cichlids (Wagner et al 2013) Analternative approach targeting the sequencing ofspecific loci using next-generation sequencingplatforms provides an efficient means of gener-ating data for loci of known genomic locationorthology size and expected level of variation(OrsquoNeill et al 2013) Markers can be targetedusing well-established PCR techniques or newerhybridization techniques and subsequentlypooled for high-throughput sequencing usingparallel-tagged sequencing of multiple individualsamples within a single sequencing run (OrsquoNeillet al 2013) A recent study of North Americantiger salamanders (Ambystoma tigrinum)demonstrated the potential for amplicon-basedparallel-tagged sequencing to rapidly generatelarge-scale genomic data for species delimitationand species tree research (OrsquoNeill et al 2013)
Although these methods provide an enormousamount of data and insight into diversification atan unprecedented scale computational limita-tions restrict the applicable analytical methodsfor large datasets although computational andanalytical advances are happening rapidly Mostrecent coalescent-based species delimitation andspecies tree models using gene trees have beenlimited to handle tens of loci for multiple indi-viduals and combining hundreds or thousands ofgene trees into a single species delimitationframework presents considerable computationalchallenges (Camargo and Sites 2013) Therecently developed method Bayes factor delim-itation (with genomic data BFD) combines adynamic programming algorithm for estimatingspecies trees that bypasses the computationallyintensive MCMC integration over gene trees toprovide a rigorous technique for species delimi-tation studies using genome-wide SNP data(Leacheacute et al 2014) Competing species delimi-tation models are compared using Bayes factorsand it appears that this approach is robust tosample sizes (ie few loci and limited samples
per species) and misspecification of the prior forpopulation size (θ) (Leacheacute et al 2014)
Fungi are an ideal model for assessing diver-gence in eukaryotes due to their simple mor-phologies small genomes broad ecological rolesand diverse lifestyles (Gladieux et al 2014)However the use of genomic data from high-throughput sequencing methods have not yet beenincluded in species delimitation studies of lichen-forming fungi This is due in part to challenges indealing with metagenomic data containing geno-mic information from a plethora of symbiontsassociated with a lichen thallus scant genomicresources and discipline-specific inertia A num-ber of lichen-forming fungal genomes are cur-rently available including Endocarponpusillum(Wang et al 2014) Cladonia spp (Park et al2013a 2014) Caloplaca flavorubescens (Parket al 2013b) and Cladonia greyi and Xanthoriaparientina through the Fungal Genomics Programat Joint Genome Institute of the United StatesDepartment of Energy (httpwwwjgidoegov)The foreseeable ongoing expansion of genomicresources for lichen-forming fungi will be centralto developing approaches for delimiting speciesusing high-throughput sequencing Specificallygenomic resources will be crucial in identifyingnovel markers (variable genes regions SNPsmicrosatellites etc) identifying conserved fungalmarkers for targeted high-throughput sequencingapproaches and references for entire genomecomparisons (Devkota et al 2014 Werth et al2013)
23 Can We Make SpeciesDelimitation in Lichen-FormingFungi Truly Integrative
The widespread availability of genetic data hascreated a biased viewpoint that only genetic datashould be used for statistical species delimitationHowever an ongoing appeal to researchers toassess species boundaries from multiple andcomplementary perspectives (phylogeneticspopulation genetics comparative morphologydevelopment ecology etc) has resulted in an
30 SD Leavitt et al
integrative taxonomic framework bringing theseconceptual and methodological perspectivestogether (Hamilton et al 2014 Fujita et al 2012Salicini et al 2011 Yeates et al 2011 Padialet al 2010 Dayrat 2005 Will et al 2005 Wiensand Penkrot 2002) In practice any study linkingdifferent kinds of data to support hypotheses ofspecies boundaries including mapping morpho-logical characters onto a molecular phylogenycan be considered integrative Integrative meth-ods for species delimitation fall across a broadspectrum ranging from verbal and qualitativeassessments of data classes to quantitative meth-ods that allow different data types to contribute tostatistical species delimitation (Yeates et al2011) In most taxonomic studies utilizing bothmolecular and morphological data expert opinionis eventually used to some degree to evaluate thefinal status of a candidate species (eg Bond andStockman 2008) In this sense many studiesusing evidence for independent data sources fordelimiting species boundaries use an iterativeapproach (Yeates et al 2011) where speciesboundaries can be tested within a hypothetico-deductive framework with diverse datasets
From a practical perspective we advocate aprocess of iterative taxonomy (sensu Yeates et al2011) to circumscribe and refine species limitsusing multiple lines of evidence This iterativeprocess involves comparisons of morphologicaldata with a phylogenetic hypothesis (ie single-locus gene tree or concatenated multilocusphylogeny) to identify the least inclusive mono-phyletic clade in the topology characterized by atleast one unambiguously diagnostic morphologi-cal character (Miralles and Vences 2013) Thisphylogenetic tree-informed approach to assessingspecies boundaries represents a common practicein studies of lichen-forming fungi Previouslyunrecognized species-level clades with corre-sponding subtle or overlooked phenotypiccharacters have been commonly observed in bothcrustose lichens [eg Acarospraceae (Wedin et al2009) Graphidaceae (Rivas Plata and Lumbsch2011 Papong et al 2009) Lecanoraceae (Leavittet al 2011a) Lecideaceae (Ruprecht et al 2010)Mycoblastaceae (Spribille et al 2011) andTeloschistaceae (Vondraacutek et al 2009 Muggia
et al 2008)] and foliose and fruticose lichens [egLobariaceae (Moncada et al 2014 McDonaldet al 2003) Parmeliaceae (Leavitt et al 2013aWirtz et al 2012 Divakar et al 2005 Molinaet al 2004 Kroken and Taylor 2001) Peltigera-ceae (OrsquoBrien et al 2009 Goffinet et al 2003)Physciaceae (Elix et al 2009 Divakar et al2007) and Sphaerophoraceae (Houmlgnabba andWedin 2003)] As a specific example Luumlckinget al (2008) used a combination of medullarychemistry and underside pigmentation in speci-mens from the Heterodermia obscurata group inCosta Rica to corroborate monophyletic clades inan ITS gene tree as species-level lineages
While this iterative approach to speciesdelimitation and taxonomy has proven valuablefor understanding species boundaries anddescribing new taxa in some groups of lichenizedfungi a posteriori examination of morphologicaland chemical features has failed to reveal diag-nostic phenotypic characters in a number ofstudies (Muggia et al 2014 Leavitt et al 2011aOtaacutelora et al 2010 OrsquoBrien et al 2009) Fur-thermore a study of widespread species in thegenus Melanelixia (Parmeliaceae) indicated thatphenotypically cryptic lichen-forming fungalspecies-level lineages may be relatively ancientand diagnosable phenotypic differences may beabsent even millions of years after the initialdivergence (Leavitt et al 2012b) The latter studyhighlights the fact that species-level lineages maycommonly exist without any observable diag-nostic phenotypic characters calling into ques-tion the impetus for a universal application ofintegrative taxonomy
In keeping with the principle that as manylines of evidence as available should be com-bined to delimit species (Dayrat 2005) the for-malized integrative taxonomic approach (ITAXMiralles and Vences 2013) uses a mtDNA guidetree and observations from different types of datathat might provide conclusive evidence for theindependence of lineages and thus their identityas different species Miralles and Vences (2013)provide a non-exhaustive list of species delimi-tation criteria to be integrated in the ITAXapproach including (a) sympatric occurrencewithout admixture as revealed by consistent
2 The Dynamic Discipline of Species Delimitation hellip 31
differences in morphological or molecular char-acters at the same geographic location (b) strongdifferences in a behavioral morphological orgenetic character known to mediate prematingisolation (c) unviability or infertility of hybrids(d) lack of gene flow across a geographicalhybrid zone (e) congruent diagnostic differencesbetween sister lineages in various unlinkedmorphological character (f) absence of haplo-type sharing in several unlinked nuclear loci and(g) a combination of criteria endashf Speciesboundaries are based on seeking the least inclu-sive monophyletic group in the mtDNA treewhich fulfills at least one of the criteria listedabove Clearly the ITAX approach is sensitive tosample size and in order to reliably support thedistinctiveness of a given species it has beenrecommended that the sampling strategy includesat least five individuals per species (Miralles andVences 2013) A similar approach could beadopted for lichenized fungi using an ITS genetopology as guide tree rather than mtDNA
A total evidence approach including concat-enation has been a common approach for inte-grating information from different sourcesincluding independent genetic markers in phy-logenetic reconstructions (Kluge 1989 Wiens1998 de Queiroz et al 1995) Proponents forconcatenation of independent data in phyloge-netic analyses argue that when combining alldata the underlying signal of speciation mayemerge even if weakly contradictory signalis contained in the individual data partitions(Gatesy et al 1999) For example establishing apreliminary perspective of species boundariesfrom multilocus sequence data using concatena-tion may provide a reasonable starting point forscreening for cryptic species and species treeinference (Šlapeta et al 2006 Leavitt et al2011a Le Gac et al 2007 Leacheacute 2009)However concatenation and consensus methodsimply a risk of obtaining inflated support forincorrect relationships and information aboutvariance in gene coalescence is lost (Degnan andRosenberg 2006 2009 Kubatko and Degnan2007 Edwards 2009) In spite of the impetus forintegrating evidence from independent data
sources into empirical species delimitation stud-ies and taxonomy (Fujita et al 2012 Padial et al2009) most currently available species delimi-tation methods are unable to accommodate non-genetic data sources in a statistical framework
231 Selecting the Appropriate Data
In the face of increasing availability of geneticdata and associated bioinformatical approachesfor delimiting species researchers should care-fully consider what information is being sacri-ficed by the failure to consider non-genetic datain species delimitation studies and whetheraccuracy could be improved by the addition ofmultiple data types Morphological data havehistorically served as a proxy to identify repro-ductively isolated groups (ie ldquospeciesrdquo) (Ray1686 Fujita et al 2012) Current methods fordelimiting species using non-genetic data (egchemistry morphology and ecology) remainwoefully understudied For example morphol-ogy-based species circumscriptions are generallybased on one or more qualitative (or quantitative)morphological characters that do not appear tooverlap with other species However ascertain-ing that a given trait is truly fixed within apopulation with statistical confidence requiresunrealistic sample sizes even when allowing forsome level of polymorphism in the diagnosticcharacter (Wiens and Servedio 2000) Now var-ious combinations of datamdashfrom morphologygenetics geography and ecologymdashare acceptedas standard information for species delimitationstudies (Ruiz-Sanchez and Sosa 2010 Ross et al2010 Edwards and Knowles 2014)
Below we briefly discuss appropriate datasources for species delimitation studies of lichen-forming fungi However homoplastic characters(similar traits that are not derived from a com-mon ancestor) are common among many traitscommonly used to circumscribe fungal taxa thebiological significance of secondary metabolitevariation remains largely unknown (Lawrey1986) and ecological niches may be difficultto adequately characterize and model due to
32 SD Leavitt et al
microhabitat requirements and data resolutionTherefore we advocate a cautious approach toselecting appropriate and relevant data forassessing species boundaries
In spite of the potential challenges and limi-tations of using phenotypic data these traits haveprovided a plethora of valuable information forunderstanding species boundaries For examplein the Melanelixia fuliginosa group (Parmelia-ceae) a morphometric analysis using color isi-dia and marginal zone free of isidia ascharacters revealed a general pattern of differ-entiation between material formerly recognizedas subspecies fuliginosa and glabratula in spiteof the fact that considerable overlap betweengroups occurs in some characters individually(Arup and Berlin 2011) Furthermore the dis-tinction of the two groups was supported by aphylogenetic analysis of the ITS marker andecological differences with M fuliginosaoccurring predominantly on rock and M glab-ratula on bark (Arup and Berlin 2011) Thisstudy of the M fuliginosa group provides a fit-ting example of using multiple independentsuites of data including ecology morphologyand genetic information to establish a robusthypothesis of species boundaries
In lichen systematics phenotypic dataincluding thallus organization secondary metab-olites mode of reproduction ascoma-type andontogeny ascus and ascospore characters havehistorically played a prominent role (Printzen2009) Ascomatal characters have traditionallyheld a major role in higher-level classification(Printzen 2009) in contrast to species-levelclassification which also tends to include a widearray of vegetative and chemical charactersCommonly assessed morphological charactersinclude thallus form and size cortical features(eg maculae and pseudocyphellae) presenceformcolor of attachment structure (eg rhizines)and reproductive mode (ascomata vs vegetativediaspores) (Printzen 2009) Different morpho-logical types of vegetative diasporesmdashcorticatedisidia and ecorticated sorediamdashand their locationare commonly used to distinguish speciesAscomatal characters including morphologylocation (laminal vs marginal) position (sessile
vs immersed) presence of thalline margins colorof an apothecial disc and presence or color ofpruina are also commonly used in speciesdelimitations of lichen-forming fungi (Printzen2009) Other important characters may includethalline characters form color size and septationof ascospores size and form and structure of ascithe hamathecium type of epihymenium andhypothecium and the type of excipulum orperidium conidiomatal characters etc (Printzen2009)
Assessments of secondary metabolites hasplayed an important role in lichen taxonomybeginning with the introduction of simple spottests by Nylander (1866a b) The use of chem-istry in lichen taxonomy has been discussed indetail in numerous reviews (Lumbsch 1998a bRogers 1989 Brodo 1986 Egan 1986 Leuckert1985 Brodo 1978 Hawksworth 1976 Culber-son 1969 1970) and we refer readers to thesevaluable sources for a more comprehensive per-spective on lichen chemistry In short extrolites(secondary metabolites) belong to various clas-ses the most common and diverse include dep-sides depsidones chlorinated xanthones andanthraquinones (Lumbsch 2002 Culberson1969) The presence or absence of specificextrolites or their replacements by another sub-stance is widely used to distinguish speciesparticularly when correlated with differences ingeographic distributions However if morpho-logical or geographical differences betweenpopulations containing different extrolites are notapparent the taxonomic significance has beendisputed with some authors distinguishing themas species and others preferring to regard them aschemical races within a species In addition tosimply using the presence or absence of extro-lites Culberson and Culberson (1976) proposedto arrange lichen substances into chemosyn-dromes of closely related substances The pres-ence or absence of these chemosyndromes maypotentially be used as characters to delimit spe-cies regarding differences involving the samechemosyndromes as intraspecific variation anddistinct chemosyndromes as evidence for inter-specific populations (Lumbsch 1994 Gowan1986)
2 The Dynamic Discipline of Species Delimitation hellip 33
Due to the fact that species delimitationstudies often incorporate a substantial biogeo-graphical or ecological component ecologicalniche modeling plays an increasing role in phy-logenetic and taxonomic research Niche mod-eling can provide evidence for ecologicalisolation between populations based on eitherconserved or divergent ecological niches andtherefore can provide additional evidence sup-porting lineage independence between putativespecies The application of ecological nichemodeling has been applied to species delimita-tion studies in a number of cases (Ruiz-Sanchezand Sosa 2010 Leacheacute et al 2009 Raxworthyet al 2007 Rissler and Apodaca 2007) althoughit has not been explicitly applied to assess speciesboundaries in lichen-forming fungi By mappingthe spatial distribution of environmental suit-ability of climatic variables for candidate speciesthe application of ecological niche modeling canbe particularly important in cases where specieshave allopatric distributions (Raxworthy et al2007)
Ecological niche modeling utilizes knownassociations between a speciesrsquo occurrencelocalities and environmental variables to defineabiotic conditions within which populations canbe maintained (Guisan and Thuiller 2005) Themethodological approach for modeling is basedon four general properties (i) The current knownspeciesrsquo localities is the dependent variable (ii)the distribution is modeled as a map composed ofgrid cells at a specified resolution (iii) a range ofenvironmental variables (eg temperature pre-cipitation and solar exposure) are collected todescribe the characteristics of each cell and (iv)classifying the degree to which each cell is eithersuitable or unsuitable for each species under arange of models (Guisan and Thuiller 2005)
Ecological data also have the potential to playan important role in understanding speciesboundaries in lichen-forming fungi For exampleNash and Zavada (1977) demonstrated that Xant-hoparmelia populations with distinct chemistriesoccurring in the northern portion of the SonoranDesert exhibit habitat selection among differentrock substrates within a region with relativelyuniform climate and topography In another case
the parmelioid species Parmelia mayi is mor-phologically indistinguishable from P saxatilisbut can be separated by bioclimatic features andgenetic and chemical characters (Molina et al2011) McCune and Printzen(2011) assess distri-butions and climatic niches of species in theLecanora varia group inwesternUSAandprovidea model that uses continental influence and annualtemperature as themajor factors predicting speciesdistributions The distribution of Usnea hirta alichen commonly used in air quality biomonitor-ing research was modeled for a section of theWhite River National Forest in central Coloradobased on the presence of U hirta at 72 biomoni-toring reference sites distributed in the inter-mountain western United States (Shrestha et al2012) The best model for predicting U hirtadistribution included four variablesmdashsolar radia-tion average monthly precipitation and averagemonthly minimum and maximum temperatures(Shrestha et al 2012) These studies supportthe potential use of ecological niche modelingmethods in species delimitation studies of lichen-forming fungi
24 Conclusions What AboutTaxonomy
In most cases species circumscription and tax-onomy requires some degree of qualitativejudgment and individual interpretation Integrat-ing multiple types of data into an empiricalframework for delimiting species boundarieswhere species boundaries can be tested within ahypothetico-deductive framework with diversedatasets can provide robust hypotheses of speciesboundaries and taxonomic stability (Yeates et al2011) However it has long been known toevolutionary biologists that distinct species donot need to have diagnosable morphologicaldifferences (Mayr 1963) and increasing avail-ability of genetic data has allowed researchers toidentify species and to rigorously test speciesboundaries with a level of precision that wasunimaginable a decade ago While analyticaladvances in statistical species delimitation havebeen largely based on genetic data the utility of
34 SD Leavitt et al
these approaches to formal taxonomy remainselusive Due to these challenges an eclecticapproach to delimiting species and cautionagainst the reliance on any single dataset ormethod is required when delimiting species
While results from many recent studies maycontradict traditional species boundaries acrossmany groups of lichen-forming fungi we areoptimistic that this research represents substantialprogress toward a more accurate perspective onspecies boundaries and diversity in fungi As aresult of ongoing research of species boundariesin lichen-forming fungi the taxonomic value ofmany phenotypes is now better understood ourunderstanding of ecological evolutionary andbiogeographic patterns has improved and we canbegin to better understand patterns of symbiontinteractions in lichens Integrating new data(including novel morphological characters andgenetic data) will be essential to accurately rep-resent species-level diversity across all groups oflichenized fungi Hopefully an improved per-spective on lichen diversity also increases ourappreciation of these incredible symbiotic sys-tems While we are strong advocates for theapplication of independent data types in devel-oping an integrative taxonomy there is anincreasing need to formally recognize the exis-tence of phenotypically cryptic species-levellineages in lichen-forming fungi (see Hibbettet al 2011) In some cases a molecular taxon-omy may provide the most practical approach toconsistent treatment of mycobiont species withinlichen groups where diagnostic morphologicalcharacters are unidentifiable or practically notfeasible (Leavitt et al 2013c d)
These are exciting times for taxonomists andphylogeneticists A closer look at lichen taxon-omy with the inclusion of new data will help usto better understand the diversity of these fasci-nating organisms accurately interpret distribu-tion patterns and play a more important role inmeaningful conservation practices Howeversome level of uncertainty will accompany pro-gress In many taxonomic groups our traditionalapproach for species identification will likelyneed to be substantially modified The search forcorroborating morphological support for cryptic
species identified using molecular data willrequire meticulous and creative approaches toassess phenotypic variation in potentially unor-thodox ways We are hopeful that lichenologistswho traditionally have been eager to include newmethods such as chromatography in their rou-tine identifications will be amenable to includemolecular techniques to their routine examina-tion of specimens for identification and classifi-cation Although this may prove difficult toachieve by single individuals especially citizenscientists that traditionally play an important rolein lichen taxonomy the increasing numberof collaborative projects in the discipline (egLumbsch et al 2011 Crespo et al 2010Gueidan et al 2009) make us optimistic thatbroad-scale collaborative approaches will facili-tate the inclusion of molecular data in lichenresearch at all levels
Acknowledgments We are indebted to various col-leagues for valuable thought-provoking discussionnotably Matthew Nelsen (University of Chicago) AnaCrespo (Universidad Complutense de Madrid) PradeepDivakar (Universidad Complutense de Madrid) BeckettSterner (The Field Museum) and Joyce Havstad (TheField Museum) We also thank anonymous reviewers whoprovided valuable comments that improved this chapterSupport by the US National Science Foundation isgratefully acknowledged (ldquoHidden diversity in parmelioidlichensrdquo DEB-0949147)
References
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Altermann S Leavitt SD Goward T Nelsen MPLumbsch HT (2014) How do you solve a problemlike Letharia A new look at cryptic species in lichen-forming fungi using Bayesian clustering and SNPSsfrom multilocus sequence data PLoS ONE 9(5)e97556 doi101371journalpone0097556
Amo de Paz G Cubas P Crespo A Elix JA Lumbsch HT(2012) Transoceanic dispersal and subsequent diver-sification on separate continents shaped diversity ofthe Xanthoparmelia pulla group (Ascomycota) PLoSONE 7(6)e39683 doi101371journalpone0039683
Arguumlello A Del Prado R Cubas P Crespo A (2007)Parmelina quercina (Parmeliaceae Lecanorales)
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includes four phylogenetically supported morphospe-cies Biol J Linn Soc 91(3)455ndash467 doi101111j1095-8312200700810x
Arup U Berlin ES (2011) A taxonomic study ofMelanelixia fuliginosa in Europe The Lichenologist43(02)89ndash97 doi101017S0024282910000678
Arup U Grube M (2000) Is Rhizoplaca (Lecanoraleslichenized Ascomycota) a monophyletic genus Can JBot 78(3)318ndash327 doi101139b00-006
Avise J Ball R (1990) Principles of genealogicalconcordance in species concepts and biological tax-onomy Oxf Surv Evol Biol 745ndash67
Baird NA Etter PD Atwood TS Currey MC Shiver ALLewis ZA Selker EU Cresko WA Johnson EA(2008) Rapid SNP discovery and genetic mappingusing sequenced RAD markers PLoS ONE 3(10)e3376 doi101371journalpone0003376
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Beaumont MA Nielsen R Robert C Hey J Gaggiotti OKnowles L Estoup A Panchal M Corander JHickerson M Sisson SA Fagundes N Chikhi LBeerli P Vitalis R Cornuet J-M Huelsenbeck J FollM Yang Z Rousset F Balding D Excoffier L (2010)In defence of model-based inference in phylogeogra-phy Mol Ecol 19(3)436ndash446 doi101111j1365-294X200904515x
Bickford D Lohman DJ Sodhi NS Ng PKL Meier RWinker K Ingram KK Das I (2007) Cryptic speciesas a window on diversity and conservation TrendsEcol Evol 22(3)148ndash155 doi101016jtree200611004
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Bond J Stockman A (2008) An integrative method fordelimiting cohesion species finding the population-species interface in a group of Californian trapdoorspiders with extreme genetic divergence and geo-graphic structuring Syst Biol 57(4)628ndash646 doi10108010635150802302443
Brodo IM (1978) Changing concepts regarding chemicaldiversity in lichens The Lichenologist 10(1)1ndash11doi101017S0024282978000031
Brodo IM (1986) Interpreting chemical variation inlichens for systematic purposes The Bryologist 89(2)132ndash138
Caley MJ Fisher R Mengersen K (2014) Global speciesrichness estimates have not converged Trends EcolEvol 29(4)187ndash188 doi101016jtree201402002
Camargo A Sites JW (2013) Species delimitation adecade after the Renaissance In Pavlinov I (ed) Thespecies problemmdashongoing issues InTech doi10577252664
Camargo A Morando M Avila LJ Sites JW (2012)Species delimitation with ABC and other coalescent-based methods a test of accuracy with simulations and
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Carstens BC Dewey TA (2010) Species delimitationusing a combined coalescent and information-theoreticapproach an example from North American Myotisbats Syst Biol 59(4)400ndash414 doi101093sysbiosyq024
Carstens BC Pelletier TA Reid NM Satler JD (2013)How to fail at species delimitation Mol Ecol 22(17)4369ndash4383 doi101111mec12413
Corander J Marttinen P (2006) Bayesian identification ofadmixture events using multi-locus molecular mark-ers Mol Ecol 15(10)2833ndash2843 doi101111j1365-294X200602994x
Corander J Waldmann P Marttinen P Sillanpaa M(2004) BAPS 2 enhanced possibilities for the analysisof genetic population structure Bioinformatics 20(15)2363ndash2369 doi101093bioinformaticsbth250
Corander J Marttinen P Mantyniemi S (2006) Bayesianidentification of stock mixtures from molecular markerdata Fish Bull 104550ndash558
Corander J Marttinen P Siren J Tang J (2008) EnhancedBayesian modelling in BAPS software for learninggenetic structures of populations BMC Bioinf 9(1)539 doi1011861471-2105-9-539
Coyne JA Orr HA (2004) Speciation Sinauer AssociatesSunderland
Cracraft J (1983) Species concepts and speciation anal-ysis Curr Ornithol 1159ndash187
Crespo A Lumbsch HT (2010) Cryptic species in lichen-forming fungi IMA Fungus 1167ndash170 doi105598imafungus2010010209
Crespo A Peacuterez-Ortega S (2009) Cryptic species andspecies pairs in lichens a discussion on the relation-ship between molecular phylogenies and morpholog-ical characters Anales del Jardin Botanico de Madrid66(S1)71ndash81 doi103989ajbm2225
Crespo A Kauff F Divakar PK del Prado R Perez-Ortega S Amo de Paz G Ferencova Z Blanco ORoca-Valiente B Nunez-Zapata J Cubas P ArguelloA Elix JA Esslinger TL Hawksworth DL MillanesA Molina MC Wedin M Ahti T Aptroot A et al(2010) Phylogenetic generic classification of parmeli-oid lichens (Parmeliaceae Ascomycota) based onmolecular morphological and chemical evidenceTaxon 59(6)1735ndash1753
Culberson WL (1969) The use of chemistry in thesystematics of the lichens Taxon 18498ndash505
Culberson WL (1970) Chemosystematics and ecology oflichen-forming fungi Annu Rev Ecol Syst 1153ndash170
Culberson CF Culberson WL (1976) Chemosyndromicvariation in lichens Syst Bot 1325ndash339
Darwin C (1859) On the origin of species by means ofnatural selection or the preservation of favoured racesin the struggle for life J Murray London
Dayrat B (2005) Towards integrative taxonomy Biol JLinn Soc 85(3)407ndash415 doi101111j1095-8312200500503x
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de Queiroz K (1998) The general lineage concept ofspecies species criteria and the process of speciationa conceptual unification and terminological recom-mendations In Howard DJ Berlocher SH (eds)Endless forms species and speciation Oxford Uni-versity Press Oxford pp 57ndash75
de Queiroz K (1999) The general lineage concept ofspecies and the defining properties of the speciescategory In Wilson RA (ed) Species new interdis-ciplinary essays MIT Press Cambridge pp 49ndash89
de Queiroz K (2007) Species concepts and speciesdelimitation Syst Biol 56(6)879ndash886 doi10108010635150701701083
de Queiroz A Donoghue MJ Kim J (1995) Separateversus combined analysis of phylogenetic evidenceAnnu Rev Ecol Syst 26(1)657ndash681 doi101146annureves26110195003301
Degnan J Rosenberg N (2006) Discordance of speciestrees with their most likely gene trees PLoS Genet 2(5)e68 doi101371journalpgen0020068
Degnan JH Rosenberg NA (2009) Gene tree discordancephylogenetic inference and the multispecies coales-cent Trends Ecol Evol 24(6)332ndash340 doihttpdxdoiorg101016jtree200901009
Del-Prado R Cubas P Lumbsch HT Divakar PK BlancoO de Paz GA Molina MC Crespo A (2010) Geneticdistances within and among species in monophyleticlineages of Parmeliaceae (Ascomycota) as a tool fortaxon delimitation Mol Phylogenet Evol 56(1)125ndash133 doi101016jympev201004014
Del-Prado R Divakar PK Crespo A (2011) Using geneticdistances in addition to ITS molecular phylogeny toidentify potential species in the Parmotrema reticul-atum complex a case study The Lichenologist 43(06)569ndash583 doi101017S0024282911000582
Del-Prado R Blanco O Lumbsch HT Divakar PK ElixJA Molina MC Crespo A (2013) Molecular phylog-eny and historical biogeography of the lichen-formingfungal genus Flavoparmelia (Ascomycota Parmelia-ceae) Taxon 62(5)928ndash939 doi101270562522
Dettman J Jacobson D Taylor J (2003a) A multilocusgenealogical approach to phylogenetic species recog-nition in the model eukaryote Neurospora Evolution57(12)2703ndash2720 doi101111j0014-38202003tb01514x
Dettman JR Jacobson DJ Turner E Pringle A TaylorJW (2003b) Reproductive isolation and phylogeneticdivergence in Neurospora comparing methods ofspecies recognition in a model eukaryote Evolution57(12)2721 doi10155403-074
Devkota S Cornejo C Werth S Chaudhary RP Sche-idegger C (2014) Characterization of microsatelliteloci in the Himalayan lichen fungus Lobaria pindar-ensis (Lobariaceae) Appl Plant Sci 2(5)1300101doi103732apps1300101
Divakar PK Molina MC Lumbsch HT Crespo A (2005)Parmelia barrenoae a new lichen species related toParmelia sulcata (Parmeliaceae) based on molecularand morphological data The Lichenologist 37(01)37ndash46 doi101017S0024282904014641
Divakar PK Amo De paz G del Prado R Esslinger TLCrespo A (2007) Upper cortex anatomy corroboratesphylogenetic hypothesis in species of Physconia(Ascomycota Lecanoromycetes) Mycol Res 111(11)1311ndash1320 doi101016jmycres200708009
Divakar PK Figueras G Hladun N Crespo A (2010)Molecular phylogenetic studies reveal an undescribedspecies within the North American concept of Mela-nelixia glabra (Parmeliaceae) Fungal Divers 42(1)47ndash55 doi101007s13225-010-0027-3
Donoghue MJ Gauthier A (2004) Implementing thephylocode Trends Ecol Evol 19(6)281ndash282 doi101016jtree200404004
Eaton DAR Ree RH (2013) Inferring phylogeny andintrogression using RADseq data an example fromflowering plants (Pedicularis Orobanchaceae) SystBiol 62(5)689ndash706 doi101093sysbiosyt032
Edwards SV (2009) Is a new and general theory ofmolecular systematics emerging Evolution 63(1)1ndash19 doi101111j1558-5646200800549x
Edwards DL Knowles LL (2014) Species detection andindividual assignment in species delimitation canintegrative data increase efficacy Proc R Soc B BiolSci 281(1777) doi101098rspb20132765
Egan RS (1986) Correlations and non-correlations ofchemical variation patterns with lichen morphologyand geography The Bryologist 8999ndash110
Elix JA Corush J Lumbsch HT (2009) Triterpenechemosyndromes and subtle morphological characterscharacterise lineages in the Physcia aipolia group inAustralia (Ascomycota) Syst Biodivers 7(04)479ndash487 doi101017S1477200009990223
Emerson KJ Merz CR Catchen JM Hohenlohe PACresko WA Bradshaw WE Holzapfel CM (2010)Resolving postglacial phylogeography using high-throughput sequencing Proc Natl Acad Sci 107(37)16196ndash16200 doi101073pnas1006538107
Ence DD Carstens BC (2011) SpedeSTEM a rapid andaccurate method for species delimitation Mol EcolResour 11(3)473ndash480 doi101111j1755-0998201002947x
EvannoGRegnaut SGoudet J (2005)Detecting thenumberof clusters of individuals using the software STRUC-TURE a simulation study Mol Ecol 14(8)2611ndash2620doi101111j1365-294X200502553x
Falush D Stephens M Pritchard JK (2003) Inference ofpopulation structure using multilocus genotype datalinked loci and correlated allele frequencies Genetics164(4)1567ndash1587 doi101111j1471-8286200701758x
Fan HH Kubatko LS (2011) Estimating species treesusing approximate Bayesian computation Mol Phylo-genet Evol 59(2)354ndash363 doi101016jympev201102019
Fernaacutendez-Mendoza F Printzen C (2013) Pleistoceneexpansion of the bipolar lichen Cetraria aculeata intothe Southern hemisphere Mol Ecol 22(7)1961ndash1983doi101111mec12210
Fernaacutendez-Mendoza F Domaschke S Garciacutea MA JordanP Martin MP Printzen C (2011) Population structure
2 The Dynamic Discipline of Species Delimitation hellip 37
of mycobionts and photobionts of the widespreadlichen Cetraria aculeata Mol Ecol 20(6)1208ndash1232doi101111j1365-294X201004993x
Fraley C Raftery A (2007) Model-based methods ofclassification Using the mclust software in chemo-metrics J Stat Softw 18i06 [Available online at httpwwwdoajorgdoajfunc=abstractampid=218544]
Fujisawa T Barraclough TG (2013) Delimiting speciesusing single-locus data and the generalized mixed yulecoalescent (GMYC) approach a revised method andevaluation on simulated datasets Syst Biol 62(5)707ndash724 doi101093sysbiosyt033
Fujita MK Leacheacute AD Burbrink FT McGuire JA MoritzC (2012) Coalescent-based species delimitation in anintegrative taxonomy Trends Ecol Evol 27(9)480ndash488 doi101016jtree201204012
Gatesy J OrsquoGrady P Baker RH (1999) Corroborationamong data sets in simultaneous analysis hiddensupport for phylogenetic relationships among higherlevel Artiodactyl taxa Cladistics 15(3)271ndash313doi101111j1096-00311999tb00268x
Gladieux P Ropars J Badouin H Branca A Aguileta Gde Vienne DM Rodriacuteguez de la Vega RC Branco SGiraud T (2014) Fungal evolutionary genomicsprovides insight into the mechanisms of adaptivedivergence in eukaryotes Mol Ecol 23(4)753ndash773doi101111mec12631
Goffinet B Miadlikowska J Goward T (2003) Phyloge-netic inferences based on nrDNA sequences supportfive morphospecies within the Peltigera didactylacomplex (Lichenized Ascomycota) The Bryologist106(3)349ndash364 doi10163901
Gowan SP (1986) Evolution of secondary natural productsin the genus Porpidia (Ascomycata Porpidiaceae) AmJ Bot 73606
Griffin PC Hoffmann AA (2014) Limited genetic diver-gence among Australian alpine Poa tussock grassescoupled with regional structuring points to ongoinggene flow and taxonomic challenges Ann Bot doi101093aobmcu017
Grube M Hawksworth DL (2007) Trouble with lichenthe re-evaluation and re-interpretation of thallus formand fruit body types in the molecular era Mycol Res111(9)1116ndash1132 doi101016jmycres200704008
Gueidan C Savi S Thues H Roux C Keller C Tibell LPrieto M Heimarsson S Breuss O Orange A FrobergL Wynns AA Navarro-Rosines P Krzewicka BPykaelae J Grube M Lutzoni F (2009) Genericclassification of the Verrucariaceae (Ascomycota)based on molecular and morphological evidencerecent progress and remaining challenges Taxon 58(1)184ndash208
Guillot G Mortier F Estoup A (2005) Geneland acomputer package for landscape genetics Mol EcolNotes 5(3)712ndash715 doi101111j1471-8286200501031x
Guillot G Renaud S Ledevin R Michaux J Claude J(2012) A unifying model for the analysis of pheno-typic genetic and geographic data Syst Biol 61(6)897ndash911 doi101093sysbiosys038
Guisan A Thuiller W (2005) Predicting species distribu-tion offering more than simple habitat models EcolLett 8(9)993ndash1009 doi101111j1461-0248200500792x
Hale ME (1990) A synopsis of the lichen genusXanthoparmelia (Vainio) Hale (AscomycotinaParmeliaceae) Smithsonian Institution Press Wash-ington DC
Hamilton CA Hendrixson BE Brewer MS Bond JE(2014) An evaluation of sampling effects on multipleDNA barcoding methods leads to an integrativeapproach for delimiting species a case study ofthe North American tarantula genus Aphonopelma(Araneae Mygalomorphae Theraphosidae) MolPhylogenet Evol 7179ndash93 doi101016jympev201311007
Hausdorf B (2011) Progress toward a general speciesconcept Evolution 65(4)923ndash931 doi101111j1558-5646201101231x
Hausdorf B Hennig C (2010) Species delimitation usingdominant and codominant multilocus markers SystBiol 59(5)491ndash503 doi101093sysbiosyq039
Hawksworth DL (1976) Lichen chemotaxonomy InBailey RH (ed) Lichenology progress and problemsAcademic London pp 139ndash184
Hebert PDN Cywinska A Ball SL deWaard JR (2003)Biological identifications through DNA barcodesProc R Soc Lond B Biol Sci 270(1512)313ndash321doi101098rspb20022218
Hebert PDN Stoeckle MY Zemlak TS Francis CM(2004) Identification of birds through DNA barcodesPLoS Biol 2(10)e312 doi101371journalpbio0020312
Heled J Drummond AJ (2010) Bayesian inference ofspecies trees from multilocus data Mol Biol Evol 27(3)570ndash580 doi101093molbevmsp274
Hey J (2006) On the failure of modern species conceptsTrends Ecol Evol 21(8)447ndash450 doi101016jtree200605011
Hibbett DS OhmanA Glotzer D NuhnMKirk P NilssonRH (2011) Progress in molecular and morphologicaltaxon discovery in Fungi and options for formalclassification on environmental sequences Fungal BiolRev 2538ndash47 doi101016jfbr201101001
Houmlgnabba F Wedin M (2003) Molecular phylogeny ofthe Sphaerophorus globosus species complex Cladis-tics 19(3)224ndash232 doi101111j1096-00312003tb00365x
Hudson RR Coyne JA (2002) Mathematical conse-quences of the genealogical species concept Evolu-tion 56(8)1557 doi101111j0014-38202002tb01467x
Huelsenbeck JP Andolfatto P Huelsenbeck ET (2011)Structurama Bayesian inference of population struc-ture Evol Bioinf Online 7(2011)55ndash59 doi104137EBOS6761
Jones G Oxelman B (2014) DISSECT an assignment-free Bayesian discovery method for species delimita-tion under the multispecies coalescent Bioinformaticsdoi101101003178
38 SD Leavitt et al
Kekkonen M Hebert PD (2014) DNA barcode-baseddelineation of putative species efficient start fortaxonomic workflows Mol Ecol Res doi1011111755-099812233
Kelly LJ Hollingsworth PM Coppins BJ Ellis CJHarrold P Tosh J Yahr R (2011) DNA barcoding oflichenized fungi demonstrates high identification suc-cess in a floristic context New Phytol 191(1)288ndash300doi101111j1469-8137201103677x
Kiss L (2012) Limits of nuclear ribosomal DNA internaltranscribed spacer (ITS) sequences as species barcodesfor fungi Proc Natl Acad Sci 109(27)E1811ndashE1811doi101073pnas1207143109
Kluge AG (1989) A concern for evidence and aphylogenetic hypothesis for relationships among Epi-crates (Boidae Serpentes) Syst Zool 38(1)7ndash25doi101093sysbio3817
Knowles LL Carstens BC (2007) Delimiting specieswithout monophyletic gene trees Syst Biol 56(6)887ndash895 doi10108010635150701701091
Kroken S Taylor JW (2001) A gene genealogical approachto recognize phylogenetic species boundaries in thelichenized fungus Letharia Mycologia 93(1)38ndash53
Kubatko LS Degnan JH (2007) Inconsistency of phylo-genetic estimates from concatenated data under coa-lescence Syst Biol 56(1)17ndash24 doi10108010635150601146041
Latch EK Dharmarajan G Glaubitz JC Rhodes OE Jr(2006) Relative performance of Bayesian clusteringsoftware for inferring population substructure andindividual assignments at low levels of populationdifferentiation Conserv Genet 7(2)295ndash302 doi101007s10592-005-9098-1
Lawrey JD (1986) Biological role of lichen substancesThe Bryologist 89(2)111ndash122
Le Gac M Hood ME Fournier E Giraud T (2007)Phylogenetic evidence of host-specific cryptic speciesin the anther smut fungus Evolution 61(1)15ndash26doi101111j1558-5646200700002x
Leacheacute AD (2009) Species tree discordance traces tophylogeographic clade boundaries in North Americanfence lizards (Sceloporus) Syst Biol 58(6)547ndash559doi101093sysbiosyp057
Leacheacute AD Fujita MK (2010) Bayesian species delim-itation in West African forest geckos (Hemidactylusfasciatus) Proc R Soc B Biol Sci 2773071ndash3077doi101098rspb20100662
Leacheacute AD Koo M Spencer C Papenfuss T Fisher RMcGuire J (2009) Quantifying ecological morpho-logical and genetic variation to delimit species in thecoast horned lizard species complex (Phrynosoma)Proc Natl Acad Sci USA 10612418ndash12423 doi101073pnas0906380106
Leacheacute AD Fujita MK Minin VN Bouckaert RR (2014)Species delimitation using genome-wide SNP dataSystematic Biology doi101093sysbiosyu018
Leavitt SD Fankhauser JD Leavitt DH Porter LDJohnson LA St Clair LL (2011a) Complex patterns ofspeciation in cosmopolitan ldquorock posyrdquo lichensmdashdiscovering and delimiting cryptic fungal species in
the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
Leavitt SD Esslinger TL Lumbsch HT (2012c) Neogene-dominated diversification in neotropical montanelichens dating divergence events in the lichen-form-ing fungal genus Oropogon (Parmeliaceae) Am J Bot99(11)1764ndash1777 doi103732ajb1200146
Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Lumbsch HT St Clair LL(2013c) DNA barcode identification of lichen-formingfungal species in the Rhizoplaca melanophthalmaspecies-complex (Lecanorales Lecanoraceae) includ-ing five new species MycoKeys 71ndash22 doi103897mycokeys74508
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Vondraacutek J Thorsten Lum-bsch H Clair LLS (2013d) Local representation ofglobal diversity in a cosmopolitan lichen-formingfungal species complex (Rhizoplaca Ascomycota)J Biogeogr 40(9)1792ndash1806 doi101111jbi12118
Leavitt SD Lumbsch HT Stenroos S St Clair LL(2013e) Pleistocene speciation in North American
2 The Dynamic Discipline of Species Delimitation hellip 39
lichenized fungi and the impact of alternative speciescircumscriptions and rates of molecular evolution ondivergence estimates PLoS ONE 8(12)e85240doi101371journalpone0085240
Leavitt SD Esslinger TL Hansen ES Divakar PK CrespoA Loomis BF Lumbsch HT (2014) DNA barcoding ofbrown Parmeliae (Parmeliaceae) species a molecularapproach for accurate specimen identification empha-sizing species in Greenland Organ Divers Evol 14(1)11ndash20 doi101007s13127-013-0147-1
Leliaert F Verbruggen H Vanormelingen P Steen FLopez-Bautista JM Zuccarello GC De Clerck O(2014) DNA-based species delimitation in algae Eur JPhycol 49(2) doi101080096702622014904524
Leuckert C (1985) Probleme der Flechten-Chemotaxon-omiemdashStoffkombinationen und ihre taxonomischeWertung Ber Deut Bot Ges 98401ndash408
Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
Lohtander K Kaumlllersjouml M Roland M Tehler A (2000)The family physciaceae in fennoscandia phylogenyinferred from its sequences Mycologia 92(4)728ndash735
Longton RE (1997) The role of bryophytes and lichens inpolar ecosystems In Woodin SJ Marquiss M (eds)Ecology of Arctic Environments Blackwell ScienceOxford pp 69ndash96 (Special publication No 13)
Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
Lumbsch HT (1994) Die Lecanora subfusca-Gruppe inAustralasien J Hattori Bot Lab 771ndash175
Lumbsch HT (1998a) Taxonomic use of metabolic data inlichen-forming fungi In Frisvad JC Bridge PDArora DK (eds) Chemical Fungal Taxonomy MarcelDekker New York pp 345ndash387
Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
Mayden RL (1997) A hierarchy of species concepts thedenouement in the saga of the species problem InClaridge MF Dawah HA Wilson MR (eds) Speciesthe units of biodiversity Chapman amp Hall Londonpp 381ndash424
Mayr E (1963) Animal species and evolution HarvardUniversity Press Cambridge
Mayr E (1970) Populations species and evolutionBelknap Press of Harvard University Press Cambridge
McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
McDonald T Miadlikowska J Lutzoni F (2003) Thelichen genus Sticta in the Great Smoky Mountains a
40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
McKay BD Mays HL Wu Y Li H Yao C-T Nishiumi IZou F (2013) An empirical comparison of character-based and coalescent-based approaches to speciesdelimitation in a young avian complex Mol Ecol 22(19)4943ndash4957 doi101111mec12446
Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
Mishler BD Brandon RN (1987) Individuality pluralismand the phylogenetic species concept Biol Philos 2397
Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
Nash TH Zavada M (1977) Population studies amongSonoran Desert species of Parmelia subg Xanthop-armelia (Parmeliaceae) Am J Bot 64(6)664ndash669
Nylander W (1866a) Circa novum in studio Lichenumcriterium chemicum Flora 49198ndash201
Nylander W (1866b) Hypochlorite of lime and hydrate ofpotash Two new criteria for the study of lichens(Tanslated and communicated by the Rev W ALeighton) Bot J Linn Soc 9358ndash365
OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
OrsquoNeill EM Schwartz R Bullock CT Williams JSShaffer HB Aguilar-Miguel X Parra-Olea G Weis-rock DW (2013) Parallel tagged amplicon sequencingreveals major lineages and phylogenetic structure inthe North American tiger salamander (Ambystomatigrinum) species complex Mol Ecol 22(1)111ndash129doi101111mec12049
Orock EA Leavitt SD Fonge BA St Clair LL LumbschHT (2012) DNA-based identification of lichen-form-ing fungi Can publicly available sequence databasesaid in lichen diversity inventories of Mount Cameroon(West Africa) The Lichenologist 44(6)833ndash839doi101017S0024282912000424
Otaacutelora MAG Martiacutenez I Aragoacuten G Molina MC (2010)Phylogeography and divergence date estimates of alichen species complex with a disjunct distributionpattern Am J Bot 97(2)216ndash223 doi103732ajb0900064
Padial J Castroviejo-Fisher S Kohler J Vila C ChaparroJ De la Riva I (2009) Deciphering the products ofevolution at the species level the need for anintegrative taxonomy Zoolog Scr 38(4)431ndash447doi101111j1463-6409200800381x
Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
Papong K Luumlcking R Thammathaworn A BoonpragobK (2009) Four new taxa of Chroodiscus (thelotremoidGraphidaceae) from Southeast Asia The Bryologist112(1)152ndash163 doi1016390007-2745-1121152
Park S-Y Choi J Kim JA Jeong M-H Kim S Lee Y-HHur J-S (2013a) Draft genome sequence of Cladoniamacilenta KoLRI003786 a lichen-forming fungusproducing biruloquinone Genome Announcements 1(5)e00695-13 doi101128genomeA00695-13
Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
Peacuterez-Ortega S Fernaacutendez-Mendoza F Raggio J VivasM Ascaso C Sancho LG Printzen C de los Riacuteos A(2012) Extreme phenotypic variation in Cetrariaaculeata (lichenized Ascomycota) adaptation or inci-dental modification Ann Bot 109(6)1133ndash1148doi101093aobmcs042
Pino-Bodas R Burgaz A Martiacuten M Lumbsch HT (2011)Phenotypical plasticity and homoplasy complicatespecies delimitation in the Cladonia gracilis group(Cladoniaceae Ascomycota) Organ Divers Evol 11(5)343ndash355 doi101007s13127-011-0062-2
Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
Pino-Bodas R Martiacuten M Burgaz A (2012b) Cladoniasubturgida and C iberica (Cladoniaceae) form asingle morphologically and chemically polymorphicspecies Mycol Prog 11(1)269ndash278 doi101007s11557-011-0746-1
Pino-Bodas R Ahti T Stenroos S Martiacuten MP BurgazAR (2013a) Multilocus approach to species recogni-tion in the Cladonia humilis complex (CladoniaceaeAscomycota) Am J Bot 100(4)664ndash678 doi103732ajb1200162
Pino-Bodas R Martiacuten AP Burgaz AR Lumbsch HT(2013b) Species delimitation in Cladonia (Ascomy-cota) a challenge to the DNA barcoding philosophyMol Ecol Resour 13(6)1058ndash1068 doi1011111755-099812086
Pons J Barraclough TG Gomez-Zurita J Cardoso ADuran DP Hazell S Kamoun S Sumlin WD VoglerAP (2006) Sequence-based species delimitation for theDNA taxonomy of undescribed insects Syst Biol 55(4)595ndash609 doi10108010635150600852011
Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
Pringle A Baker DM Platt JL Wares JP Latgeacute JPTaylor JW (2005) Cryptic speciation in the cosmo-politan and clonal human pathogenic fungus Asper-gillus fumigatus Evolution 59(9)1886ndash1899 doi10155404-2411
Printzen C (2009) Lichen systematics the role ofmorphological and molecular data to reconstructphylogenetic relationships Progress in botany vol71 Springer Berlin pp 233ndash275
Pritchard JK Stephens M Donnelly P (2000) Inference ofpopulation structure using multilocus genotype dataGenetics 155(2)945ndash959
Puillandre N Lambert A Brouillet S Achaz G (2012)ABGD automatic barcode gap discovery for primaryspecies delimitation Mol Ecol 21(8)1864ndash1877doi101111j1365-294X201105239x
Rannala B Yang Z (2003) Bayes estimation of speciesdivergence times and ancestral population sizes usingDNA sequences from multiple loci Genetics1641645ndash1656
Raxworthy CJ Ingram CM Rabibisoa N Pearson RG(2007) Applications of ecological niche modelingfor species delimitation a review and empiricalevaluation using day geckos (Phelsuma) fromMadagascar Syst Biol 56(6)907ndash923 doi10108010635150701775111
Ray J (1686) Historia planarum vol 1 Clark LondonReese Naeligsborg R Ekman S Tibell L (2007) Molecular
phylogeny of the genus Lecania (Ramalinaceaelichenized Ascomycota) Mycol Res 111(5)581ndash591doi101016jmycres200703001
Reid N Carstens B (2012) Phylogenetic estimation errorcan decrease the accuracy of species delimitation aBayesian implementation of the general mixed Yule-coalescent model BMC Evol Biol 12(1)196 doi1011861471-2148-12-196
Rissler L Apodaca J (2007) Adding more ecology intospecies delimitation ecological niche models andphylogeography help define cryptic species in theBlack Salamander (Aneides flavipunctatus) Syst Biol56924ndash942 doi10108010635150701703063
Rivas Plata E Luumlcking R (2013) High diversity ofGraphidaceae (lichenized Ascomycota Ostropales) inAmazonian Peruacute Fungal Divers 58(1)13ndash32 doi101007s13225-012-0172-y
Rivas Plata E Lumbsch HT (2011) Parallel evolution andphenotypic divergence in lichenized fungi a casestudy in the lichen-forming fungal family Graphida-ceae (Ascomycota Lecanoromycetes Ostropales)Mol Phylogenet Evol 61(1)45ndash63 doi101016jympev201104025
Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
Ross KG Gotzek D Ascunce MS Shoemaker DD (2010)Species delimitation a case study in a problematic anttaxon Syst Biol 59(2)162ndash184 doi101093sysbiosyp089
Rubin BER Ree RH Moreau CS (2012) Inferringphylogenies from RAD sequence data PLoS ONE 7(4)e33394 doi101371journalpone0033394
Rubinoff D (2006) Utility of mitochondrial DNA barcodesin species conservation Conserv Biol 20(4)1026ndash1033 doi101111j1523-1739200600372x
Ruiz-Sanchez E Sosa V (2010) Delimiting speciesboundaries within the Neotropical bamboo Otatea(Poaceae Bambusoideae) using molecular morpho-logical and ecological data Mol Phylogenet Evol 54(2)344ndash356 doi101016jympev200910035
Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
morphological characters Antarctic Sci 22 (SpecialIssue 06)727ndash741 doi101017S0954102010000477
Salicini I Ibaacutentildeez C Juste J (2011) Multilocus phylogenyand species delimitation within the Nattererrsquos batspecies complex in the Western Palearctic MolPhylogenet Evol 61(3)888ndash898 doi101016jympev201108010
Satler JD Carstens BC Hedin M (2013) Multilocusspecies delimitation in a complex of morphologicallyconserved trapdoor spiders (Mygalomorphae Antro-diaetidae Aliatypus) Syst Biol 62(6)805ndash823 doi101093sysbiosyt041
Schlick-Steiner BC Steiner FM Seifert B Stauffer CChristian E Crozier RH (2010) Integrative taxonomya multisource approach to exploring biodiversityAnnu Rev Entomol 55(1)421ndash438 doi101146annurev-ento-112408-085432
Schoch CL Seifert KA Huhndorf S Robert V SpougeJL Levesque CA Chen W Fungal Barcoding Con-sortium (2012) Nuclear ribosomal internal transcribedspacer (ITS) region as a universal DNA barcodemarker for Fungi Proc Natl Acad Sci 109(16)6241ndash6246 doi101073pnas1117018109
Seacuterusiaux E Villarreal AJC Wheeler T Goffinet B(2011) Recent origin active speciation and dispersalfor the lichen genus Nephroma (Peltigerales) inMacaronesia J Biogeogr 38(6)1138ndash1151 doi101111j1365-2699201002469x
Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
Simpson GG (1951) The species concept Evolution5285ndash298
Sites JW Marshall JC (2003) Delimiting species a renais-sance issue in systematic biology Trends Ecol Evol18462ndash470 doi101016S0169-5347(03)00184-8
Sites JW Marshall JC (2004) Operational criteria fordelimiting species Annu Rev Ecol Evol Syst 35(1)199ndash227 doi101146annurevecolsys35112202130128
Šlapeta J Loacutepez-Garciacutea P Moreira D (2006) Globaldispersal and ancient cryptic species in the smallestmarine eukaryotes Mol Biol Evol 23(1)23ndash29doi101093molbevmsj001
Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
Talavera G Dincă V Vila R (2013) Factors affectingspecies delimitations with the GMYC model insightsfrom a butterfly survey Methods Ecol Evol 4(12)1101ndash1110 doi1011112041-210X12107
Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
Tewksbury J Anderson JGT Bakker JD Billo TJDunwiddie PW Groom MJ Hampton SE HermanSG Levey DJ Machnicki NJ del Rio CM PowerME Rowell K Salomon AK Stacey L TrombulakSC Wheeler TA (2014) Natural historyrsquos place inscience and society BioScience doi101093bioscibiu032
Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
Werth S Cornejo C Scheidegger C (2013) Characteriza-tion of microsatellite loci in the lichen fungus Lobariapulmonaria (Lobariaceae) Appl Plant Sci 1(2)1200290 doi103732apps1200290
Westberg M Arup U Kaumlrnefelt I (2007) Phylogeneticstudies in the Candelariaceae (lichenized Ascomy-cota) based on nuclear ITS DNA sequence dataMycol Res 111(11)1277ndash1284 doi101017S0953756204002102
Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
2 The Dynamic Discipline of Species Delimitation hellip 43
(Lepidoptera Lycaenidae) Front Zoo 4(1)8 doi1011861742-9994-4-8
Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
points for species delimitation using single-genetopologies
The ldquoSpecies Delimitationrdquo plug-in to theGeneious software provides statistical approachesfor exploring species boundaries in single-genetopologies (Masters et al 2011) Using a priorispecimen assignments to putative species theldquoSpecies Delimitationrdquo plug-in computes statis-tics relating to the probability of the observedmonophyly or exclusivity having occurred bychance in a coalescent process and assesses thewithin- and between-species genetic distances inorder to infer the probability with which membersof a putative species might be identified suc-cessfully with tree-based methods An importantcontribution of the ldquoSpecies Delimitationrdquo plug-in is that it provides an objective means for usersto assess putative species within an empiricalstatistic-based framework rather than qualitativeevaluations of what level of hierarchy constitutesa species in a phylogeny
In contrast to simple sequence similaritythresholds (OTU-picking) for delimiting putativeevolutionarily significant units the AutomaticBarcode Gap Discovery (ABGD) method is anautomated procedure that sorts sequences intohypothetical species based on the existence of abarcode gap observed whenever intraspecificgenetic distances are less than those amongorganisms from different species (Puillandreet al 2012) Ultimately ABGD is a fast simplemethod that can be used to group individualsrepresented in a single-locus sequence alignmentinto candidate species that should be comple-mented with other lines of evidence in an inte-grative taxonomic approach (Kekkonen andHebert 2014 Puillandre et al 2012)
In general the ABGD GMYC and PTP ana-lytical protocols using single-locus data arerepeatable and computationally relatively fastproviding a valuable starting point for a pre-liminary perspective into species boundaries inunderstudied groups that can be validated withsubsequent studies (Kekkonen and Hebert 2014)Similarly analyses of single-locus data can beused to corroborate traditional phenotype-basedspecies boundaries and identify candidate speciesthat have previously been hidden within nominal
species However single-locus species delimita-tionmethodsmaybeparticularly prone to failure inrecognizing significant proportions of species-level biodiversity due to the strict criterion forreciprocal monophyly or alternatively providespurious inflations of estimated species diversitybased on genetic differences that donot correspondto species-level lineages For example recentestimates suggest that the incidence of species-level gene tree paraphyly is approximately 20 (Ross 2014) suggesting that analyses of single-locus datasets would likely fail to accurately deli-mit species in one in every five cases In contrastrecent empirical studies suggest that in some casesthe GMYC provides a striking overestimate ofspecies diversity (Miralles and Vences 2013)
The internal transcribed spacer region (ITS)has played a central role in molecular phyloge-netic studies of lichenized fungi and has recentlybeen adopted as the official barcoding marker forfungi (Schoch et al 2012) In many cases the ITSis sufficiently variable to resolve species bound-aries for lichenized fungi (Schoch et al 2012Kelly et al 2011) although accurate specimenidentification using sequence-based identificationapproaches requires a thoroughly curated refer-ence database (Leavitt et al 2014 Orock et al2012 Kelly et al 2011) In spite of the generalutility of the ITS marker a number of issues maypotentially limit its effective use in speciesdelimitation studies including the potential forintragenomic variation of the nuclear ribosomaltandem repeat and difficulties in aligning ITSsequences from divergent taxa (Kiss 2012)Because the ITS has been formally adopted as theofficial barcoding marker for fungi we recom-mend that species delimitation studies attempt toinclude this region for consistency across studiesHowever we recognize that in some taxonomicgroups the inclusion of the ITS may be prob-lematic and therefore suggest a careful screeningof other markers to identify appropriate loci forresolving species-level relationships
Ultimately the success of any single-locusspecies delimitation method largely depends onthe evolutionary history of the species groupunder study and the variability of the selectedmarker (Fig 22) Species delimitation will be
2 The Dynamic Discipline of Species Delimitation hellip 25
more difficult in more recently diverged lineagesand in cases with some level of interspecific geneflow relative to older well-diverged lineages(eg Leavitt et al 2012c 2013e) In the end wereemphasize that single-locus data alone shouldonly be used to provide a preliminary perspectiveof species boundaries and not as the sole evi-dence in species circumscriptions
222 Sampling Across the GenomeMultilocus Sequence Dataand Genome-Wide Markers
Although single-locus methods can provide anefficient approach for preliminary large-scaleassessments of species diversity there are
significant limitations particularly in recentlydiverged species groups where retention ofancestral polymorphisms and incomplete lineagesorting will likely result in different neutral locihaving unique gene topologies that do not mirrorthe speciation process (Knowles and Carstens2007 Heled and Drummond 2010 Taylor et al2000) In contrast to single-locus and strictlyphenotype-based approaches for species delimi-tation analyses of genetic data collected fromindependent genomic regions can provide robusthypotheses of species boundaries with increasingconfidence (Satler et al 2013 Zhang et al 2011Yang and Rannala 2010) Sequences from mul-tiple independent loci provide an importantsource of data for species delimitation studiesincluding recently developed models that
Fig 22 A simplified diagram illustrating the process ofspeciation through time in a single gene history andresulting gene topologies sampled at two points in thespeciation history (modified from Leliaert et al 2014)aEach dot represents a distinct gene copy and each row onenon-overlapping generation with lines connecting genecopies to their ancestors in the previous generation (onerow below) dashed diagonal lines represent reproductivebarriers two hypothetical sampling intervals at differentpoints in the speciation history are shown Tx and Ty
species delimitation methods using single-locus data areeffective only when species are reciprocally monophyleticin the sampled gene tree genetic clustering and coalescent-based species delimitation methods can circumscribespecies when species may not be monophyletic in sampledgenetic loci b Gene topology representing sampledhaplotypes at time Tx (shown in panel a) hypotheticalspecies are reciprocally monophyletic c Gene topologyrepresenting sampled haplotypes at time Ty (shown in panela) hypothetical species are para- and polyphyletic
26 SD Leavitt et al
combine individual gene genealogies and speciesphylogenies via modeling the coalescent historyof markers (Yang and Rannala 2010 Edwards2009 Knowles and Carstens 2007) As aresponse to advances in sequencing technologiesbioinformatical approaches for multimarker spe-cies delimitation analyses continue to be devel-oped (Table 21 Camargo and Sites 2013)
Long-term reproductive isolation of candidatespecies can be assessed with multilocus sequencedata by evaluating genealogical concordance ofunlinked markers (Baum and Shaw 1995 Aviseand Ball 1990) Within species the mixingeffects of recombination cause unlinked loci tohave distinct genealogical histories but geneticdrift and long-term divergence leads to concor-dant genealogical histories at loci across thegenome Relationships of individuals belongingto distinct candidate species can be evaluatedusing gene genealogies (eg haplotype networksor single-gene topologies) to identify lineagesthat exhibit genealogical exclusivity acrossunlinked neutral loci (Hudson and Coyne 2002Dettman et al 2003b Avise and Ball 1990) Thepresence of the same clades in the majority ofsingle-locus genealogies is taken as evidence thatthe clades represent reproductively isolated lin-eages (Dettman et al 2003a Pringle et al 2005)In practice the criteria of reciprocal monophylyand genealogical concordance of unlinked lociprovide a conservative approach for assessingspecies boundaries due to the fact that a sub-stantial amount of time is required after the initialdivergence of species until ancestral polymor-phisms have fully sorted (Knowles and Carstens2007 Hudson and Coyne 2002) Consequentlygroups with recent divergence histories willlikely go undiscovered under a genealogicalconcordance criterion due to the fact that speciesboundaries likely are not reflected in genegenealogies For example it would take morethan 1 million years after speciation before spe-cies would be delimited if 15 loci were sampledin species with an effective population size (Ne)of 100000 assuming one generation a yearunder a strict reciprocal monophyly criterion(Knowles and Carstens 2007) The amount oftime required for a species to be recognized using
a criterion of reciprocal monophyly increasesproportionally with increasing population sizes(Hudson and Coyne 2002) In a number ofstudies of lichen-forming fungi a genealogicalconcordance criterion has been used to bothdelimit previously unrecognized species-levellineages and validate some conventional pheno-type-based species (Leavitt et al 2012a c2013b Molina et al 2011 Kroken and Taylor2001)
Although analyses of molecular sequence datarelying on reciprocal monophyly or fixed char-acter differences for species delimitation canserve as important criteria for identifying speciesthese criteria are not commonly met acrossmultiple loci particularly in recent speciationhistories (Fig 22) To accommodate theobserved conflict among genealogies from mul-tiple loci with the underlying speciation historythe recent merge of coalescent theory with phy-logenetics has driven a major paradigm shift inspecies delimitation and molecular systematics ingeneral (Fujita et al 2012 Edwards 2009) Themultispecies coalescent model can be applied togenealogical histories from multiple independentloci to assemble separate coalescent processesoccurring in populations into a species tree(Degnan and Rosenberg 2009 Rannala andYang 2003) Within this coalescent-basedframework multiple individuals can be assignedto a single speciespopulation and the speciationhistory of ancestral and descendant species-levellineages can be inferred as a ldquospecies treerdquo incontrast to estimating gene genealogies fromindividual samples (Degnan and Rosenberg2006 2009 Rannala and Yang 2003)
A number of multispecies coalescent-basedspecies delimitation methods have recently beenintroduced offering an exciting framework forempirically assessing species boundaries byselecting the best species tree model from a set ofalternative models representing differenthypotheses of species boundaries (Table 21)For example SpeDeSTEM (Ence and Carstens2011) finds the maximum likelihood values for aspecies tree assuming all putative species areseparate lineages and for alternative species treeswhere two or more species are collapsed into a
2 The Dynamic Discipline of Species Delimitation hellip 27
single lineage The best fitting model is thenselected using the Akaike information criterionunder the assumption of constant populationsizes and fixed gene topologies across the speciestree The species delimitation program BayesianPhylogenetic and Phylogeography (BPampPYangand Rannala 2010) accommodates gene treeuncertainty and variable population sizes andsamples from the Bayesian posterior distributionof species delimitation models using reversible-jump Markov chain Monte Carlo (rjMCMC)sampling BPampP requires a user-provided guidetree with resolution finer than the species leveland evaluates alternative modes derived from allpossible subtrees that are generated by collapsingor splitting nodes on the guide tree (Yang andRannala 2010 Rannala and Yang 2003) Theproportion of time spent on each model is pro-portional to the posterior probability of themodel ie ldquospeciation probabilitiesrdquo WhileBPampP ranks among the most popular coalescent-based species delimitation programs (Fujita et al2012) misspecifications of the guide tree andorthe prior distribution for population size (θ) canresult in strong support for models containing aninflated number of species (Leacheacute and Fujita2010 Zhang et al 2011) The nonparametricheuristic species delimitation approach imple-mented in the program Brownie (OrsquoMeara 2010)jointly sorts anonymous samples into species andinfers a species tree from input gene trees fromdifferent loci assuming that for a speciationevent the corresponding nodes on gene trees willbe more consistent with each than the diver-gences within species However finding both theoptimum species tree and species boundariesremains computationally challenging andBrownie has been shown to frequently yieldincorrect results (OrsquoMeara 2010)
Most recently available coalescent-basedspecies delimitation methods require individualassignments to a species or population a priori Ina number of scenarios correct assignments ofsamples to species may be difficult includingpresence of cryptic species incongruencebetween conventional species and moleculardata or simply the fact that accurate specimenidentification in complex groups with subtle or
difficult to discern diagnostic characters is anontrivial task (Leavitt et al 2013e) Statisticalmethods for assessing individual assignment andspecies detection prior to coalescent-based spe-cies delimitation and species tree reconstructionand species provide a more objective approachfor understanding species boundaries
Population assignment tests using a variety ofclustering algorithms using genetic data offer auseful approach for grouping individuals intoputative reproductively isolated groups (Table 21Hausdorf and Hennig 2010 Corander et al2004 2008 Falush et al 2003 Pritchard et al2000) particularly for species delimitation prob-lems that exist at the interface of traditionalpopulation genetic and phylogenetic analyses(Carstens et al 2013 Weisrock et al 2010Knowles and Carstens 2007)
The programs STRUCTURE (Falush et al2003 Pritchard et al 2000) and STRUCTURA-MA (Huelsenbeck et al 2011) cluster samplesinto populations by minimizing HardyndashWeinbergdisequilibrium for a given number of populationclusters using unlinked genetic markers In gen-eral unlinked markers are not available for mostgroups of lichen-forming fungi and a numberof studies have used information from multilocussequence data for STRUCTURE analyses(Leavitt et al 2011a b c 2013e Fernaacutendez-Mendoza and Printzen 2013 Fernaacutendez-Mendoza et al 2011) Varying approaches havebeen implemented to convert DNA sequencedata to allelic data for Bayesian clustering (seeOrsquoNeill et al 2013) STRUCTURE is expectedto perform well when there is sufficient inde-pendence across regions such that linkage dis-equilibrium within regions does not dominate thedata (STRUCTURE manual) but can also beeffective using multilocus sequence data andtreating all SNPs as independent loci regardlessof physical linkage within each locus (OrsquoNeillet al 2013 Falush et al 2003) A recent study ofthe lichen-forming genus Letharia showed thatBayesian clustering implemented in the programSTRUCTURE was generally able to recover thesame putative Letharia lineages circumscribedemploying a gene genealogical approach inKroken and Taylorrsquos iconic species delimitation
28 SD Leavitt et al
study (Altermann et al 2014 Kroken and Taylor2001) Altermann et al (2014) show that popu-lation assignments were largely consistent acrossa range of scenarios including extensiveamounts of missing data the exclusion of SNPsfrom variable markers and inferences based onSNPs from as few as three gene regions Incontrast to STRUCTURE and STRUCTURA-MA the program BAPS (Corander et al 2008)can explicitly infer genetic structure using hap-loid sequence data or other linked geneticmarkers in the ldquoclustering with linked locirdquomodel Another advantage of BAPS is that theprogram is much more computationally efficientthan STRUCTURE or STRUCTURAMA Sim-ulation studies indicate that both BAPS andSTRUCTURE perform well at low levels ofpopulation differentiation and when clusters arenot well differentiated (Latch et al 2006)
In practice inferring the most appropriatelevel of population structure using Bayesianclustering algorithms remains challenging (Latchet al 2006 Evanno et al 2005) BAPS andSTRUCTURAMA can infer both individualassignments and the most likely number ofgenetic clusters (Corander et al 2008 Huelsen-beck et al 2011) and the ad hoc ΔK statisticproposed by Evanno et al (2005) provides anobjective approach for identifying the uppermosthierarchical level of structure However carefulconsideration of other levels of populationstructure may ultimately reveal more biologicallymeaning results and researchers should examineindividual assignments across a range of geneticgroupings Some markers generated fromindependent genomic regions such as SNPs andfast-evolving microsatellites can be used to dis-tinguish fine-scale population structuring on thebasis of allele frequencies and species delimita-tion analyses based on these markers may implythe risk of severe taxonomic oversplitting Inthese cases validation approaches such asBPampP and SpeDeSTEM and corroborationamong different species delimitation approachescan provide perspective between intraspecificpopulation structure and species-level clusters
Another limitation of clustering approaches isthat they do not assess or take into account
evolutionarily divergences and relationshipsamong population clusters Coalescent-basedspecies tree methods (discussed below) provide ameans to assess the diversification histories ofpopulations inferred from clustering analysesTherefore a potential working protocol for aninformed species delimitation study that takesinto account population structure could consist offirst applying a genetic clustering analysis under apopulation genetics criterion (eg BAPSSTRUCTURE STRUCTURAMA) to identifygenetically distinct population clusters that can beconsidered ldquocandidate speciesrdquo From these can-didate species a species tree can be inferred forfocal group using coalescent-based species treereconstruction methods (eg BEAST) Subse-quently a coalescent-based validation methodcan be applied to assess whether the distinctpopulation clusters represent independent evolu-tionary lineages (eg BPampP) This multistepapproach would provide a consistent and standardcriterion for distinguishing between population-and species-level lineages (Camargo and Sites2013) and has been applied to a number of casesincluding lichen-forming fungi (Leavitt et al2011a b c 2013e Leacheacute and Fujita 2010)
High-throughput sequencing methods providethe means to effectively sample hundreds tothousands of loci from across a species genome fora large number of species and continue to revolu-tionize studies that can be performed even in non-model organisms Targeted high-throughputsequencing approaches such as anchored phy-logenomics transcriptome sequencing reduced-representation genomic library sequencing(restriction-site-associated DNA sequencingRAD-Seq and genotype-by-sequencing GBS)and high-throughput amplicon sequencing pro-vide important insight into species boundaries fora number groups although none of these approa-ches has yet been applied to studies of lichenizedfungi
For example restriction-site-associated(RAD-Seq) DNA can simultaneously detect andgenotype thousands of genome-wide SNPs byfocusing the sequencing effort on a reducedrepresentation of the entire genome (Baird et al2008) and has been successfully applied to
2 The Dynamic Discipline of Species Delimitation hellip 29
intraspecies (Lewis et al 2007 Miller et al 2007Emerson et al 2010) and interspecies studies(Rubin et al 2012 Eaton and Ree 2013 Wagneret al 2013) This approach has provided strikinginsight into the recent adaption radiation of LakeVictoria cichlids (Wagner et al 2013) Analternative approach targeting the sequencing ofspecific loci using next-generation sequencingplatforms provides an efficient means of gener-ating data for loci of known genomic locationorthology size and expected level of variation(OrsquoNeill et al 2013) Markers can be targetedusing well-established PCR techniques or newerhybridization techniques and subsequentlypooled for high-throughput sequencing usingparallel-tagged sequencing of multiple individualsamples within a single sequencing run (OrsquoNeillet al 2013) A recent study of North Americantiger salamanders (Ambystoma tigrinum)demonstrated the potential for amplicon-basedparallel-tagged sequencing to rapidly generatelarge-scale genomic data for species delimitationand species tree research (OrsquoNeill et al 2013)
Although these methods provide an enormousamount of data and insight into diversification atan unprecedented scale computational limita-tions restrict the applicable analytical methodsfor large datasets although computational andanalytical advances are happening rapidly Mostrecent coalescent-based species delimitation andspecies tree models using gene trees have beenlimited to handle tens of loci for multiple indi-viduals and combining hundreds or thousands ofgene trees into a single species delimitationframework presents considerable computationalchallenges (Camargo and Sites 2013) Therecently developed method Bayes factor delim-itation (with genomic data BFD) combines adynamic programming algorithm for estimatingspecies trees that bypasses the computationallyintensive MCMC integration over gene trees toprovide a rigorous technique for species delimi-tation studies using genome-wide SNP data(Leacheacute et al 2014) Competing species delimi-tation models are compared using Bayes factorsand it appears that this approach is robust tosample sizes (ie few loci and limited samples
per species) and misspecification of the prior forpopulation size (θ) (Leacheacute et al 2014)
Fungi are an ideal model for assessing diver-gence in eukaryotes due to their simple mor-phologies small genomes broad ecological rolesand diverse lifestyles (Gladieux et al 2014)However the use of genomic data from high-throughput sequencing methods have not yet beenincluded in species delimitation studies of lichen-forming fungi This is due in part to challenges indealing with metagenomic data containing geno-mic information from a plethora of symbiontsassociated with a lichen thallus scant genomicresources and discipline-specific inertia A num-ber of lichen-forming fungal genomes are cur-rently available including Endocarponpusillum(Wang et al 2014) Cladonia spp (Park et al2013a 2014) Caloplaca flavorubescens (Parket al 2013b) and Cladonia greyi and Xanthoriaparientina through the Fungal Genomics Programat Joint Genome Institute of the United StatesDepartment of Energy (httpwwwjgidoegov)The foreseeable ongoing expansion of genomicresources for lichen-forming fungi will be centralto developing approaches for delimiting speciesusing high-throughput sequencing Specificallygenomic resources will be crucial in identifyingnovel markers (variable genes regions SNPsmicrosatellites etc) identifying conserved fungalmarkers for targeted high-throughput sequencingapproaches and references for entire genomecomparisons (Devkota et al 2014 Werth et al2013)
23 Can We Make SpeciesDelimitation in Lichen-FormingFungi Truly Integrative
The widespread availability of genetic data hascreated a biased viewpoint that only genetic datashould be used for statistical species delimitationHowever an ongoing appeal to researchers toassess species boundaries from multiple andcomplementary perspectives (phylogeneticspopulation genetics comparative morphologydevelopment ecology etc) has resulted in an
30 SD Leavitt et al
integrative taxonomic framework bringing theseconceptual and methodological perspectivestogether (Hamilton et al 2014 Fujita et al 2012Salicini et al 2011 Yeates et al 2011 Padialet al 2010 Dayrat 2005 Will et al 2005 Wiensand Penkrot 2002) In practice any study linkingdifferent kinds of data to support hypotheses ofspecies boundaries including mapping morpho-logical characters onto a molecular phylogenycan be considered integrative Integrative meth-ods for species delimitation fall across a broadspectrum ranging from verbal and qualitativeassessments of data classes to quantitative meth-ods that allow different data types to contribute tostatistical species delimitation (Yeates et al2011) In most taxonomic studies utilizing bothmolecular and morphological data expert opinionis eventually used to some degree to evaluate thefinal status of a candidate species (eg Bond andStockman 2008) In this sense many studiesusing evidence for independent data sources fordelimiting species boundaries use an iterativeapproach (Yeates et al 2011) where speciesboundaries can be tested within a hypothetico-deductive framework with diverse datasets
From a practical perspective we advocate aprocess of iterative taxonomy (sensu Yeates et al2011) to circumscribe and refine species limitsusing multiple lines of evidence This iterativeprocess involves comparisons of morphologicaldata with a phylogenetic hypothesis (ie single-locus gene tree or concatenated multilocusphylogeny) to identify the least inclusive mono-phyletic clade in the topology characterized by atleast one unambiguously diagnostic morphologi-cal character (Miralles and Vences 2013) Thisphylogenetic tree-informed approach to assessingspecies boundaries represents a common practicein studies of lichen-forming fungi Previouslyunrecognized species-level clades with corre-sponding subtle or overlooked phenotypiccharacters have been commonly observed in bothcrustose lichens [eg Acarospraceae (Wedin et al2009) Graphidaceae (Rivas Plata and Lumbsch2011 Papong et al 2009) Lecanoraceae (Leavittet al 2011a) Lecideaceae (Ruprecht et al 2010)Mycoblastaceae (Spribille et al 2011) andTeloschistaceae (Vondraacutek et al 2009 Muggia
et al 2008)] and foliose and fruticose lichens [egLobariaceae (Moncada et al 2014 McDonaldet al 2003) Parmeliaceae (Leavitt et al 2013aWirtz et al 2012 Divakar et al 2005 Molinaet al 2004 Kroken and Taylor 2001) Peltigera-ceae (OrsquoBrien et al 2009 Goffinet et al 2003)Physciaceae (Elix et al 2009 Divakar et al2007) and Sphaerophoraceae (Houmlgnabba andWedin 2003)] As a specific example Luumlckinget al (2008) used a combination of medullarychemistry and underside pigmentation in speci-mens from the Heterodermia obscurata group inCosta Rica to corroborate monophyletic clades inan ITS gene tree as species-level lineages
While this iterative approach to speciesdelimitation and taxonomy has proven valuablefor understanding species boundaries anddescribing new taxa in some groups of lichenizedfungi a posteriori examination of morphologicaland chemical features has failed to reveal diag-nostic phenotypic characters in a number ofstudies (Muggia et al 2014 Leavitt et al 2011aOtaacutelora et al 2010 OrsquoBrien et al 2009) Fur-thermore a study of widespread species in thegenus Melanelixia (Parmeliaceae) indicated thatphenotypically cryptic lichen-forming fungalspecies-level lineages may be relatively ancientand diagnosable phenotypic differences may beabsent even millions of years after the initialdivergence (Leavitt et al 2012b) The latter studyhighlights the fact that species-level lineages maycommonly exist without any observable diag-nostic phenotypic characters calling into ques-tion the impetus for a universal application ofintegrative taxonomy
In keeping with the principle that as manylines of evidence as available should be com-bined to delimit species (Dayrat 2005) the for-malized integrative taxonomic approach (ITAXMiralles and Vences 2013) uses a mtDNA guidetree and observations from different types of datathat might provide conclusive evidence for theindependence of lineages and thus their identityas different species Miralles and Vences (2013)provide a non-exhaustive list of species delimi-tation criteria to be integrated in the ITAXapproach including (a) sympatric occurrencewithout admixture as revealed by consistent
2 The Dynamic Discipline of Species Delimitation hellip 31
differences in morphological or molecular char-acters at the same geographic location (b) strongdifferences in a behavioral morphological orgenetic character known to mediate prematingisolation (c) unviability or infertility of hybrids(d) lack of gene flow across a geographicalhybrid zone (e) congruent diagnostic differencesbetween sister lineages in various unlinkedmorphological character (f) absence of haplo-type sharing in several unlinked nuclear loci and(g) a combination of criteria endashf Speciesboundaries are based on seeking the least inclu-sive monophyletic group in the mtDNA treewhich fulfills at least one of the criteria listedabove Clearly the ITAX approach is sensitive tosample size and in order to reliably support thedistinctiveness of a given species it has beenrecommended that the sampling strategy includesat least five individuals per species (Miralles andVences 2013) A similar approach could beadopted for lichenized fungi using an ITS genetopology as guide tree rather than mtDNA
A total evidence approach including concat-enation has been a common approach for inte-grating information from different sourcesincluding independent genetic markers in phy-logenetic reconstructions (Kluge 1989 Wiens1998 de Queiroz et al 1995) Proponents forconcatenation of independent data in phyloge-netic analyses argue that when combining alldata the underlying signal of speciation mayemerge even if weakly contradictory signalis contained in the individual data partitions(Gatesy et al 1999) For example establishing apreliminary perspective of species boundariesfrom multilocus sequence data using concatena-tion may provide a reasonable starting point forscreening for cryptic species and species treeinference (Šlapeta et al 2006 Leavitt et al2011a Le Gac et al 2007 Leacheacute 2009)However concatenation and consensus methodsimply a risk of obtaining inflated support forincorrect relationships and information aboutvariance in gene coalescence is lost (Degnan andRosenberg 2006 2009 Kubatko and Degnan2007 Edwards 2009) In spite of the impetus forintegrating evidence from independent data
sources into empirical species delimitation stud-ies and taxonomy (Fujita et al 2012 Padial et al2009) most currently available species delimi-tation methods are unable to accommodate non-genetic data sources in a statistical framework
231 Selecting the Appropriate Data
In the face of increasing availability of geneticdata and associated bioinformatical approachesfor delimiting species researchers should care-fully consider what information is being sacri-ficed by the failure to consider non-genetic datain species delimitation studies and whetheraccuracy could be improved by the addition ofmultiple data types Morphological data havehistorically served as a proxy to identify repro-ductively isolated groups (ie ldquospeciesrdquo) (Ray1686 Fujita et al 2012) Current methods fordelimiting species using non-genetic data (egchemistry morphology and ecology) remainwoefully understudied For example morphol-ogy-based species circumscriptions are generallybased on one or more qualitative (or quantitative)morphological characters that do not appear tooverlap with other species However ascertain-ing that a given trait is truly fixed within apopulation with statistical confidence requiresunrealistic sample sizes even when allowing forsome level of polymorphism in the diagnosticcharacter (Wiens and Servedio 2000) Now var-ious combinations of datamdashfrom morphologygenetics geography and ecologymdashare acceptedas standard information for species delimitationstudies (Ruiz-Sanchez and Sosa 2010 Ross et al2010 Edwards and Knowles 2014)
Below we briefly discuss appropriate datasources for species delimitation studies of lichen-forming fungi However homoplastic characters(similar traits that are not derived from a com-mon ancestor) are common among many traitscommonly used to circumscribe fungal taxa thebiological significance of secondary metabolitevariation remains largely unknown (Lawrey1986) and ecological niches may be difficultto adequately characterize and model due to
32 SD Leavitt et al
microhabitat requirements and data resolutionTherefore we advocate a cautious approach toselecting appropriate and relevant data forassessing species boundaries
In spite of the potential challenges and limi-tations of using phenotypic data these traits haveprovided a plethora of valuable information forunderstanding species boundaries For examplein the Melanelixia fuliginosa group (Parmelia-ceae) a morphometric analysis using color isi-dia and marginal zone free of isidia ascharacters revealed a general pattern of differ-entiation between material formerly recognizedas subspecies fuliginosa and glabratula in spiteof the fact that considerable overlap betweengroups occurs in some characters individually(Arup and Berlin 2011) Furthermore the dis-tinction of the two groups was supported by aphylogenetic analysis of the ITS marker andecological differences with M fuliginosaoccurring predominantly on rock and M glab-ratula on bark (Arup and Berlin 2011) Thisstudy of the M fuliginosa group provides a fit-ting example of using multiple independentsuites of data including ecology morphologyand genetic information to establish a robusthypothesis of species boundaries
In lichen systematics phenotypic dataincluding thallus organization secondary metab-olites mode of reproduction ascoma-type andontogeny ascus and ascospore characters havehistorically played a prominent role (Printzen2009) Ascomatal characters have traditionallyheld a major role in higher-level classification(Printzen 2009) in contrast to species-levelclassification which also tends to include a widearray of vegetative and chemical charactersCommonly assessed morphological charactersinclude thallus form and size cortical features(eg maculae and pseudocyphellae) presenceformcolor of attachment structure (eg rhizines)and reproductive mode (ascomata vs vegetativediaspores) (Printzen 2009) Different morpho-logical types of vegetative diasporesmdashcorticatedisidia and ecorticated sorediamdashand their locationare commonly used to distinguish speciesAscomatal characters including morphologylocation (laminal vs marginal) position (sessile
vs immersed) presence of thalline margins colorof an apothecial disc and presence or color ofpruina are also commonly used in speciesdelimitations of lichen-forming fungi (Printzen2009) Other important characters may includethalline characters form color size and septationof ascospores size and form and structure of ascithe hamathecium type of epihymenium andhypothecium and the type of excipulum orperidium conidiomatal characters etc (Printzen2009)
Assessments of secondary metabolites hasplayed an important role in lichen taxonomybeginning with the introduction of simple spottests by Nylander (1866a b) The use of chem-istry in lichen taxonomy has been discussed indetail in numerous reviews (Lumbsch 1998a bRogers 1989 Brodo 1986 Egan 1986 Leuckert1985 Brodo 1978 Hawksworth 1976 Culber-son 1969 1970) and we refer readers to thesevaluable sources for a more comprehensive per-spective on lichen chemistry In short extrolites(secondary metabolites) belong to various clas-ses the most common and diverse include dep-sides depsidones chlorinated xanthones andanthraquinones (Lumbsch 2002 Culberson1969) The presence or absence of specificextrolites or their replacements by another sub-stance is widely used to distinguish speciesparticularly when correlated with differences ingeographic distributions However if morpho-logical or geographical differences betweenpopulations containing different extrolites are notapparent the taxonomic significance has beendisputed with some authors distinguishing themas species and others preferring to regard them aschemical races within a species In addition tosimply using the presence or absence of extro-lites Culberson and Culberson (1976) proposedto arrange lichen substances into chemosyn-dromes of closely related substances The pres-ence or absence of these chemosyndromes maypotentially be used as characters to delimit spe-cies regarding differences involving the samechemosyndromes as intraspecific variation anddistinct chemosyndromes as evidence for inter-specific populations (Lumbsch 1994 Gowan1986)
2 The Dynamic Discipline of Species Delimitation hellip 33
Due to the fact that species delimitationstudies often incorporate a substantial biogeo-graphical or ecological component ecologicalniche modeling plays an increasing role in phy-logenetic and taxonomic research Niche mod-eling can provide evidence for ecologicalisolation between populations based on eitherconserved or divergent ecological niches andtherefore can provide additional evidence sup-porting lineage independence between putativespecies The application of ecological nichemodeling has been applied to species delimita-tion studies in a number of cases (Ruiz-Sanchezand Sosa 2010 Leacheacute et al 2009 Raxworthyet al 2007 Rissler and Apodaca 2007) althoughit has not been explicitly applied to assess speciesboundaries in lichen-forming fungi By mappingthe spatial distribution of environmental suit-ability of climatic variables for candidate speciesthe application of ecological niche modeling canbe particularly important in cases where specieshave allopatric distributions (Raxworthy et al2007)
Ecological niche modeling utilizes knownassociations between a speciesrsquo occurrencelocalities and environmental variables to defineabiotic conditions within which populations canbe maintained (Guisan and Thuiller 2005) Themethodological approach for modeling is basedon four general properties (i) The current knownspeciesrsquo localities is the dependent variable (ii)the distribution is modeled as a map composed ofgrid cells at a specified resolution (iii) a range ofenvironmental variables (eg temperature pre-cipitation and solar exposure) are collected todescribe the characteristics of each cell and (iv)classifying the degree to which each cell is eithersuitable or unsuitable for each species under arange of models (Guisan and Thuiller 2005)
Ecological data also have the potential to playan important role in understanding speciesboundaries in lichen-forming fungi For exampleNash and Zavada (1977) demonstrated that Xant-hoparmelia populations with distinct chemistriesoccurring in the northern portion of the SonoranDesert exhibit habitat selection among differentrock substrates within a region with relativelyuniform climate and topography In another case
the parmelioid species Parmelia mayi is mor-phologically indistinguishable from P saxatilisbut can be separated by bioclimatic features andgenetic and chemical characters (Molina et al2011) McCune and Printzen(2011) assess distri-butions and climatic niches of species in theLecanora varia group inwesternUSAandprovidea model that uses continental influence and annualtemperature as themajor factors predicting speciesdistributions The distribution of Usnea hirta alichen commonly used in air quality biomonitor-ing research was modeled for a section of theWhite River National Forest in central Coloradobased on the presence of U hirta at 72 biomoni-toring reference sites distributed in the inter-mountain western United States (Shrestha et al2012) The best model for predicting U hirtadistribution included four variablesmdashsolar radia-tion average monthly precipitation and averagemonthly minimum and maximum temperatures(Shrestha et al 2012) These studies supportthe potential use of ecological niche modelingmethods in species delimitation studies of lichen-forming fungi
24 Conclusions What AboutTaxonomy
In most cases species circumscription and tax-onomy requires some degree of qualitativejudgment and individual interpretation Integrat-ing multiple types of data into an empiricalframework for delimiting species boundarieswhere species boundaries can be tested within ahypothetico-deductive framework with diversedatasets can provide robust hypotheses of speciesboundaries and taxonomic stability (Yeates et al2011) However it has long been known toevolutionary biologists that distinct species donot need to have diagnosable morphologicaldifferences (Mayr 1963) and increasing avail-ability of genetic data has allowed researchers toidentify species and to rigorously test speciesboundaries with a level of precision that wasunimaginable a decade ago While analyticaladvances in statistical species delimitation havebeen largely based on genetic data the utility of
34 SD Leavitt et al
these approaches to formal taxonomy remainselusive Due to these challenges an eclecticapproach to delimiting species and cautionagainst the reliance on any single dataset ormethod is required when delimiting species
While results from many recent studies maycontradict traditional species boundaries acrossmany groups of lichen-forming fungi we areoptimistic that this research represents substantialprogress toward a more accurate perspective onspecies boundaries and diversity in fungi As aresult of ongoing research of species boundariesin lichen-forming fungi the taxonomic value ofmany phenotypes is now better understood ourunderstanding of ecological evolutionary andbiogeographic patterns has improved and we canbegin to better understand patterns of symbiontinteractions in lichens Integrating new data(including novel morphological characters andgenetic data) will be essential to accurately rep-resent species-level diversity across all groups oflichenized fungi Hopefully an improved per-spective on lichen diversity also increases ourappreciation of these incredible symbiotic sys-tems While we are strong advocates for theapplication of independent data types in devel-oping an integrative taxonomy there is anincreasing need to formally recognize the exis-tence of phenotypically cryptic species-levellineages in lichen-forming fungi (see Hibbettet al 2011) In some cases a molecular taxon-omy may provide the most practical approach toconsistent treatment of mycobiont species withinlichen groups where diagnostic morphologicalcharacters are unidentifiable or practically notfeasible (Leavitt et al 2013c d)
These are exciting times for taxonomists andphylogeneticists A closer look at lichen taxon-omy with the inclusion of new data will help usto better understand the diversity of these fasci-nating organisms accurately interpret distribu-tion patterns and play a more important role inmeaningful conservation practices Howeversome level of uncertainty will accompany pro-gress In many taxonomic groups our traditionalapproach for species identification will likelyneed to be substantially modified The search forcorroborating morphological support for cryptic
species identified using molecular data willrequire meticulous and creative approaches toassess phenotypic variation in potentially unor-thodox ways We are hopeful that lichenologistswho traditionally have been eager to include newmethods such as chromatography in their rou-tine identifications will be amenable to includemolecular techniques to their routine examina-tion of specimens for identification and classifi-cation Although this may prove difficult toachieve by single individuals especially citizenscientists that traditionally play an important rolein lichen taxonomy the increasing numberof collaborative projects in the discipline (egLumbsch et al 2011 Crespo et al 2010Gueidan et al 2009) make us optimistic thatbroad-scale collaborative approaches will facili-tate the inclusion of molecular data in lichenresearch at all levels
Acknowledgments We are indebted to various col-leagues for valuable thought-provoking discussionnotably Matthew Nelsen (University of Chicago) AnaCrespo (Universidad Complutense de Madrid) PradeepDivakar (Universidad Complutense de Madrid) BeckettSterner (The Field Museum) and Joyce Havstad (TheField Museum) We also thank anonymous reviewers whoprovided valuable comments that improved this chapterSupport by the US National Science Foundation isgratefully acknowledged (ldquoHidden diversity in parmelioidlichensrdquo DEB-0949147)
References
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Altermann S Leavitt SD Goward T Nelsen MPLumbsch HT (2014) How do you solve a problemlike Letharia A new look at cryptic species in lichen-forming fungi using Bayesian clustering and SNPSsfrom multilocus sequence data PLoS ONE 9(5)e97556 doi101371journalpone0097556
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Brodo IM (1986) Interpreting chemical variation inlichens for systematic purposes The Bryologist 89(2)132ndash138
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Carstens BC Pelletier TA Reid NM Satler JD (2013)How to fail at species delimitation Mol Ecol 22(17)4369ndash4383 doi101111mec12413
Corander J Marttinen P (2006) Bayesian identification ofadmixture events using multi-locus molecular mark-ers Mol Ecol 15(10)2833ndash2843 doi101111j1365-294X200602994x
Corander J Waldmann P Marttinen P Sillanpaa M(2004) BAPS 2 enhanced possibilities for the analysisof genetic population structure Bioinformatics 20(15)2363ndash2369 doi101093bioinformaticsbth250
Corander J Marttinen P Mantyniemi S (2006) Bayesianidentification of stock mixtures from molecular markerdata Fish Bull 104550ndash558
Corander J Marttinen P Siren J Tang J (2008) EnhancedBayesian modelling in BAPS software for learninggenetic structures of populations BMC Bioinf 9(1)539 doi1011861471-2105-9-539
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Cracraft J (1983) Species concepts and speciation anal-ysis Curr Ornithol 1159ndash187
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Crespo A Peacuterez-Ortega S (2009) Cryptic species andspecies pairs in lichens a discussion on the relation-ship between molecular phylogenies and morpholog-ical characters Anales del Jardin Botanico de Madrid66(S1)71ndash81 doi103989ajbm2225
Crespo A Kauff F Divakar PK del Prado R Perez-Ortega S Amo de Paz G Ferencova Z Blanco ORoca-Valiente B Nunez-Zapata J Cubas P ArguelloA Elix JA Esslinger TL Hawksworth DL MillanesA Molina MC Wedin M Ahti T Aptroot A et al(2010) Phylogenetic generic classification of parmeli-oid lichens (Parmeliaceae Ascomycota) based onmolecular morphological and chemical evidenceTaxon 59(6)1735ndash1753
Culberson WL (1969) The use of chemistry in thesystematics of the lichens Taxon 18498ndash505
Culberson WL (1970) Chemosystematics and ecology oflichen-forming fungi Annu Rev Ecol Syst 1153ndash170
Culberson CF Culberson WL (1976) Chemosyndromicvariation in lichens Syst Bot 1325ndash339
Darwin C (1859) On the origin of species by means ofnatural selection or the preservation of favoured racesin the struggle for life J Murray London
Dayrat B (2005) Towards integrative taxonomy Biol JLinn Soc 85(3)407ndash415 doi101111j1095-8312200500503x
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de Queiroz K (1998) The general lineage concept ofspecies species criteria and the process of speciationa conceptual unification and terminological recom-mendations In Howard DJ Berlocher SH (eds)Endless forms species and speciation Oxford Uni-versity Press Oxford pp 57ndash75
de Queiroz K (1999) The general lineage concept ofspecies and the defining properties of the speciescategory In Wilson RA (ed) Species new interdis-ciplinary essays MIT Press Cambridge pp 49ndash89
de Queiroz K (2007) Species concepts and speciesdelimitation Syst Biol 56(6)879ndash886 doi10108010635150701701083
de Queiroz A Donoghue MJ Kim J (1995) Separateversus combined analysis of phylogenetic evidenceAnnu Rev Ecol Syst 26(1)657ndash681 doi101146annureves26110195003301
Degnan J Rosenberg N (2006) Discordance of speciestrees with their most likely gene trees PLoS Genet 2(5)e68 doi101371journalpgen0020068
Degnan JH Rosenberg NA (2009) Gene tree discordancephylogenetic inference and the multispecies coales-cent Trends Ecol Evol 24(6)332ndash340 doihttpdxdoiorg101016jtree200901009
Del-Prado R Cubas P Lumbsch HT Divakar PK BlancoO de Paz GA Molina MC Crespo A (2010) Geneticdistances within and among species in monophyleticlineages of Parmeliaceae (Ascomycota) as a tool fortaxon delimitation Mol Phylogenet Evol 56(1)125ndash133 doi101016jympev201004014
Del-Prado R Divakar PK Crespo A (2011) Using geneticdistances in addition to ITS molecular phylogeny toidentify potential species in the Parmotrema reticul-atum complex a case study The Lichenologist 43(06)569ndash583 doi101017S0024282911000582
Del-Prado R Blanco O Lumbsch HT Divakar PK ElixJA Molina MC Crespo A (2013) Molecular phylog-eny and historical biogeography of the lichen-formingfungal genus Flavoparmelia (Ascomycota Parmelia-ceae) Taxon 62(5)928ndash939 doi101270562522
Dettman J Jacobson D Taylor J (2003a) A multilocusgenealogical approach to phylogenetic species recog-nition in the model eukaryote Neurospora Evolution57(12)2703ndash2720 doi101111j0014-38202003tb01514x
Dettman JR Jacobson DJ Turner E Pringle A TaylorJW (2003b) Reproductive isolation and phylogeneticdivergence in Neurospora comparing methods ofspecies recognition in a model eukaryote Evolution57(12)2721 doi10155403-074
Devkota S Cornejo C Werth S Chaudhary RP Sche-idegger C (2014) Characterization of microsatelliteloci in the Himalayan lichen fungus Lobaria pindar-ensis (Lobariaceae) Appl Plant Sci 2(5)1300101doi103732apps1300101
Divakar PK Molina MC Lumbsch HT Crespo A (2005)Parmelia barrenoae a new lichen species related toParmelia sulcata (Parmeliaceae) based on molecularand morphological data The Lichenologist 37(01)37ndash46 doi101017S0024282904014641
Divakar PK Amo De paz G del Prado R Esslinger TLCrespo A (2007) Upper cortex anatomy corroboratesphylogenetic hypothesis in species of Physconia(Ascomycota Lecanoromycetes) Mycol Res 111(11)1311ndash1320 doi101016jmycres200708009
Divakar PK Figueras G Hladun N Crespo A (2010)Molecular phylogenetic studies reveal an undescribedspecies within the North American concept of Mela-nelixia glabra (Parmeliaceae) Fungal Divers 42(1)47ndash55 doi101007s13225-010-0027-3
Donoghue MJ Gauthier A (2004) Implementing thephylocode Trends Ecol Evol 19(6)281ndash282 doi101016jtree200404004
Eaton DAR Ree RH (2013) Inferring phylogeny andintrogression using RADseq data an example fromflowering plants (Pedicularis Orobanchaceae) SystBiol 62(5)689ndash706 doi101093sysbiosyt032
Edwards SV (2009) Is a new and general theory ofmolecular systematics emerging Evolution 63(1)1ndash19 doi101111j1558-5646200800549x
Edwards DL Knowles LL (2014) Species detection andindividual assignment in species delimitation canintegrative data increase efficacy Proc R Soc B BiolSci 281(1777) doi101098rspb20132765
Egan RS (1986) Correlations and non-correlations ofchemical variation patterns with lichen morphologyand geography The Bryologist 8999ndash110
Elix JA Corush J Lumbsch HT (2009) Triterpenechemosyndromes and subtle morphological characterscharacterise lineages in the Physcia aipolia group inAustralia (Ascomycota) Syst Biodivers 7(04)479ndash487 doi101017S1477200009990223
Emerson KJ Merz CR Catchen JM Hohenlohe PACresko WA Bradshaw WE Holzapfel CM (2010)Resolving postglacial phylogeography using high-throughput sequencing Proc Natl Acad Sci 107(37)16196ndash16200 doi101073pnas1006538107
Ence DD Carstens BC (2011) SpedeSTEM a rapid andaccurate method for species delimitation Mol EcolResour 11(3)473ndash480 doi101111j1755-0998201002947x
EvannoGRegnaut SGoudet J (2005)Detecting thenumberof clusters of individuals using the software STRUC-TURE a simulation study Mol Ecol 14(8)2611ndash2620doi101111j1365-294X200502553x
Falush D Stephens M Pritchard JK (2003) Inference ofpopulation structure using multilocus genotype datalinked loci and correlated allele frequencies Genetics164(4)1567ndash1587 doi101111j1471-8286200701758x
Fan HH Kubatko LS (2011) Estimating species treesusing approximate Bayesian computation Mol Phylo-genet Evol 59(2)354ndash363 doi101016jympev201102019
Fernaacutendez-Mendoza F Printzen C (2013) Pleistoceneexpansion of the bipolar lichen Cetraria aculeata intothe Southern hemisphere Mol Ecol 22(7)1961ndash1983doi101111mec12210
Fernaacutendez-Mendoza F Domaschke S Garciacutea MA JordanP Martin MP Printzen C (2011) Population structure
2 The Dynamic Discipline of Species Delimitation hellip 37
of mycobionts and photobionts of the widespreadlichen Cetraria aculeata Mol Ecol 20(6)1208ndash1232doi101111j1365-294X201004993x
Fraley C Raftery A (2007) Model-based methods ofclassification Using the mclust software in chemo-metrics J Stat Softw 18i06 [Available online at httpwwwdoajorgdoajfunc=abstractampid=218544]
Fujisawa T Barraclough TG (2013) Delimiting speciesusing single-locus data and the generalized mixed yulecoalescent (GMYC) approach a revised method andevaluation on simulated datasets Syst Biol 62(5)707ndash724 doi101093sysbiosyt033
Fujita MK Leacheacute AD Burbrink FT McGuire JA MoritzC (2012) Coalescent-based species delimitation in anintegrative taxonomy Trends Ecol Evol 27(9)480ndash488 doi101016jtree201204012
Gatesy J OrsquoGrady P Baker RH (1999) Corroborationamong data sets in simultaneous analysis hiddensupport for phylogenetic relationships among higherlevel Artiodactyl taxa Cladistics 15(3)271ndash313doi101111j1096-00311999tb00268x
Gladieux P Ropars J Badouin H Branca A Aguileta Gde Vienne DM Rodriacuteguez de la Vega RC Branco SGiraud T (2014) Fungal evolutionary genomicsprovides insight into the mechanisms of adaptivedivergence in eukaryotes Mol Ecol 23(4)753ndash773doi101111mec12631
Goffinet B Miadlikowska J Goward T (2003) Phyloge-netic inferences based on nrDNA sequences supportfive morphospecies within the Peltigera didactylacomplex (Lichenized Ascomycota) The Bryologist106(3)349ndash364 doi10163901
Gowan SP (1986) Evolution of secondary natural productsin the genus Porpidia (Ascomycata Porpidiaceae) AmJ Bot 73606
Griffin PC Hoffmann AA (2014) Limited genetic diver-gence among Australian alpine Poa tussock grassescoupled with regional structuring points to ongoinggene flow and taxonomic challenges Ann Bot doi101093aobmcu017
Grube M Hawksworth DL (2007) Trouble with lichenthe re-evaluation and re-interpretation of thallus formand fruit body types in the molecular era Mycol Res111(9)1116ndash1132 doi101016jmycres200704008
Gueidan C Savi S Thues H Roux C Keller C Tibell LPrieto M Heimarsson S Breuss O Orange A FrobergL Wynns AA Navarro-Rosines P Krzewicka BPykaelae J Grube M Lutzoni F (2009) Genericclassification of the Verrucariaceae (Ascomycota)based on molecular and morphological evidencerecent progress and remaining challenges Taxon 58(1)184ndash208
Guillot G Mortier F Estoup A (2005) Geneland acomputer package for landscape genetics Mol EcolNotes 5(3)712ndash715 doi101111j1471-8286200501031x
Guillot G Renaud S Ledevin R Michaux J Claude J(2012) A unifying model for the analysis of pheno-typic genetic and geographic data Syst Biol 61(6)897ndash911 doi101093sysbiosys038
Guisan A Thuiller W (2005) Predicting species distribu-tion offering more than simple habitat models EcolLett 8(9)993ndash1009 doi101111j1461-0248200500792x
Hale ME (1990) A synopsis of the lichen genusXanthoparmelia (Vainio) Hale (AscomycotinaParmeliaceae) Smithsonian Institution Press Wash-ington DC
Hamilton CA Hendrixson BE Brewer MS Bond JE(2014) An evaluation of sampling effects on multipleDNA barcoding methods leads to an integrativeapproach for delimiting species a case study ofthe North American tarantula genus Aphonopelma(Araneae Mygalomorphae Theraphosidae) MolPhylogenet Evol 7179ndash93 doi101016jympev201311007
Hausdorf B (2011) Progress toward a general speciesconcept Evolution 65(4)923ndash931 doi101111j1558-5646201101231x
Hausdorf B Hennig C (2010) Species delimitation usingdominant and codominant multilocus markers SystBiol 59(5)491ndash503 doi101093sysbiosyq039
Hawksworth DL (1976) Lichen chemotaxonomy InBailey RH (ed) Lichenology progress and problemsAcademic London pp 139ndash184
Hebert PDN Cywinska A Ball SL deWaard JR (2003)Biological identifications through DNA barcodesProc R Soc Lond B Biol Sci 270(1512)313ndash321doi101098rspb20022218
Hebert PDN Stoeckle MY Zemlak TS Francis CM(2004) Identification of birds through DNA barcodesPLoS Biol 2(10)e312 doi101371journalpbio0020312
Heled J Drummond AJ (2010) Bayesian inference ofspecies trees from multilocus data Mol Biol Evol 27(3)570ndash580 doi101093molbevmsp274
Hey J (2006) On the failure of modern species conceptsTrends Ecol Evol 21(8)447ndash450 doi101016jtree200605011
Hibbett DS OhmanA Glotzer D NuhnMKirk P NilssonRH (2011) Progress in molecular and morphologicaltaxon discovery in Fungi and options for formalclassification on environmental sequences Fungal BiolRev 2538ndash47 doi101016jfbr201101001
Houmlgnabba F Wedin M (2003) Molecular phylogeny ofthe Sphaerophorus globosus species complex Cladis-tics 19(3)224ndash232 doi101111j1096-00312003tb00365x
Hudson RR Coyne JA (2002) Mathematical conse-quences of the genealogical species concept Evolu-tion 56(8)1557 doi101111j0014-38202002tb01467x
Huelsenbeck JP Andolfatto P Huelsenbeck ET (2011)Structurama Bayesian inference of population struc-ture Evol Bioinf Online 7(2011)55ndash59 doi104137EBOS6761
Jones G Oxelman B (2014) DISSECT an assignment-free Bayesian discovery method for species delimita-tion under the multispecies coalescent Bioinformaticsdoi101101003178
38 SD Leavitt et al
Kekkonen M Hebert PD (2014) DNA barcode-baseddelineation of putative species efficient start fortaxonomic workflows Mol Ecol Res doi1011111755-099812233
Kelly LJ Hollingsworth PM Coppins BJ Ellis CJHarrold P Tosh J Yahr R (2011) DNA barcoding oflichenized fungi demonstrates high identification suc-cess in a floristic context New Phytol 191(1)288ndash300doi101111j1469-8137201103677x
Kiss L (2012) Limits of nuclear ribosomal DNA internaltranscribed spacer (ITS) sequences as species barcodesfor fungi Proc Natl Acad Sci 109(27)E1811ndashE1811doi101073pnas1207143109
Kluge AG (1989) A concern for evidence and aphylogenetic hypothesis for relationships among Epi-crates (Boidae Serpentes) Syst Zool 38(1)7ndash25doi101093sysbio3817
Knowles LL Carstens BC (2007) Delimiting specieswithout monophyletic gene trees Syst Biol 56(6)887ndash895 doi10108010635150701701091
Kroken S Taylor JW (2001) A gene genealogical approachto recognize phylogenetic species boundaries in thelichenized fungus Letharia Mycologia 93(1)38ndash53
Kubatko LS Degnan JH (2007) Inconsistency of phylo-genetic estimates from concatenated data under coa-lescence Syst Biol 56(1)17ndash24 doi10108010635150601146041
Latch EK Dharmarajan G Glaubitz JC Rhodes OE Jr(2006) Relative performance of Bayesian clusteringsoftware for inferring population substructure andindividual assignments at low levels of populationdifferentiation Conserv Genet 7(2)295ndash302 doi101007s10592-005-9098-1
Lawrey JD (1986) Biological role of lichen substancesThe Bryologist 89(2)111ndash122
Le Gac M Hood ME Fournier E Giraud T (2007)Phylogenetic evidence of host-specific cryptic speciesin the anther smut fungus Evolution 61(1)15ndash26doi101111j1558-5646200700002x
Leacheacute AD (2009) Species tree discordance traces tophylogeographic clade boundaries in North Americanfence lizards (Sceloporus) Syst Biol 58(6)547ndash559doi101093sysbiosyp057
Leacheacute AD Fujita MK (2010) Bayesian species delim-itation in West African forest geckos (Hemidactylusfasciatus) Proc R Soc B Biol Sci 2773071ndash3077doi101098rspb20100662
Leacheacute AD Koo M Spencer C Papenfuss T Fisher RMcGuire J (2009) Quantifying ecological morpho-logical and genetic variation to delimit species in thecoast horned lizard species complex (Phrynosoma)Proc Natl Acad Sci USA 10612418ndash12423 doi101073pnas0906380106
Leacheacute AD Fujita MK Minin VN Bouckaert RR (2014)Species delimitation using genome-wide SNP dataSystematic Biology doi101093sysbiosyu018
Leavitt SD Fankhauser JD Leavitt DH Porter LDJohnson LA St Clair LL (2011a) Complex patterns ofspeciation in cosmopolitan ldquorock posyrdquo lichensmdashdiscovering and delimiting cryptic fungal species in
the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
Leavitt SD Esslinger TL Lumbsch HT (2012c) Neogene-dominated diversification in neotropical montanelichens dating divergence events in the lichen-form-ing fungal genus Oropogon (Parmeliaceae) Am J Bot99(11)1764ndash1777 doi103732ajb1200146
Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Lumbsch HT St Clair LL(2013c) DNA barcode identification of lichen-formingfungal species in the Rhizoplaca melanophthalmaspecies-complex (Lecanorales Lecanoraceae) includ-ing five new species MycoKeys 71ndash22 doi103897mycokeys74508
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Vondraacutek J Thorsten Lum-bsch H Clair LLS (2013d) Local representation ofglobal diversity in a cosmopolitan lichen-formingfungal species complex (Rhizoplaca Ascomycota)J Biogeogr 40(9)1792ndash1806 doi101111jbi12118
Leavitt SD Lumbsch HT Stenroos S St Clair LL(2013e) Pleistocene speciation in North American
2 The Dynamic Discipline of Species Delimitation hellip 39
lichenized fungi and the impact of alternative speciescircumscriptions and rates of molecular evolution ondivergence estimates PLoS ONE 8(12)e85240doi101371journalpone0085240
Leavitt SD Esslinger TL Hansen ES Divakar PK CrespoA Loomis BF Lumbsch HT (2014) DNA barcoding ofbrown Parmeliae (Parmeliaceae) species a molecularapproach for accurate specimen identification empha-sizing species in Greenland Organ Divers Evol 14(1)11ndash20 doi101007s13127-013-0147-1
Leliaert F Verbruggen H Vanormelingen P Steen FLopez-Bautista JM Zuccarello GC De Clerck O(2014) DNA-based species delimitation in algae Eur JPhycol 49(2) doi101080096702622014904524
Leuckert C (1985) Probleme der Flechten-Chemotaxon-omiemdashStoffkombinationen und ihre taxonomischeWertung Ber Deut Bot Ges 98401ndash408
Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
Lohtander K Kaumlllersjouml M Roland M Tehler A (2000)The family physciaceae in fennoscandia phylogenyinferred from its sequences Mycologia 92(4)728ndash735
Longton RE (1997) The role of bryophytes and lichens inpolar ecosystems In Woodin SJ Marquiss M (eds)Ecology of Arctic Environments Blackwell ScienceOxford pp 69ndash96 (Special publication No 13)
Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
Lumbsch HT (1994) Die Lecanora subfusca-Gruppe inAustralasien J Hattori Bot Lab 771ndash175
Lumbsch HT (1998a) Taxonomic use of metabolic data inlichen-forming fungi In Frisvad JC Bridge PDArora DK (eds) Chemical Fungal Taxonomy MarcelDekker New York pp 345ndash387
Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
Mayden RL (1997) A hierarchy of species concepts thedenouement in the saga of the species problem InClaridge MF Dawah HA Wilson MR (eds) Speciesthe units of biodiversity Chapman amp Hall Londonpp 381ndash424
Mayr E (1963) Animal species and evolution HarvardUniversity Press Cambridge
Mayr E (1970) Populations species and evolutionBelknap Press of Harvard University Press Cambridge
McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
McDonald T Miadlikowska J Lutzoni F (2003) Thelichen genus Sticta in the Great Smoky Mountains a
40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
McKay BD Mays HL Wu Y Li H Yao C-T Nishiumi IZou F (2013) An empirical comparison of character-based and coalescent-based approaches to speciesdelimitation in a young avian complex Mol Ecol 22(19)4943ndash4957 doi101111mec12446
Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
Mishler BD Brandon RN (1987) Individuality pluralismand the phylogenetic species concept Biol Philos 2397
Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
Nash TH Zavada M (1977) Population studies amongSonoran Desert species of Parmelia subg Xanthop-armelia (Parmeliaceae) Am J Bot 64(6)664ndash669
Nylander W (1866a) Circa novum in studio Lichenumcriterium chemicum Flora 49198ndash201
Nylander W (1866b) Hypochlorite of lime and hydrate ofpotash Two new criteria for the study of lichens(Tanslated and communicated by the Rev W ALeighton) Bot J Linn Soc 9358ndash365
OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
OrsquoNeill EM Schwartz R Bullock CT Williams JSShaffer HB Aguilar-Miguel X Parra-Olea G Weis-rock DW (2013) Parallel tagged amplicon sequencingreveals major lineages and phylogenetic structure inthe North American tiger salamander (Ambystomatigrinum) species complex Mol Ecol 22(1)111ndash129doi101111mec12049
Orock EA Leavitt SD Fonge BA St Clair LL LumbschHT (2012) DNA-based identification of lichen-form-ing fungi Can publicly available sequence databasesaid in lichen diversity inventories of Mount Cameroon(West Africa) The Lichenologist 44(6)833ndash839doi101017S0024282912000424
Otaacutelora MAG Martiacutenez I Aragoacuten G Molina MC (2010)Phylogeography and divergence date estimates of alichen species complex with a disjunct distributionpattern Am J Bot 97(2)216ndash223 doi103732ajb0900064
Padial J Castroviejo-Fisher S Kohler J Vila C ChaparroJ De la Riva I (2009) Deciphering the products ofevolution at the species level the need for anintegrative taxonomy Zoolog Scr 38(4)431ndash447doi101111j1463-6409200800381x
Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
Papong K Luumlcking R Thammathaworn A BoonpragobK (2009) Four new taxa of Chroodiscus (thelotremoidGraphidaceae) from Southeast Asia The Bryologist112(1)152ndash163 doi1016390007-2745-1121152
Park S-Y Choi J Kim JA Jeong M-H Kim S Lee Y-HHur J-S (2013a) Draft genome sequence of Cladoniamacilenta KoLRI003786 a lichen-forming fungusproducing biruloquinone Genome Announcements 1(5)e00695-13 doi101128genomeA00695-13
Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
Peacuterez-Ortega S Fernaacutendez-Mendoza F Raggio J VivasM Ascaso C Sancho LG Printzen C de los Riacuteos A(2012) Extreme phenotypic variation in Cetrariaaculeata (lichenized Ascomycota) adaptation or inci-dental modification Ann Bot 109(6)1133ndash1148doi101093aobmcs042
Pino-Bodas R Burgaz A Martiacuten M Lumbsch HT (2011)Phenotypical plasticity and homoplasy complicatespecies delimitation in the Cladonia gracilis group(Cladoniaceae Ascomycota) Organ Divers Evol 11(5)343ndash355 doi101007s13127-011-0062-2
Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
Pino-Bodas R Martiacuten M Burgaz A (2012b) Cladoniasubturgida and C iberica (Cladoniaceae) form asingle morphologically and chemically polymorphicspecies Mycol Prog 11(1)269ndash278 doi101007s11557-011-0746-1
Pino-Bodas R Ahti T Stenroos S Martiacuten MP BurgazAR (2013a) Multilocus approach to species recogni-tion in the Cladonia humilis complex (CladoniaceaeAscomycota) Am J Bot 100(4)664ndash678 doi103732ajb1200162
Pino-Bodas R Martiacuten AP Burgaz AR Lumbsch HT(2013b) Species delimitation in Cladonia (Ascomy-cota) a challenge to the DNA barcoding philosophyMol Ecol Resour 13(6)1058ndash1068 doi1011111755-099812086
Pons J Barraclough TG Gomez-Zurita J Cardoso ADuran DP Hazell S Kamoun S Sumlin WD VoglerAP (2006) Sequence-based species delimitation for theDNA taxonomy of undescribed insects Syst Biol 55(4)595ndash609 doi10108010635150600852011
Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
Pringle A Baker DM Platt JL Wares JP Latgeacute JPTaylor JW (2005) Cryptic speciation in the cosmo-politan and clonal human pathogenic fungus Asper-gillus fumigatus Evolution 59(9)1886ndash1899 doi10155404-2411
Printzen C (2009) Lichen systematics the role ofmorphological and molecular data to reconstructphylogenetic relationships Progress in botany vol71 Springer Berlin pp 233ndash275
Pritchard JK Stephens M Donnelly P (2000) Inference ofpopulation structure using multilocus genotype dataGenetics 155(2)945ndash959
Puillandre N Lambert A Brouillet S Achaz G (2012)ABGD automatic barcode gap discovery for primaryspecies delimitation Mol Ecol 21(8)1864ndash1877doi101111j1365-294X201105239x
Rannala B Yang Z (2003) Bayes estimation of speciesdivergence times and ancestral population sizes usingDNA sequences from multiple loci Genetics1641645ndash1656
Raxworthy CJ Ingram CM Rabibisoa N Pearson RG(2007) Applications of ecological niche modelingfor species delimitation a review and empiricalevaluation using day geckos (Phelsuma) fromMadagascar Syst Biol 56(6)907ndash923 doi10108010635150701775111
Ray J (1686) Historia planarum vol 1 Clark LondonReese Naeligsborg R Ekman S Tibell L (2007) Molecular
phylogeny of the genus Lecania (Ramalinaceaelichenized Ascomycota) Mycol Res 111(5)581ndash591doi101016jmycres200703001
Reid N Carstens B (2012) Phylogenetic estimation errorcan decrease the accuracy of species delimitation aBayesian implementation of the general mixed Yule-coalescent model BMC Evol Biol 12(1)196 doi1011861471-2148-12-196
Rissler L Apodaca J (2007) Adding more ecology intospecies delimitation ecological niche models andphylogeography help define cryptic species in theBlack Salamander (Aneides flavipunctatus) Syst Biol56924ndash942 doi10108010635150701703063
Rivas Plata E Luumlcking R (2013) High diversity ofGraphidaceae (lichenized Ascomycota Ostropales) inAmazonian Peruacute Fungal Divers 58(1)13ndash32 doi101007s13225-012-0172-y
Rivas Plata E Lumbsch HT (2011) Parallel evolution andphenotypic divergence in lichenized fungi a casestudy in the lichen-forming fungal family Graphida-ceae (Ascomycota Lecanoromycetes Ostropales)Mol Phylogenet Evol 61(1)45ndash63 doi101016jympev201104025
Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
Ross KG Gotzek D Ascunce MS Shoemaker DD (2010)Species delimitation a case study in a problematic anttaxon Syst Biol 59(2)162ndash184 doi101093sysbiosyp089
Rubin BER Ree RH Moreau CS (2012) Inferringphylogenies from RAD sequence data PLoS ONE 7(4)e33394 doi101371journalpone0033394
Rubinoff D (2006) Utility of mitochondrial DNA barcodesin species conservation Conserv Biol 20(4)1026ndash1033 doi101111j1523-1739200600372x
Ruiz-Sanchez E Sosa V (2010) Delimiting speciesboundaries within the Neotropical bamboo Otatea(Poaceae Bambusoideae) using molecular morpho-logical and ecological data Mol Phylogenet Evol 54(2)344ndash356 doi101016jympev200910035
Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
morphological characters Antarctic Sci 22 (SpecialIssue 06)727ndash741 doi101017S0954102010000477
Salicini I Ibaacutentildeez C Juste J (2011) Multilocus phylogenyand species delimitation within the Nattererrsquos batspecies complex in the Western Palearctic MolPhylogenet Evol 61(3)888ndash898 doi101016jympev201108010
Satler JD Carstens BC Hedin M (2013) Multilocusspecies delimitation in a complex of morphologicallyconserved trapdoor spiders (Mygalomorphae Antro-diaetidae Aliatypus) Syst Biol 62(6)805ndash823 doi101093sysbiosyt041
Schlick-Steiner BC Steiner FM Seifert B Stauffer CChristian E Crozier RH (2010) Integrative taxonomya multisource approach to exploring biodiversityAnnu Rev Entomol 55(1)421ndash438 doi101146annurev-ento-112408-085432
Schoch CL Seifert KA Huhndorf S Robert V SpougeJL Levesque CA Chen W Fungal Barcoding Con-sortium (2012) Nuclear ribosomal internal transcribedspacer (ITS) region as a universal DNA barcodemarker for Fungi Proc Natl Acad Sci 109(16)6241ndash6246 doi101073pnas1117018109
Seacuterusiaux E Villarreal AJC Wheeler T Goffinet B(2011) Recent origin active speciation and dispersalfor the lichen genus Nephroma (Peltigerales) inMacaronesia J Biogeogr 38(6)1138ndash1151 doi101111j1365-2699201002469x
Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
Simpson GG (1951) The species concept Evolution5285ndash298
Sites JW Marshall JC (2003) Delimiting species a renais-sance issue in systematic biology Trends Ecol Evol18462ndash470 doi101016S0169-5347(03)00184-8
Sites JW Marshall JC (2004) Operational criteria fordelimiting species Annu Rev Ecol Evol Syst 35(1)199ndash227 doi101146annurevecolsys35112202130128
Šlapeta J Loacutepez-Garciacutea P Moreira D (2006) Globaldispersal and ancient cryptic species in the smallestmarine eukaryotes Mol Biol Evol 23(1)23ndash29doi101093molbevmsj001
Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
Talavera G Dincă V Vila R (2013) Factors affectingspecies delimitations with the GMYC model insightsfrom a butterfly survey Methods Ecol Evol 4(12)1101ndash1110 doi1011112041-210X12107
Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
Tewksbury J Anderson JGT Bakker JD Billo TJDunwiddie PW Groom MJ Hampton SE HermanSG Levey DJ Machnicki NJ del Rio CM PowerME Rowell K Salomon AK Stacey L TrombulakSC Wheeler TA (2014) Natural historyrsquos place inscience and society BioScience doi101093bioscibiu032
Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
Werth S Cornejo C Scheidegger C (2013) Characteriza-tion of microsatellite loci in the lichen fungus Lobariapulmonaria (Lobariaceae) Appl Plant Sci 1(2)1200290 doi103732apps1200290
Westberg M Arup U Kaumlrnefelt I (2007) Phylogeneticstudies in the Candelariaceae (lichenized Ascomy-cota) based on nuclear ITS DNA sequence dataMycol Res 111(11)1277ndash1284 doi101017S0953756204002102
Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
2 The Dynamic Discipline of Species Delimitation hellip 43
(Lepidoptera Lycaenidae) Front Zoo 4(1)8 doi1011861742-9994-4-8
Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
more difficult in more recently diverged lineagesand in cases with some level of interspecific geneflow relative to older well-diverged lineages(eg Leavitt et al 2012c 2013e) In the end wereemphasize that single-locus data alone shouldonly be used to provide a preliminary perspectiveof species boundaries and not as the sole evi-dence in species circumscriptions
222 Sampling Across the GenomeMultilocus Sequence Dataand Genome-Wide Markers
Although single-locus methods can provide anefficient approach for preliminary large-scaleassessments of species diversity there are
significant limitations particularly in recentlydiverged species groups where retention ofancestral polymorphisms and incomplete lineagesorting will likely result in different neutral locihaving unique gene topologies that do not mirrorthe speciation process (Knowles and Carstens2007 Heled and Drummond 2010 Taylor et al2000) In contrast to single-locus and strictlyphenotype-based approaches for species delimi-tation analyses of genetic data collected fromindependent genomic regions can provide robusthypotheses of species boundaries with increasingconfidence (Satler et al 2013 Zhang et al 2011Yang and Rannala 2010) Sequences from mul-tiple independent loci provide an importantsource of data for species delimitation studiesincluding recently developed models that
Fig 22 A simplified diagram illustrating the process ofspeciation through time in a single gene history andresulting gene topologies sampled at two points in thespeciation history (modified from Leliaert et al 2014)aEach dot represents a distinct gene copy and each row onenon-overlapping generation with lines connecting genecopies to their ancestors in the previous generation (onerow below) dashed diagonal lines represent reproductivebarriers two hypothetical sampling intervals at differentpoints in the speciation history are shown Tx and Ty
species delimitation methods using single-locus data areeffective only when species are reciprocally monophyleticin the sampled gene tree genetic clustering and coalescent-based species delimitation methods can circumscribespecies when species may not be monophyletic in sampledgenetic loci b Gene topology representing sampledhaplotypes at time Tx (shown in panel a) hypotheticalspecies are reciprocally monophyletic c Gene topologyrepresenting sampled haplotypes at time Ty (shown in panela) hypothetical species are para- and polyphyletic
26 SD Leavitt et al
combine individual gene genealogies and speciesphylogenies via modeling the coalescent historyof markers (Yang and Rannala 2010 Edwards2009 Knowles and Carstens 2007) As aresponse to advances in sequencing technologiesbioinformatical approaches for multimarker spe-cies delimitation analyses continue to be devel-oped (Table 21 Camargo and Sites 2013)
Long-term reproductive isolation of candidatespecies can be assessed with multilocus sequencedata by evaluating genealogical concordance ofunlinked markers (Baum and Shaw 1995 Aviseand Ball 1990) Within species the mixingeffects of recombination cause unlinked loci tohave distinct genealogical histories but geneticdrift and long-term divergence leads to concor-dant genealogical histories at loci across thegenome Relationships of individuals belongingto distinct candidate species can be evaluatedusing gene genealogies (eg haplotype networksor single-gene topologies) to identify lineagesthat exhibit genealogical exclusivity acrossunlinked neutral loci (Hudson and Coyne 2002Dettman et al 2003b Avise and Ball 1990) Thepresence of the same clades in the majority ofsingle-locus genealogies is taken as evidence thatthe clades represent reproductively isolated lin-eages (Dettman et al 2003a Pringle et al 2005)In practice the criteria of reciprocal monophylyand genealogical concordance of unlinked lociprovide a conservative approach for assessingspecies boundaries due to the fact that a sub-stantial amount of time is required after the initialdivergence of species until ancestral polymor-phisms have fully sorted (Knowles and Carstens2007 Hudson and Coyne 2002) Consequentlygroups with recent divergence histories willlikely go undiscovered under a genealogicalconcordance criterion due to the fact that speciesboundaries likely are not reflected in genegenealogies For example it would take morethan 1 million years after speciation before spe-cies would be delimited if 15 loci were sampledin species with an effective population size (Ne)of 100000 assuming one generation a yearunder a strict reciprocal monophyly criterion(Knowles and Carstens 2007) The amount oftime required for a species to be recognized using
a criterion of reciprocal monophyly increasesproportionally with increasing population sizes(Hudson and Coyne 2002) In a number ofstudies of lichen-forming fungi a genealogicalconcordance criterion has been used to bothdelimit previously unrecognized species-levellineages and validate some conventional pheno-type-based species (Leavitt et al 2012a c2013b Molina et al 2011 Kroken and Taylor2001)
Although analyses of molecular sequence datarelying on reciprocal monophyly or fixed char-acter differences for species delimitation canserve as important criteria for identifying speciesthese criteria are not commonly met acrossmultiple loci particularly in recent speciationhistories (Fig 22) To accommodate theobserved conflict among genealogies from mul-tiple loci with the underlying speciation historythe recent merge of coalescent theory with phy-logenetics has driven a major paradigm shift inspecies delimitation and molecular systematics ingeneral (Fujita et al 2012 Edwards 2009) Themultispecies coalescent model can be applied togenealogical histories from multiple independentloci to assemble separate coalescent processesoccurring in populations into a species tree(Degnan and Rosenberg 2009 Rannala andYang 2003) Within this coalescent-basedframework multiple individuals can be assignedto a single speciespopulation and the speciationhistory of ancestral and descendant species-levellineages can be inferred as a ldquospecies treerdquo incontrast to estimating gene genealogies fromindividual samples (Degnan and Rosenberg2006 2009 Rannala and Yang 2003)
A number of multispecies coalescent-basedspecies delimitation methods have recently beenintroduced offering an exciting framework forempirically assessing species boundaries byselecting the best species tree model from a set ofalternative models representing differenthypotheses of species boundaries (Table 21)For example SpeDeSTEM (Ence and Carstens2011) finds the maximum likelihood values for aspecies tree assuming all putative species areseparate lineages and for alternative species treeswhere two or more species are collapsed into a
2 The Dynamic Discipline of Species Delimitation hellip 27
single lineage The best fitting model is thenselected using the Akaike information criterionunder the assumption of constant populationsizes and fixed gene topologies across the speciestree The species delimitation program BayesianPhylogenetic and Phylogeography (BPampPYangand Rannala 2010) accommodates gene treeuncertainty and variable population sizes andsamples from the Bayesian posterior distributionof species delimitation models using reversible-jump Markov chain Monte Carlo (rjMCMC)sampling BPampP requires a user-provided guidetree with resolution finer than the species leveland evaluates alternative modes derived from allpossible subtrees that are generated by collapsingor splitting nodes on the guide tree (Yang andRannala 2010 Rannala and Yang 2003) Theproportion of time spent on each model is pro-portional to the posterior probability of themodel ie ldquospeciation probabilitiesrdquo WhileBPampP ranks among the most popular coalescent-based species delimitation programs (Fujita et al2012) misspecifications of the guide tree andorthe prior distribution for population size (θ) canresult in strong support for models containing aninflated number of species (Leacheacute and Fujita2010 Zhang et al 2011) The nonparametricheuristic species delimitation approach imple-mented in the program Brownie (OrsquoMeara 2010)jointly sorts anonymous samples into species andinfers a species tree from input gene trees fromdifferent loci assuming that for a speciationevent the corresponding nodes on gene trees willbe more consistent with each than the diver-gences within species However finding both theoptimum species tree and species boundariesremains computationally challenging andBrownie has been shown to frequently yieldincorrect results (OrsquoMeara 2010)
Most recently available coalescent-basedspecies delimitation methods require individualassignments to a species or population a priori Ina number of scenarios correct assignments ofsamples to species may be difficult includingpresence of cryptic species incongruencebetween conventional species and moleculardata or simply the fact that accurate specimenidentification in complex groups with subtle or
difficult to discern diagnostic characters is anontrivial task (Leavitt et al 2013e) Statisticalmethods for assessing individual assignment andspecies detection prior to coalescent-based spe-cies delimitation and species tree reconstructionand species provide a more objective approachfor understanding species boundaries
Population assignment tests using a variety ofclustering algorithms using genetic data offer auseful approach for grouping individuals intoputative reproductively isolated groups (Table 21Hausdorf and Hennig 2010 Corander et al2004 2008 Falush et al 2003 Pritchard et al2000) particularly for species delimitation prob-lems that exist at the interface of traditionalpopulation genetic and phylogenetic analyses(Carstens et al 2013 Weisrock et al 2010Knowles and Carstens 2007)
The programs STRUCTURE (Falush et al2003 Pritchard et al 2000) and STRUCTURA-MA (Huelsenbeck et al 2011) cluster samplesinto populations by minimizing HardyndashWeinbergdisequilibrium for a given number of populationclusters using unlinked genetic markers In gen-eral unlinked markers are not available for mostgroups of lichen-forming fungi and a numberof studies have used information from multilocussequence data for STRUCTURE analyses(Leavitt et al 2011a b c 2013e Fernaacutendez-Mendoza and Printzen 2013 Fernaacutendez-Mendoza et al 2011) Varying approaches havebeen implemented to convert DNA sequencedata to allelic data for Bayesian clustering (seeOrsquoNeill et al 2013) STRUCTURE is expectedto perform well when there is sufficient inde-pendence across regions such that linkage dis-equilibrium within regions does not dominate thedata (STRUCTURE manual) but can also beeffective using multilocus sequence data andtreating all SNPs as independent loci regardlessof physical linkage within each locus (OrsquoNeillet al 2013 Falush et al 2003) A recent study ofthe lichen-forming genus Letharia showed thatBayesian clustering implemented in the programSTRUCTURE was generally able to recover thesame putative Letharia lineages circumscribedemploying a gene genealogical approach inKroken and Taylorrsquos iconic species delimitation
28 SD Leavitt et al
study (Altermann et al 2014 Kroken and Taylor2001) Altermann et al (2014) show that popu-lation assignments were largely consistent acrossa range of scenarios including extensiveamounts of missing data the exclusion of SNPsfrom variable markers and inferences based onSNPs from as few as three gene regions Incontrast to STRUCTURE and STRUCTURA-MA the program BAPS (Corander et al 2008)can explicitly infer genetic structure using hap-loid sequence data or other linked geneticmarkers in the ldquoclustering with linked locirdquomodel Another advantage of BAPS is that theprogram is much more computationally efficientthan STRUCTURE or STRUCTURAMA Sim-ulation studies indicate that both BAPS andSTRUCTURE perform well at low levels ofpopulation differentiation and when clusters arenot well differentiated (Latch et al 2006)
In practice inferring the most appropriatelevel of population structure using Bayesianclustering algorithms remains challenging (Latchet al 2006 Evanno et al 2005) BAPS andSTRUCTURAMA can infer both individualassignments and the most likely number ofgenetic clusters (Corander et al 2008 Huelsen-beck et al 2011) and the ad hoc ΔK statisticproposed by Evanno et al (2005) provides anobjective approach for identifying the uppermosthierarchical level of structure However carefulconsideration of other levels of populationstructure may ultimately reveal more biologicallymeaning results and researchers should examineindividual assignments across a range of geneticgroupings Some markers generated fromindependent genomic regions such as SNPs andfast-evolving microsatellites can be used to dis-tinguish fine-scale population structuring on thebasis of allele frequencies and species delimita-tion analyses based on these markers may implythe risk of severe taxonomic oversplitting Inthese cases validation approaches such asBPampP and SpeDeSTEM and corroborationamong different species delimitation approachescan provide perspective between intraspecificpopulation structure and species-level clusters
Another limitation of clustering approaches isthat they do not assess or take into account
evolutionarily divergences and relationshipsamong population clusters Coalescent-basedspecies tree methods (discussed below) provide ameans to assess the diversification histories ofpopulations inferred from clustering analysesTherefore a potential working protocol for aninformed species delimitation study that takesinto account population structure could consist offirst applying a genetic clustering analysis under apopulation genetics criterion (eg BAPSSTRUCTURE STRUCTURAMA) to identifygenetically distinct population clusters that can beconsidered ldquocandidate speciesrdquo From these can-didate species a species tree can be inferred forfocal group using coalescent-based species treereconstruction methods (eg BEAST) Subse-quently a coalescent-based validation methodcan be applied to assess whether the distinctpopulation clusters represent independent evolu-tionary lineages (eg BPampP) This multistepapproach would provide a consistent and standardcriterion for distinguishing between population-and species-level lineages (Camargo and Sites2013) and has been applied to a number of casesincluding lichen-forming fungi (Leavitt et al2011a b c 2013e Leacheacute and Fujita 2010)
High-throughput sequencing methods providethe means to effectively sample hundreds tothousands of loci from across a species genome fora large number of species and continue to revolu-tionize studies that can be performed even in non-model organisms Targeted high-throughputsequencing approaches such as anchored phy-logenomics transcriptome sequencing reduced-representation genomic library sequencing(restriction-site-associated DNA sequencingRAD-Seq and genotype-by-sequencing GBS)and high-throughput amplicon sequencing pro-vide important insight into species boundaries fora number groups although none of these approa-ches has yet been applied to studies of lichenizedfungi
For example restriction-site-associated(RAD-Seq) DNA can simultaneously detect andgenotype thousands of genome-wide SNPs byfocusing the sequencing effort on a reducedrepresentation of the entire genome (Baird et al2008) and has been successfully applied to
2 The Dynamic Discipline of Species Delimitation hellip 29
intraspecies (Lewis et al 2007 Miller et al 2007Emerson et al 2010) and interspecies studies(Rubin et al 2012 Eaton and Ree 2013 Wagneret al 2013) This approach has provided strikinginsight into the recent adaption radiation of LakeVictoria cichlids (Wagner et al 2013) Analternative approach targeting the sequencing ofspecific loci using next-generation sequencingplatforms provides an efficient means of gener-ating data for loci of known genomic locationorthology size and expected level of variation(OrsquoNeill et al 2013) Markers can be targetedusing well-established PCR techniques or newerhybridization techniques and subsequentlypooled for high-throughput sequencing usingparallel-tagged sequencing of multiple individualsamples within a single sequencing run (OrsquoNeillet al 2013) A recent study of North Americantiger salamanders (Ambystoma tigrinum)demonstrated the potential for amplicon-basedparallel-tagged sequencing to rapidly generatelarge-scale genomic data for species delimitationand species tree research (OrsquoNeill et al 2013)
Although these methods provide an enormousamount of data and insight into diversification atan unprecedented scale computational limita-tions restrict the applicable analytical methodsfor large datasets although computational andanalytical advances are happening rapidly Mostrecent coalescent-based species delimitation andspecies tree models using gene trees have beenlimited to handle tens of loci for multiple indi-viduals and combining hundreds or thousands ofgene trees into a single species delimitationframework presents considerable computationalchallenges (Camargo and Sites 2013) Therecently developed method Bayes factor delim-itation (with genomic data BFD) combines adynamic programming algorithm for estimatingspecies trees that bypasses the computationallyintensive MCMC integration over gene trees toprovide a rigorous technique for species delimi-tation studies using genome-wide SNP data(Leacheacute et al 2014) Competing species delimi-tation models are compared using Bayes factorsand it appears that this approach is robust tosample sizes (ie few loci and limited samples
per species) and misspecification of the prior forpopulation size (θ) (Leacheacute et al 2014)
Fungi are an ideal model for assessing diver-gence in eukaryotes due to their simple mor-phologies small genomes broad ecological rolesand diverse lifestyles (Gladieux et al 2014)However the use of genomic data from high-throughput sequencing methods have not yet beenincluded in species delimitation studies of lichen-forming fungi This is due in part to challenges indealing with metagenomic data containing geno-mic information from a plethora of symbiontsassociated with a lichen thallus scant genomicresources and discipline-specific inertia A num-ber of lichen-forming fungal genomes are cur-rently available including Endocarponpusillum(Wang et al 2014) Cladonia spp (Park et al2013a 2014) Caloplaca flavorubescens (Parket al 2013b) and Cladonia greyi and Xanthoriaparientina through the Fungal Genomics Programat Joint Genome Institute of the United StatesDepartment of Energy (httpwwwjgidoegov)The foreseeable ongoing expansion of genomicresources for lichen-forming fungi will be centralto developing approaches for delimiting speciesusing high-throughput sequencing Specificallygenomic resources will be crucial in identifyingnovel markers (variable genes regions SNPsmicrosatellites etc) identifying conserved fungalmarkers for targeted high-throughput sequencingapproaches and references for entire genomecomparisons (Devkota et al 2014 Werth et al2013)
23 Can We Make SpeciesDelimitation in Lichen-FormingFungi Truly Integrative
The widespread availability of genetic data hascreated a biased viewpoint that only genetic datashould be used for statistical species delimitationHowever an ongoing appeal to researchers toassess species boundaries from multiple andcomplementary perspectives (phylogeneticspopulation genetics comparative morphologydevelopment ecology etc) has resulted in an
30 SD Leavitt et al
integrative taxonomic framework bringing theseconceptual and methodological perspectivestogether (Hamilton et al 2014 Fujita et al 2012Salicini et al 2011 Yeates et al 2011 Padialet al 2010 Dayrat 2005 Will et al 2005 Wiensand Penkrot 2002) In practice any study linkingdifferent kinds of data to support hypotheses ofspecies boundaries including mapping morpho-logical characters onto a molecular phylogenycan be considered integrative Integrative meth-ods for species delimitation fall across a broadspectrum ranging from verbal and qualitativeassessments of data classes to quantitative meth-ods that allow different data types to contribute tostatistical species delimitation (Yeates et al2011) In most taxonomic studies utilizing bothmolecular and morphological data expert opinionis eventually used to some degree to evaluate thefinal status of a candidate species (eg Bond andStockman 2008) In this sense many studiesusing evidence for independent data sources fordelimiting species boundaries use an iterativeapproach (Yeates et al 2011) where speciesboundaries can be tested within a hypothetico-deductive framework with diverse datasets
From a practical perspective we advocate aprocess of iterative taxonomy (sensu Yeates et al2011) to circumscribe and refine species limitsusing multiple lines of evidence This iterativeprocess involves comparisons of morphologicaldata with a phylogenetic hypothesis (ie single-locus gene tree or concatenated multilocusphylogeny) to identify the least inclusive mono-phyletic clade in the topology characterized by atleast one unambiguously diagnostic morphologi-cal character (Miralles and Vences 2013) Thisphylogenetic tree-informed approach to assessingspecies boundaries represents a common practicein studies of lichen-forming fungi Previouslyunrecognized species-level clades with corre-sponding subtle or overlooked phenotypiccharacters have been commonly observed in bothcrustose lichens [eg Acarospraceae (Wedin et al2009) Graphidaceae (Rivas Plata and Lumbsch2011 Papong et al 2009) Lecanoraceae (Leavittet al 2011a) Lecideaceae (Ruprecht et al 2010)Mycoblastaceae (Spribille et al 2011) andTeloschistaceae (Vondraacutek et al 2009 Muggia
et al 2008)] and foliose and fruticose lichens [egLobariaceae (Moncada et al 2014 McDonaldet al 2003) Parmeliaceae (Leavitt et al 2013aWirtz et al 2012 Divakar et al 2005 Molinaet al 2004 Kroken and Taylor 2001) Peltigera-ceae (OrsquoBrien et al 2009 Goffinet et al 2003)Physciaceae (Elix et al 2009 Divakar et al2007) and Sphaerophoraceae (Houmlgnabba andWedin 2003)] As a specific example Luumlckinget al (2008) used a combination of medullarychemistry and underside pigmentation in speci-mens from the Heterodermia obscurata group inCosta Rica to corroborate monophyletic clades inan ITS gene tree as species-level lineages
While this iterative approach to speciesdelimitation and taxonomy has proven valuablefor understanding species boundaries anddescribing new taxa in some groups of lichenizedfungi a posteriori examination of morphologicaland chemical features has failed to reveal diag-nostic phenotypic characters in a number ofstudies (Muggia et al 2014 Leavitt et al 2011aOtaacutelora et al 2010 OrsquoBrien et al 2009) Fur-thermore a study of widespread species in thegenus Melanelixia (Parmeliaceae) indicated thatphenotypically cryptic lichen-forming fungalspecies-level lineages may be relatively ancientand diagnosable phenotypic differences may beabsent even millions of years after the initialdivergence (Leavitt et al 2012b) The latter studyhighlights the fact that species-level lineages maycommonly exist without any observable diag-nostic phenotypic characters calling into ques-tion the impetus for a universal application ofintegrative taxonomy
In keeping with the principle that as manylines of evidence as available should be com-bined to delimit species (Dayrat 2005) the for-malized integrative taxonomic approach (ITAXMiralles and Vences 2013) uses a mtDNA guidetree and observations from different types of datathat might provide conclusive evidence for theindependence of lineages and thus their identityas different species Miralles and Vences (2013)provide a non-exhaustive list of species delimi-tation criteria to be integrated in the ITAXapproach including (a) sympatric occurrencewithout admixture as revealed by consistent
2 The Dynamic Discipline of Species Delimitation hellip 31
differences in morphological or molecular char-acters at the same geographic location (b) strongdifferences in a behavioral morphological orgenetic character known to mediate prematingisolation (c) unviability or infertility of hybrids(d) lack of gene flow across a geographicalhybrid zone (e) congruent diagnostic differencesbetween sister lineages in various unlinkedmorphological character (f) absence of haplo-type sharing in several unlinked nuclear loci and(g) a combination of criteria endashf Speciesboundaries are based on seeking the least inclu-sive monophyletic group in the mtDNA treewhich fulfills at least one of the criteria listedabove Clearly the ITAX approach is sensitive tosample size and in order to reliably support thedistinctiveness of a given species it has beenrecommended that the sampling strategy includesat least five individuals per species (Miralles andVences 2013) A similar approach could beadopted for lichenized fungi using an ITS genetopology as guide tree rather than mtDNA
A total evidence approach including concat-enation has been a common approach for inte-grating information from different sourcesincluding independent genetic markers in phy-logenetic reconstructions (Kluge 1989 Wiens1998 de Queiroz et al 1995) Proponents forconcatenation of independent data in phyloge-netic analyses argue that when combining alldata the underlying signal of speciation mayemerge even if weakly contradictory signalis contained in the individual data partitions(Gatesy et al 1999) For example establishing apreliminary perspective of species boundariesfrom multilocus sequence data using concatena-tion may provide a reasonable starting point forscreening for cryptic species and species treeinference (Šlapeta et al 2006 Leavitt et al2011a Le Gac et al 2007 Leacheacute 2009)However concatenation and consensus methodsimply a risk of obtaining inflated support forincorrect relationships and information aboutvariance in gene coalescence is lost (Degnan andRosenberg 2006 2009 Kubatko and Degnan2007 Edwards 2009) In spite of the impetus forintegrating evidence from independent data
sources into empirical species delimitation stud-ies and taxonomy (Fujita et al 2012 Padial et al2009) most currently available species delimi-tation methods are unable to accommodate non-genetic data sources in a statistical framework
231 Selecting the Appropriate Data
In the face of increasing availability of geneticdata and associated bioinformatical approachesfor delimiting species researchers should care-fully consider what information is being sacri-ficed by the failure to consider non-genetic datain species delimitation studies and whetheraccuracy could be improved by the addition ofmultiple data types Morphological data havehistorically served as a proxy to identify repro-ductively isolated groups (ie ldquospeciesrdquo) (Ray1686 Fujita et al 2012) Current methods fordelimiting species using non-genetic data (egchemistry morphology and ecology) remainwoefully understudied For example morphol-ogy-based species circumscriptions are generallybased on one or more qualitative (or quantitative)morphological characters that do not appear tooverlap with other species However ascertain-ing that a given trait is truly fixed within apopulation with statistical confidence requiresunrealistic sample sizes even when allowing forsome level of polymorphism in the diagnosticcharacter (Wiens and Servedio 2000) Now var-ious combinations of datamdashfrom morphologygenetics geography and ecologymdashare acceptedas standard information for species delimitationstudies (Ruiz-Sanchez and Sosa 2010 Ross et al2010 Edwards and Knowles 2014)
Below we briefly discuss appropriate datasources for species delimitation studies of lichen-forming fungi However homoplastic characters(similar traits that are not derived from a com-mon ancestor) are common among many traitscommonly used to circumscribe fungal taxa thebiological significance of secondary metabolitevariation remains largely unknown (Lawrey1986) and ecological niches may be difficultto adequately characterize and model due to
32 SD Leavitt et al
microhabitat requirements and data resolutionTherefore we advocate a cautious approach toselecting appropriate and relevant data forassessing species boundaries
In spite of the potential challenges and limi-tations of using phenotypic data these traits haveprovided a plethora of valuable information forunderstanding species boundaries For examplein the Melanelixia fuliginosa group (Parmelia-ceae) a morphometric analysis using color isi-dia and marginal zone free of isidia ascharacters revealed a general pattern of differ-entiation between material formerly recognizedas subspecies fuliginosa and glabratula in spiteof the fact that considerable overlap betweengroups occurs in some characters individually(Arup and Berlin 2011) Furthermore the dis-tinction of the two groups was supported by aphylogenetic analysis of the ITS marker andecological differences with M fuliginosaoccurring predominantly on rock and M glab-ratula on bark (Arup and Berlin 2011) Thisstudy of the M fuliginosa group provides a fit-ting example of using multiple independentsuites of data including ecology morphologyand genetic information to establish a robusthypothesis of species boundaries
In lichen systematics phenotypic dataincluding thallus organization secondary metab-olites mode of reproduction ascoma-type andontogeny ascus and ascospore characters havehistorically played a prominent role (Printzen2009) Ascomatal characters have traditionallyheld a major role in higher-level classification(Printzen 2009) in contrast to species-levelclassification which also tends to include a widearray of vegetative and chemical charactersCommonly assessed morphological charactersinclude thallus form and size cortical features(eg maculae and pseudocyphellae) presenceformcolor of attachment structure (eg rhizines)and reproductive mode (ascomata vs vegetativediaspores) (Printzen 2009) Different morpho-logical types of vegetative diasporesmdashcorticatedisidia and ecorticated sorediamdashand their locationare commonly used to distinguish speciesAscomatal characters including morphologylocation (laminal vs marginal) position (sessile
vs immersed) presence of thalline margins colorof an apothecial disc and presence or color ofpruina are also commonly used in speciesdelimitations of lichen-forming fungi (Printzen2009) Other important characters may includethalline characters form color size and septationof ascospores size and form and structure of ascithe hamathecium type of epihymenium andhypothecium and the type of excipulum orperidium conidiomatal characters etc (Printzen2009)
Assessments of secondary metabolites hasplayed an important role in lichen taxonomybeginning with the introduction of simple spottests by Nylander (1866a b) The use of chem-istry in lichen taxonomy has been discussed indetail in numerous reviews (Lumbsch 1998a bRogers 1989 Brodo 1986 Egan 1986 Leuckert1985 Brodo 1978 Hawksworth 1976 Culber-son 1969 1970) and we refer readers to thesevaluable sources for a more comprehensive per-spective on lichen chemistry In short extrolites(secondary metabolites) belong to various clas-ses the most common and diverse include dep-sides depsidones chlorinated xanthones andanthraquinones (Lumbsch 2002 Culberson1969) The presence or absence of specificextrolites or their replacements by another sub-stance is widely used to distinguish speciesparticularly when correlated with differences ingeographic distributions However if morpho-logical or geographical differences betweenpopulations containing different extrolites are notapparent the taxonomic significance has beendisputed with some authors distinguishing themas species and others preferring to regard them aschemical races within a species In addition tosimply using the presence or absence of extro-lites Culberson and Culberson (1976) proposedto arrange lichen substances into chemosyn-dromes of closely related substances The pres-ence or absence of these chemosyndromes maypotentially be used as characters to delimit spe-cies regarding differences involving the samechemosyndromes as intraspecific variation anddistinct chemosyndromes as evidence for inter-specific populations (Lumbsch 1994 Gowan1986)
2 The Dynamic Discipline of Species Delimitation hellip 33
Due to the fact that species delimitationstudies often incorporate a substantial biogeo-graphical or ecological component ecologicalniche modeling plays an increasing role in phy-logenetic and taxonomic research Niche mod-eling can provide evidence for ecologicalisolation between populations based on eitherconserved or divergent ecological niches andtherefore can provide additional evidence sup-porting lineage independence between putativespecies The application of ecological nichemodeling has been applied to species delimita-tion studies in a number of cases (Ruiz-Sanchezand Sosa 2010 Leacheacute et al 2009 Raxworthyet al 2007 Rissler and Apodaca 2007) althoughit has not been explicitly applied to assess speciesboundaries in lichen-forming fungi By mappingthe spatial distribution of environmental suit-ability of climatic variables for candidate speciesthe application of ecological niche modeling canbe particularly important in cases where specieshave allopatric distributions (Raxworthy et al2007)
Ecological niche modeling utilizes knownassociations between a speciesrsquo occurrencelocalities and environmental variables to defineabiotic conditions within which populations canbe maintained (Guisan and Thuiller 2005) Themethodological approach for modeling is basedon four general properties (i) The current knownspeciesrsquo localities is the dependent variable (ii)the distribution is modeled as a map composed ofgrid cells at a specified resolution (iii) a range ofenvironmental variables (eg temperature pre-cipitation and solar exposure) are collected todescribe the characteristics of each cell and (iv)classifying the degree to which each cell is eithersuitable or unsuitable for each species under arange of models (Guisan and Thuiller 2005)
Ecological data also have the potential to playan important role in understanding speciesboundaries in lichen-forming fungi For exampleNash and Zavada (1977) demonstrated that Xant-hoparmelia populations with distinct chemistriesoccurring in the northern portion of the SonoranDesert exhibit habitat selection among differentrock substrates within a region with relativelyuniform climate and topography In another case
the parmelioid species Parmelia mayi is mor-phologically indistinguishable from P saxatilisbut can be separated by bioclimatic features andgenetic and chemical characters (Molina et al2011) McCune and Printzen(2011) assess distri-butions and climatic niches of species in theLecanora varia group inwesternUSAandprovidea model that uses continental influence and annualtemperature as themajor factors predicting speciesdistributions The distribution of Usnea hirta alichen commonly used in air quality biomonitor-ing research was modeled for a section of theWhite River National Forest in central Coloradobased on the presence of U hirta at 72 biomoni-toring reference sites distributed in the inter-mountain western United States (Shrestha et al2012) The best model for predicting U hirtadistribution included four variablesmdashsolar radia-tion average monthly precipitation and averagemonthly minimum and maximum temperatures(Shrestha et al 2012) These studies supportthe potential use of ecological niche modelingmethods in species delimitation studies of lichen-forming fungi
24 Conclusions What AboutTaxonomy
In most cases species circumscription and tax-onomy requires some degree of qualitativejudgment and individual interpretation Integrat-ing multiple types of data into an empiricalframework for delimiting species boundarieswhere species boundaries can be tested within ahypothetico-deductive framework with diversedatasets can provide robust hypotheses of speciesboundaries and taxonomic stability (Yeates et al2011) However it has long been known toevolutionary biologists that distinct species donot need to have diagnosable morphologicaldifferences (Mayr 1963) and increasing avail-ability of genetic data has allowed researchers toidentify species and to rigorously test speciesboundaries with a level of precision that wasunimaginable a decade ago While analyticaladvances in statistical species delimitation havebeen largely based on genetic data the utility of
34 SD Leavitt et al
these approaches to formal taxonomy remainselusive Due to these challenges an eclecticapproach to delimiting species and cautionagainst the reliance on any single dataset ormethod is required when delimiting species
While results from many recent studies maycontradict traditional species boundaries acrossmany groups of lichen-forming fungi we areoptimistic that this research represents substantialprogress toward a more accurate perspective onspecies boundaries and diversity in fungi As aresult of ongoing research of species boundariesin lichen-forming fungi the taxonomic value ofmany phenotypes is now better understood ourunderstanding of ecological evolutionary andbiogeographic patterns has improved and we canbegin to better understand patterns of symbiontinteractions in lichens Integrating new data(including novel morphological characters andgenetic data) will be essential to accurately rep-resent species-level diversity across all groups oflichenized fungi Hopefully an improved per-spective on lichen diversity also increases ourappreciation of these incredible symbiotic sys-tems While we are strong advocates for theapplication of independent data types in devel-oping an integrative taxonomy there is anincreasing need to formally recognize the exis-tence of phenotypically cryptic species-levellineages in lichen-forming fungi (see Hibbettet al 2011) In some cases a molecular taxon-omy may provide the most practical approach toconsistent treatment of mycobiont species withinlichen groups where diagnostic morphologicalcharacters are unidentifiable or practically notfeasible (Leavitt et al 2013c d)
These are exciting times for taxonomists andphylogeneticists A closer look at lichen taxon-omy with the inclusion of new data will help usto better understand the diversity of these fasci-nating organisms accurately interpret distribu-tion patterns and play a more important role inmeaningful conservation practices Howeversome level of uncertainty will accompany pro-gress In many taxonomic groups our traditionalapproach for species identification will likelyneed to be substantially modified The search forcorroborating morphological support for cryptic
species identified using molecular data willrequire meticulous and creative approaches toassess phenotypic variation in potentially unor-thodox ways We are hopeful that lichenologistswho traditionally have been eager to include newmethods such as chromatography in their rou-tine identifications will be amenable to includemolecular techniques to their routine examina-tion of specimens for identification and classifi-cation Although this may prove difficult toachieve by single individuals especially citizenscientists that traditionally play an important rolein lichen taxonomy the increasing numberof collaborative projects in the discipline (egLumbsch et al 2011 Crespo et al 2010Gueidan et al 2009) make us optimistic thatbroad-scale collaborative approaches will facili-tate the inclusion of molecular data in lichenresearch at all levels
Acknowledgments We are indebted to various col-leagues for valuable thought-provoking discussionnotably Matthew Nelsen (University of Chicago) AnaCrespo (Universidad Complutense de Madrid) PradeepDivakar (Universidad Complutense de Madrid) BeckettSterner (The Field Museum) and Joyce Havstad (TheField Museum) We also thank anonymous reviewers whoprovided valuable comments that improved this chapterSupport by the US National Science Foundation isgratefully acknowledged (ldquoHidden diversity in parmelioidlichensrdquo DEB-0949147)
References
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Altermann S Leavitt SD Goward T Nelsen MPLumbsch HT (2014) How do you solve a problemlike Letharia A new look at cryptic species in lichen-forming fungi using Bayesian clustering and SNPSsfrom multilocus sequence data PLoS ONE 9(5)e97556 doi101371journalpone0097556
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2 The Dynamic Discipline of Species Delimitation hellip 35
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Corander J Marttinen P (2006) Bayesian identification ofadmixture events using multi-locus molecular mark-ers Mol Ecol 15(10)2833ndash2843 doi101111j1365-294X200602994x
Corander J Waldmann P Marttinen P Sillanpaa M(2004) BAPS 2 enhanced possibilities for the analysisof genetic population structure Bioinformatics 20(15)2363ndash2369 doi101093bioinformaticsbth250
Corander J Marttinen P Mantyniemi S (2006) Bayesianidentification of stock mixtures from molecular markerdata Fish Bull 104550ndash558
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Cracraft J (1983) Species concepts and speciation anal-ysis Curr Ornithol 1159ndash187
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Culberson WL (1969) The use of chemistry in thesystematics of the lichens Taxon 18498ndash505
Culberson WL (1970) Chemosystematics and ecology oflichen-forming fungi Annu Rev Ecol Syst 1153ndash170
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Darwin C (1859) On the origin of species by means ofnatural selection or the preservation of favoured racesin the struggle for life J Murray London
Dayrat B (2005) Towards integrative taxonomy Biol JLinn Soc 85(3)407ndash415 doi101111j1095-8312200500503x
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de Queiroz K (1998) The general lineage concept ofspecies species criteria and the process of speciationa conceptual unification and terminological recom-mendations In Howard DJ Berlocher SH (eds)Endless forms species and speciation Oxford Uni-versity Press Oxford pp 57ndash75
de Queiroz K (1999) The general lineage concept ofspecies and the defining properties of the speciescategory In Wilson RA (ed) Species new interdis-ciplinary essays MIT Press Cambridge pp 49ndash89
de Queiroz K (2007) Species concepts and speciesdelimitation Syst Biol 56(6)879ndash886 doi10108010635150701701083
de Queiroz A Donoghue MJ Kim J (1995) Separateversus combined analysis of phylogenetic evidenceAnnu Rev Ecol Syst 26(1)657ndash681 doi101146annureves26110195003301
Degnan J Rosenberg N (2006) Discordance of speciestrees with their most likely gene trees PLoS Genet 2(5)e68 doi101371journalpgen0020068
Degnan JH Rosenberg NA (2009) Gene tree discordancephylogenetic inference and the multispecies coales-cent Trends Ecol Evol 24(6)332ndash340 doihttpdxdoiorg101016jtree200901009
Del-Prado R Cubas P Lumbsch HT Divakar PK BlancoO de Paz GA Molina MC Crespo A (2010) Geneticdistances within and among species in monophyleticlineages of Parmeliaceae (Ascomycota) as a tool fortaxon delimitation Mol Phylogenet Evol 56(1)125ndash133 doi101016jympev201004014
Del-Prado R Divakar PK Crespo A (2011) Using geneticdistances in addition to ITS molecular phylogeny toidentify potential species in the Parmotrema reticul-atum complex a case study The Lichenologist 43(06)569ndash583 doi101017S0024282911000582
Del-Prado R Blanco O Lumbsch HT Divakar PK ElixJA Molina MC Crespo A (2013) Molecular phylog-eny and historical biogeography of the lichen-formingfungal genus Flavoparmelia (Ascomycota Parmelia-ceae) Taxon 62(5)928ndash939 doi101270562522
Dettman J Jacobson D Taylor J (2003a) A multilocusgenealogical approach to phylogenetic species recog-nition in the model eukaryote Neurospora Evolution57(12)2703ndash2720 doi101111j0014-38202003tb01514x
Dettman JR Jacobson DJ Turner E Pringle A TaylorJW (2003b) Reproductive isolation and phylogeneticdivergence in Neurospora comparing methods ofspecies recognition in a model eukaryote Evolution57(12)2721 doi10155403-074
Devkota S Cornejo C Werth S Chaudhary RP Sche-idegger C (2014) Characterization of microsatelliteloci in the Himalayan lichen fungus Lobaria pindar-ensis (Lobariaceae) Appl Plant Sci 2(5)1300101doi103732apps1300101
Divakar PK Molina MC Lumbsch HT Crespo A (2005)Parmelia barrenoae a new lichen species related toParmelia sulcata (Parmeliaceae) based on molecularand morphological data The Lichenologist 37(01)37ndash46 doi101017S0024282904014641
Divakar PK Amo De paz G del Prado R Esslinger TLCrespo A (2007) Upper cortex anatomy corroboratesphylogenetic hypothesis in species of Physconia(Ascomycota Lecanoromycetes) Mycol Res 111(11)1311ndash1320 doi101016jmycres200708009
Divakar PK Figueras G Hladun N Crespo A (2010)Molecular phylogenetic studies reveal an undescribedspecies within the North American concept of Mela-nelixia glabra (Parmeliaceae) Fungal Divers 42(1)47ndash55 doi101007s13225-010-0027-3
Donoghue MJ Gauthier A (2004) Implementing thephylocode Trends Ecol Evol 19(6)281ndash282 doi101016jtree200404004
Eaton DAR Ree RH (2013) Inferring phylogeny andintrogression using RADseq data an example fromflowering plants (Pedicularis Orobanchaceae) SystBiol 62(5)689ndash706 doi101093sysbiosyt032
Edwards SV (2009) Is a new and general theory ofmolecular systematics emerging Evolution 63(1)1ndash19 doi101111j1558-5646200800549x
Edwards DL Knowles LL (2014) Species detection andindividual assignment in species delimitation canintegrative data increase efficacy Proc R Soc B BiolSci 281(1777) doi101098rspb20132765
Egan RS (1986) Correlations and non-correlations ofchemical variation patterns with lichen morphologyand geography The Bryologist 8999ndash110
Elix JA Corush J Lumbsch HT (2009) Triterpenechemosyndromes and subtle morphological characterscharacterise lineages in the Physcia aipolia group inAustralia (Ascomycota) Syst Biodivers 7(04)479ndash487 doi101017S1477200009990223
Emerson KJ Merz CR Catchen JM Hohenlohe PACresko WA Bradshaw WE Holzapfel CM (2010)Resolving postglacial phylogeography using high-throughput sequencing Proc Natl Acad Sci 107(37)16196ndash16200 doi101073pnas1006538107
Ence DD Carstens BC (2011) SpedeSTEM a rapid andaccurate method for species delimitation Mol EcolResour 11(3)473ndash480 doi101111j1755-0998201002947x
EvannoGRegnaut SGoudet J (2005)Detecting thenumberof clusters of individuals using the software STRUC-TURE a simulation study Mol Ecol 14(8)2611ndash2620doi101111j1365-294X200502553x
Falush D Stephens M Pritchard JK (2003) Inference ofpopulation structure using multilocus genotype datalinked loci and correlated allele frequencies Genetics164(4)1567ndash1587 doi101111j1471-8286200701758x
Fan HH Kubatko LS (2011) Estimating species treesusing approximate Bayesian computation Mol Phylo-genet Evol 59(2)354ndash363 doi101016jympev201102019
Fernaacutendez-Mendoza F Printzen C (2013) Pleistoceneexpansion of the bipolar lichen Cetraria aculeata intothe Southern hemisphere Mol Ecol 22(7)1961ndash1983doi101111mec12210
Fernaacutendez-Mendoza F Domaschke S Garciacutea MA JordanP Martin MP Printzen C (2011) Population structure
2 The Dynamic Discipline of Species Delimitation hellip 37
of mycobionts and photobionts of the widespreadlichen Cetraria aculeata Mol Ecol 20(6)1208ndash1232doi101111j1365-294X201004993x
Fraley C Raftery A (2007) Model-based methods ofclassification Using the mclust software in chemo-metrics J Stat Softw 18i06 [Available online at httpwwwdoajorgdoajfunc=abstractampid=218544]
Fujisawa T Barraclough TG (2013) Delimiting speciesusing single-locus data and the generalized mixed yulecoalescent (GMYC) approach a revised method andevaluation on simulated datasets Syst Biol 62(5)707ndash724 doi101093sysbiosyt033
Fujita MK Leacheacute AD Burbrink FT McGuire JA MoritzC (2012) Coalescent-based species delimitation in anintegrative taxonomy Trends Ecol Evol 27(9)480ndash488 doi101016jtree201204012
Gatesy J OrsquoGrady P Baker RH (1999) Corroborationamong data sets in simultaneous analysis hiddensupport for phylogenetic relationships among higherlevel Artiodactyl taxa Cladistics 15(3)271ndash313doi101111j1096-00311999tb00268x
Gladieux P Ropars J Badouin H Branca A Aguileta Gde Vienne DM Rodriacuteguez de la Vega RC Branco SGiraud T (2014) Fungal evolutionary genomicsprovides insight into the mechanisms of adaptivedivergence in eukaryotes Mol Ecol 23(4)753ndash773doi101111mec12631
Goffinet B Miadlikowska J Goward T (2003) Phyloge-netic inferences based on nrDNA sequences supportfive morphospecies within the Peltigera didactylacomplex (Lichenized Ascomycota) The Bryologist106(3)349ndash364 doi10163901
Gowan SP (1986) Evolution of secondary natural productsin the genus Porpidia (Ascomycata Porpidiaceae) AmJ Bot 73606
Griffin PC Hoffmann AA (2014) Limited genetic diver-gence among Australian alpine Poa tussock grassescoupled with regional structuring points to ongoinggene flow and taxonomic challenges Ann Bot doi101093aobmcu017
Grube M Hawksworth DL (2007) Trouble with lichenthe re-evaluation and re-interpretation of thallus formand fruit body types in the molecular era Mycol Res111(9)1116ndash1132 doi101016jmycres200704008
Gueidan C Savi S Thues H Roux C Keller C Tibell LPrieto M Heimarsson S Breuss O Orange A FrobergL Wynns AA Navarro-Rosines P Krzewicka BPykaelae J Grube M Lutzoni F (2009) Genericclassification of the Verrucariaceae (Ascomycota)based on molecular and morphological evidencerecent progress and remaining challenges Taxon 58(1)184ndash208
Guillot G Mortier F Estoup A (2005) Geneland acomputer package for landscape genetics Mol EcolNotes 5(3)712ndash715 doi101111j1471-8286200501031x
Guillot G Renaud S Ledevin R Michaux J Claude J(2012) A unifying model for the analysis of pheno-typic genetic and geographic data Syst Biol 61(6)897ndash911 doi101093sysbiosys038
Guisan A Thuiller W (2005) Predicting species distribu-tion offering more than simple habitat models EcolLett 8(9)993ndash1009 doi101111j1461-0248200500792x
Hale ME (1990) A synopsis of the lichen genusXanthoparmelia (Vainio) Hale (AscomycotinaParmeliaceae) Smithsonian Institution Press Wash-ington DC
Hamilton CA Hendrixson BE Brewer MS Bond JE(2014) An evaluation of sampling effects on multipleDNA barcoding methods leads to an integrativeapproach for delimiting species a case study ofthe North American tarantula genus Aphonopelma(Araneae Mygalomorphae Theraphosidae) MolPhylogenet Evol 7179ndash93 doi101016jympev201311007
Hausdorf B (2011) Progress toward a general speciesconcept Evolution 65(4)923ndash931 doi101111j1558-5646201101231x
Hausdorf B Hennig C (2010) Species delimitation usingdominant and codominant multilocus markers SystBiol 59(5)491ndash503 doi101093sysbiosyq039
Hawksworth DL (1976) Lichen chemotaxonomy InBailey RH (ed) Lichenology progress and problemsAcademic London pp 139ndash184
Hebert PDN Cywinska A Ball SL deWaard JR (2003)Biological identifications through DNA barcodesProc R Soc Lond B Biol Sci 270(1512)313ndash321doi101098rspb20022218
Hebert PDN Stoeckle MY Zemlak TS Francis CM(2004) Identification of birds through DNA barcodesPLoS Biol 2(10)e312 doi101371journalpbio0020312
Heled J Drummond AJ (2010) Bayesian inference ofspecies trees from multilocus data Mol Biol Evol 27(3)570ndash580 doi101093molbevmsp274
Hey J (2006) On the failure of modern species conceptsTrends Ecol Evol 21(8)447ndash450 doi101016jtree200605011
Hibbett DS OhmanA Glotzer D NuhnMKirk P NilssonRH (2011) Progress in molecular and morphologicaltaxon discovery in Fungi and options for formalclassification on environmental sequences Fungal BiolRev 2538ndash47 doi101016jfbr201101001
Houmlgnabba F Wedin M (2003) Molecular phylogeny ofthe Sphaerophorus globosus species complex Cladis-tics 19(3)224ndash232 doi101111j1096-00312003tb00365x
Hudson RR Coyne JA (2002) Mathematical conse-quences of the genealogical species concept Evolu-tion 56(8)1557 doi101111j0014-38202002tb01467x
Huelsenbeck JP Andolfatto P Huelsenbeck ET (2011)Structurama Bayesian inference of population struc-ture Evol Bioinf Online 7(2011)55ndash59 doi104137EBOS6761
Jones G Oxelman B (2014) DISSECT an assignment-free Bayesian discovery method for species delimita-tion under the multispecies coalescent Bioinformaticsdoi101101003178
38 SD Leavitt et al
Kekkonen M Hebert PD (2014) DNA barcode-baseddelineation of putative species efficient start fortaxonomic workflows Mol Ecol Res doi1011111755-099812233
Kelly LJ Hollingsworth PM Coppins BJ Ellis CJHarrold P Tosh J Yahr R (2011) DNA barcoding oflichenized fungi demonstrates high identification suc-cess in a floristic context New Phytol 191(1)288ndash300doi101111j1469-8137201103677x
Kiss L (2012) Limits of nuclear ribosomal DNA internaltranscribed spacer (ITS) sequences as species barcodesfor fungi Proc Natl Acad Sci 109(27)E1811ndashE1811doi101073pnas1207143109
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Knowles LL Carstens BC (2007) Delimiting specieswithout monophyletic gene trees Syst Biol 56(6)887ndash895 doi10108010635150701701091
Kroken S Taylor JW (2001) A gene genealogical approachto recognize phylogenetic species boundaries in thelichenized fungus Letharia Mycologia 93(1)38ndash53
Kubatko LS Degnan JH (2007) Inconsistency of phylo-genetic estimates from concatenated data under coa-lescence Syst Biol 56(1)17ndash24 doi10108010635150601146041
Latch EK Dharmarajan G Glaubitz JC Rhodes OE Jr(2006) Relative performance of Bayesian clusteringsoftware for inferring population substructure andindividual assignments at low levels of populationdifferentiation Conserv Genet 7(2)295ndash302 doi101007s10592-005-9098-1
Lawrey JD (1986) Biological role of lichen substancesThe Bryologist 89(2)111ndash122
Le Gac M Hood ME Fournier E Giraud T (2007)Phylogenetic evidence of host-specific cryptic speciesin the anther smut fungus Evolution 61(1)15ndash26doi101111j1558-5646200700002x
Leacheacute AD (2009) Species tree discordance traces tophylogeographic clade boundaries in North Americanfence lizards (Sceloporus) Syst Biol 58(6)547ndash559doi101093sysbiosyp057
Leacheacute AD Fujita MK (2010) Bayesian species delim-itation in West African forest geckos (Hemidactylusfasciatus) Proc R Soc B Biol Sci 2773071ndash3077doi101098rspb20100662
Leacheacute AD Koo M Spencer C Papenfuss T Fisher RMcGuire J (2009) Quantifying ecological morpho-logical and genetic variation to delimit species in thecoast horned lizard species complex (Phrynosoma)Proc Natl Acad Sci USA 10612418ndash12423 doi101073pnas0906380106
Leacheacute AD Fujita MK Minin VN Bouckaert RR (2014)Species delimitation using genome-wide SNP dataSystematic Biology doi101093sysbiosyu018
Leavitt SD Fankhauser JD Leavitt DH Porter LDJohnson LA St Clair LL (2011a) Complex patterns ofspeciation in cosmopolitan ldquorock posyrdquo lichensmdashdiscovering and delimiting cryptic fungal species in
the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
Leavitt SD Esslinger TL Lumbsch HT (2012c) Neogene-dominated diversification in neotropical montanelichens dating divergence events in the lichen-form-ing fungal genus Oropogon (Parmeliaceae) Am J Bot99(11)1764ndash1777 doi103732ajb1200146
Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Lumbsch HT St Clair LL(2013c) DNA barcode identification of lichen-formingfungal species in the Rhizoplaca melanophthalmaspecies-complex (Lecanorales Lecanoraceae) includ-ing five new species MycoKeys 71ndash22 doi103897mycokeys74508
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Vondraacutek J Thorsten Lum-bsch H Clair LLS (2013d) Local representation ofglobal diversity in a cosmopolitan lichen-formingfungal species complex (Rhizoplaca Ascomycota)J Biogeogr 40(9)1792ndash1806 doi101111jbi12118
Leavitt SD Lumbsch HT Stenroos S St Clair LL(2013e) Pleistocene speciation in North American
2 The Dynamic Discipline of Species Delimitation hellip 39
lichenized fungi and the impact of alternative speciescircumscriptions and rates of molecular evolution ondivergence estimates PLoS ONE 8(12)e85240doi101371journalpone0085240
Leavitt SD Esslinger TL Hansen ES Divakar PK CrespoA Loomis BF Lumbsch HT (2014) DNA barcoding ofbrown Parmeliae (Parmeliaceae) species a molecularapproach for accurate specimen identification empha-sizing species in Greenland Organ Divers Evol 14(1)11ndash20 doi101007s13127-013-0147-1
Leliaert F Verbruggen H Vanormelingen P Steen FLopez-Bautista JM Zuccarello GC De Clerck O(2014) DNA-based species delimitation in algae Eur JPhycol 49(2) doi101080096702622014904524
Leuckert C (1985) Probleme der Flechten-Chemotaxon-omiemdashStoffkombinationen und ihre taxonomischeWertung Ber Deut Bot Ges 98401ndash408
Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
Lohtander K Kaumlllersjouml M Roland M Tehler A (2000)The family physciaceae in fennoscandia phylogenyinferred from its sequences Mycologia 92(4)728ndash735
Longton RE (1997) The role of bryophytes and lichens inpolar ecosystems In Woodin SJ Marquiss M (eds)Ecology of Arctic Environments Blackwell ScienceOxford pp 69ndash96 (Special publication No 13)
Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
Lumbsch HT (1994) Die Lecanora subfusca-Gruppe inAustralasien J Hattori Bot Lab 771ndash175
Lumbsch HT (1998a) Taxonomic use of metabolic data inlichen-forming fungi In Frisvad JC Bridge PDArora DK (eds) Chemical Fungal Taxonomy MarcelDekker New York pp 345ndash387
Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
Mayden RL (1997) A hierarchy of species concepts thedenouement in the saga of the species problem InClaridge MF Dawah HA Wilson MR (eds) Speciesthe units of biodiversity Chapman amp Hall Londonpp 381ndash424
Mayr E (1963) Animal species and evolution HarvardUniversity Press Cambridge
Mayr E (1970) Populations species and evolutionBelknap Press of Harvard University Press Cambridge
McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
McDonald T Miadlikowska J Lutzoni F (2003) Thelichen genus Sticta in the Great Smoky Mountains a
40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
McKay BD Mays HL Wu Y Li H Yao C-T Nishiumi IZou F (2013) An empirical comparison of character-based and coalescent-based approaches to speciesdelimitation in a young avian complex Mol Ecol 22(19)4943ndash4957 doi101111mec12446
Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
Mishler BD Brandon RN (1987) Individuality pluralismand the phylogenetic species concept Biol Philos 2397
Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
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Nylander W (1866a) Circa novum in studio Lichenumcriterium chemicum Flora 49198ndash201
Nylander W (1866b) Hypochlorite of lime and hydrate ofpotash Two new criteria for the study of lichens(Tanslated and communicated by the Rev W ALeighton) Bot J Linn Soc 9358ndash365
OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
OrsquoNeill EM Schwartz R Bullock CT Williams JSShaffer HB Aguilar-Miguel X Parra-Olea G Weis-rock DW (2013) Parallel tagged amplicon sequencingreveals major lineages and phylogenetic structure inthe North American tiger salamander (Ambystomatigrinum) species complex Mol Ecol 22(1)111ndash129doi101111mec12049
Orock EA Leavitt SD Fonge BA St Clair LL LumbschHT (2012) DNA-based identification of lichen-form-ing fungi Can publicly available sequence databasesaid in lichen diversity inventories of Mount Cameroon(West Africa) The Lichenologist 44(6)833ndash839doi101017S0024282912000424
Otaacutelora MAG Martiacutenez I Aragoacuten G Molina MC (2010)Phylogeography and divergence date estimates of alichen species complex with a disjunct distributionpattern Am J Bot 97(2)216ndash223 doi103732ajb0900064
Padial J Castroviejo-Fisher S Kohler J Vila C ChaparroJ De la Riva I (2009) Deciphering the products ofevolution at the species level the need for anintegrative taxonomy Zoolog Scr 38(4)431ndash447doi101111j1463-6409200800381x
Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
Papong K Luumlcking R Thammathaworn A BoonpragobK (2009) Four new taxa of Chroodiscus (thelotremoidGraphidaceae) from Southeast Asia The Bryologist112(1)152ndash163 doi1016390007-2745-1121152
Park S-Y Choi J Kim JA Jeong M-H Kim S Lee Y-HHur J-S (2013a) Draft genome sequence of Cladoniamacilenta KoLRI003786 a lichen-forming fungusproducing biruloquinone Genome Announcements 1(5)e00695-13 doi101128genomeA00695-13
Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
Peacuterez-Ortega S Fernaacutendez-Mendoza F Raggio J VivasM Ascaso C Sancho LG Printzen C de los Riacuteos A(2012) Extreme phenotypic variation in Cetrariaaculeata (lichenized Ascomycota) adaptation or inci-dental modification Ann Bot 109(6)1133ndash1148doi101093aobmcs042
Pino-Bodas R Burgaz A Martiacuten M Lumbsch HT (2011)Phenotypical plasticity and homoplasy complicatespecies delimitation in the Cladonia gracilis group(Cladoniaceae Ascomycota) Organ Divers Evol 11(5)343ndash355 doi101007s13127-011-0062-2
Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
Pino-Bodas R Martiacuten M Burgaz A (2012b) Cladoniasubturgida and C iberica (Cladoniaceae) form asingle morphologically and chemically polymorphicspecies Mycol Prog 11(1)269ndash278 doi101007s11557-011-0746-1
Pino-Bodas R Ahti T Stenroos S Martiacuten MP BurgazAR (2013a) Multilocus approach to species recogni-tion in the Cladonia humilis complex (CladoniaceaeAscomycota) Am J Bot 100(4)664ndash678 doi103732ajb1200162
Pino-Bodas R Martiacuten AP Burgaz AR Lumbsch HT(2013b) Species delimitation in Cladonia (Ascomy-cota) a challenge to the DNA barcoding philosophyMol Ecol Resour 13(6)1058ndash1068 doi1011111755-099812086
Pons J Barraclough TG Gomez-Zurita J Cardoso ADuran DP Hazell S Kamoun S Sumlin WD VoglerAP (2006) Sequence-based species delimitation for theDNA taxonomy of undescribed insects Syst Biol 55(4)595ndash609 doi10108010635150600852011
Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
Pringle A Baker DM Platt JL Wares JP Latgeacute JPTaylor JW (2005) Cryptic speciation in the cosmo-politan and clonal human pathogenic fungus Asper-gillus fumigatus Evolution 59(9)1886ndash1899 doi10155404-2411
Printzen C (2009) Lichen systematics the role ofmorphological and molecular data to reconstructphylogenetic relationships Progress in botany vol71 Springer Berlin pp 233ndash275
Pritchard JK Stephens M Donnelly P (2000) Inference ofpopulation structure using multilocus genotype dataGenetics 155(2)945ndash959
Puillandre N Lambert A Brouillet S Achaz G (2012)ABGD automatic barcode gap discovery for primaryspecies delimitation Mol Ecol 21(8)1864ndash1877doi101111j1365-294X201105239x
Rannala B Yang Z (2003) Bayes estimation of speciesdivergence times and ancestral population sizes usingDNA sequences from multiple loci Genetics1641645ndash1656
Raxworthy CJ Ingram CM Rabibisoa N Pearson RG(2007) Applications of ecological niche modelingfor species delimitation a review and empiricalevaluation using day geckos (Phelsuma) fromMadagascar Syst Biol 56(6)907ndash923 doi10108010635150701775111
Ray J (1686) Historia planarum vol 1 Clark LondonReese Naeligsborg R Ekman S Tibell L (2007) Molecular
phylogeny of the genus Lecania (Ramalinaceaelichenized Ascomycota) Mycol Res 111(5)581ndash591doi101016jmycres200703001
Reid N Carstens B (2012) Phylogenetic estimation errorcan decrease the accuracy of species delimitation aBayesian implementation of the general mixed Yule-coalescent model BMC Evol Biol 12(1)196 doi1011861471-2148-12-196
Rissler L Apodaca J (2007) Adding more ecology intospecies delimitation ecological niche models andphylogeography help define cryptic species in theBlack Salamander (Aneides flavipunctatus) Syst Biol56924ndash942 doi10108010635150701703063
Rivas Plata E Luumlcking R (2013) High diversity ofGraphidaceae (lichenized Ascomycota Ostropales) inAmazonian Peruacute Fungal Divers 58(1)13ndash32 doi101007s13225-012-0172-y
Rivas Plata E Lumbsch HT (2011) Parallel evolution andphenotypic divergence in lichenized fungi a casestudy in the lichen-forming fungal family Graphida-ceae (Ascomycota Lecanoromycetes Ostropales)Mol Phylogenet Evol 61(1)45ndash63 doi101016jympev201104025
Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
Ross KG Gotzek D Ascunce MS Shoemaker DD (2010)Species delimitation a case study in a problematic anttaxon Syst Biol 59(2)162ndash184 doi101093sysbiosyp089
Rubin BER Ree RH Moreau CS (2012) Inferringphylogenies from RAD sequence data PLoS ONE 7(4)e33394 doi101371journalpone0033394
Rubinoff D (2006) Utility of mitochondrial DNA barcodesin species conservation Conserv Biol 20(4)1026ndash1033 doi101111j1523-1739200600372x
Ruiz-Sanchez E Sosa V (2010) Delimiting speciesboundaries within the Neotropical bamboo Otatea(Poaceae Bambusoideae) using molecular morpho-logical and ecological data Mol Phylogenet Evol 54(2)344ndash356 doi101016jympev200910035
Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
morphological characters Antarctic Sci 22 (SpecialIssue 06)727ndash741 doi101017S0954102010000477
Salicini I Ibaacutentildeez C Juste J (2011) Multilocus phylogenyand species delimitation within the Nattererrsquos batspecies complex in the Western Palearctic MolPhylogenet Evol 61(3)888ndash898 doi101016jympev201108010
Satler JD Carstens BC Hedin M (2013) Multilocusspecies delimitation in a complex of morphologicallyconserved trapdoor spiders (Mygalomorphae Antro-diaetidae Aliatypus) Syst Biol 62(6)805ndash823 doi101093sysbiosyt041
Schlick-Steiner BC Steiner FM Seifert B Stauffer CChristian E Crozier RH (2010) Integrative taxonomya multisource approach to exploring biodiversityAnnu Rev Entomol 55(1)421ndash438 doi101146annurev-ento-112408-085432
Schoch CL Seifert KA Huhndorf S Robert V SpougeJL Levesque CA Chen W Fungal Barcoding Con-sortium (2012) Nuclear ribosomal internal transcribedspacer (ITS) region as a universal DNA barcodemarker for Fungi Proc Natl Acad Sci 109(16)6241ndash6246 doi101073pnas1117018109
Seacuterusiaux E Villarreal AJC Wheeler T Goffinet B(2011) Recent origin active speciation and dispersalfor the lichen genus Nephroma (Peltigerales) inMacaronesia J Biogeogr 38(6)1138ndash1151 doi101111j1365-2699201002469x
Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
Simpson GG (1951) The species concept Evolution5285ndash298
Sites JW Marshall JC (2003) Delimiting species a renais-sance issue in systematic biology Trends Ecol Evol18462ndash470 doi101016S0169-5347(03)00184-8
Sites JW Marshall JC (2004) Operational criteria fordelimiting species Annu Rev Ecol Evol Syst 35(1)199ndash227 doi101146annurevecolsys35112202130128
Šlapeta J Loacutepez-Garciacutea P Moreira D (2006) Globaldispersal and ancient cryptic species in the smallestmarine eukaryotes Mol Biol Evol 23(1)23ndash29doi101093molbevmsj001
Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
Talavera G Dincă V Vila R (2013) Factors affectingspecies delimitations with the GMYC model insightsfrom a butterfly survey Methods Ecol Evol 4(12)1101ndash1110 doi1011112041-210X12107
Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
Tewksbury J Anderson JGT Bakker JD Billo TJDunwiddie PW Groom MJ Hampton SE HermanSG Levey DJ Machnicki NJ del Rio CM PowerME Rowell K Salomon AK Stacey L TrombulakSC Wheeler TA (2014) Natural historyrsquos place inscience and society BioScience doi101093bioscibiu032
Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
Werth S Cornejo C Scheidegger C (2013) Characteriza-tion of microsatellite loci in the lichen fungus Lobariapulmonaria (Lobariaceae) Appl Plant Sci 1(2)1200290 doi103732apps1200290
Westberg M Arup U Kaumlrnefelt I (2007) Phylogeneticstudies in the Candelariaceae (lichenized Ascomy-cota) based on nuclear ITS DNA sequence dataMycol Res 111(11)1277ndash1284 doi101017S0953756204002102
Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
2 The Dynamic Discipline of Species Delimitation hellip 43
(Lepidoptera Lycaenidae) Front Zoo 4(1)8 doi1011861742-9994-4-8
Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
combine individual gene genealogies and speciesphylogenies via modeling the coalescent historyof markers (Yang and Rannala 2010 Edwards2009 Knowles and Carstens 2007) As aresponse to advances in sequencing technologiesbioinformatical approaches for multimarker spe-cies delimitation analyses continue to be devel-oped (Table 21 Camargo and Sites 2013)
Long-term reproductive isolation of candidatespecies can be assessed with multilocus sequencedata by evaluating genealogical concordance ofunlinked markers (Baum and Shaw 1995 Aviseand Ball 1990) Within species the mixingeffects of recombination cause unlinked loci tohave distinct genealogical histories but geneticdrift and long-term divergence leads to concor-dant genealogical histories at loci across thegenome Relationships of individuals belongingto distinct candidate species can be evaluatedusing gene genealogies (eg haplotype networksor single-gene topologies) to identify lineagesthat exhibit genealogical exclusivity acrossunlinked neutral loci (Hudson and Coyne 2002Dettman et al 2003b Avise and Ball 1990) Thepresence of the same clades in the majority ofsingle-locus genealogies is taken as evidence thatthe clades represent reproductively isolated lin-eages (Dettman et al 2003a Pringle et al 2005)In practice the criteria of reciprocal monophylyand genealogical concordance of unlinked lociprovide a conservative approach for assessingspecies boundaries due to the fact that a sub-stantial amount of time is required after the initialdivergence of species until ancestral polymor-phisms have fully sorted (Knowles and Carstens2007 Hudson and Coyne 2002) Consequentlygroups with recent divergence histories willlikely go undiscovered under a genealogicalconcordance criterion due to the fact that speciesboundaries likely are not reflected in genegenealogies For example it would take morethan 1 million years after speciation before spe-cies would be delimited if 15 loci were sampledin species with an effective population size (Ne)of 100000 assuming one generation a yearunder a strict reciprocal monophyly criterion(Knowles and Carstens 2007) The amount oftime required for a species to be recognized using
a criterion of reciprocal monophyly increasesproportionally with increasing population sizes(Hudson and Coyne 2002) In a number ofstudies of lichen-forming fungi a genealogicalconcordance criterion has been used to bothdelimit previously unrecognized species-levellineages and validate some conventional pheno-type-based species (Leavitt et al 2012a c2013b Molina et al 2011 Kroken and Taylor2001)
Although analyses of molecular sequence datarelying on reciprocal monophyly or fixed char-acter differences for species delimitation canserve as important criteria for identifying speciesthese criteria are not commonly met acrossmultiple loci particularly in recent speciationhistories (Fig 22) To accommodate theobserved conflict among genealogies from mul-tiple loci with the underlying speciation historythe recent merge of coalescent theory with phy-logenetics has driven a major paradigm shift inspecies delimitation and molecular systematics ingeneral (Fujita et al 2012 Edwards 2009) Themultispecies coalescent model can be applied togenealogical histories from multiple independentloci to assemble separate coalescent processesoccurring in populations into a species tree(Degnan and Rosenberg 2009 Rannala andYang 2003) Within this coalescent-basedframework multiple individuals can be assignedto a single speciespopulation and the speciationhistory of ancestral and descendant species-levellineages can be inferred as a ldquospecies treerdquo incontrast to estimating gene genealogies fromindividual samples (Degnan and Rosenberg2006 2009 Rannala and Yang 2003)
A number of multispecies coalescent-basedspecies delimitation methods have recently beenintroduced offering an exciting framework forempirically assessing species boundaries byselecting the best species tree model from a set ofalternative models representing differenthypotheses of species boundaries (Table 21)For example SpeDeSTEM (Ence and Carstens2011) finds the maximum likelihood values for aspecies tree assuming all putative species areseparate lineages and for alternative species treeswhere two or more species are collapsed into a
2 The Dynamic Discipline of Species Delimitation hellip 27
single lineage The best fitting model is thenselected using the Akaike information criterionunder the assumption of constant populationsizes and fixed gene topologies across the speciestree The species delimitation program BayesianPhylogenetic and Phylogeography (BPampPYangand Rannala 2010) accommodates gene treeuncertainty and variable population sizes andsamples from the Bayesian posterior distributionof species delimitation models using reversible-jump Markov chain Monte Carlo (rjMCMC)sampling BPampP requires a user-provided guidetree with resolution finer than the species leveland evaluates alternative modes derived from allpossible subtrees that are generated by collapsingor splitting nodes on the guide tree (Yang andRannala 2010 Rannala and Yang 2003) Theproportion of time spent on each model is pro-portional to the posterior probability of themodel ie ldquospeciation probabilitiesrdquo WhileBPampP ranks among the most popular coalescent-based species delimitation programs (Fujita et al2012) misspecifications of the guide tree andorthe prior distribution for population size (θ) canresult in strong support for models containing aninflated number of species (Leacheacute and Fujita2010 Zhang et al 2011) The nonparametricheuristic species delimitation approach imple-mented in the program Brownie (OrsquoMeara 2010)jointly sorts anonymous samples into species andinfers a species tree from input gene trees fromdifferent loci assuming that for a speciationevent the corresponding nodes on gene trees willbe more consistent with each than the diver-gences within species However finding both theoptimum species tree and species boundariesremains computationally challenging andBrownie has been shown to frequently yieldincorrect results (OrsquoMeara 2010)
Most recently available coalescent-basedspecies delimitation methods require individualassignments to a species or population a priori Ina number of scenarios correct assignments ofsamples to species may be difficult includingpresence of cryptic species incongruencebetween conventional species and moleculardata or simply the fact that accurate specimenidentification in complex groups with subtle or
difficult to discern diagnostic characters is anontrivial task (Leavitt et al 2013e) Statisticalmethods for assessing individual assignment andspecies detection prior to coalescent-based spe-cies delimitation and species tree reconstructionand species provide a more objective approachfor understanding species boundaries
Population assignment tests using a variety ofclustering algorithms using genetic data offer auseful approach for grouping individuals intoputative reproductively isolated groups (Table 21Hausdorf and Hennig 2010 Corander et al2004 2008 Falush et al 2003 Pritchard et al2000) particularly for species delimitation prob-lems that exist at the interface of traditionalpopulation genetic and phylogenetic analyses(Carstens et al 2013 Weisrock et al 2010Knowles and Carstens 2007)
The programs STRUCTURE (Falush et al2003 Pritchard et al 2000) and STRUCTURA-MA (Huelsenbeck et al 2011) cluster samplesinto populations by minimizing HardyndashWeinbergdisequilibrium for a given number of populationclusters using unlinked genetic markers In gen-eral unlinked markers are not available for mostgroups of lichen-forming fungi and a numberof studies have used information from multilocussequence data for STRUCTURE analyses(Leavitt et al 2011a b c 2013e Fernaacutendez-Mendoza and Printzen 2013 Fernaacutendez-Mendoza et al 2011) Varying approaches havebeen implemented to convert DNA sequencedata to allelic data for Bayesian clustering (seeOrsquoNeill et al 2013) STRUCTURE is expectedto perform well when there is sufficient inde-pendence across regions such that linkage dis-equilibrium within regions does not dominate thedata (STRUCTURE manual) but can also beeffective using multilocus sequence data andtreating all SNPs as independent loci regardlessof physical linkage within each locus (OrsquoNeillet al 2013 Falush et al 2003) A recent study ofthe lichen-forming genus Letharia showed thatBayesian clustering implemented in the programSTRUCTURE was generally able to recover thesame putative Letharia lineages circumscribedemploying a gene genealogical approach inKroken and Taylorrsquos iconic species delimitation
28 SD Leavitt et al
study (Altermann et al 2014 Kroken and Taylor2001) Altermann et al (2014) show that popu-lation assignments were largely consistent acrossa range of scenarios including extensiveamounts of missing data the exclusion of SNPsfrom variable markers and inferences based onSNPs from as few as three gene regions Incontrast to STRUCTURE and STRUCTURA-MA the program BAPS (Corander et al 2008)can explicitly infer genetic structure using hap-loid sequence data or other linked geneticmarkers in the ldquoclustering with linked locirdquomodel Another advantage of BAPS is that theprogram is much more computationally efficientthan STRUCTURE or STRUCTURAMA Sim-ulation studies indicate that both BAPS andSTRUCTURE perform well at low levels ofpopulation differentiation and when clusters arenot well differentiated (Latch et al 2006)
In practice inferring the most appropriatelevel of population structure using Bayesianclustering algorithms remains challenging (Latchet al 2006 Evanno et al 2005) BAPS andSTRUCTURAMA can infer both individualassignments and the most likely number ofgenetic clusters (Corander et al 2008 Huelsen-beck et al 2011) and the ad hoc ΔK statisticproposed by Evanno et al (2005) provides anobjective approach for identifying the uppermosthierarchical level of structure However carefulconsideration of other levels of populationstructure may ultimately reveal more biologicallymeaning results and researchers should examineindividual assignments across a range of geneticgroupings Some markers generated fromindependent genomic regions such as SNPs andfast-evolving microsatellites can be used to dis-tinguish fine-scale population structuring on thebasis of allele frequencies and species delimita-tion analyses based on these markers may implythe risk of severe taxonomic oversplitting Inthese cases validation approaches such asBPampP and SpeDeSTEM and corroborationamong different species delimitation approachescan provide perspective between intraspecificpopulation structure and species-level clusters
Another limitation of clustering approaches isthat they do not assess or take into account
evolutionarily divergences and relationshipsamong population clusters Coalescent-basedspecies tree methods (discussed below) provide ameans to assess the diversification histories ofpopulations inferred from clustering analysesTherefore a potential working protocol for aninformed species delimitation study that takesinto account population structure could consist offirst applying a genetic clustering analysis under apopulation genetics criterion (eg BAPSSTRUCTURE STRUCTURAMA) to identifygenetically distinct population clusters that can beconsidered ldquocandidate speciesrdquo From these can-didate species a species tree can be inferred forfocal group using coalescent-based species treereconstruction methods (eg BEAST) Subse-quently a coalescent-based validation methodcan be applied to assess whether the distinctpopulation clusters represent independent evolu-tionary lineages (eg BPampP) This multistepapproach would provide a consistent and standardcriterion for distinguishing between population-and species-level lineages (Camargo and Sites2013) and has been applied to a number of casesincluding lichen-forming fungi (Leavitt et al2011a b c 2013e Leacheacute and Fujita 2010)
High-throughput sequencing methods providethe means to effectively sample hundreds tothousands of loci from across a species genome fora large number of species and continue to revolu-tionize studies that can be performed even in non-model organisms Targeted high-throughputsequencing approaches such as anchored phy-logenomics transcriptome sequencing reduced-representation genomic library sequencing(restriction-site-associated DNA sequencingRAD-Seq and genotype-by-sequencing GBS)and high-throughput amplicon sequencing pro-vide important insight into species boundaries fora number groups although none of these approa-ches has yet been applied to studies of lichenizedfungi
For example restriction-site-associated(RAD-Seq) DNA can simultaneously detect andgenotype thousands of genome-wide SNPs byfocusing the sequencing effort on a reducedrepresentation of the entire genome (Baird et al2008) and has been successfully applied to
2 The Dynamic Discipline of Species Delimitation hellip 29
intraspecies (Lewis et al 2007 Miller et al 2007Emerson et al 2010) and interspecies studies(Rubin et al 2012 Eaton and Ree 2013 Wagneret al 2013) This approach has provided strikinginsight into the recent adaption radiation of LakeVictoria cichlids (Wagner et al 2013) Analternative approach targeting the sequencing ofspecific loci using next-generation sequencingplatforms provides an efficient means of gener-ating data for loci of known genomic locationorthology size and expected level of variation(OrsquoNeill et al 2013) Markers can be targetedusing well-established PCR techniques or newerhybridization techniques and subsequentlypooled for high-throughput sequencing usingparallel-tagged sequencing of multiple individualsamples within a single sequencing run (OrsquoNeillet al 2013) A recent study of North Americantiger salamanders (Ambystoma tigrinum)demonstrated the potential for amplicon-basedparallel-tagged sequencing to rapidly generatelarge-scale genomic data for species delimitationand species tree research (OrsquoNeill et al 2013)
Although these methods provide an enormousamount of data and insight into diversification atan unprecedented scale computational limita-tions restrict the applicable analytical methodsfor large datasets although computational andanalytical advances are happening rapidly Mostrecent coalescent-based species delimitation andspecies tree models using gene trees have beenlimited to handle tens of loci for multiple indi-viduals and combining hundreds or thousands ofgene trees into a single species delimitationframework presents considerable computationalchallenges (Camargo and Sites 2013) Therecently developed method Bayes factor delim-itation (with genomic data BFD) combines adynamic programming algorithm for estimatingspecies trees that bypasses the computationallyintensive MCMC integration over gene trees toprovide a rigorous technique for species delimi-tation studies using genome-wide SNP data(Leacheacute et al 2014) Competing species delimi-tation models are compared using Bayes factorsand it appears that this approach is robust tosample sizes (ie few loci and limited samples
per species) and misspecification of the prior forpopulation size (θ) (Leacheacute et al 2014)
Fungi are an ideal model for assessing diver-gence in eukaryotes due to their simple mor-phologies small genomes broad ecological rolesand diverse lifestyles (Gladieux et al 2014)However the use of genomic data from high-throughput sequencing methods have not yet beenincluded in species delimitation studies of lichen-forming fungi This is due in part to challenges indealing with metagenomic data containing geno-mic information from a plethora of symbiontsassociated with a lichen thallus scant genomicresources and discipline-specific inertia A num-ber of lichen-forming fungal genomes are cur-rently available including Endocarponpusillum(Wang et al 2014) Cladonia spp (Park et al2013a 2014) Caloplaca flavorubescens (Parket al 2013b) and Cladonia greyi and Xanthoriaparientina through the Fungal Genomics Programat Joint Genome Institute of the United StatesDepartment of Energy (httpwwwjgidoegov)The foreseeable ongoing expansion of genomicresources for lichen-forming fungi will be centralto developing approaches for delimiting speciesusing high-throughput sequencing Specificallygenomic resources will be crucial in identifyingnovel markers (variable genes regions SNPsmicrosatellites etc) identifying conserved fungalmarkers for targeted high-throughput sequencingapproaches and references for entire genomecomparisons (Devkota et al 2014 Werth et al2013)
23 Can We Make SpeciesDelimitation in Lichen-FormingFungi Truly Integrative
The widespread availability of genetic data hascreated a biased viewpoint that only genetic datashould be used for statistical species delimitationHowever an ongoing appeal to researchers toassess species boundaries from multiple andcomplementary perspectives (phylogeneticspopulation genetics comparative morphologydevelopment ecology etc) has resulted in an
30 SD Leavitt et al
integrative taxonomic framework bringing theseconceptual and methodological perspectivestogether (Hamilton et al 2014 Fujita et al 2012Salicini et al 2011 Yeates et al 2011 Padialet al 2010 Dayrat 2005 Will et al 2005 Wiensand Penkrot 2002) In practice any study linkingdifferent kinds of data to support hypotheses ofspecies boundaries including mapping morpho-logical characters onto a molecular phylogenycan be considered integrative Integrative meth-ods for species delimitation fall across a broadspectrum ranging from verbal and qualitativeassessments of data classes to quantitative meth-ods that allow different data types to contribute tostatistical species delimitation (Yeates et al2011) In most taxonomic studies utilizing bothmolecular and morphological data expert opinionis eventually used to some degree to evaluate thefinal status of a candidate species (eg Bond andStockman 2008) In this sense many studiesusing evidence for independent data sources fordelimiting species boundaries use an iterativeapproach (Yeates et al 2011) where speciesboundaries can be tested within a hypothetico-deductive framework with diverse datasets
From a practical perspective we advocate aprocess of iterative taxonomy (sensu Yeates et al2011) to circumscribe and refine species limitsusing multiple lines of evidence This iterativeprocess involves comparisons of morphologicaldata with a phylogenetic hypothesis (ie single-locus gene tree or concatenated multilocusphylogeny) to identify the least inclusive mono-phyletic clade in the topology characterized by atleast one unambiguously diagnostic morphologi-cal character (Miralles and Vences 2013) Thisphylogenetic tree-informed approach to assessingspecies boundaries represents a common practicein studies of lichen-forming fungi Previouslyunrecognized species-level clades with corre-sponding subtle or overlooked phenotypiccharacters have been commonly observed in bothcrustose lichens [eg Acarospraceae (Wedin et al2009) Graphidaceae (Rivas Plata and Lumbsch2011 Papong et al 2009) Lecanoraceae (Leavittet al 2011a) Lecideaceae (Ruprecht et al 2010)Mycoblastaceae (Spribille et al 2011) andTeloschistaceae (Vondraacutek et al 2009 Muggia
et al 2008)] and foliose and fruticose lichens [egLobariaceae (Moncada et al 2014 McDonaldet al 2003) Parmeliaceae (Leavitt et al 2013aWirtz et al 2012 Divakar et al 2005 Molinaet al 2004 Kroken and Taylor 2001) Peltigera-ceae (OrsquoBrien et al 2009 Goffinet et al 2003)Physciaceae (Elix et al 2009 Divakar et al2007) and Sphaerophoraceae (Houmlgnabba andWedin 2003)] As a specific example Luumlckinget al (2008) used a combination of medullarychemistry and underside pigmentation in speci-mens from the Heterodermia obscurata group inCosta Rica to corroborate monophyletic clades inan ITS gene tree as species-level lineages
While this iterative approach to speciesdelimitation and taxonomy has proven valuablefor understanding species boundaries anddescribing new taxa in some groups of lichenizedfungi a posteriori examination of morphologicaland chemical features has failed to reveal diag-nostic phenotypic characters in a number ofstudies (Muggia et al 2014 Leavitt et al 2011aOtaacutelora et al 2010 OrsquoBrien et al 2009) Fur-thermore a study of widespread species in thegenus Melanelixia (Parmeliaceae) indicated thatphenotypically cryptic lichen-forming fungalspecies-level lineages may be relatively ancientand diagnosable phenotypic differences may beabsent even millions of years after the initialdivergence (Leavitt et al 2012b) The latter studyhighlights the fact that species-level lineages maycommonly exist without any observable diag-nostic phenotypic characters calling into ques-tion the impetus for a universal application ofintegrative taxonomy
In keeping with the principle that as manylines of evidence as available should be com-bined to delimit species (Dayrat 2005) the for-malized integrative taxonomic approach (ITAXMiralles and Vences 2013) uses a mtDNA guidetree and observations from different types of datathat might provide conclusive evidence for theindependence of lineages and thus their identityas different species Miralles and Vences (2013)provide a non-exhaustive list of species delimi-tation criteria to be integrated in the ITAXapproach including (a) sympatric occurrencewithout admixture as revealed by consistent
2 The Dynamic Discipline of Species Delimitation hellip 31
differences in morphological or molecular char-acters at the same geographic location (b) strongdifferences in a behavioral morphological orgenetic character known to mediate prematingisolation (c) unviability or infertility of hybrids(d) lack of gene flow across a geographicalhybrid zone (e) congruent diagnostic differencesbetween sister lineages in various unlinkedmorphological character (f) absence of haplo-type sharing in several unlinked nuclear loci and(g) a combination of criteria endashf Speciesboundaries are based on seeking the least inclu-sive monophyletic group in the mtDNA treewhich fulfills at least one of the criteria listedabove Clearly the ITAX approach is sensitive tosample size and in order to reliably support thedistinctiveness of a given species it has beenrecommended that the sampling strategy includesat least five individuals per species (Miralles andVences 2013) A similar approach could beadopted for lichenized fungi using an ITS genetopology as guide tree rather than mtDNA
A total evidence approach including concat-enation has been a common approach for inte-grating information from different sourcesincluding independent genetic markers in phy-logenetic reconstructions (Kluge 1989 Wiens1998 de Queiroz et al 1995) Proponents forconcatenation of independent data in phyloge-netic analyses argue that when combining alldata the underlying signal of speciation mayemerge even if weakly contradictory signalis contained in the individual data partitions(Gatesy et al 1999) For example establishing apreliminary perspective of species boundariesfrom multilocus sequence data using concatena-tion may provide a reasonable starting point forscreening for cryptic species and species treeinference (Šlapeta et al 2006 Leavitt et al2011a Le Gac et al 2007 Leacheacute 2009)However concatenation and consensus methodsimply a risk of obtaining inflated support forincorrect relationships and information aboutvariance in gene coalescence is lost (Degnan andRosenberg 2006 2009 Kubatko and Degnan2007 Edwards 2009) In spite of the impetus forintegrating evidence from independent data
sources into empirical species delimitation stud-ies and taxonomy (Fujita et al 2012 Padial et al2009) most currently available species delimi-tation methods are unable to accommodate non-genetic data sources in a statistical framework
231 Selecting the Appropriate Data
In the face of increasing availability of geneticdata and associated bioinformatical approachesfor delimiting species researchers should care-fully consider what information is being sacri-ficed by the failure to consider non-genetic datain species delimitation studies and whetheraccuracy could be improved by the addition ofmultiple data types Morphological data havehistorically served as a proxy to identify repro-ductively isolated groups (ie ldquospeciesrdquo) (Ray1686 Fujita et al 2012) Current methods fordelimiting species using non-genetic data (egchemistry morphology and ecology) remainwoefully understudied For example morphol-ogy-based species circumscriptions are generallybased on one or more qualitative (or quantitative)morphological characters that do not appear tooverlap with other species However ascertain-ing that a given trait is truly fixed within apopulation with statistical confidence requiresunrealistic sample sizes even when allowing forsome level of polymorphism in the diagnosticcharacter (Wiens and Servedio 2000) Now var-ious combinations of datamdashfrom morphologygenetics geography and ecologymdashare acceptedas standard information for species delimitationstudies (Ruiz-Sanchez and Sosa 2010 Ross et al2010 Edwards and Knowles 2014)
Below we briefly discuss appropriate datasources for species delimitation studies of lichen-forming fungi However homoplastic characters(similar traits that are not derived from a com-mon ancestor) are common among many traitscommonly used to circumscribe fungal taxa thebiological significance of secondary metabolitevariation remains largely unknown (Lawrey1986) and ecological niches may be difficultto adequately characterize and model due to
32 SD Leavitt et al
microhabitat requirements and data resolutionTherefore we advocate a cautious approach toselecting appropriate and relevant data forassessing species boundaries
In spite of the potential challenges and limi-tations of using phenotypic data these traits haveprovided a plethora of valuable information forunderstanding species boundaries For examplein the Melanelixia fuliginosa group (Parmelia-ceae) a morphometric analysis using color isi-dia and marginal zone free of isidia ascharacters revealed a general pattern of differ-entiation between material formerly recognizedas subspecies fuliginosa and glabratula in spiteof the fact that considerable overlap betweengroups occurs in some characters individually(Arup and Berlin 2011) Furthermore the dis-tinction of the two groups was supported by aphylogenetic analysis of the ITS marker andecological differences with M fuliginosaoccurring predominantly on rock and M glab-ratula on bark (Arup and Berlin 2011) Thisstudy of the M fuliginosa group provides a fit-ting example of using multiple independentsuites of data including ecology morphologyand genetic information to establish a robusthypothesis of species boundaries
In lichen systematics phenotypic dataincluding thallus organization secondary metab-olites mode of reproduction ascoma-type andontogeny ascus and ascospore characters havehistorically played a prominent role (Printzen2009) Ascomatal characters have traditionallyheld a major role in higher-level classification(Printzen 2009) in contrast to species-levelclassification which also tends to include a widearray of vegetative and chemical charactersCommonly assessed morphological charactersinclude thallus form and size cortical features(eg maculae and pseudocyphellae) presenceformcolor of attachment structure (eg rhizines)and reproductive mode (ascomata vs vegetativediaspores) (Printzen 2009) Different morpho-logical types of vegetative diasporesmdashcorticatedisidia and ecorticated sorediamdashand their locationare commonly used to distinguish speciesAscomatal characters including morphologylocation (laminal vs marginal) position (sessile
vs immersed) presence of thalline margins colorof an apothecial disc and presence or color ofpruina are also commonly used in speciesdelimitations of lichen-forming fungi (Printzen2009) Other important characters may includethalline characters form color size and septationof ascospores size and form and structure of ascithe hamathecium type of epihymenium andhypothecium and the type of excipulum orperidium conidiomatal characters etc (Printzen2009)
Assessments of secondary metabolites hasplayed an important role in lichen taxonomybeginning with the introduction of simple spottests by Nylander (1866a b) The use of chem-istry in lichen taxonomy has been discussed indetail in numerous reviews (Lumbsch 1998a bRogers 1989 Brodo 1986 Egan 1986 Leuckert1985 Brodo 1978 Hawksworth 1976 Culber-son 1969 1970) and we refer readers to thesevaluable sources for a more comprehensive per-spective on lichen chemistry In short extrolites(secondary metabolites) belong to various clas-ses the most common and diverse include dep-sides depsidones chlorinated xanthones andanthraquinones (Lumbsch 2002 Culberson1969) The presence or absence of specificextrolites or their replacements by another sub-stance is widely used to distinguish speciesparticularly when correlated with differences ingeographic distributions However if morpho-logical or geographical differences betweenpopulations containing different extrolites are notapparent the taxonomic significance has beendisputed with some authors distinguishing themas species and others preferring to regard them aschemical races within a species In addition tosimply using the presence or absence of extro-lites Culberson and Culberson (1976) proposedto arrange lichen substances into chemosyn-dromes of closely related substances The pres-ence or absence of these chemosyndromes maypotentially be used as characters to delimit spe-cies regarding differences involving the samechemosyndromes as intraspecific variation anddistinct chemosyndromes as evidence for inter-specific populations (Lumbsch 1994 Gowan1986)
2 The Dynamic Discipline of Species Delimitation hellip 33
Due to the fact that species delimitationstudies often incorporate a substantial biogeo-graphical or ecological component ecologicalniche modeling plays an increasing role in phy-logenetic and taxonomic research Niche mod-eling can provide evidence for ecologicalisolation between populations based on eitherconserved or divergent ecological niches andtherefore can provide additional evidence sup-porting lineage independence between putativespecies The application of ecological nichemodeling has been applied to species delimita-tion studies in a number of cases (Ruiz-Sanchezand Sosa 2010 Leacheacute et al 2009 Raxworthyet al 2007 Rissler and Apodaca 2007) althoughit has not been explicitly applied to assess speciesboundaries in lichen-forming fungi By mappingthe spatial distribution of environmental suit-ability of climatic variables for candidate speciesthe application of ecological niche modeling canbe particularly important in cases where specieshave allopatric distributions (Raxworthy et al2007)
Ecological niche modeling utilizes knownassociations between a speciesrsquo occurrencelocalities and environmental variables to defineabiotic conditions within which populations canbe maintained (Guisan and Thuiller 2005) Themethodological approach for modeling is basedon four general properties (i) The current knownspeciesrsquo localities is the dependent variable (ii)the distribution is modeled as a map composed ofgrid cells at a specified resolution (iii) a range ofenvironmental variables (eg temperature pre-cipitation and solar exposure) are collected todescribe the characteristics of each cell and (iv)classifying the degree to which each cell is eithersuitable or unsuitable for each species under arange of models (Guisan and Thuiller 2005)
Ecological data also have the potential to playan important role in understanding speciesboundaries in lichen-forming fungi For exampleNash and Zavada (1977) demonstrated that Xant-hoparmelia populations with distinct chemistriesoccurring in the northern portion of the SonoranDesert exhibit habitat selection among differentrock substrates within a region with relativelyuniform climate and topography In another case
the parmelioid species Parmelia mayi is mor-phologically indistinguishable from P saxatilisbut can be separated by bioclimatic features andgenetic and chemical characters (Molina et al2011) McCune and Printzen(2011) assess distri-butions and climatic niches of species in theLecanora varia group inwesternUSAandprovidea model that uses continental influence and annualtemperature as themajor factors predicting speciesdistributions The distribution of Usnea hirta alichen commonly used in air quality biomonitor-ing research was modeled for a section of theWhite River National Forest in central Coloradobased on the presence of U hirta at 72 biomoni-toring reference sites distributed in the inter-mountain western United States (Shrestha et al2012) The best model for predicting U hirtadistribution included four variablesmdashsolar radia-tion average monthly precipitation and averagemonthly minimum and maximum temperatures(Shrestha et al 2012) These studies supportthe potential use of ecological niche modelingmethods in species delimitation studies of lichen-forming fungi
24 Conclusions What AboutTaxonomy
In most cases species circumscription and tax-onomy requires some degree of qualitativejudgment and individual interpretation Integrat-ing multiple types of data into an empiricalframework for delimiting species boundarieswhere species boundaries can be tested within ahypothetico-deductive framework with diversedatasets can provide robust hypotheses of speciesboundaries and taxonomic stability (Yeates et al2011) However it has long been known toevolutionary biologists that distinct species donot need to have diagnosable morphologicaldifferences (Mayr 1963) and increasing avail-ability of genetic data has allowed researchers toidentify species and to rigorously test speciesboundaries with a level of precision that wasunimaginable a decade ago While analyticaladvances in statistical species delimitation havebeen largely based on genetic data the utility of
34 SD Leavitt et al
these approaches to formal taxonomy remainselusive Due to these challenges an eclecticapproach to delimiting species and cautionagainst the reliance on any single dataset ormethod is required when delimiting species
While results from many recent studies maycontradict traditional species boundaries acrossmany groups of lichen-forming fungi we areoptimistic that this research represents substantialprogress toward a more accurate perspective onspecies boundaries and diversity in fungi As aresult of ongoing research of species boundariesin lichen-forming fungi the taxonomic value ofmany phenotypes is now better understood ourunderstanding of ecological evolutionary andbiogeographic patterns has improved and we canbegin to better understand patterns of symbiontinteractions in lichens Integrating new data(including novel morphological characters andgenetic data) will be essential to accurately rep-resent species-level diversity across all groups oflichenized fungi Hopefully an improved per-spective on lichen diversity also increases ourappreciation of these incredible symbiotic sys-tems While we are strong advocates for theapplication of independent data types in devel-oping an integrative taxonomy there is anincreasing need to formally recognize the exis-tence of phenotypically cryptic species-levellineages in lichen-forming fungi (see Hibbettet al 2011) In some cases a molecular taxon-omy may provide the most practical approach toconsistent treatment of mycobiont species withinlichen groups where diagnostic morphologicalcharacters are unidentifiable or practically notfeasible (Leavitt et al 2013c d)
These are exciting times for taxonomists andphylogeneticists A closer look at lichen taxon-omy with the inclusion of new data will help usto better understand the diversity of these fasci-nating organisms accurately interpret distribu-tion patterns and play a more important role inmeaningful conservation practices Howeversome level of uncertainty will accompany pro-gress In many taxonomic groups our traditionalapproach for species identification will likelyneed to be substantially modified The search forcorroborating morphological support for cryptic
species identified using molecular data willrequire meticulous and creative approaches toassess phenotypic variation in potentially unor-thodox ways We are hopeful that lichenologistswho traditionally have been eager to include newmethods such as chromatography in their rou-tine identifications will be amenable to includemolecular techniques to their routine examina-tion of specimens for identification and classifi-cation Although this may prove difficult toachieve by single individuals especially citizenscientists that traditionally play an important rolein lichen taxonomy the increasing numberof collaborative projects in the discipline (egLumbsch et al 2011 Crespo et al 2010Gueidan et al 2009) make us optimistic thatbroad-scale collaborative approaches will facili-tate the inclusion of molecular data in lichenresearch at all levels
Acknowledgments We are indebted to various col-leagues for valuable thought-provoking discussionnotably Matthew Nelsen (University of Chicago) AnaCrespo (Universidad Complutense de Madrid) PradeepDivakar (Universidad Complutense de Madrid) BeckettSterner (The Field Museum) and Joyce Havstad (TheField Museum) We also thank anonymous reviewers whoprovided valuable comments that improved this chapterSupport by the US National Science Foundation isgratefully acknowledged (ldquoHidden diversity in parmelioidlichensrdquo DEB-0949147)
References
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Altermann S Leavitt SD Goward T Nelsen MPLumbsch HT (2014) How do you solve a problemlike Letharia A new look at cryptic species in lichen-forming fungi using Bayesian clustering and SNPSsfrom multilocus sequence data PLoS ONE 9(5)e97556 doi101371journalpone0097556
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2 The Dynamic Discipline of Species Delimitation hellip 35
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Corander J Marttinen P Mantyniemi S (2006) Bayesianidentification of stock mixtures from molecular markerdata Fish Bull 104550ndash558
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Culberson WL (1969) The use of chemistry in thesystematics of the lichens Taxon 18498ndash505
Culberson WL (1970) Chemosystematics and ecology oflichen-forming fungi Annu Rev Ecol Syst 1153ndash170
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Darwin C (1859) On the origin of species by means ofnatural selection or the preservation of favoured racesin the struggle for life J Murray London
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de Queiroz K (1998) The general lineage concept ofspecies species criteria and the process of speciationa conceptual unification and terminological recom-mendations In Howard DJ Berlocher SH (eds)Endless forms species and speciation Oxford Uni-versity Press Oxford pp 57ndash75
de Queiroz K (1999) The general lineage concept ofspecies and the defining properties of the speciescategory In Wilson RA (ed) Species new interdis-ciplinary essays MIT Press Cambridge pp 49ndash89
de Queiroz K (2007) Species concepts and speciesdelimitation Syst Biol 56(6)879ndash886 doi10108010635150701701083
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Degnan JH Rosenberg NA (2009) Gene tree discordancephylogenetic inference and the multispecies coales-cent Trends Ecol Evol 24(6)332ndash340 doihttpdxdoiorg101016jtree200901009
Del-Prado R Cubas P Lumbsch HT Divakar PK BlancoO de Paz GA Molina MC Crespo A (2010) Geneticdistances within and among species in monophyleticlineages of Parmeliaceae (Ascomycota) as a tool fortaxon delimitation Mol Phylogenet Evol 56(1)125ndash133 doi101016jympev201004014
Del-Prado R Divakar PK Crespo A (2011) Using geneticdistances in addition to ITS molecular phylogeny toidentify potential species in the Parmotrema reticul-atum complex a case study The Lichenologist 43(06)569ndash583 doi101017S0024282911000582
Del-Prado R Blanco O Lumbsch HT Divakar PK ElixJA Molina MC Crespo A (2013) Molecular phylog-eny and historical biogeography of the lichen-formingfungal genus Flavoparmelia (Ascomycota Parmelia-ceae) Taxon 62(5)928ndash939 doi101270562522
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Dettman JR Jacobson DJ Turner E Pringle A TaylorJW (2003b) Reproductive isolation and phylogeneticdivergence in Neurospora comparing methods ofspecies recognition in a model eukaryote Evolution57(12)2721 doi10155403-074
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Divakar PK Molina MC Lumbsch HT Crespo A (2005)Parmelia barrenoae a new lichen species related toParmelia sulcata (Parmeliaceae) based on molecularand morphological data The Lichenologist 37(01)37ndash46 doi101017S0024282904014641
Divakar PK Amo De paz G del Prado R Esslinger TLCrespo A (2007) Upper cortex anatomy corroboratesphylogenetic hypothesis in species of Physconia(Ascomycota Lecanoromycetes) Mycol Res 111(11)1311ndash1320 doi101016jmycres200708009
Divakar PK Figueras G Hladun N Crespo A (2010)Molecular phylogenetic studies reveal an undescribedspecies within the North American concept of Mela-nelixia glabra (Parmeliaceae) Fungal Divers 42(1)47ndash55 doi101007s13225-010-0027-3
Donoghue MJ Gauthier A (2004) Implementing thephylocode Trends Ecol Evol 19(6)281ndash282 doi101016jtree200404004
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Edwards SV (2009) Is a new and general theory ofmolecular systematics emerging Evolution 63(1)1ndash19 doi101111j1558-5646200800549x
Edwards DL Knowles LL (2014) Species detection andindividual assignment in species delimitation canintegrative data increase efficacy Proc R Soc B BiolSci 281(1777) doi101098rspb20132765
Egan RS (1986) Correlations and non-correlations ofchemical variation patterns with lichen morphologyand geography The Bryologist 8999ndash110
Elix JA Corush J Lumbsch HT (2009) Triterpenechemosyndromes and subtle morphological characterscharacterise lineages in the Physcia aipolia group inAustralia (Ascomycota) Syst Biodivers 7(04)479ndash487 doi101017S1477200009990223
Emerson KJ Merz CR Catchen JM Hohenlohe PACresko WA Bradshaw WE Holzapfel CM (2010)Resolving postglacial phylogeography using high-throughput sequencing Proc Natl Acad Sci 107(37)16196ndash16200 doi101073pnas1006538107
Ence DD Carstens BC (2011) SpedeSTEM a rapid andaccurate method for species delimitation Mol EcolResour 11(3)473ndash480 doi101111j1755-0998201002947x
EvannoGRegnaut SGoudet J (2005)Detecting thenumberof clusters of individuals using the software STRUC-TURE a simulation study Mol Ecol 14(8)2611ndash2620doi101111j1365-294X200502553x
Falush D Stephens M Pritchard JK (2003) Inference ofpopulation structure using multilocus genotype datalinked loci and correlated allele frequencies Genetics164(4)1567ndash1587 doi101111j1471-8286200701758x
Fan HH Kubatko LS (2011) Estimating species treesusing approximate Bayesian computation Mol Phylo-genet Evol 59(2)354ndash363 doi101016jympev201102019
Fernaacutendez-Mendoza F Printzen C (2013) Pleistoceneexpansion of the bipolar lichen Cetraria aculeata intothe Southern hemisphere Mol Ecol 22(7)1961ndash1983doi101111mec12210
Fernaacutendez-Mendoza F Domaschke S Garciacutea MA JordanP Martin MP Printzen C (2011) Population structure
2 The Dynamic Discipline of Species Delimitation hellip 37
of mycobionts and photobionts of the widespreadlichen Cetraria aculeata Mol Ecol 20(6)1208ndash1232doi101111j1365-294X201004993x
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Fujita MK Leacheacute AD Burbrink FT McGuire JA MoritzC (2012) Coalescent-based species delimitation in anintegrative taxonomy Trends Ecol Evol 27(9)480ndash488 doi101016jtree201204012
Gatesy J OrsquoGrady P Baker RH (1999) Corroborationamong data sets in simultaneous analysis hiddensupport for phylogenetic relationships among higherlevel Artiodactyl taxa Cladistics 15(3)271ndash313doi101111j1096-00311999tb00268x
Gladieux P Ropars J Badouin H Branca A Aguileta Gde Vienne DM Rodriacuteguez de la Vega RC Branco SGiraud T (2014) Fungal evolutionary genomicsprovides insight into the mechanisms of adaptivedivergence in eukaryotes Mol Ecol 23(4)753ndash773doi101111mec12631
Goffinet B Miadlikowska J Goward T (2003) Phyloge-netic inferences based on nrDNA sequences supportfive morphospecies within the Peltigera didactylacomplex (Lichenized Ascomycota) The Bryologist106(3)349ndash364 doi10163901
Gowan SP (1986) Evolution of secondary natural productsin the genus Porpidia (Ascomycata Porpidiaceae) AmJ Bot 73606
Griffin PC Hoffmann AA (2014) Limited genetic diver-gence among Australian alpine Poa tussock grassescoupled with regional structuring points to ongoinggene flow and taxonomic challenges Ann Bot doi101093aobmcu017
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Gueidan C Savi S Thues H Roux C Keller C Tibell LPrieto M Heimarsson S Breuss O Orange A FrobergL Wynns AA Navarro-Rosines P Krzewicka BPykaelae J Grube M Lutzoni F (2009) Genericclassification of the Verrucariaceae (Ascomycota)based on molecular and morphological evidencerecent progress and remaining challenges Taxon 58(1)184ndash208
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Guillot G Renaud S Ledevin R Michaux J Claude J(2012) A unifying model for the analysis of pheno-typic genetic and geographic data Syst Biol 61(6)897ndash911 doi101093sysbiosys038
Guisan A Thuiller W (2005) Predicting species distribu-tion offering more than simple habitat models EcolLett 8(9)993ndash1009 doi101111j1461-0248200500792x
Hale ME (1990) A synopsis of the lichen genusXanthoparmelia (Vainio) Hale (AscomycotinaParmeliaceae) Smithsonian Institution Press Wash-ington DC
Hamilton CA Hendrixson BE Brewer MS Bond JE(2014) An evaluation of sampling effects on multipleDNA barcoding methods leads to an integrativeapproach for delimiting species a case study ofthe North American tarantula genus Aphonopelma(Araneae Mygalomorphae Theraphosidae) MolPhylogenet Evol 7179ndash93 doi101016jympev201311007
Hausdorf B (2011) Progress toward a general speciesconcept Evolution 65(4)923ndash931 doi101111j1558-5646201101231x
Hausdorf B Hennig C (2010) Species delimitation usingdominant and codominant multilocus markers SystBiol 59(5)491ndash503 doi101093sysbiosyq039
Hawksworth DL (1976) Lichen chemotaxonomy InBailey RH (ed) Lichenology progress and problemsAcademic London pp 139ndash184
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Hebert PDN Stoeckle MY Zemlak TS Francis CM(2004) Identification of birds through DNA barcodesPLoS Biol 2(10)e312 doi101371journalpbio0020312
Heled J Drummond AJ (2010) Bayesian inference ofspecies trees from multilocus data Mol Biol Evol 27(3)570ndash580 doi101093molbevmsp274
Hey J (2006) On the failure of modern species conceptsTrends Ecol Evol 21(8)447ndash450 doi101016jtree200605011
Hibbett DS OhmanA Glotzer D NuhnMKirk P NilssonRH (2011) Progress in molecular and morphologicaltaxon discovery in Fungi and options for formalclassification on environmental sequences Fungal BiolRev 2538ndash47 doi101016jfbr201101001
Houmlgnabba F Wedin M (2003) Molecular phylogeny ofthe Sphaerophorus globosus species complex Cladis-tics 19(3)224ndash232 doi101111j1096-00312003tb00365x
Hudson RR Coyne JA (2002) Mathematical conse-quences of the genealogical species concept Evolu-tion 56(8)1557 doi101111j0014-38202002tb01467x
Huelsenbeck JP Andolfatto P Huelsenbeck ET (2011)Structurama Bayesian inference of population struc-ture Evol Bioinf Online 7(2011)55ndash59 doi104137EBOS6761
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Leavitt SD Fankhauser JD Leavitt DH Porter LDJohnson LA St Clair LL (2011a) Complex patterns ofspeciation in cosmopolitan ldquorock posyrdquo lichensmdashdiscovering and delimiting cryptic fungal species in
the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
Leavitt SD Esslinger TL Lumbsch HT (2012c) Neogene-dominated diversification in neotropical montanelichens dating divergence events in the lichen-form-ing fungal genus Oropogon (Parmeliaceae) Am J Bot99(11)1764ndash1777 doi103732ajb1200146
Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Lumbsch HT St Clair LL(2013c) DNA barcode identification of lichen-formingfungal species in the Rhizoplaca melanophthalmaspecies-complex (Lecanorales Lecanoraceae) includ-ing five new species MycoKeys 71ndash22 doi103897mycokeys74508
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Vondraacutek J Thorsten Lum-bsch H Clair LLS (2013d) Local representation ofglobal diversity in a cosmopolitan lichen-formingfungal species complex (Rhizoplaca Ascomycota)J Biogeogr 40(9)1792ndash1806 doi101111jbi12118
Leavitt SD Lumbsch HT Stenroos S St Clair LL(2013e) Pleistocene speciation in North American
2 The Dynamic Discipline of Species Delimitation hellip 39
lichenized fungi and the impact of alternative speciescircumscriptions and rates of molecular evolution ondivergence estimates PLoS ONE 8(12)e85240doi101371journalpone0085240
Leavitt SD Esslinger TL Hansen ES Divakar PK CrespoA Loomis BF Lumbsch HT (2014) DNA barcoding ofbrown Parmeliae (Parmeliaceae) species a molecularapproach for accurate specimen identification empha-sizing species in Greenland Organ Divers Evol 14(1)11ndash20 doi101007s13127-013-0147-1
Leliaert F Verbruggen H Vanormelingen P Steen FLopez-Bautista JM Zuccarello GC De Clerck O(2014) DNA-based species delimitation in algae Eur JPhycol 49(2) doi101080096702622014904524
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Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
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Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
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Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
Mayden RL (1997) A hierarchy of species concepts thedenouement in the saga of the species problem InClaridge MF Dawah HA Wilson MR (eds) Speciesthe units of biodiversity Chapman amp Hall Londonpp 381ndash424
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McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
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40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
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Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
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Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
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OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
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Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
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Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
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Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
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Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
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Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
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Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
morphological characters Antarctic Sci 22 (SpecialIssue 06)727ndash741 doi101017S0954102010000477
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Seacuterusiaux E Villarreal AJC Wheeler T Goffinet B(2011) Recent origin active speciation and dispersalfor the lichen genus Nephroma (Peltigerales) inMacaronesia J Biogeogr 38(6)1138ndash1151 doi101111j1365-2699201002469x
Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
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Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
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Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
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Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
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Westberg M Arup U Kaumlrnefelt I (2007) Phylogeneticstudies in the Candelariaceae (lichenized Ascomy-cota) based on nuclear ITS DNA sequence dataMycol Res 111(11)1277ndash1284 doi101017S0953756204002102
Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
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(Lepidoptera Lycaenidae) Front Zoo 4(1)8 doi1011861742-9994-4-8
Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
single lineage The best fitting model is thenselected using the Akaike information criterionunder the assumption of constant populationsizes and fixed gene topologies across the speciestree The species delimitation program BayesianPhylogenetic and Phylogeography (BPampPYangand Rannala 2010) accommodates gene treeuncertainty and variable population sizes andsamples from the Bayesian posterior distributionof species delimitation models using reversible-jump Markov chain Monte Carlo (rjMCMC)sampling BPampP requires a user-provided guidetree with resolution finer than the species leveland evaluates alternative modes derived from allpossible subtrees that are generated by collapsingor splitting nodes on the guide tree (Yang andRannala 2010 Rannala and Yang 2003) Theproportion of time spent on each model is pro-portional to the posterior probability of themodel ie ldquospeciation probabilitiesrdquo WhileBPampP ranks among the most popular coalescent-based species delimitation programs (Fujita et al2012) misspecifications of the guide tree andorthe prior distribution for population size (θ) canresult in strong support for models containing aninflated number of species (Leacheacute and Fujita2010 Zhang et al 2011) The nonparametricheuristic species delimitation approach imple-mented in the program Brownie (OrsquoMeara 2010)jointly sorts anonymous samples into species andinfers a species tree from input gene trees fromdifferent loci assuming that for a speciationevent the corresponding nodes on gene trees willbe more consistent with each than the diver-gences within species However finding both theoptimum species tree and species boundariesremains computationally challenging andBrownie has been shown to frequently yieldincorrect results (OrsquoMeara 2010)
Most recently available coalescent-basedspecies delimitation methods require individualassignments to a species or population a priori Ina number of scenarios correct assignments ofsamples to species may be difficult includingpresence of cryptic species incongruencebetween conventional species and moleculardata or simply the fact that accurate specimenidentification in complex groups with subtle or
difficult to discern diagnostic characters is anontrivial task (Leavitt et al 2013e) Statisticalmethods for assessing individual assignment andspecies detection prior to coalescent-based spe-cies delimitation and species tree reconstructionand species provide a more objective approachfor understanding species boundaries
Population assignment tests using a variety ofclustering algorithms using genetic data offer auseful approach for grouping individuals intoputative reproductively isolated groups (Table 21Hausdorf and Hennig 2010 Corander et al2004 2008 Falush et al 2003 Pritchard et al2000) particularly for species delimitation prob-lems that exist at the interface of traditionalpopulation genetic and phylogenetic analyses(Carstens et al 2013 Weisrock et al 2010Knowles and Carstens 2007)
The programs STRUCTURE (Falush et al2003 Pritchard et al 2000) and STRUCTURA-MA (Huelsenbeck et al 2011) cluster samplesinto populations by minimizing HardyndashWeinbergdisequilibrium for a given number of populationclusters using unlinked genetic markers In gen-eral unlinked markers are not available for mostgroups of lichen-forming fungi and a numberof studies have used information from multilocussequence data for STRUCTURE analyses(Leavitt et al 2011a b c 2013e Fernaacutendez-Mendoza and Printzen 2013 Fernaacutendez-Mendoza et al 2011) Varying approaches havebeen implemented to convert DNA sequencedata to allelic data for Bayesian clustering (seeOrsquoNeill et al 2013) STRUCTURE is expectedto perform well when there is sufficient inde-pendence across regions such that linkage dis-equilibrium within regions does not dominate thedata (STRUCTURE manual) but can also beeffective using multilocus sequence data andtreating all SNPs as independent loci regardlessof physical linkage within each locus (OrsquoNeillet al 2013 Falush et al 2003) A recent study ofthe lichen-forming genus Letharia showed thatBayesian clustering implemented in the programSTRUCTURE was generally able to recover thesame putative Letharia lineages circumscribedemploying a gene genealogical approach inKroken and Taylorrsquos iconic species delimitation
28 SD Leavitt et al
study (Altermann et al 2014 Kroken and Taylor2001) Altermann et al (2014) show that popu-lation assignments were largely consistent acrossa range of scenarios including extensiveamounts of missing data the exclusion of SNPsfrom variable markers and inferences based onSNPs from as few as three gene regions Incontrast to STRUCTURE and STRUCTURA-MA the program BAPS (Corander et al 2008)can explicitly infer genetic structure using hap-loid sequence data or other linked geneticmarkers in the ldquoclustering with linked locirdquomodel Another advantage of BAPS is that theprogram is much more computationally efficientthan STRUCTURE or STRUCTURAMA Sim-ulation studies indicate that both BAPS andSTRUCTURE perform well at low levels ofpopulation differentiation and when clusters arenot well differentiated (Latch et al 2006)
In practice inferring the most appropriatelevel of population structure using Bayesianclustering algorithms remains challenging (Latchet al 2006 Evanno et al 2005) BAPS andSTRUCTURAMA can infer both individualassignments and the most likely number ofgenetic clusters (Corander et al 2008 Huelsen-beck et al 2011) and the ad hoc ΔK statisticproposed by Evanno et al (2005) provides anobjective approach for identifying the uppermosthierarchical level of structure However carefulconsideration of other levels of populationstructure may ultimately reveal more biologicallymeaning results and researchers should examineindividual assignments across a range of geneticgroupings Some markers generated fromindependent genomic regions such as SNPs andfast-evolving microsatellites can be used to dis-tinguish fine-scale population structuring on thebasis of allele frequencies and species delimita-tion analyses based on these markers may implythe risk of severe taxonomic oversplitting Inthese cases validation approaches such asBPampP and SpeDeSTEM and corroborationamong different species delimitation approachescan provide perspective between intraspecificpopulation structure and species-level clusters
Another limitation of clustering approaches isthat they do not assess or take into account
evolutionarily divergences and relationshipsamong population clusters Coalescent-basedspecies tree methods (discussed below) provide ameans to assess the diversification histories ofpopulations inferred from clustering analysesTherefore a potential working protocol for aninformed species delimitation study that takesinto account population structure could consist offirst applying a genetic clustering analysis under apopulation genetics criterion (eg BAPSSTRUCTURE STRUCTURAMA) to identifygenetically distinct population clusters that can beconsidered ldquocandidate speciesrdquo From these can-didate species a species tree can be inferred forfocal group using coalescent-based species treereconstruction methods (eg BEAST) Subse-quently a coalescent-based validation methodcan be applied to assess whether the distinctpopulation clusters represent independent evolu-tionary lineages (eg BPampP) This multistepapproach would provide a consistent and standardcriterion for distinguishing between population-and species-level lineages (Camargo and Sites2013) and has been applied to a number of casesincluding lichen-forming fungi (Leavitt et al2011a b c 2013e Leacheacute and Fujita 2010)
High-throughput sequencing methods providethe means to effectively sample hundreds tothousands of loci from across a species genome fora large number of species and continue to revolu-tionize studies that can be performed even in non-model organisms Targeted high-throughputsequencing approaches such as anchored phy-logenomics transcriptome sequencing reduced-representation genomic library sequencing(restriction-site-associated DNA sequencingRAD-Seq and genotype-by-sequencing GBS)and high-throughput amplicon sequencing pro-vide important insight into species boundaries fora number groups although none of these approa-ches has yet been applied to studies of lichenizedfungi
For example restriction-site-associated(RAD-Seq) DNA can simultaneously detect andgenotype thousands of genome-wide SNPs byfocusing the sequencing effort on a reducedrepresentation of the entire genome (Baird et al2008) and has been successfully applied to
2 The Dynamic Discipline of Species Delimitation hellip 29
intraspecies (Lewis et al 2007 Miller et al 2007Emerson et al 2010) and interspecies studies(Rubin et al 2012 Eaton and Ree 2013 Wagneret al 2013) This approach has provided strikinginsight into the recent adaption radiation of LakeVictoria cichlids (Wagner et al 2013) Analternative approach targeting the sequencing ofspecific loci using next-generation sequencingplatforms provides an efficient means of gener-ating data for loci of known genomic locationorthology size and expected level of variation(OrsquoNeill et al 2013) Markers can be targetedusing well-established PCR techniques or newerhybridization techniques and subsequentlypooled for high-throughput sequencing usingparallel-tagged sequencing of multiple individualsamples within a single sequencing run (OrsquoNeillet al 2013) A recent study of North Americantiger salamanders (Ambystoma tigrinum)demonstrated the potential for amplicon-basedparallel-tagged sequencing to rapidly generatelarge-scale genomic data for species delimitationand species tree research (OrsquoNeill et al 2013)
Although these methods provide an enormousamount of data and insight into diversification atan unprecedented scale computational limita-tions restrict the applicable analytical methodsfor large datasets although computational andanalytical advances are happening rapidly Mostrecent coalescent-based species delimitation andspecies tree models using gene trees have beenlimited to handle tens of loci for multiple indi-viduals and combining hundreds or thousands ofgene trees into a single species delimitationframework presents considerable computationalchallenges (Camargo and Sites 2013) Therecently developed method Bayes factor delim-itation (with genomic data BFD) combines adynamic programming algorithm for estimatingspecies trees that bypasses the computationallyintensive MCMC integration over gene trees toprovide a rigorous technique for species delimi-tation studies using genome-wide SNP data(Leacheacute et al 2014) Competing species delimi-tation models are compared using Bayes factorsand it appears that this approach is robust tosample sizes (ie few loci and limited samples
per species) and misspecification of the prior forpopulation size (θ) (Leacheacute et al 2014)
Fungi are an ideal model for assessing diver-gence in eukaryotes due to their simple mor-phologies small genomes broad ecological rolesand diverse lifestyles (Gladieux et al 2014)However the use of genomic data from high-throughput sequencing methods have not yet beenincluded in species delimitation studies of lichen-forming fungi This is due in part to challenges indealing with metagenomic data containing geno-mic information from a plethora of symbiontsassociated with a lichen thallus scant genomicresources and discipline-specific inertia A num-ber of lichen-forming fungal genomes are cur-rently available including Endocarponpusillum(Wang et al 2014) Cladonia spp (Park et al2013a 2014) Caloplaca flavorubescens (Parket al 2013b) and Cladonia greyi and Xanthoriaparientina through the Fungal Genomics Programat Joint Genome Institute of the United StatesDepartment of Energy (httpwwwjgidoegov)The foreseeable ongoing expansion of genomicresources for lichen-forming fungi will be centralto developing approaches for delimiting speciesusing high-throughput sequencing Specificallygenomic resources will be crucial in identifyingnovel markers (variable genes regions SNPsmicrosatellites etc) identifying conserved fungalmarkers for targeted high-throughput sequencingapproaches and references for entire genomecomparisons (Devkota et al 2014 Werth et al2013)
23 Can We Make SpeciesDelimitation in Lichen-FormingFungi Truly Integrative
The widespread availability of genetic data hascreated a biased viewpoint that only genetic datashould be used for statistical species delimitationHowever an ongoing appeal to researchers toassess species boundaries from multiple andcomplementary perspectives (phylogeneticspopulation genetics comparative morphologydevelopment ecology etc) has resulted in an
30 SD Leavitt et al
integrative taxonomic framework bringing theseconceptual and methodological perspectivestogether (Hamilton et al 2014 Fujita et al 2012Salicini et al 2011 Yeates et al 2011 Padialet al 2010 Dayrat 2005 Will et al 2005 Wiensand Penkrot 2002) In practice any study linkingdifferent kinds of data to support hypotheses ofspecies boundaries including mapping morpho-logical characters onto a molecular phylogenycan be considered integrative Integrative meth-ods for species delimitation fall across a broadspectrum ranging from verbal and qualitativeassessments of data classes to quantitative meth-ods that allow different data types to contribute tostatistical species delimitation (Yeates et al2011) In most taxonomic studies utilizing bothmolecular and morphological data expert opinionis eventually used to some degree to evaluate thefinal status of a candidate species (eg Bond andStockman 2008) In this sense many studiesusing evidence for independent data sources fordelimiting species boundaries use an iterativeapproach (Yeates et al 2011) where speciesboundaries can be tested within a hypothetico-deductive framework with diverse datasets
From a practical perspective we advocate aprocess of iterative taxonomy (sensu Yeates et al2011) to circumscribe and refine species limitsusing multiple lines of evidence This iterativeprocess involves comparisons of morphologicaldata with a phylogenetic hypothesis (ie single-locus gene tree or concatenated multilocusphylogeny) to identify the least inclusive mono-phyletic clade in the topology characterized by atleast one unambiguously diagnostic morphologi-cal character (Miralles and Vences 2013) Thisphylogenetic tree-informed approach to assessingspecies boundaries represents a common practicein studies of lichen-forming fungi Previouslyunrecognized species-level clades with corre-sponding subtle or overlooked phenotypiccharacters have been commonly observed in bothcrustose lichens [eg Acarospraceae (Wedin et al2009) Graphidaceae (Rivas Plata and Lumbsch2011 Papong et al 2009) Lecanoraceae (Leavittet al 2011a) Lecideaceae (Ruprecht et al 2010)Mycoblastaceae (Spribille et al 2011) andTeloschistaceae (Vondraacutek et al 2009 Muggia
et al 2008)] and foliose and fruticose lichens [egLobariaceae (Moncada et al 2014 McDonaldet al 2003) Parmeliaceae (Leavitt et al 2013aWirtz et al 2012 Divakar et al 2005 Molinaet al 2004 Kroken and Taylor 2001) Peltigera-ceae (OrsquoBrien et al 2009 Goffinet et al 2003)Physciaceae (Elix et al 2009 Divakar et al2007) and Sphaerophoraceae (Houmlgnabba andWedin 2003)] As a specific example Luumlckinget al (2008) used a combination of medullarychemistry and underside pigmentation in speci-mens from the Heterodermia obscurata group inCosta Rica to corroborate monophyletic clades inan ITS gene tree as species-level lineages
While this iterative approach to speciesdelimitation and taxonomy has proven valuablefor understanding species boundaries anddescribing new taxa in some groups of lichenizedfungi a posteriori examination of morphologicaland chemical features has failed to reveal diag-nostic phenotypic characters in a number ofstudies (Muggia et al 2014 Leavitt et al 2011aOtaacutelora et al 2010 OrsquoBrien et al 2009) Fur-thermore a study of widespread species in thegenus Melanelixia (Parmeliaceae) indicated thatphenotypically cryptic lichen-forming fungalspecies-level lineages may be relatively ancientand diagnosable phenotypic differences may beabsent even millions of years after the initialdivergence (Leavitt et al 2012b) The latter studyhighlights the fact that species-level lineages maycommonly exist without any observable diag-nostic phenotypic characters calling into ques-tion the impetus for a universal application ofintegrative taxonomy
In keeping with the principle that as manylines of evidence as available should be com-bined to delimit species (Dayrat 2005) the for-malized integrative taxonomic approach (ITAXMiralles and Vences 2013) uses a mtDNA guidetree and observations from different types of datathat might provide conclusive evidence for theindependence of lineages and thus their identityas different species Miralles and Vences (2013)provide a non-exhaustive list of species delimi-tation criteria to be integrated in the ITAXapproach including (a) sympatric occurrencewithout admixture as revealed by consistent
2 The Dynamic Discipline of Species Delimitation hellip 31
differences in morphological or molecular char-acters at the same geographic location (b) strongdifferences in a behavioral morphological orgenetic character known to mediate prematingisolation (c) unviability or infertility of hybrids(d) lack of gene flow across a geographicalhybrid zone (e) congruent diagnostic differencesbetween sister lineages in various unlinkedmorphological character (f) absence of haplo-type sharing in several unlinked nuclear loci and(g) a combination of criteria endashf Speciesboundaries are based on seeking the least inclu-sive monophyletic group in the mtDNA treewhich fulfills at least one of the criteria listedabove Clearly the ITAX approach is sensitive tosample size and in order to reliably support thedistinctiveness of a given species it has beenrecommended that the sampling strategy includesat least five individuals per species (Miralles andVences 2013) A similar approach could beadopted for lichenized fungi using an ITS genetopology as guide tree rather than mtDNA
A total evidence approach including concat-enation has been a common approach for inte-grating information from different sourcesincluding independent genetic markers in phy-logenetic reconstructions (Kluge 1989 Wiens1998 de Queiroz et al 1995) Proponents forconcatenation of independent data in phyloge-netic analyses argue that when combining alldata the underlying signal of speciation mayemerge even if weakly contradictory signalis contained in the individual data partitions(Gatesy et al 1999) For example establishing apreliminary perspective of species boundariesfrom multilocus sequence data using concatena-tion may provide a reasonable starting point forscreening for cryptic species and species treeinference (Šlapeta et al 2006 Leavitt et al2011a Le Gac et al 2007 Leacheacute 2009)However concatenation and consensus methodsimply a risk of obtaining inflated support forincorrect relationships and information aboutvariance in gene coalescence is lost (Degnan andRosenberg 2006 2009 Kubatko and Degnan2007 Edwards 2009) In spite of the impetus forintegrating evidence from independent data
sources into empirical species delimitation stud-ies and taxonomy (Fujita et al 2012 Padial et al2009) most currently available species delimi-tation methods are unable to accommodate non-genetic data sources in a statistical framework
231 Selecting the Appropriate Data
In the face of increasing availability of geneticdata and associated bioinformatical approachesfor delimiting species researchers should care-fully consider what information is being sacri-ficed by the failure to consider non-genetic datain species delimitation studies and whetheraccuracy could be improved by the addition ofmultiple data types Morphological data havehistorically served as a proxy to identify repro-ductively isolated groups (ie ldquospeciesrdquo) (Ray1686 Fujita et al 2012) Current methods fordelimiting species using non-genetic data (egchemistry morphology and ecology) remainwoefully understudied For example morphol-ogy-based species circumscriptions are generallybased on one or more qualitative (or quantitative)morphological characters that do not appear tooverlap with other species However ascertain-ing that a given trait is truly fixed within apopulation with statistical confidence requiresunrealistic sample sizes even when allowing forsome level of polymorphism in the diagnosticcharacter (Wiens and Servedio 2000) Now var-ious combinations of datamdashfrom morphologygenetics geography and ecologymdashare acceptedas standard information for species delimitationstudies (Ruiz-Sanchez and Sosa 2010 Ross et al2010 Edwards and Knowles 2014)
Below we briefly discuss appropriate datasources for species delimitation studies of lichen-forming fungi However homoplastic characters(similar traits that are not derived from a com-mon ancestor) are common among many traitscommonly used to circumscribe fungal taxa thebiological significance of secondary metabolitevariation remains largely unknown (Lawrey1986) and ecological niches may be difficultto adequately characterize and model due to
32 SD Leavitt et al
microhabitat requirements and data resolutionTherefore we advocate a cautious approach toselecting appropriate and relevant data forassessing species boundaries
In spite of the potential challenges and limi-tations of using phenotypic data these traits haveprovided a plethora of valuable information forunderstanding species boundaries For examplein the Melanelixia fuliginosa group (Parmelia-ceae) a morphometric analysis using color isi-dia and marginal zone free of isidia ascharacters revealed a general pattern of differ-entiation between material formerly recognizedas subspecies fuliginosa and glabratula in spiteof the fact that considerable overlap betweengroups occurs in some characters individually(Arup and Berlin 2011) Furthermore the dis-tinction of the two groups was supported by aphylogenetic analysis of the ITS marker andecological differences with M fuliginosaoccurring predominantly on rock and M glab-ratula on bark (Arup and Berlin 2011) Thisstudy of the M fuliginosa group provides a fit-ting example of using multiple independentsuites of data including ecology morphologyand genetic information to establish a robusthypothesis of species boundaries
In lichen systematics phenotypic dataincluding thallus organization secondary metab-olites mode of reproduction ascoma-type andontogeny ascus and ascospore characters havehistorically played a prominent role (Printzen2009) Ascomatal characters have traditionallyheld a major role in higher-level classification(Printzen 2009) in contrast to species-levelclassification which also tends to include a widearray of vegetative and chemical charactersCommonly assessed morphological charactersinclude thallus form and size cortical features(eg maculae and pseudocyphellae) presenceformcolor of attachment structure (eg rhizines)and reproductive mode (ascomata vs vegetativediaspores) (Printzen 2009) Different morpho-logical types of vegetative diasporesmdashcorticatedisidia and ecorticated sorediamdashand their locationare commonly used to distinguish speciesAscomatal characters including morphologylocation (laminal vs marginal) position (sessile
vs immersed) presence of thalline margins colorof an apothecial disc and presence or color ofpruina are also commonly used in speciesdelimitations of lichen-forming fungi (Printzen2009) Other important characters may includethalline characters form color size and septationof ascospores size and form and structure of ascithe hamathecium type of epihymenium andhypothecium and the type of excipulum orperidium conidiomatal characters etc (Printzen2009)
Assessments of secondary metabolites hasplayed an important role in lichen taxonomybeginning with the introduction of simple spottests by Nylander (1866a b) The use of chem-istry in lichen taxonomy has been discussed indetail in numerous reviews (Lumbsch 1998a bRogers 1989 Brodo 1986 Egan 1986 Leuckert1985 Brodo 1978 Hawksworth 1976 Culber-son 1969 1970) and we refer readers to thesevaluable sources for a more comprehensive per-spective on lichen chemistry In short extrolites(secondary metabolites) belong to various clas-ses the most common and diverse include dep-sides depsidones chlorinated xanthones andanthraquinones (Lumbsch 2002 Culberson1969) The presence or absence of specificextrolites or their replacements by another sub-stance is widely used to distinguish speciesparticularly when correlated with differences ingeographic distributions However if morpho-logical or geographical differences betweenpopulations containing different extrolites are notapparent the taxonomic significance has beendisputed with some authors distinguishing themas species and others preferring to regard them aschemical races within a species In addition tosimply using the presence or absence of extro-lites Culberson and Culberson (1976) proposedto arrange lichen substances into chemosyn-dromes of closely related substances The pres-ence or absence of these chemosyndromes maypotentially be used as characters to delimit spe-cies regarding differences involving the samechemosyndromes as intraspecific variation anddistinct chemosyndromes as evidence for inter-specific populations (Lumbsch 1994 Gowan1986)
2 The Dynamic Discipline of Species Delimitation hellip 33
Due to the fact that species delimitationstudies often incorporate a substantial biogeo-graphical or ecological component ecologicalniche modeling plays an increasing role in phy-logenetic and taxonomic research Niche mod-eling can provide evidence for ecologicalisolation between populations based on eitherconserved or divergent ecological niches andtherefore can provide additional evidence sup-porting lineage independence between putativespecies The application of ecological nichemodeling has been applied to species delimita-tion studies in a number of cases (Ruiz-Sanchezand Sosa 2010 Leacheacute et al 2009 Raxworthyet al 2007 Rissler and Apodaca 2007) althoughit has not been explicitly applied to assess speciesboundaries in lichen-forming fungi By mappingthe spatial distribution of environmental suit-ability of climatic variables for candidate speciesthe application of ecological niche modeling canbe particularly important in cases where specieshave allopatric distributions (Raxworthy et al2007)
Ecological niche modeling utilizes knownassociations between a speciesrsquo occurrencelocalities and environmental variables to defineabiotic conditions within which populations canbe maintained (Guisan and Thuiller 2005) Themethodological approach for modeling is basedon four general properties (i) The current knownspeciesrsquo localities is the dependent variable (ii)the distribution is modeled as a map composed ofgrid cells at a specified resolution (iii) a range ofenvironmental variables (eg temperature pre-cipitation and solar exposure) are collected todescribe the characteristics of each cell and (iv)classifying the degree to which each cell is eithersuitable or unsuitable for each species under arange of models (Guisan and Thuiller 2005)
Ecological data also have the potential to playan important role in understanding speciesboundaries in lichen-forming fungi For exampleNash and Zavada (1977) demonstrated that Xant-hoparmelia populations with distinct chemistriesoccurring in the northern portion of the SonoranDesert exhibit habitat selection among differentrock substrates within a region with relativelyuniform climate and topography In another case
the parmelioid species Parmelia mayi is mor-phologically indistinguishable from P saxatilisbut can be separated by bioclimatic features andgenetic and chemical characters (Molina et al2011) McCune and Printzen(2011) assess distri-butions and climatic niches of species in theLecanora varia group inwesternUSAandprovidea model that uses continental influence and annualtemperature as themajor factors predicting speciesdistributions The distribution of Usnea hirta alichen commonly used in air quality biomonitor-ing research was modeled for a section of theWhite River National Forest in central Coloradobased on the presence of U hirta at 72 biomoni-toring reference sites distributed in the inter-mountain western United States (Shrestha et al2012) The best model for predicting U hirtadistribution included four variablesmdashsolar radia-tion average monthly precipitation and averagemonthly minimum and maximum temperatures(Shrestha et al 2012) These studies supportthe potential use of ecological niche modelingmethods in species delimitation studies of lichen-forming fungi
24 Conclusions What AboutTaxonomy
In most cases species circumscription and tax-onomy requires some degree of qualitativejudgment and individual interpretation Integrat-ing multiple types of data into an empiricalframework for delimiting species boundarieswhere species boundaries can be tested within ahypothetico-deductive framework with diversedatasets can provide robust hypotheses of speciesboundaries and taxonomic stability (Yeates et al2011) However it has long been known toevolutionary biologists that distinct species donot need to have diagnosable morphologicaldifferences (Mayr 1963) and increasing avail-ability of genetic data has allowed researchers toidentify species and to rigorously test speciesboundaries with a level of precision that wasunimaginable a decade ago While analyticaladvances in statistical species delimitation havebeen largely based on genetic data the utility of
34 SD Leavitt et al
these approaches to formal taxonomy remainselusive Due to these challenges an eclecticapproach to delimiting species and cautionagainst the reliance on any single dataset ormethod is required when delimiting species
While results from many recent studies maycontradict traditional species boundaries acrossmany groups of lichen-forming fungi we areoptimistic that this research represents substantialprogress toward a more accurate perspective onspecies boundaries and diversity in fungi As aresult of ongoing research of species boundariesin lichen-forming fungi the taxonomic value ofmany phenotypes is now better understood ourunderstanding of ecological evolutionary andbiogeographic patterns has improved and we canbegin to better understand patterns of symbiontinteractions in lichens Integrating new data(including novel morphological characters andgenetic data) will be essential to accurately rep-resent species-level diversity across all groups oflichenized fungi Hopefully an improved per-spective on lichen diversity also increases ourappreciation of these incredible symbiotic sys-tems While we are strong advocates for theapplication of independent data types in devel-oping an integrative taxonomy there is anincreasing need to formally recognize the exis-tence of phenotypically cryptic species-levellineages in lichen-forming fungi (see Hibbettet al 2011) In some cases a molecular taxon-omy may provide the most practical approach toconsistent treatment of mycobiont species withinlichen groups where diagnostic morphologicalcharacters are unidentifiable or practically notfeasible (Leavitt et al 2013c d)
These are exciting times for taxonomists andphylogeneticists A closer look at lichen taxon-omy with the inclusion of new data will help usto better understand the diversity of these fasci-nating organisms accurately interpret distribu-tion patterns and play a more important role inmeaningful conservation practices Howeversome level of uncertainty will accompany pro-gress In many taxonomic groups our traditionalapproach for species identification will likelyneed to be substantially modified The search forcorroborating morphological support for cryptic
species identified using molecular data willrequire meticulous and creative approaches toassess phenotypic variation in potentially unor-thodox ways We are hopeful that lichenologistswho traditionally have been eager to include newmethods such as chromatography in their rou-tine identifications will be amenable to includemolecular techniques to their routine examina-tion of specimens for identification and classifi-cation Although this may prove difficult toachieve by single individuals especially citizenscientists that traditionally play an important rolein lichen taxonomy the increasing numberof collaborative projects in the discipline (egLumbsch et al 2011 Crespo et al 2010Gueidan et al 2009) make us optimistic thatbroad-scale collaborative approaches will facili-tate the inclusion of molecular data in lichenresearch at all levels
Acknowledgments We are indebted to various col-leagues for valuable thought-provoking discussionnotably Matthew Nelsen (University of Chicago) AnaCrespo (Universidad Complutense de Madrid) PradeepDivakar (Universidad Complutense de Madrid) BeckettSterner (The Field Museum) and Joyce Havstad (TheField Museum) We also thank anonymous reviewers whoprovided valuable comments that improved this chapterSupport by the US National Science Foundation isgratefully acknowledged (ldquoHidden diversity in parmelioidlichensrdquo DEB-0949147)
References
Ahti T Hawksworth DL (2005) Xanthoparmelia steno-phylla the correct name for X somloeumlnsis one of themost widespread usnic acid containing species of thegenus The Lichenologist 37(4)363ndash366 doi101017S0024282905015197
Altermann S Leavitt SD Goward T Nelsen MPLumbsch HT (2014) How do you solve a problemlike Letharia A new look at cryptic species in lichen-forming fungi using Bayesian clustering and SNPSsfrom multilocus sequence data PLoS ONE 9(5)e97556 doi101371journalpone0097556
Amo de Paz G Cubas P Crespo A Elix JA Lumbsch HT(2012) Transoceanic dispersal and subsequent diver-sification on separate continents shaped diversity ofthe Xanthoparmelia pulla group (Ascomycota) PLoSONE 7(6)e39683 doi101371journalpone0039683
Arguumlello A Del Prado R Cubas P Crespo A (2007)Parmelina quercina (Parmeliaceae Lecanorales)
2 The Dynamic Discipline of Species Delimitation hellip 35
includes four phylogenetically supported morphospe-cies Biol J Linn Soc 91(3)455ndash467 doi101111j1095-8312200700810x
Arup U Berlin ES (2011) A taxonomic study ofMelanelixia fuliginosa in Europe The Lichenologist43(02)89ndash97 doi101017S0024282910000678
Arup U Grube M (2000) Is Rhizoplaca (Lecanoraleslichenized Ascomycota) a monophyletic genus Can JBot 78(3)318ndash327 doi101139b00-006
Avise J Ball R (1990) Principles of genealogicalconcordance in species concepts and biological tax-onomy Oxf Surv Evol Biol 745ndash67
Baird NA Etter PD Atwood TS Currey MC Shiver ALLewis ZA Selker EU Cresko WA Johnson EA(2008) Rapid SNP discovery and genetic mappingusing sequenced RAD markers PLoS ONE 3(10)e3376 doi101371journalpone0003376
Baum DA Shaw KL (1995) Genealogical perspectives onthe species problem In Hoch PC Stephenson AG(eds) Experimental and molecular approaches to plantbiosystematics Missouri Botanical Garden St Louispp 289ndash303
Beaumont MA Nielsen R Robert C Hey J Gaggiotti OKnowles L Estoup A Panchal M Corander JHickerson M Sisson SA Fagundes N Chikhi LBeerli P Vitalis R Cornuet J-M Huelsenbeck J FollM Yang Z Rousset F Balding D Excoffier L (2010)In defence of model-based inference in phylogeogra-phy Mol Ecol 19(3)436ndash446 doi101111j1365-294X200904515x
Bickford D Lohman DJ Sodhi NS Ng PKL Meier RWinker K Ingram KK Das I (2007) Cryptic speciesas a window on diversity and conservation TrendsEcol Evol 22(3)148ndash155 doi101016jtree200611004
Bonan GB Shugart HH (1989) Environmental factors andecological processes in boreal forests Annu Rev EcolSyst 20(1989)1ndash28
Bond J Stockman A (2008) An integrative method fordelimiting cohesion species finding the population-species interface in a group of Californian trapdoorspiders with extreme genetic divergence and geo-graphic structuring Syst Biol 57(4)628ndash646 doi10108010635150802302443
Brodo IM (1978) Changing concepts regarding chemicaldiversity in lichens The Lichenologist 10(1)1ndash11doi101017S0024282978000031
Brodo IM (1986) Interpreting chemical variation inlichens for systematic purposes The Bryologist 89(2)132ndash138
Caley MJ Fisher R Mengersen K (2014) Global speciesrichness estimates have not converged Trends EcolEvol 29(4)187ndash188 doi101016jtree201402002
Camargo A Sites JW (2013) Species delimitation adecade after the Renaissance In Pavlinov I (ed) Thespecies problemmdashongoing issues InTech doi10577252664
Camargo A Morando M Avila LJ Sites JW (2012)Species delimitation with ABC and other coalescent-based methods a test of accuracy with simulations and
an empirical example with lizards of the Liolaemusdarwinii complex (Squamata Liolaemidae) Evolution66(9)2834ndash2849 doi101111j1558-5646201201640x
Carstens BC Dewey TA (2010) Species delimitationusing a combined coalescent and information-theoreticapproach an example from North American Myotisbats Syst Biol 59(4)400ndash414 doi101093sysbiosyq024
Carstens BC Pelletier TA Reid NM Satler JD (2013)How to fail at species delimitation Mol Ecol 22(17)4369ndash4383 doi101111mec12413
Corander J Marttinen P (2006) Bayesian identification ofadmixture events using multi-locus molecular mark-ers Mol Ecol 15(10)2833ndash2843 doi101111j1365-294X200602994x
Corander J Waldmann P Marttinen P Sillanpaa M(2004) BAPS 2 enhanced possibilities for the analysisof genetic population structure Bioinformatics 20(15)2363ndash2369 doi101093bioinformaticsbth250
Corander J Marttinen P Mantyniemi S (2006) Bayesianidentification of stock mixtures from molecular markerdata Fish Bull 104550ndash558
Corander J Marttinen P Siren J Tang J (2008) EnhancedBayesian modelling in BAPS software for learninggenetic structures of populations BMC Bioinf 9(1)539 doi1011861471-2105-9-539
Coyne JA Orr HA (2004) Speciation Sinauer AssociatesSunderland
Cracraft J (1983) Species concepts and speciation anal-ysis Curr Ornithol 1159ndash187
Crespo A Lumbsch HT (2010) Cryptic species in lichen-forming fungi IMA Fungus 1167ndash170 doi105598imafungus2010010209
Crespo A Peacuterez-Ortega S (2009) Cryptic species andspecies pairs in lichens a discussion on the relation-ship between molecular phylogenies and morpholog-ical characters Anales del Jardin Botanico de Madrid66(S1)71ndash81 doi103989ajbm2225
Crespo A Kauff F Divakar PK del Prado R Perez-Ortega S Amo de Paz G Ferencova Z Blanco ORoca-Valiente B Nunez-Zapata J Cubas P ArguelloA Elix JA Esslinger TL Hawksworth DL MillanesA Molina MC Wedin M Ahti T Aptroot A et al(2010) Phylogenetic generic classification of parmeli-oid lichens (Parmeliaceae Ascomycota) based onmolecular morphological and chemical evidenceTaxon 59(6)1735ndash1753
Culberson WL (1969) The use of chemistry in thesystematics of the lichens Taxon 18498ndash505
Culberson WL (1970) Chemosystematics and ecology oflichen-forming fungi Annu Rev Ecol Syst 1153ndash170
Culberson CF Culberson WL (1976) Chemosyndromicvariation in lichens Syst Bot 1325ndash339
Darwin C (1859) On the origin of species by means ofnatural selection or the preservation of favoured racesin the struggle for life J Murray London
Dayrat B (2005) Towards integrative taxonomy Biol JLinn Soc 85(3)407ndash415 doi101111j1095-8312200500503x
36 SD Leavitt et al
de Queiroz K (1998) The general lineage concept ofspecies species criteria and the process of speciationa conceptual unification and terminological recom-mendations In Howard DJ Berlocher SH (eds)Endless forms species and speciation Oxford Uni-versity Press Oxford pp 57ndash75
de Queiroz K (1999) The general lineage concept ofspecies and the defining properties of the speciescategory In Wilson RA (ed) Species new interdis-ciplinary essays MIT Press Cambridge pp 49ndash89
de Queiroz K (2007) Species concepts and speciesdelimitation Syst Biol 56(6)879ndash886 doi10108010635150701701083
de Queiroz A Donoghue MJ Kim J (1995) Separateversus combined analysis of phylogenetic evidenceAnnu Rev Ecol Syst 26(1)657ndash681 doi101146annureves26110195003301
Degnan J Rosenberg N (2006) Discordance of speciestrees with their most likely gene trees PLoS Genet 2(5)e68 doi101371journalpgen0020068
Degnan JH Rosenberg NA (2009) Gene tree discordancephylogenetic inference and the multispecies coales-cent Trends Ecol Evol 24(6)332ndash340 doihttpdxdoiorg101016jtree200901009
Del-Prado R Cubas P Lumbsch HT Divakar PK BlancoO de Paz GA Molina MC Crespo A (2010) Geneticdistances within and among species in monophyleticlineages of Parmeliaceae (Ascomycota) as a tool fortaxon delimitation Mol Phylogenet Evol 56(1)125ndash133 doi101016jympev201004014
Del-Prado R Divakar PK Crespo A (2011) Using geneticdistances in addition to ITS molecular phylogeny toidentify potential species in the Parmotrema reticul-atum complex a case study The Lichenologist 43(06)569ndash583 doi101017S0024282911000582
Del-Prado R Blanco O Lumbsch HT Divakar PK ElixJA Molina MC Crespo A (2013) Molecular phylog-eny and historical biogeography of the lichen-formingfungal genus Flavoparmelia (Ascomycota Parmelia-ceae) Taxon 62(5)928ndash939 doi101270562522
Dettman J Jacobson D Taylor J (2003a) A multilocusgenealogical approach to phylogenetic species recog-nition in the model eukaryote Neurospora Evolution57(12)2703ndash2720 doi101111j0014-38202003tb01514x
Dettman JR Jacobson DJ Turner E Pringle A TaylorJW (2003b) Reproductive isolation and phylogeneticdivergence in Neurospora comparing methods ofspecies recognition in a model eukaryote Evolution57(12)2721 doi10155403-074
Devkota S Cornejo C Werth S Chaudhary RP Sche-idegger C (2014) Characterization of microsatelliteloci in the Himalayan lichen fungus Lobaria pindar-ensis (Lobariaceae) Appl Plant Sci 2(5)1300101doi103732apps1300101
Divakar PK Molina MC Lumbsch HT Crespo A (2005)Parmelia barrenoae a new lichen species related toParmelia sulcata (Parmeliaceae) based on molecularand morphological data The Lichenologist 37(01)37ndash46 doi101017S0024282904014641
Divakar PK Amo De paz G del Prado R Esslinger TLCrespo A (2007) Upper cortex anatomy corroboratesphylogenetic hypothesis in species of Physconia(Ascomycota Lecanoromycetes) Mycol Res 111(11)1311ndash1320 doi101016jmycres200708009
Divakar PK Figueras G Hladun N Crespo A (2010)Molecular phylogenetic studies reveal an undescribedspecies within the North American concept of Mela-nelixia glabra (Parmeliaceae) Fungal Divers 42(1)47ndash55 doi101007s13225-010-0027-3
Donoghue MJ Gauthier A (2004) Implementing thephylocode Trends Ecol Evol 19(6)281ndash282 doi101016jtree200404004
Eaton DAR Ree RH (2013) Inferring phylogeny andintrogression using RADseq data an example fromflowering plants (Pedicularis Orobanchaceae) SystBiol 62(5)689ndash706 doi101093sysbiosyt032
Edwards SV (2009) Is a new and general theory ofmolecular systematics emerging Evolution 63(1)1ndash19 doi101111j1558-5646200800549x
Edwards DL Knowles LL (2014) Species detection andindividual assignment in species delimitation canintegrative data increase efficacy Proc R Soc B BiolSci 281(1777) doi101098rspb20132765
Egan RS (1986) Correlations and non-correlations ofchemical variation patterns with lichen morphologyand geography The Bryologist 8999ndash110
Elix JA Corush J Lumbsch HT (2009) Triterpenechemosyndromes and subtle morphological characterscharacterise lineages in the Physcia aipolia group inAustralia (Ascomycota) Syst Biodivers 7(04)479ndash487 doi101017S1477200009990223
Emerson KJ Merz CR Catchen JM Hohenlohe PACresko WA Bradshaw WE Holzapfel CM (2010)Resolving postglacial phylogeography using high-throughput sequencing Proc Natl Acad Sci 107(37)16196ndash16200 doi101073pnas1006538107
Ence DD Carstens BC (2011) SpedeSTEM a rapid andaccurate method for species delimitation Mol EcolResour 11(3)473ndash480 doi101111j1755-0998201002947x
EvannoGRegnaut SGoudet J (2005)Detecting thenumberof clusters of individuals using the software STRUC-TURE a simulation study Mol Ecol 14(8)2611ndash2620doi101111j1365-294X200502553x
Falush D Stephens M Pritchard JK (2003) Inference ofpopulation structure using multilocus genotype datalinked loci and correlated allele frequencies Genetics164(4)1567ndash1587 doi101111j1471-8286200701758x
Fan HH Kubatko LS (2011) Estimating species treesusing approximate Bayesian computation Mol Phylo-genet Evol 59(2)354ndash363 doi101016jympev201102019
Fernaacutendez-Mendoza F Printzen C (2013) Pleistoceneexpansion of the bipolar lichen Cetraria aculeata intothe Southern hemisphere Mol Ecol 22(7)1961ndash1983doi101111mec12210
Fernaacutendez-Mendoza F Domaschke S Garciacutea MA JordanP Martin MP Printzen C (2011) Population structure
2 The Dynamic Discipline of Species Delimitation hellip 37
of mycobionts and photobionts of the widespreadlichen Cetraria aculeata Mol Ecol 20(6)1208ndash1232doi101111j1365-294X201004993x
Fraley C Raftery A (2007) Model-based methods ofclassification Using the mclust software in chemo-metrics J Stat Softw 18i06 [Available online at httpwwwdoajorgdoajfunc=abstractampid=218544]
Fujisawa T Barraclough TG (2013) Delimiting speciesusing single-locus data and the generalized mixed yulecoalescent (GMYC) approach a revised method andevaluation on simulated datasets Syst Biol 62(5)707ndash724 doi101093sysbiosyt033
Fujita MK Leacheacute AD Burbrink FT McGuire JA MoritzC (2012) Coalescent-based species delimitation in anintegrative taxonomy Trends Ecol Evol 27(9)480ndash488 doi101016jtree201204012
Gatesy J OrsquoGrady P Baker RH (1999) Corroborationamong data sets in simultaneous analysis hiddensupport for phylogenetic relationships among higherlevel Artiodactyl taxa Cladistics 15(3)271ndash313doi101111j1096-00311999tb00268x
Gladieux P Ropars J Badouin H Branca A Aguileta Gde Vienne DM Rodriacuteguez de la Vega RC Branco SGiraud T (2014) Fungal evolutionary genomicsprovides insight into the mechanisms of adaptivedivergence in eukaryotes Mol Ecol 23(4)753ndash773doi101111mec12631
Goffinet B Miadlikowska J Goward T (2003) Phyloge-netic inferences based on nrDNA sequences supportfive morphospecies within the Peltigera didactylacomplex (Lichenized Ascomycota) The Bryologist106(3)349ndash364 doi10163901
Gowan SP (1986) Evolution of secondary natural productsin the genus Porpidia (Ascomycata Porpidiaceae) AmJ Bot 73606
Griffin PC Hoffmann AA (2014) Limited genetic diver-gence among Australian alpine Poa tussock grassescoupled with regional structuring points to ongoinggene flow and taxonomic challenges Ann Bot doi101093aobmcu017
Grube M Hawksworth DL (2007) Trouble with lichenthe re-evaluation and re-interpretation of thallus formand fruit body types in the molecular era Mycol Res111(9)1116ndash1132 doi101016jmycres200704008
Gueidan C Savi S Thues H Roux C Keller C Tibell LPrieto M Heimarsson S Breuss O Orange A FrobergL Wynns AA Navarro-Rosines P Krzewicka BPykaelae J Grube M Lutzoni F (2009) Genericclassification of the Verrucariaceae (Ascomycota)based on molecular and morphological evidencerecent progress and remaining challenges Taxon 58(1)184ndash208
Guillot G Mortier F Estoup A (2005) Geneland acomputer package for landscape genetics Mol EcolNotes 5(3)712ndash715 doi101111j1471-8286200501031x
Guillot G Renaud S Ledevin R Michaux J Claude J(2012) A unifying model for the analysis of pheno-typic genetic and geographic data Syst Biol 61(6)897ndash911 doi101093sysbiosys038
Guisan A Thuiller W (2005) Predicting species distribu-tion offering more than simple habitat models EcolLett 8(9)993ndash1009 doi101111j1461-0248200500792x
Hale ME (1990) A synopsis of the lichen genusXanthoparmelia (Vainio) Hale (AscomycotinaParmeliaceae) Smithsonian Institution Press Wash-ington DC
Hamilton CA Hendrixson BE Brewer MS Bond JE(2014) An evaluation of sampling effects on multipleDNA barcoding methods leads to an integrativeapproach for delimiting species a case study ofthe North American tarantula genus Aphonopelma(Araneae Mygalomorphae Theraphosidae) MolPhylogenet Evol 7179ndash93 doi101016jympev201311007
Hausdorf B (2011) Progress toward a general speciesconcept Evolution 65(4)923ndash931 doi101111j1558-5646201101231x
Hausdorf B Hennig C (2010) Species delimitation usingdominant and codominant multilocus markers SystBiol 59(5)491ndash503 doi101093sysbiosyq039
Hawksworth DL (1976) Lichen chemotaxonomy InBailey RH (ed) Lichenology progress and problemsAcademic London pp 139ndash184
Hebert PDN Cywinska A Ball SL deWaard JR (2003)Biological identifications through DNA barcodesProc R Soc Lond B Biol Sci 270(1512)313ndash321doi101098rspb20022218
Hebert PDN Stoeckle MY Zemlak TS Francis CM(2004) Identification of birds through DNA barcodesPLoS Biol 2(10)e312 doi101371journalpbio0020312
Heled J Drummond AJ (2010) Bayesian inference ofspecies trees from multilocus data Mol Biol Evol 27(3)570ndash580 doi101093molbevmsp274
Hey J (2006) On the failure of modern species conceptsTrends Ecol Evol 21(8)447ndash450 doi101016jtree200605011
Hibbett DS OhmanA Glotzer D NuhnMKirk P NilssonRH (2011) Progress in molecular and morphologicaltaxon discovery in Fungi and options for formalclassification on environmental sequences Fungal BiolRev 2538ndash47 doi101016jfbr201101001
Houmlgnabba F Wedin M (2003) Molecular phylogeny ofthe Sphaerophorus globosus species complex Cladis-tics 19(3)224ndash232 doi101111j1096-00312003tb00365x
Hudson RR Coyne JA (2002) Mathematical conse-quences of the genealogical species concept Evolu-tion 56(8)1557 doi101111j0014-38202002tb01467x
Huelsenbeck JP Andolfatto P Huelsenbeck ET (2011)Structurama Bayesian inference of population struc-ture Evol Bioinf Online 7(2011)55ndash59 doi104137EBOS6761
Jones G Oxelman B (2014) DISSECT an assignment-free Bayesian discovery method for species delimita-tion under the multispecies coalescent Bioinformaticsdoi101101003178
38 SD Leavitt et al
Kekkonen M Hebert PD (2014) DNA barcode-baseddelineation of putative species efficient start fortaxonomic workflows Mol Ecol Res doi1011111755-099812233
Kelly LJ Hollingsworth PM Coppins BJ Ellis CJHarrold P Tosh J Yahr R (2011) DNA barcoding oflichenized fungi demonstrates high identification suc-cess in a floristic context New Phytol 191(1)288ndash300doi101111j1469-8137201103677x
Kiss L (2012) Limits of nuclear ribosomal DNA internaltranscribed spacer (ITS) sequences as species barcodesfor fungi Proc Natl Acad Sci 109(27)E1811ndashE1811doi101073pnas1207143109
Kluge AG (1989) A concern for evidence and aphylogenetic hypothesis for relationships among Epi-crates (Boidae Serpentes) Syst Zool 38(1)7ndash25doi101093sysbio3817
Knowles LL Carstens BC (2007) Delimiting specieswithout monophyletic gene trees Syst Biol 56(6)887ndash895 doi10108010635150701701091
Kroken S Taylor JW (2001) A gene genealogical approachto recognize phylogenetic species boundaries in thelichenized fungus Letharia Mycologia 93(1)38ndash53
Kubatko LS Degnan JH (2007) Inconsistency of phylo-genetic estimates from concatenated data under coa-lescence Syst Biol 56(1)17ndash24 doi10108010635150601146041
Latch EK Dharmarajan G Glaubitz JC Rhodes OE Jr(2006) Relative performance of Bayesian clusteringsoftware for inferring population substructure andindividual assignments at low levels of populationdifferentiation Conserv Genet 7(2)295ndash302 doi101007s10592-005-9098-1
Lawrey JD (1986) Biological role of lichen substancesThe Bryologist 89(2)111ndash122
Le Gac M Hood ME Fournier E Giraud T (2007)Phylogenetic evidence of host-specific cryptic speciesin the anther smut fungus Evolution 61(1)15ndash26doi101111j1558-5646200700002x
Leacheacute AD (2009) Species tree discordance traces tophylogeographic clade boundaries in North Americanfence lizards (Sceloporus) Syst Biol 58(6)547ndash559doi101093sysbiosyp057
Leacheacute AD Fujita MK (2010) Bayesian species delim-itation in West African forest geckos (Hemidactylusfasciatus) Proc R Soc B Biol Sci 2773071ndash3077doi101098rspb20100662
Leacheacute AD Koo M Spencer C Papenfuss T Fisher RMcGuire J (2009) Quantifying ecological morpho-logical and genetic variation to delimit species in thecoast horned lizard species complex (Phrynosoma)Proc Natl Acad Sci USA 10612418ndash12423 doi101073pnas0906380106
Leacheacute AD Fujita MK Minin VN Bouckaert RR (2014)Species delimitation using genome-wide SNP dataSystematic Biology doi101093sysbiosyu018
Leavitt SD Fankhauser JD Leavitt DH Porter LDJohnson LA St Clair LL (2011a) Complex patterns ofspeciation in cosmopolitan ldquorock posyrdquo lichensmdashdiscovering and delimiting cryptic fungal species in
the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
Leavitt SD Esslinger TL Lumbsch HT (2012c) Neogene-dominated diversification in neotropical montanelichens dating divergence events in the lichen-form-ing fungal genus Oropogon (Parmeliaceae) Am J Bot99(11)1764ndash1777 doi103732ajb1200146
Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Lumbsch HT St Clair LL(2013c) DNA barcode identification of lichen-formingfungal species in the Rhizoplaca melanophthalmaspecies-complex (Lecanorales Lecanoraceae) includ-ing five new species MycoKeys 71ndash22 doi103897mycokeys74508
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Vondraacutek J Thorsten Lum-bsch H Clair LLS (2013d) Local representation ofglobal diversity in a cosmopolitan lichen-formingfungal species complex (Rhizoplaca Ascomycota)J Biogeogr 40(9)1792ndash1806 doi101111jbi12118
Leavitt SD Lumbsch HT Stenroos S St Clair LL(2013e) Pleistocene speciation in North American
2 The Dynamic Discipline of Species Delimitation hellip 39
lichenized fungi and the impact of alternative speciescircumscriptions and rates of molecular evolution ondivergence estimates PLoS ONE 8(12)e85240doi101371journalpone0085240
Leavitt SD Esslinger TL Hansen ES Divakar PK CrespoA Loomis BF Lumbsch HT (2014) DNA barcoding ofbrown Parmeliae (Parmeliaceae) species a molecularapproach for accurate specimen identification empha-sizing species in Greenland Organ Divers Evol 14(1)11ndash20 doi101007s13127-013-0147-1
Leliaert F Verbruggen H Vanormelingen P Steen FLopez-Bautista JM Zuccarello GC De Clerck O(2014) DNA-based species delimitation in algae Eur JPhycol 49(2) doi101080096702622014904524
Leuckert C (1985) Probleme der Flechten-Chemotaxon-omiemdashStoffkombinationen und ihre taxonomischeWertung Ber Deut Bot Ges 98401ndash408
Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
Lohtander K Kaumlllersjouml M Roland M Tehler A (2000)The family physciaceae in fennoscandia phylogenyinferred from its sequences Mycologia 92(4)728ndash735
Longton RE (1997) The role of bryophytes and lichens inpolar ecosystems In Woodin SJ Marquiss M (eds)Ecology of Arctic Environments Blackwell ScienceOxford pp 69ndash96 (Special publication No 13)
Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
Lumbsch HT (1994) Die Lecanora subfusca-Gruppe inAustralasien J Hattori Bot Lab 771ndash175
Lumbsch HT (1998a) Taxonomic use of metabolic data inlichen-forming fungi In Frisvad JC Bridge PDArora DK (eds) Chemical Fungal Taxonomy MarcelDekker New York pp 345ndash387
Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
Mayden RL (1997) A hierarchy of species concepts thedenouement in the saga of the species problem InClaridge MF Dawah HA Wilson MR (eds) Speciesthe units of biodiversity Chapman amp Hall Londonpp 381ndash424
Mayr E (1963) Animal species and evolution HarvardUniversity Press Cambridge
Mayr E (1970) Populations species and evolutionBelknap Press of Harvard University Press Cambridge
McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
McDonald T Miadlikowska J Lutzoni F (2003) Thelichen genus Sticta in the Great Smoky Mountains a
40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
McKay BD Mays HL Wu Y Li H Yao C-T Nishiumi IZou F (2013) An empirical comparison of character-based and coalescent-based approaches to speciesdelimitation in a young avian complex Mol Ecol 22(19)4943ndash4957 doi101111mec12446
Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
Mishler BD Brandon RN (1987) Individuality pluralismand the phylogenetic species concept Biol Philos 2397
Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
Nash TH Zavada M (1977) Population studies amongSonoran Desert species of Parmelia subg Xanthop-armelia (Parmeliaceae) Am J Bot 64(6)664ndash669
Nylander W (1866a) Circa novum in studio Lichenumcriterium chemicum Flora 49198ndash201
Nylander W (1866b) Hypochlorite of lime and hydrate ofpotash Two new criteria for the study of lichens(Tanslated and communicated by the Rev W ALeighton) Bot J Linn Soc 9358ndash365
OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
OrsquoNeill EM Schwartz R Bullock CT Williams JSShaffer HB Aguilar-Miguel X Parra-Olea G Weis-rock DW (2013) Parallel tagged amplicon sequencingreveals major lineages and phylogenetic structure inthe North American tiger salamander (Ambystomatigrinum) species complex Mol Ecol 22(1)111ndash129doi101111mec12049
Orock EA Leavitt SD Fonge BA St Clair LL LumbschHT (2012) DNA-based identification of lichen-form-ing fungi Can publicly available sequence databasesaid in lichen diversity inventories of Mount Cameroon(West Africa) The Lichenologist 44(6)833ndash839doi101017S0024282912000424
Otaacutelora MAG Martiacutenez I Aragoacuten G Molina MC (2010)Phylogeography and divergence date estimates of alichen species complex with a disjunct distributionpattern Am J Bot 97(2)216ndash223 doi103732ajb0900064
Padial J Castroviejo-Fisher S Kohler J Vila C ChaparroJ De la Riva I (2009) Deciphering the products ofevolution at the species level the need for anintegrative taxonomy Zoolog Scr 38(4)431ndash447doi101111j1463-6409200800381x
Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
Papong K Luumlcking R Thammathaworn A BoonpragobK (2009) Four new taxa of Chroodiscus (thelotremoidGraphidaceae) from Southeast Asia The Bryologist112(1)152ndash163 doi1016390007-2745-1121152
Park S-Y Choi J Kim JA Jeong M-H Kim S Lee Y-HHur J-S (2013a) Draft genome sequence of Cladoniamacilenta KoLRI003786 a lichen-forming fungusproducing biruloquinone Genome Announcements 1(5)e00695-13 doi101128genomeA00695-13
Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
Peacuterez-Ortega S Fernaacutendez-Mendoza F Raggio J VivasM Ascaso C Sancho LG Printzen C de los Riacuteos A(2012) Extreme phenotypic variation in Cetrariaaculeata (lichenized Ascomycota) adaptation or inci-dental modification Ann Bot 109(6)1133ndash1148doi101093aobmcs042
Pino-Bodas R Burgaz A Martiacuten M Lumbsch HT (2011)Phenotypical plasticity and homoplasy complicatespecies delimitation in the Cladonia gracilis group(Cladoniaceae Ascomycota) Organ Divers Evol 11(5)343ndash355 doi101007s13127-011-0062-2
Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
Pino-Bodas R Martiacuten M Burgaz A (2012b) Cladoniasubturgida and C iberica (Cladoniaceae) form asingle morphologically and chemically polymorphicspecies Mycol Prog 11(1)269ndash278 doi101007s11557-011-0746-1
Pino-Bodas R Ahti T Stenroos S Martiacuten MP BurgazAR (2013a) Multilocus approach to species recogni-tion in the Cladonia humilis complex (CladoniaceaeAscomycota) Am J Bot 100(4)664ndash678 doi103732ajb1200162
Pino-Bodas R Martiacuten AP Burgaz AR Lumbsch HT(2013b) Species delimitation in Cladonia (Ascomy-cota) a challenge to the DNA barcoding philosophyMol Ecol Resour 13(6)1058ndash1068 doi1011111755-099812086
Pons J Barraclough TG Gomez-Zurita J Cardoso ADuran DP Hazell S Kamoun S Sumlin WD VoglerAP (2006) Sequence-based species delimitation for theDNA taxonomy of undescribed insects Syst Biol 55(4)595ndash609 doi10108010635150600852011
Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
Pringle A Baker DM Platt JL Wares JP Latgeacute JPTaylor JW (2005) Cryptic speciation in the cosmo-politan and clonal human pathogenic fungus Asper-gillus fumigatus Evolution 59(9)1886ndash1899 doi10155404-2411
Printzen C (2009) Lichen systematics the role ofmorphological and molecular data to reconstructphylogenetic relationships Progress in botany vol71 Springer Berlin pp 233ndash275
Pritchard JK Stephens M Donnelly P (2000) Inference ofpopulation structure using multilocus genotype dataGenetics 155(2)945ndash959
Puillandre N Lambert A Brouillet S Achaz G (2012)ABGD automatic barcode gap discovery for primaryspecies delimitation Mol Ecol 21(8)1864ndash1877doi101111j1365-294X201105239x
Rannala B Yang Z (2003) Bayes estimation of speciesdivergence times and ancestral population sizes usingDNA sequences from multiple loci Genetics1641645ndash1656
Raxworthy CJ Ingram CM Rabibisoa N Pearson RG(2007) Applications of ecological niche modelingfor species delimitation a review and empiricalevaluation using day geckos (Phelsuma) fromMadagascar Syst Biol 56(6)907ndash923 doi10108010635150701775111
Ray J (1686) Historia planarum vol 1 Clark LondonReese Naeligsborg R Ekman S Tibell L (2007) Molecular
phylogeny of the genus Lecania (Ramalinaceaelichenized Ascomycota) Mycol Res 111(5)581ndash591doi101016jmycres200703001
Reid N Carstens B (2012) Phylogenetic estimation errorcan decrease the accuracy of species delimitation aBayesian implementation of the general mixed Yule-coalescent model BMC Evol Biol 12(1)196 doi1011861471-2148-12-196
Rissler L Apodaca J (2007) Adding more ecology intospecies delimitation ecological niche models andphylogeography help define cryptic species in theBlack Salamander (Aneides flavipunctatus) Syst Biol56924ndash942 doi10108010635150701703063
Rivas Plata E Luumlcking R (2013) High diversity ofGraphidaceae (lichenized Ascomycota Ostropales) inAmazonian Peruacute Fungal Divers 58(1)13ndash32 doi101007s13225-012-0172-y
Rivas Plata E Lumbsch HT (2011) Parallel evolution andphenotypic divergence in lichenized fungi a casestudy in the lichen-forming fungal family Graphida-ceae (Ascomycota Lecanoromycetes Ostropales)Mol Phylogenet Evol 61(1)45ndash63 doi101016jympev201104025
Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
Ross KG Gotzek D Ascunce MS Shoemaker DD (2010)Species delimitation a case study in a problematic anttaxon Syst Biol 59(2)162ndash184 doi101093sysbiosyp089
Rubin BER Ree RH Moreau CS (2012) Inferringphylogenies from RAD sequence data PLoS ONE 7(4)e33394 doi101371journalpone0033394
Rubinoff D (2006) Utility of mitochondrial DNA barcodesin species conservation Conserv Biol 20(4)1026ndash1033 doi101111j1523-1739200600372x
Ruiz-Sanchez E Sosa V (2010) Delimiting speciesboundaries within the Neotropical bamboo Otatea(Poaceae Bambusoideae) using molecular morpho-logical and ecological data Mol Phylogenet Evol 54(2)344ndash356 doi101016jympev200910035
Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
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Salicini I Ibaacutentildeez C Juste J (2011) Multilocus phylogenyand species delimitation within the Nattererrsquos batspecies complex in the Western Palearctic MolPhylogenet Evol 61(3)888ndash898 doi101016jympev201108010
Satler JD Carstens BC Hedin M (2013) Multilocusspecies delimitation in a complex of morphologicallyconserved trapdoor spiders (Mygalomorphae Antro-diaetidae Aliatypus) Syst Biol 62(6)805ndash823 doi101093sysbiosyt041
Schlick-Steiner BC Steiner FM Seifert B Stauffer CChristian E Crozier RH (2010) Integrative taxonomya multisource approach to exploring biodiversityAnnu Rev Entomol 55(1)421ndash438 doi101146annurev-ento-112408-085432
Schoch CL Seifert KA Huhndorf S Robert V SpougeJL Levesque CA Chen W Fungal Barcoding Con-sortium (2012) Nuclear ribosomal internal transcribedspacer (ITS) region as a universal DNA barcodemarker for Fungi Proc Natl Acad Sci 109(16)6241ndash6246 doi101073pnas1117018109
Seacuterusiaux E Villarreal AJC Wheeler T Goffinet B(2011) Recent origin active speciation and dispersalfor the lichen genus Nephroma (Peltigerales) inMacaronesia J Biogeogr 38(6)1138ndash1151 doi101111j1365-2699201002469x
Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
Simpson GG (1951) The species concept Evolution5285ndash298
Sites JW Marshall JC (2003) Delimiting species a renais-sance issue in systematic biology Trends Ecol Evol18462ndash470 doi101016S0169-5347(03)00184-8
Sites JW Marshall JC (2004) Operational criteria fordelimiting species Annu Rev Ecol Evol Syst 35(1)199ndash227 doi101146annurevecolsys35112202130128
Šlapeta J Loacutepez-Garciacutea P Moreira D (2006) Globaldispersal and ancient cryptic species in the smallestmarine eukaryotes Mol Biol Evol 23(1)23ndash29doi101093molbevmsj001
Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
Talavera G Dincă V Vila R (2013) Factors affectingspecies delimitations with the GMYC model insightsfrom a butterfly survey Methods Ecol Evol 4(12)1101ndash1110 doi1011112041-210X12107
Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
Tewksbury J Anderson JGT Bakker JD Billo TJDunwiddie PW Groom MJ Hampton SE HermanSG Levey DJ Machnicki NJ del Rio CM PowerME Rowell K Salomon AK Stacey L TrombulakSC Wheeler TA (2014) Natural historyrsquos place inscience and society BioScience doi101093bioscibiu032
Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
Werth S Cornejo C Scheidegger C (2013) Characteriza-tion of microsatellite loci in the lichen fungus Lobariapulmonaria (Lobariaceae) Appl Plant Sci 1(2)1200290 doi103732apps1200290
Westberg M Arup U Kaumlrnefelt I (2007) Phylogeneticstudies in the Candelariaceae (lichenized Ascomy-cota) based on nuclear ITS DNA sequence dataMycol Res 111(11)1277ndash1284 doi101017S0953756204002102
Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
2 The Dynamic Discipline of Species Delimitation hellip 43
(Lepidoptera Lycaenidae) Front Zoo 4(1)8 doi1011861742-9994-4-8
Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
study (Altermann et al 2014 Kroken and Taylor2001) Altermann et al (2014) show that popu-lation assignments were largely consistent acrossa range of scenarios including extensiveamounts of missing data the exclusion of SNPsfrom variable markers and inferences based onSNPs from as few as three gene regions Incontrast to STRUCTURE and STRUCTURA-MA the program BAPS (Corander et al 2008)can explicitly infer genetic structure using hap-loid sequence data or other linked geneticmarkers in the ldquoclustering with linked locirdquomodel Another advantage of BAPS is that theprogram is much more computationally efficientthan STRUCTURE or STRUCTURAMA Sim-ulation studies indicate that both BAPS andSTRUCTURE perform well at low levels ofpopulation differentiation and when clusters arenot well differentiated (Latch et al 2006)
In practice inferring the most appropriatelevel of population structure using Bayesianclustering algorithms remains challenging (Latchet al 2006 Evanno et al 2005) BAPS andSTRUCTURAMA can infer both individualassignments and the most likely number ofgenetic clusters (Corander et al 2008 Huelsen-beck et al 2011) and the ad hoc ΔK statisticproposed by Evanno et al (2005) provides anobjective approach for identifying the uppermosthierarchical level of structure However carefulconsideration of other levels of populationstructure may ultimately reveal more biologicallymeaning results and researchers should examineindividual assignments across a range of geneticgroupings Some markers generated fromindependent genomic regions such as SNPs andfast-evolving microsatellites can be used to dis-tinguish fine-scale population structuring on thebasis of allele frequencies and species delimita-tion analyses based on these markers may implythe risk of severe taxonomic oversplitting Inthese cases validation approaches such asBPampP and SpeDeSTEM and corroborationamong different species delimitation approachescan provide perspective between intraspecificpopulation structure and species-level clusters
Another limitation of clustering approaches isthat they do not assess or take into account
evolutionarily divergences and relationshipsamong population clusters Coalescent-basedspecies tree methods (discussed below) provide ameans to assess the diversification histories ofpopulations inferred from clustering analysesTherefore a potential working protocol for aninformed species delimitation study that takesinto account population structure could consist offirst applying a genetic clustering analysis under apopulation genetics criterion (eg BAPSSTRUCTURE STRUCTURAMA) to identifygenetically distinct population clusters that can beconsidered ldquocandidate speciesrdquo From these can-didate species a species tree can be inferred forfocal group using coalescent-based species treereconstruction methods (eg BEAST) Subse-quently a coalescent-based validation methodcan be applied to assess whether the distinctpopulation clusters represent independent evolu-tionary lineages (eg BPampP) This multistepapproach would provide a consistent and standardcriterion for distinguishing between population-and species-level lineages (Camargo and Sites2013) and has been applied to a number of casesincluding lichen-forming fungi (Leavitt et al2011a b c 2013e Leacheacute and Fujita 2010)
High-throughput sequencing methods providethe means to effectively sample hundreds tothousands of loci from across a species genome fora large number of species and continue to revolu-tionize studies that can be performed even in non-model organisms Targeted high-throughputsequencing approaches such as anchored phy-logenomics transcriptome sequencing reduced-representation genomic library sequencing(restriction-site-associated DNA sequencingRAD-Seq and genotype-by-sequencing GBS)and high-throughput amplicon sequencing pro-vide important insight into species boundaries fora number groups although none of these approa-ches has yet been applied to studies of lichenizedfungi
For example restriction-site-associated(RAD-Seq) DNA can simultaneously detect andgenotype thousands of genome-wide SNPs byfocusing the sequencing effort on a reducedrepresentation of the entire genome (Baird et al2008) and has been successfully applied to
2 The Dynamic Discipline of Species Delimitation hellip 29
intraspecies (Lewis et al 2007 Miller et al 2007Emerson et al 2010) and interspecies studies(Rubin et al 2012 Eaton and Ree 2013 Wagneret al 2013) This approach has provided strikinginsight into the recent adaption radiation of LakeVictoria cichlids (Wagner et al 2013) Analternative approach targeting the sequencing ofspecific loci using next-generation sequencingplatforms provides an efficient means of gener-ating data for loci of known genomic locationorthology size and expected level of variation(OrsquoNeill et al 2013) Markers can be targetedusing well-established PCR techniques or newerhybridization techniques and subsequentlypooled for high-throughput sequencing usingparallel-tagged sequencing of multiple individualsamples within a single sequencing run (OrsquoNeillet al 2013) A recent study of North Americantiger salamanders (Ambystoma tigrinum)demonstrated the potential for amplicon-basedparallel-tagged sequencing to rapidly generatelarge-scale genomic data for species delimitationand species tree research (OrsquoNeill et al 2013)
Although these methods provide an enormousamount of data and insight into diversification atan unprecedented scale computational limita-tions restrict the applicable analytical methodsfor large datasets although computational andanalytical advances are happening rapidly Mostrecent coalescent-based species delimitation andspecies tree models using gene trees have beenlimited to handle tens of loci for multiple indi-viduals and combining hundreds or thousands ofgene trees into a single species delimitationframework presents considerable computationalchallenges (Camargo and Sites 2013) Therecently developed method Bayes factor delim-itation (with genomic data BFD) combines adynamic programming algorithm for estimatingspecies trees that bypasses the computationallyintensive MCMC integration over gene trees toprovide a rigorous technique for species delimi-tation studies using genome-wide SNP data(Leacheacute et al 2014) Competing species delimi-tation models are compared using Bayes factorsand it appears that this approach is robust tosample sizes (ie few loci and limited samples
per species) and misspecification of the prior forpopulation size (θ) (Leacheacute et al 2014)
Fungi are an ideal model for assessing diver-gence in eukaryotes due to their simple mor-phologies small genomes broad ecological rolesand diverse lifestyles (Gladieux et al 2014)However the use of genomic data from high-throughput sequencing methods have not yet beenincluded in species delimitation studies of lichen-forming fungi This is due in part to challenges indealing with metagenomic data containing geno-mic information from a plethora of symbiontsassociated with a lichen thallus scant genomicresources and discipline-specific inertia A num-ber of lichen-forming fungal genomes are cur-rently available including Endocarponpusillum(Wang et al 2014) Cladonia spp (Park et al2013a 2014) Caloplaca flavorubescens (Parket al 2013b) and Cladonia greyi and Xanthoriaparientina through the Fungal Genomics Programat Joint Genome Institute of the United StatesDepartment of Energy (httpwwwjgidoegov)The foreseeable ongoing expansion of genomicresources for lichen-forming fungi will be centralto developing approaches for delimiting speciesusing high-throughput sequencing Specificallygenomic resources will be crucial in identifyingnovel markers (variable genes regions SNPsmicrosatellites etc) identifying conserved fungalmarkers for targeted high-throughput sequencingapproaches and references for entire genomecomparisons (Devkota et al 2014 Werth et al2013)
23 Can We Make SpeciesDelimitation in Lichen-FormingFungi Truly Integrative
The widespread availability of genetic data hascreated a biased viewpoint that only genetic datashould be used for statistical species delimitationHowever an ongoing appeal to researchers toassess species boundaries from multiple andcomplementary perspectives (phylogeneticspopulation genetics comparative morphologydevelopment ecology etc) has resulted in an
30 SD Leavitt et al
integrative taxonomic framework bringing theseconceptual and methodological perspectivestogether (Hamilton et al 2014 Fujita et al 2012Salicini et al 2011 Yeates et al 2011 Padialet al 2010 Dayrat 2005 Will et al 2005 Wiensand Penkrot 2002) In practice any study linkingdifferent kinds of data to support hypotheses ofspecies boundaries including mapping morpho-logical characters onto a molecular phylogenycan be considered integrative Integrative meth-ods for species delimitation fall across a broadspectrum ranging from verbal and qualitativeassessments of data classes to quantitative meth-ods that allow different data types to contribute tostatistical species delimitation (Yeates et al2011) In most taxonomic studies utilizing bothmolecular and morphological data expert opinionis eventually used to some degree to evaluate thefinal status of a candidate species (eg Bond andStockman 2008) In this sense many studiesusing evidence for independent data sources fordelimiting species boundaries use an iterativeapproach (Yeates et al 2011) where speciesboundaries can be tested within a hypothetico-deductive framework with diverse datasets
From a practical perspective we advocate aprocess of iterative taxonomy (sensu Yeates et al2011) to circumscribe and refine species limitsusing multiple lines of evidence This iterativeprocess involves comparisons of morphologicaldata with a phylogenetic hypothesis (ie single-locus gene tree or concatenated multilocusphylogeny) to identify the least inclusive mono-phyletic clade in the topology characterized by atleast one unambiguously diagnostic morphologi-cal character (Miralles and Vences 2013) Thisphylogenetic tree-informed approach to assessingspecies boundaries represents a common practicein studies of lichen-forming fungi Previouslyunrecognized species-level clades with corre-sponding subtle or overlooked phenotypiccharacters have been commonly observed in bothcrustose lichens [eg Acarospraceae (Wedin et al2009) Graphidaceae (Rivas Plata and Lumbsch2011 Papong et al 2009) Lecanoraceae (Leavittet al 2011a) Lecideaceae (Ruprecht et al 2010)Mycoblastaceae (Spribille et al 2011) andTeloschistaceae (Vondraacutek et al 2009 Muggia
et al 2008)] and foliose and fruticose lichens [egLobariaceae (Moncada et al 2014 McDonaldet al 2003) Parmeliaceae (Leavitt et al 2013aWirtz et al 2012 Divakar et al 2005 Molinaet al 2004 Kroken and Taylor 2001) Peltigera-ceae (OrsquoBrien et al 2009 Goffinet et al 2003)Physciaceae (Elix et al 2009 Divakar et al2007) and Sphaerophoraceae (Houmlgnabba andWedin 2003)] As a specific example Luumlckinget al (2008) used a combination of medullarychemistry and underside pigmentation in speci-mens from the Heterodermia obscurata group inCosta Rica to corroborate monophyletic clades inan ITS gene tree as species-level lineages
While this iterative approach to speciesdelimitation and taxonomy has proven valuablefor understanding species boundaries anddescribing new taxa in some groups of lichenizedfungi a posteriori examination of morphologicaland chemical features has failed to reveal diag-nostic phenotypic characters in a number ofstudies (Muggia et al 2014 Leavitt et al 2011aOtaacutelora et al 2010 OrsquoBrien et al 2009) Fur-thermore a study of widespread species in thegenus Melanelixia (Parmeliaceae) indicated thatphenotypically cryptic lichen-forming fungalspecies-level lineages may be relatively ancientand diagnosable phenotypic differences may beabsent even millions of years after the initialdivergence (Leavitt et al 2012b) The latter studyhighlights the fact that species-level lineages maycommonly exist without any observable diag-nostic phenotypic characters calling into ques-tion the impetus for a universal application ofintegrative taxonomy
In keeping with the principle that as manylines of evidence as available should be com-bined to delimit species (Dayrat 2005) the for-malized integrative taxonomic approach (ITAXMiralles and Vences 2013) uses a mtDNA guidetree and observations from different types of datathat might provide conclusive evidence for theindependence of lineages and thus their identityas different species Miralles and Vences (2013)provide a non-exhaustive list of species delimi-tation criteria to be integrated in the ITAXapproach including (a) sympatric occurrencewithout admixture as revealed by consistent
2 The Dynamic Discipline of Species Delimitation hellip 31
differences in morphological or molecular char-acters at the same geographic location (b) strongdifferences in a behavioral morphological orgenetic character known to mediate prematingisolation (c) unviability or infertility of hybrids(d) lack of gene flow across a geographicalhybrid zone (e) congruent diagnostic differencesbetween sister lineages in various unlinkedmorphological character (f) absence of haplo-type sharing in several unlinked nuclear loci and(g) a combination of criteria endashf Speciesboundaries are based on seeking the least inclu-sive monophyletic group in the mtDNA treewhich fulfills at least one of the criteria listedabove Clearly the ITAX approach is sensitive tosample size and in order to reliably support thedistinctiveness of a given species it has beenrecommended that the sampling strategy includesat least five individuals per species (Miralles andVences 2013) A similar approach could beadopted for lichenized fungi using an ITS genetopology as guide tree rather than mtDNA
A total evidence approach including concat-enation has been a common approach for inte-grating information from different sourcesincluding independent genetic markers in phy-logenetic reconstructions (Kluge 1989 Wiens1998 de Queiroz et al 1995) Proponents forconcatenation of independent data in phyloge-netic analyses argue that when combining alldata the underlying signal of speciation mayemerge even if weakly contradictory signalis contained in the individual data partitions(Gatesy et al 1999) For example establishing apreliminary perspective of species boundariesfrom multilocus sequence data using concatena-tion may provide a reasonable starting point forscreening for cryptic species and species treeinference (Šlapeta et al 2006 Leavitt et al2011a Le Gac et al 2007 Leacheacute 2009)However concatenation and consensus methodsimply a risk of obtaining inflated support forincorrect relationships and information aboutvariance in gene coalescence is lost (Degnan andRosenberg 2006 2009 Kubatko and Degnan2007 Edwards 2009) In spite of the impetus forintegrating evidence from independent data
sources into empirical species delimitation stud-ies and taxonomy (Fujita et al 2012 Padial et al2009) most currently available species delimi-tation methods are unable to accommodate non-genetic data sources in a statistical framework
231 Selecting the Appropriate Data
In the face of increasing availability of geneticdata and associated bioinformatical approachesfor delimiting species researchers should care-fully consider what information is being sacri-ficed by the failure to consider non-genetic datain species delimitation studies and whetheraccuracy could be improved by the addition ofmultiple data types Morphological data havehistorically served as a proxy to identify repro-ductively isolated groups (ie ldquospeciesrdquo) (Ray1686 Fujita et al 2012) Current methods fordelimiting species using non-genetic data (egchemistry morphology and ecology) remainwoefully understudied For example morphol-ogy-based species circumscriptions are generallybased on one or more qualitative (or quantitative)morphological characters that do not appear tooverlap with other species However ascertain-ing that a given trait is truly fixed within apopulation with statistical confidence requiresunrealistic sample sizes even when allowing forsome level of polymorphism in the diagnosticcharacter (Wiens and Servedio 2000) Now var-ious combinations of datamdashfrom morphologygenetics geography and ecologymdashare acceptedas standard information for species delimitationstudies (Ruiz-Sanchez and Sosa 2010 Ross et al2010 Edwards and Knowles 2014)
Below we briefly discuss appropriate datasources for species delimitation studies of lichen-forming fungi However homoplastic characters(similar traits that are not derived from a com-mon ancestor) are common among many traitscommonly used to circumscribe fungal taxa thebiological significance of secondary metabolitevariation remains largely unknown (Lawrey1986) and ecological niches may be difficultto adequately characterize and model due to
32 SD Leavitt et al
microhabitat requirements and data resolutionTherefore we advocate a cautious approach toselecting appropriate and relevant data forassessing species boundaries
In spite of the potential challenges and limi-tations of using phenotypic data these traits haveprovided a plethora of valuable information forunderstanding species boundaries For examplein the Melanelixia fuliginosa group (Parmelia-ceae) a morphometric analysis using color isi-dia and marginal zone free of isidia ascharacters revealed a general pattern of differ-entiation between material formerly recognizedas subspecies fuliginosa and glabratula in spiteof the fact that considerable overlap betweengroups occurs in some characters individually(Arup and Berlin 2011) Furthermore the dis-tinction of the two groups was supported by aphylogenetic analysis of the ITS marker andecological differences with M fuliginosaoccurring predominantly on rock and M glab-ratula on bark (Arup and Berlin 2011) Thisstudy of the M fuliginosa group provides a fit-ting example of using multiple independentsuites of data including ecology morphologyand genetic information to establish a robusthypothesis of species boundaries
In lichen systematics phenotypic dataincluding thallus organization secondary metab-olites mode of reproduction ascoma-type andontogeny ascus and ascospore characters havehistorically played a prominent role (Printzen2009) Ascomatal characters have traditionallyheld a major role in higher-level classification(Printzen 2009) in contrast to species-levelclassification which also tends to include a widearray of vegetative and chemical charactersCommonly assessed morphological charactersinclude thallus form and size cortical features(eg maculae and pseudocyphellae) presenceformcolor of attachment structure (eg rhizines)and reproductive mode (ascomata vs vegetativediaspores) (Printzen 2009) Different morpho-logical types of vegetative diasporesmdashcorticatedisidia and ecorticated sorediamdashand their locationare commonly used to distinguish speciesAscomatal characters including morphologylocation (laminal vs marginal) position (sessile
vs immersed) presence of thalline margins colorof an apothecial disc and presence or color ofpruina are also commonly used in speciesdelimitations of lichen-forming fungi (Printzen2009) Other important characters may includethalline characters form color size and septationof ascospores size and form and structure of ascithe hamathecium type of epihymenium andhypothecium and the type of excipulum orperidium conidiomatal characters etc (Printzen2009)
Assessments of secondary metabolites hasplayed an important role in lichen taxonomybeginning with the introduction of simple spottests by Nylander (1866a b) The use of chem-istry in lichen taxonomy has been discussed indetail in numerous reviews (Lumbsch 1998a bRogers 1989 Brodo 1986 Egan 1986 Leuckert1985 Brodo 1978 Hawksworth 1976 Culber-son 1969 1970) and we refer readers to thesevaluable sources for a more comprehensive per-spective on lichen chemistry In short extrolites(secondary metabolites) belong to various clas-ses the most common and diverse include dep-sides depsidones chlorinated xanthones andanthraquinones (Lumbsch 2002 Culberson1969) The presence or absence of specificextrolites or their replacements by another sub-stance is widely used to distinguish speciesparticularly when correlated with differences ingeographic distributions However if morpho-logical or geographical differences betweenpopulations containing different extrolites are notapparent the taxonomic significance has beendisputed with some authors distinguishing themas species and others preferring to regard them aschemical races within a species In addition tosimply using the presence or absence of extro-lites Culberson and Culberson (1976) proposedto arrange lichen substances into chemosyn-dromes of closely related substances The pres-ence or absence of these chemosyndromes maypotentially be used as characters to delimit spe-cies regarding differences involving the samechemosyndromes as intraspecific variation anddistinct chemosyndromes as evidence for inter-specific populations (Lumbsch 1994 Gowan1986)
2 The Dynamic Discipline of Species Delimitation hellip 33
Due to the fact that species delimitationstudies often incorporate a substantial biogeo-graphical or ecological component ecologicalniche modeling plays an increasing role in phy-logenetic and taxonomic research Niche mod-eling can provide evidence for ecologicalisolation between populations based on eitherconserved or divergent ecological niches andtherefore can provide additional evidence sup-porting lineage independence between putativespecies The application of ecological nichemodeling has been applied to species delimita-tion studies in a number of cases (Ruiz-Sanchezand Sosa 2010 Leacheacute et al 2009 Raxworthyet al 2007 Rissler and Apodaca 2007) althoughit has not been explicitly applied to assess speciesboundaries in lichen-forming fungi By mappingthe spatial distribution of environmental suit-ability of climatic variables for candidate speciesthe application of ecological niche modeling canbe particularly important in cases where specieshave allopatric distributions (Raxworthy et al2007)
Ecological niche modeling utilizes knownassociations between a speciesrsquo occurrencelocalities and environmental variables to defineabiotic conditions within which populations canbe maintained (Guisan and Thuiller 2005) Themethodological approach for modeling is basedon four general properties (i) The current knownspeciesrsquo localities is the dependent variable (ii)the distribution is modeled as a map composed ofgrid cells at a specified resolution (iii) a range ofenvironmental variables (eg temperature pre-cipitation and solar exposure) are collected todescribe the characteristics of each cell and (iv)classifying the degree to which each cell is eithersuitable or unsuitable for each species under arange of models (Guisan and Thuiller 2005)
Ecological data also have the potential to playan important role in understanding speciesboundaries in lichen-forming fungi For exampleNash and Zavada (1977) demonstrated that Xant-hoparmelia populations with distinct chemistriesoccurring in the northern portion of the SonoranDesert exhibit habitat selection among differentrock substrates within a region with relativelyuniform climate and topography In another case
the parmelioid species Parmelia mayi is mor-phologically indistinguishable from P saxatilisbut can be separated by bioclimatic features andgenetic and chemical characters (Molina et al2011) McCune and Printzen(2011) assess distri-butions and climatic niches of species in theLecanora varia group inwesternUSAandprovidea model that uses continental influence and annualtemperature as themajor factors predicting speciesdistributions The distribution of Usnea hirta alichen commonly used in air quality biomonitor-ing research was modeled for a section of theWhite River National Forest in central Coloradobased on the presence of U hirta at 72 biomoni-toring reference sites distributed in the inter-mountain western United States (Shrestha et al2012) The best model for predicting U hirtadistribution included four variablesmdashsolar radia-tion average monthly precipitation and averagemonthly minimum and maximum temperatures(Shrestha et al 2012) These studies supportthe potential use of ecological niche modelingmethods in species delimitation studies of lichen-forming fungi
24 Conclusions What AboutTaxonomy
In most cases species circumscription and tax-onomy requires some degree of qualitativejudgment and individual interpretation Integrat-ing multiple types of data into an empiricalframework for delimiting species boundarieswhere species boundaries can be tested within ahypothetico-deductive framework with diversedatasets can provide robust hypotheses of speciesboundaries and taxonomic stability (Yeates et al2011) However it has long been known toevolutionary biologists that distinct species donot need to have diagnosable morphologicaldifferences (Mayr 1963) and increasing avail-ability of genetic data has allowed researchers toidentify species and to rigorously test speciesboundaries with a level of precision that wasunimaginable a decade ago While analyticaladvances in statistical species delimitation havebeen largely based on genetic data the utility of
34 SD Leavitt et al
these approaches to formal taxonomy remainselusive Due to these challenges an eclecticapproach to delimiting species and cautionagainst the reliance on any single dataset ormethod is required when delimiting species
While results from many recent studies maycontradict traditional species boundaries acrossmany groups of lichen-forming fungi we areoptimistic that this research represents substantialprogress toward a more accurate perspective onspecies boundaries and diversity in fungi As aresult of ongoing research of species boundariesin lichen-forming fungi the taxonomic value ofmany phenotypes is now better understood ourunderstanding of ecological evolutionary andbiogeographic patterns has improved and we canbegin to better understand patterns of symbiontinteractions in lichens Integrating new data(including novel morphological characters andgenetic data) will be essential to accurately rep-resent species-level diversity across all groups oflichenized fungi Hopefully an improved per-spective on lichen diversity also increases ourappreciation of these incredible symbiotic sys-tems While we are strong advocates for theapplication of independent data types in devel-oping an integrative taxonomy there is anincreasing need to formally recognize the exis-tence of phenotypically cryptic species-levellineages in lichen-forming fungi (see Hibbettet al 2011) In some cases a molecular taxon-omy may provide the most practical approach toconsistent treatment of mycobiont species withinlichen groups where diagnostic morphologicalcharacters are unidentifiable or practically notfeasible (Leavitt et al 2013c d)
These are exciting times for taxonomists andphylogeneticists A closer look at lichen taxon-omy with the inclusion of new data will help usto better understand the diversity of these fasci-nating organisms accurately interpret distribu-tion patterns and play a more important role inmeaningful conservation practices Howeversome level of uncertainty will accompany pro-gress In many taxonomic groups our traditionalapproach for species identification will likelyneed to be substantially modified The search forcorroborating morphological support for cryptic
species identified using molecular data willrequire meticulous and creative approaches toassess phenotypic variation in potentially unor-thodox ways We are hopeful that lichenologistswho traditionally have been eager to include newmethods such as chromatography in their rou-tine identifications will be amenable to includemolecular techniques to their routine examina-tion of specimens for identification and classifi-cation Although this may prove difficult toachieve by single individuals especially citizenscientists that traditionally play an important rolein lichen taxonomy the increasing numberof collaborative projects in the discipline (egLumbsch et al 2011 Crespo et al 2010Gueidan et al 2009) make us optimistic thatbroad-scale collaborative approaches will facili-tate the inclusion of molecular data in lichenresearch at all levels
Acknowledgments We are indebted to various col-leagues for valuable thought-provoking discussionnotably Matthew Nelsen (University of Chicago) AnaCrespo (Universidad Complutense de Madrid) PradeepDivakar (Universidad Complutense de Madrid) BeckettSterner (The Field Museum) and Joyce Havstad (TheField Museum) We also thank anonymous reviewers whoprovided valuable comments that improved this chapterSupport by the US National Science Foundation isgratefully acknowledged (ldquoHidden diversity in parmelioidlichensrdquo DEB-0949147)
References
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Altermann S Leavitt SD Goward T Nelsen MPLumbsch HT (2014) How do you solve a problemlike Letharia A new look at cryptic species in lichen-forming fungi using Bayesian clustering and SNPSsfrom multilocus sequence data PLoS ONE 9(5)e97556 doi101371journalpone0097556
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2 The Dynamic Discipline of Species Delimitation hellip 35
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Corander J Marttinen P (2006) Bayesian identification ofadmixture events using multi-locus molecular mark-ers Mol Ecol 15(10)2833ndash2843 doi101111j1365-294X200602994x
Corander J Waldmann P Marttinen P Sillanpaa M(2004) BAPS 2 enhanced possibilities for the analysisof genetic population structure Bioinformatics 20(15)2363ndash2369 doi101093bioinformaticsbth250
Corander J Marttinen P Mantyniemi S (2006) Bayesianidentification of stock mixtures from molecular markerdata Fish Bull 104550ndash558
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Cracraft J (1983) Species concepts and speciation anal-ysis Curr Ornithol 1159ndash187
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Culberson WL (1969) The use of chemistry in thesystematics of the lichens Taxon 18498ndash505
Culberson WL (1970) Chemosystematics and ecology oflichen-forming fungi Annu Rev Ecol Syst 1153ndash170
Culberson CF Culberson WL (1976) Chemosyndromicvariation in lichens Syst Bot 1325ndash339
Darwin C (1859) On the origin of species by means ofnatural selection or the preservation of favoured racesin the struggle for life J Murray London
Dayrat B (2005) Towards integrative taxonomy Biol JLinn Soc 85(3)407ndash415 doi101111j1095-8312200500503x
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de Queiroz K (1998) The general lineage concept ofspecies species criteria and the process of speciationa conceptual unification and terminological recom-mendations In Howard DJ Berlocher SH (eds)Endless forms species and speciation Oxford Uni-versity Press Oxford pp 57ndash75
de Queiroz K (1999) The general lineage concept ofspecies and the defining properties of the speciescategory In Wilson RA (ed) Species new interdis-ciplinary essays MIT Press Cambridge pp 49ndash89
de Queiroz K (2007) Species concepts and speciesdelimitation Syst Biol 56(6)879ndash886 doi10108010635150701701083
de Queiroz A Donoghue MJ Kim J (1995) Separateversus combined analysis of phylogenetic evidenceAnnu Rev Ecol Syst 26(1)657ndash681 doi101146annureves26110195003301
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Degnan JH Rosenberg NA (2009) Gene tree discordancephylogenetic inference and the multispecies coales-cent Trends Ecol Evol 24(6)332ndash340 doihttpdxdoiorg101016jtree200901009
Del-Prado R Cubas P Lumbsch HT Divakar PK BlancoO de Paz GA Molina MC Crespo A (2010) Geneticdistances within and among species in monophyleticlineages of Parmeliaceae (Ascomycota) as a tool fortaxon delimitation Mol Phylogenet Evol 56(1)125ndash133 doi101016jympev201004014
Del-Prado R Divakar PK Crespo A (2011) Using geneticdistances in addition to ITS molecular phylogeny toidentify potential species in the Parmotrema reticul-atum complex a case study The Lichenologist 43(06)569ndash583 doi101017S0024282911000582
Del-Prado R Blanco O Lumbsch HT Divakar PK ElixJA Molina MC Crespo A (2013) Molecular phylog-eny and historical biogeography of the lichen-formingfungal genus Flavoparmelia (Ascomycota Parmelia-ceae) Taxon 62(5)928ndash939 doi101270562522
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Dettman JR Jacobson DJ Turner E Pringle A TaylorJW (2003b) Reproductive isolation and phylogeneticdivergence in Neurospora comparing methods ofspecies recognition in a model eukaryote Evolution57(12)2721 doi10155403-074
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Divakar PK Molina MC Lumbsch HT Crespo A (2005)Parmelia barrenoae a new lichen species related toParmelia sulcata (Parmeliaceae) based on molecularand morphological data The Lichenologist 37(01)37ndash46 doi101017S0024282904014641
Divakar PK Amo De paz G del Prado R Esslinger TLCrespo A (2007) Upper cortex anatomy corroboratesphylogenetic hypothesis in species of Physconia(Ascomycota Lecanoromycetes) Mycol Res 111(11)1311ndash1320 doi101016jmycres200708009
Divakar PK Figueras G Hladun N Crespo A (2010)Molecular phylogenetic studies reveal an undescribedspecies within the North American concept of Mela-nelixia glabra (Parmeliaceae) Fungal Divers 42(1)47ndash55 doi101007s13225-010-0027-3
Donoghue MJ Gauthier A (2004) Implementing thephylocode Trends Ecol Evol 19(6)281ndash282 doi101016jtree200404004
Eaton DAR Ree RH (2013) Inferring phylogeny andintrogression using RADseq data an example fromflowering plants (Pedicularis Orobanchaceae) SystBiol 62(5)689ndash706 doi101093sysbiosyt032
Edwards SV (2009) Is a new and general theory ofmolecular systematics emerging Evolution 63(1)1ndash19 doi101111j1558-5646200800549x
Edwards DL Knowles LL (2014) Species detection andindividual assignment in species delimitation canintegrative data increase efficacy Proc R Soc B BiolSci 281(1777) doi101098rspb20132765
Egan RS (1986) Correlations and non-correlations ofchemical variation patterns with lichen morphologyand geography The Bryologist 8999ndash110
Elix JA Corush J Lumbsch HT (2009) Triterpenechemosyndromes and subtle morphological characterscharacterise lineages in the Physcia aipolia group inAustralia (Ascomycota) Syst Biodivers 7(04)479ndash487 doi101017S1477200009990223
Emerson KJ Merz CR Catchen JM Hohenlohe PACresko WA Bradshaw WE Holzapfel CM (2010)Resolving postglacial phylogeography using high-throughput sequencing Proc Natl Acad Sci 107(37)16196ndash16200 doi101073pnas1006538107
Ence DD Carstens BC (2011) SpedeSTEM a rapid andaccurate method for species delimitation Mol EcolResour 11(3)473ndash480 doi101111j1755-0998201002947x
EvannoGRegnaut SGoudet J (2005)Detecting thenumberof clusters of individuals using the software STRUC-TURE a simulation study Mol Ecol 14(8)2611ndash2620doi101111j1365-294X200502553x
Falush D Stephens M Pritchard JK (2003) Inference ofpopulation structure using multilocus genotype datalinked loci and correlated allele frequencies Genetics164(4)1567ndash1587 doi101111j1471-8286200701758x
Fan HH Kubatko LS (2011) Estimating species treesusing approximate Bayesian computation Mol Phylo-genet Evol 59(2)354ndash363 doi101016jympev201102019
Fernaacutendez-Mendoza F Printzen C (2013) Pleistoceneexpansion of the bipolar lichen Cetraria aculeata intothe Southern hemisphere Mol Ecol 22(7)1961ndash1983doi101111mec12210
Fernaacutendez-Mendoza F Domaschke S Garciacutea MA JordanP Martin MP Printzen C (2011) Population structure
2 The Dynamic Discipline of Species Delimitation hellip 37
of mycobionts and photobionts of the widespreadlichen Cetraria aculeata Mol Ecol 20(6)1208ndash1232doi101111j1365-294X201004993x
Fraley C Raftery A (2007) Model-based methods ofclassification Using the mclust software in chemo-metrics J Stat Softw 18i06 [Available online at httpwwwdoajorgdoajfunc=abstractampid=218544]
Fujisawa T Barraclough TG (2013) Delimiting speciesusing single-locus data and the generalized mixed yulecoalescent (GMYC) approach a revised method andevaluation on simulated datasets Syst Biol 62(5)707ndash724 doi101093sysbiosyt033
Fujita MK Leacheacute AD Burbrink FT McGuire JA MoritzC (2012) Coalescent-based species delimitation in anintegrative taxonomy Trends Ecol Evol 27(9)480ndash488 doi101016jtree201204012
Gatesy J OrsquoGrady P Baker RH (1999) Corroborationamong data sets in simultaneous analysis hiddensupport for phylogenetic relationships among higherlevel Artiodactyl taxa Cladistics 15(3)271ndash313doi101111j1096-00311999tb00268x
Gladieux P Ropars J Badouin H Branca A Aguileta Gde Vienne DM Rodriacuteguez de la Vega RC Branco SGiraud T (2014) Fungal evolutionary genomicsprovides insight into the mechanisms of adaptivedivergence in eukaryotes Mol Ecol 23(4)753ndash773doi101111mec12631
Goffinet B Miadlikowska J Goward T (2003) Phyloge-netic inferences based on nrDNA sequences supportfive morphospecies within the Peltigera didactylacomplex (Lichenized Ascomycota) The Bryologist106(3)349ndash364 doi10163901
Gowan SP (1986) Evolution of secondary natural productsin the genus Porpidia (Ascomycata Porpidiaceae) AmJ Bot 73606
Griffin PC Hoffmann AA (2014) Limited genetic diver-gence among Australian alpine Poa tussock grassescoupled with regional structuring points to ongoinggene flow and taxonomic challenges Ann Bot doi101093aobmcu017
Grube M Hawksworth DL (2007) Trouble with lichenthe re-evaluation and re-interpretation of thallus formand fruit body types in the molecular era Mycol Res111(9)1116ndash1132 doi101016jmycres200704008
Gueidan C Savi S Thues H Roux C Keller C Tibell LPrieto M Heimarsson S Breuss O Orange A FrobergL Wynns AA Navarro-Rosines P Krzewicka BPykaelae J Grube M Lutzoni F (2009) Genericclassification of the Verrucariaceae (Ascomycota)based on molecular and morphological evidencerecent progress and remaining challenges Taxon 58(1)184ndash208
Guillot G Mortier F Estoup A (2005) Geneland acomputer package for landscape genetics Mol EcolNotes 5(3)712ndash715 doi101111j1471-8286200501031x
Guillot G Renaud S Ledevin R Michaux J Claude J(2012) A unifying model for the analysis of pheno-typic genetic and geographic data Syst Biol 61(6)897ndash911 doi101093sysbiosys038
Guisan A Thuiller W (2005) Predicting species distribu-tion offering more than simple habitat models EcolLett 8(9)993ndash1009 doi101111j1461-0248200500792x
Hale ME (1990) A synopsis of the lichen genusXanthoparmelia (Vainio) Hale (AscomycotinaParmeliaceae) Smithsonian Institution Press Wash-ington DC
Hamilton CA Hendrixson BE Brewer MS Bond JE(2014) An evaluation of sampling effects on multipleDNA barcoding methods leads to an integrativeapproach for delimiting species a case study ofthe North American tarantula genus Aphonopelma(Araneae Mygalomorphae Theraphosidae) MolPhylogenet Evol 7179ndash93 doi101016jympev201311007
Hausdorf B (2011) Progress toward a general speciesconcept Evolution 65(4)923ndash931 doi101111j1558-5646201101231x
Hausdorf B Hennig C (2010) Species delimitation usingdominant and codominant multilocus markers SystBiol 59(5)491ndash503 doi101093sysbiosyq039
Hawksworth DL (1976) Lichen chemotaxonomy InBailey RH (ed) Lichenology progress and problemsAcademic London pp 139ndash184
Hebert PDN Cywinska A Ball SL deWaard JR (2003)Biological identifications through DNA barcodesProc R Soc Lond B Biol Sci 270(1512)313ndash321doi101098rspb20022218
Hebert PDN Stoeckle MY Zemlak TS Francis CM(2004) Identification of birds through DNA barcodesPLoS Biol 2(10)e312 doi101371journalpbio0020312
Heled J Drummond AJ (2010) Bayesian inference ofspecies trees from multilocus data Mol Biol Evol 27(3)570ndash580 doi101093molbevmsp274
Hey J (2006) On the failure of modern species conceptsTrends Ecol Evol 21(8)447ndash450 doi101016jtree200605011
Hibbett DS OhmanA Glotzer D NuhnMKirk P NilssonRH (2011) Progress in molecular and morphologicaltaxon discovery in Fungi and options for formalclassification on environmental sequences Fungal BiolRev 2538ndash47 doi101016jfbr201101001
Houmlgnabba F Wedin M (2003) Molecular phylogeny ofthe Sphaerophorus globosus species complex Cladis-tics 19(3)224ndash232 doi101111j1096-00312003tb00365x
Hudson RR Coyne JA (2002) Mathematical conse-quences of the genealogical species concept Evolu-tion 56(8)1557 doi101111j0014-38202002tb01467x
Huelsenbeck JP Andolfatto P Huelsenbeck ET (2011)Structurama Bayesian inference of population struc-ture Evol Bioinf Online 7(2011)55ndash59 doi104137EBOS6761
Jones G Oxelman B (2014) DISSECT an assignment-free Bayesian discovery method for species delimita-tion under the multispecies coalescent Bioinformaticsdoi101101003178
38 SD Leavitt et al
Kekkonen M Hebert PD (2014) DNA barcode-baseddelineation of putative species efficient start fortaxonomic workflows Mol Ecol Res doi1011111755-099812233
Kelly LJ Hollingsworth PM Coppins BJ Ellis CJHarrold P Tosh J Yahr R (2011) DNA barcoding oflichenized fungi demonstrates high identification suc-cess in a floristic context New Phytol 191(1)288ndash300doi101111j1469-8137201103677x
Kiss L (2012) Limits of nuclear ribosomal DNA internaltranscribed spacer (ITS) sequences as species barcodesfor fungi Proc Natl Acad Sci 109(27)E1811ndashE1811doi101073pnas1207143109
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Knowles LL Carstens BC (2007) Delimiting specieswithout monophyletic gene trees Syst Biol 56(6)887ndash895 doi10108010635150701701091
Kroken S Taylor JW (2001) A gene genealogical approachto recognize phylogenetic species boundaries in thelichenized fungus Letharia Mycologia 93(1)38ndash53
Kubatko LS Degnan JH (2007) Inconsistency of phylo-genetic estimates from concatenated data under coa-lescence Syst Biol 56(1)17ndash24 doi10108010635150601146041
Latch EK Dharmarajan G Glaubitz JC Rhodes OE Jr(2006) Relative performance of Bayesian clusteringsoftware for inferring population substructure andindividual assignments at low levels of populationdifferentiation Conserv Genet 7(2)295ndash302 doi101007s10592-005-9098-1
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Le Gac M Hood ME Fournier E Giraud T (2007)Phylogenetic evidence of host-specific cryptic speciesin the anther smut fungus Evolution 61(1)15ndash26doi101111j1558-5646200700002x
Leacheacute AD (2009) Species tree discordance traces tophylogeographic clade boundaries in North Americanfence lizards (Sceloporus) Syst Biol 58(6)547ndash559doi101093sysbiosyp057
Leacheacute AD Fujita MK (2010) Bayesian species delim-itation in West African forest geckos (Hemidactylusfasciatus) Proc R Soc B Biol Sci 2773071ndash3077doi101098rspb20100662
Leacheacute AD Koo M Spencer C Papenfuss T Fisher RMcGuire J (2009) Quantifying ecological morpho-logical and genetic variation to delimit species in thecoast horned lizard species complex (Phrynosoma)Proc Natl Acad Sci USA 10612418ndash12423 doi101073pnas0906380106
Leacheacute AD Fujita MK Minin VN Bouckaert RR (2014)Species delimitation using genome-wide SNP dataSystematic Biology doi101093sysbiosyu018
Leavitt SD Fankhauser JD Leavitt DH Porter LDJohnson LA St Clair LL (2011a) Complex patterns ofspeciation in cosmopolitan ldquorock posyrdquo lichensmdashdiscovering and delimiting cryptic fungal species in
the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
Leavitt SD Esslinger TL Lumbsch HT (2012c) Neogene-dominated diversification in neotropical montanelichens dating divergence events in the lichen-form-ing fungal genus Oropogon (Parmeliaceae) Am J Bot99(11)1764ndash1777 doi103732ajb1200146
Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Lumbsch HT St Clair LL(2013c) DNA barcode identification of lichen-formingfungal species in the Rhizoplaca melanophthalmaspecies-complex (Lecanorales Lecanoraceae) includ-ing five new species MycoKeys 71ndash22 doi103897mycokeys74508
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Vondraacutek J Thorsten Lum-bsch H Clair LLS (2013d) Local representation ofglobal diversity in a cosmopolitan lichen-formingfungal species complex (Rhizoplaca Ascomycota)J Biogeogr 40(9)1792ndash1806 doi101111jbi12118
Leavitt SD Lumbsch HT Stenroos S St Clair LL(2013e) Pleistocene speciation in North American
2 The Dynamic Discipline of Species Delimitation hellip 39
lichenized fungi and the impact of alternative speciescircumscriptions and rates of molecular evolution ondivergence estimates PLoS ONE 8(12)e85240doi101371journalpone0085240
Leavitt SD Esslinger TL Hansen ES Divakar PK CrespoA Loomis BF Lumbsch HT (2014) DNA barcoding ofbrown Parmeliae (Parmeliaceae) species a molecularapproach for accurate specimen identification empha-sizing species in Greenland Organ Divers Evol 14(1)11ndash20 doi101007s13127-013-0147-1
Leliaert F Verbruggen H Vanormelingen P Steen FLopez-Bautista JM Zuccarello GC De Clerck O(2014) DNA-based species delimitation in algae Eur JPhycol 49(2) doi101080096702622014904524
Leuckert C (1985) Probleme der Flechten-Chemotaxon-omiemdashStoffkombinationen und ihre taxonomischeWertung Ber Deut Bot Ges 98401ndash408
Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
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Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
Lumbsch HT (1994) Die Lecanora subfusca-Gruppe inAustralasien J Hattori Bot Lab 771ndash175
Lumbsch HT (1998a) Taxonomic use of metabolic data inlichen-forming fungi In Frisvad JC Bridge PDArora DK (eds) Chemical Fungal Taxonomy MarcelDekker New York pp 345ndash387
Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
Mayden RL (1997) A hierarchy of species concepts thedenouement in the saga of the species problem InClaridge MF Dawah HA Wilson MR (eds) Speciesthe units of biodiversity Chapman amp Hall Londonpp 381ndash424
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McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
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40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
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Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
Mishler BD Brandon RN (1987) Individuality pluralismand the phylogenetic species concept Biol Philos 2397
Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
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Nylander W (1866a) Circa novum in studio Lichenumcriterium chemicum Flora 49198ndash201
Nylander W (1866b) Hypochlorite of lime and hydrate ofpotash Two new criteria for the study of lichens(Tanslated and communicated by the Rev W ALeighton) Bot J Linn Soc 9358ndash365
OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
OrsquoNeill EM Schwartz R Bullock CT Williams JSShaffer HB Aguilar-Miguel X Parra-Olea G Weis-rock DW (2013) Parallel tagged amplicon sequencingreveals major lineages and phylogenetic structure inthe North American tiger salamander (Ambystomatigrinum) species complex Mol Ecol 22(1)111ndash129doi101111mec12049
Orock EA Leavitt SD Fonge BA St Clair LL LumbschHT (2012) DNA-based identification of lichen-form-ing fungi Can publicly available sequence databasesaid in lichen diversity inventories of Mount Cameroon(West Africa) The Lichenologist 44(6)833ndash839doi101017S0024282912000424
Otaacutelora MAG Martiacutenez I Aragoacuten G Molina MC (2010)Phylogeography and divergence date estimates of alichen species complex with a disjunct distributionpattern Am J Bot 97(2)216ndash223 doi103732ajb0900064
Padial J Castroviejo-Fisher S Kohler J Vila C ChaparroJ De la Riva I (2009) Deciphering the products ofevolution at the species level the need for anintegrative taxonomy Zoolog Scr 38(4)431ndash447doi101111j1463-6409200800381x
Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
Papong K Luumlcking R Thammathaworn A BoonpragobK (2009) Four new taxa of Chroodiscus (thelotremoidGraphidaceae) from Southeast Asia The Bryologist112(1)152ndash163 doi1016390007-2745-1121152
Park S-Y Choi J Kim JA Jeong M-H Kim S Lee Y-HHur J-S (2013a) Draft genome sequence of Cladoniamacilenta KoLRI003786 a lichen-forming fungusproducing biruloquinone Genome Announcements 1(5)e00695-13 doi101128genomeA00695-13
Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
Peacuterez-Ortega S Fernaacutendez-Mendoza F Raggio J VivasM Ascaso C Sancho LG Printzen C de los Riacuteos A(2012) Extreme phenotypic variation in Cetrariaaculeata (lichenized Ascomycota) adaptation or inci-dental modification Ann Bot 109(6)1133ndash1148doi101093aobmcs042
Pino-Bodas R Burgaz A Martiacuten M Lumbsch HT (2011)Phenotypical plasticity and homoplasy complicatespecies delimitation in the Cladonia gracilis group(Cladoniaceae Ascomycota) Organ Divers Evol 11(5)343ndash355 doi101007s13127-011-0062-2
Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
Pino-Bodas R Martiacuten M Burgaz A (2012b) Cladoniasubturgida and C iberica (Cladoniaceae) form asingle morphologically and chemically polymorphicspecies Mycol Prog 11(1)269ndash278 doi101007s11557-011-0746-1
Pino-Bodas R Ahti T Stenroos S Martiacuten MP BurgazAR (2013a) Multilocus approach to species recogni-tion in the Cladonia humilis complex (CladoniaceaeAscomycota) Am J Bot 100(4)664ndash678 doi103732ajb1200162
Pino-Bodas R Martiacuten AP Burgaz AR Lumbsch HT(2013b) Species delimitation in Cladonia (Ascomy-cota) a challenge to the DNA barcoding philosophyMol Ecol Resour 13(6)1058ndash1068 doi1011111755-099812086
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Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
Pringle A Baker DM Platt JL Wares JP Latgeacute JPTaylor JW (2005) Cryptic speciation in the cosmo-politan and clonal human pathogenic fungus Asper-gillus fumigatus Evolution 59(9)1886ndash1899 doi10155404-2411
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Pritchard JK Stephens M Donnelly P (2000) Inference ofpopulation structure using multilocus genotype dataGenetics 155(2)945ndash959
Puillandre N Lambert A Brouillet S Achaz G (2012)ABGD automatic barcode gap discovery for primaryspecies delimitation Mol Ecol 21(8)1864ndash1877doi101111j1365-294X201105239x
Rannala B Yang Z (2003) Bayes estimation of speciesdivergence times and ancestral population sizes usingDNA sequences from multiple loci Genetics1641645ndash1656
Raxworthy CJ Ingram CM Rabibisoa N Pearson RG(2007) Applications of ecological niche modelingfor species delimitation a review and empiricalevaluation using day geckos (Phelsuma) fromMadagascar Syst Biol 56(6)907ndash923 doi10108010635150701775111
Ray J (1686) Historia planarum vol 1 Clark LondonReese Naeligsborg R Ekman S Tibell L (2007) Molecular
phylogeny of the genus Lecania (Ramalinaceaelichenized Ascomycota) Mycol Res 111(5)581ndash591doi101016jmycres200703001
Reid N Carstens B (2012) Phylogenetic estimation errorcan decrease the accuracy of species delimitation aBayesian implementation of the general mixed Yule-coalescent model BMC Evol Biol 12(1)196 doi1011861471-2148-12-196
Rissler L Apodaca J (2007) Adding more ecology intospecies delimitation ecological niche models andphylogeography help define cryptic species in theBlack Salamander (Aneides flavipunctatus) Syst Biol56924ndash942 doi10108010635150701703063
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Rivas Plata E Lumbsch HT (2011) Parallel evolution andphenotypic divergence in lichenized fungi a casestudy in the lichen-forming fungal family Graphida-ceae (Ascomycota Lecanoromycetes Ostropales)Mol Phylogenet Evol 61(1)45ndash63 doi101016jympev201104025
Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
Ross KG Gotzek D Ascunce MS Shoemaker DD (2010)Species delimitation a case study in a problematic anttaxon Syst Biol 59(2)162ndash184 doi101093sysbiosyp089
Rubin BER Ree RH Moreau CS (2012) Inferringphylogenies from RAD sequence data PLoS ONE 7(4)e33394 doi101371journalpone0033394
Rubinoff D (2006) Utility of mitochondrial DNA barcodesin species conservation Conserv Biol 20(4)1026ndash1033 doi101111j1523-1739200600372x
Ruiz-Sanchez E Sosa V (2010) Delimiting speciesboundaries within the Neotropical bamboo Otatea(Poaceae Bambusoideae) using molecular morpho-logical and ecological data Mol Phylogenet Evol 54(2)344ndash356 doi101016jympev200910035
Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
morphological characters Antarctic Sci 22 (SpecialIssue 06)727ndash741 doi101017S0954102010000477
Salicini I Ibaacutentildeez C Juste J (2011) Multilocus phylogenyand species delimitation within the Nattererrsquos batspecies complex in the Western Palearctic MolPhylogenet Evol 61(3)888ndash898 doi101016jympev201108010
Satler JD Carstens BC Hedin M (2013) Multilocusspecies delimitation in a complex of morphologicallyconserved trapdoor spiders (Mygalomorphae Antro-diaetidae Aliatypus) Syst Biol 62(6)805ndash823 doi101093sysbiosyt041
Schlick-Steiner BC Steiner FM Seifert B Stauffer CChristian E Crozier RH (2010) Integrative taxonomya multisource approach to exploring biodiversityAnnu Rev Entomol 55(1)421ndash438 doi101146annurev-ento-112408-085432
Schoch CL Seifert KA Huhndorf S Robert V SpougeJL Levesque CA Chen W Fungal Barcoding Con-sortium (2012) Nuclear ribosomal internal transcribedspacer (ITS) region as a universal DNA barcodemarker for Fungi Proc Natl Acad Sci 109(16)6241ndash6246 doi101073pnas1117018109
Seacuterusiaux E Villarreal AJC Wheeler T Goffinet B(2011) Recent origin active speciation and dispersalfor the lichen genus Nephroma (Peltigerales) inMacaronesia J Biogeogr 38(6)1138ndash1151 doi101111j1365-2699201002469x
Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
Simpson GG (1951) The species concept Evolution5285ndash298
Sites JW Marshall JC (2003) Delimiting species a renais-sance issue in systematic biology Trends Ecol Evol18462ndash470 doi101016S0169-5347(03)00184-8
Sites JW Marshall JC (2004) Operational criteria fordelimiting species Annu Rev Ecol Evol Syst 35(1)199ndash227 doi101146annurevecolsys35112202130128
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Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
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Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
Tewksbury J Anderson JGT Bakker JD Billo TJDunwiddie PW Groom MJ Hampton SE HermanSG Levey DJ Machnicki NJ del Rio CM PowerME Rowell K Salomon AK Stacey L TrombulakSC Wheeler TA (2014) Natural historyrsquos place inscience and society BioScience doi101093bioscibiu032
Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
Werth S Cornejo C Scheidegger C (2013) Characteriza-tion of microsatellite loci in the lichen fungus Lobariapulmonaria (Lobariaceae) Appl Plant Sci 1(2)1200290 doi103732apps1200290
Westberg M Arup U Kaumlrnefelt I (2007) Phylogeneticstudies in the Candelariaceae (lichenized Ascomy-cota) based on nuclear ITS DNA sequence dataMycol Res 111(11)1277ndash1284 doi101017S0953756204002102
Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
2 The Dynamic Discipline of Species Delimitation hellip 43
(Lepidoptera Lycaenidae) Front Zoo 4(1)8 doi1011861742-9994-4-8
Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
intraspecies (Lewis et al 2007 Miller et al 2007Emerson et al 2010) and interspecies studies(Rubin et al 2012 Eaton and Ree 2013 Wagneret al 2013) This approach has provided strikinginsight into the recent adaption radiation of LakeVictoria cichlids (Wagner et al 2013) Analternative approach targeting the sequencing ofspecific loci using next-generation sequencingplatforms provides an efficient means of gener-ating data for loci of known genomic locationorthology size and expected level of variation(OrsquoNeill et al 2013) Markers can be targetedusing well-established PCR techniques or newerhybridization techniques and subsequentlypooled for high-throughput sequencing usingparallel-tagged sequencing of multiple individualsamples within a single sequencing run (OrsquoNeillet al 2013) A recent study of North Americantiger salamanders (Ambystoma tigrinum)demonstrated the potential for amplicon-basedparallel-tagged sequencing to rapidly generatelarge-scale genomic data for species delimitationand species tree research (OrsquoNeill et al 2013)
Although these methods provide an enormousamount of data and insight into diversification atan unprecedented scale computational limita-tions restrict the applicable analytical methodsfor large datasets although computational andanalytical advances are happening rapidly Mostrecent coalescent-based species delimitation andspecies tree models using gene trees have beenlimited to handle tens of loci for multiple indi-viduals and combining hundreds or thousands ofgene trees into a single species delimitationframework presents considerable computationalchallenges (Camargo and Sites 2013) Therecently developed method Bayes factor delim-itation (with genomic data BFD) combines adynamic programming algorithm for estimatingspecies trees that bypasses the computationallyintensive MCMC integration over gene trees toprovide a rigorous technique for species delimi-tation studies using genome-wide SNP data(Leacheacute et al 2014) Competing species delimi-tation models are compared using Bayes factorsand it appears that this approach is robust tosample sizes (ie few loci and limited samples
per species) and misspecification of the prior forpopulation size (θ) (Leacheacute et al 2014)
Fungi are an ideal model for assessing diver-gence in eukaryotes due to their simple mor-phologies small genomes broad ecological rolesand diverse lifestyles (Gladieux et al 2014)However the use of genomic data from high-throughput sequencing methods have not yet beenincluded in species delimitation studies of lichen-forming fungi This is due in part to challenges indealing with metagenomic data containing geno-mic information from a plethora of symbiontsassociated with a lichen thallus scant genomicresources and discipline-specific inertia A num-ber of lichen-forming fungal genomes are cur-rently available including Endocarponpusillum(Wang et al 2014) Cladonia spp (Park et al2013a 2014) Caloplaca flavorubescens (Parket al 2013b) and Cladonia greyi and Xanthoriaparientina through the Fungal Genomics Programat Joint Genome Institute of the United StatesDepartment of Energy (httpwwwjgidoegov)The foreseeable ongoing expansion of genomicresources for lichen-forming fungi will be centralto developing approaches for delimiting speciesusing high-throughput sequencing Specificallygenomic resources will be crucial in identifyingnovel markers (variable genes regions SNPsmicrosatellites etc) identifying conserved fungalmarkers for targeted high-throughput sequencingapproaches and references for entire genomecomparisons (Devkota et al 2014 Werth et al2013)
23 Can We Make SpeciesDelimitation in Lichen-FormingFungi Truly Integrative
The widespread availability of genetic data hascreated a biased viewpoint that only genetic datashould be used for statistical species delimitationHowever an ongoing appeal to researchers toassess species boundaries from multiple andcomplementary perspectives (phylogeneticspopulation genetics comparative morphologydevelopment ecology etc) has resulted in an
30 SD Leavitt et al
integrative taxonomic framework bringing theseconceptual and methodological perspectivestogether (Hamilton et al 2014 Fujita et al 2012Salicini et al 2011 Yeates et al 2011 Padialet al 2010 Dayrat 2005 Will et al 2005 Wiensand Penkrot 2002) In practice any study linkingdifferent kinds of data to support hypotheses ofspecies boundaries including mapping morpho-logical characters onto a molecular phylogenycan be considered integrative Integrative meth-ods for species delimitation fall across a broadspectrum ranging from verbal and qualitativeassessments of data classes to quantitative meth-ods that allow different data types to contribute tostatistical species delimitation (Yeates et al2011) In most taxonomic studies utilizing bothmolecular and morphological data expert opinionis eventually used to some degree to evaluate thefinal status of a candidate species (eg Bond andStockman 2008) In this sense many studiesusing evidence for independent data sources fordelimiting species boundaries use an iterativeapproach (Yeates et al 2011) where speciesboundaries can be tested within a hypothetico-deductive framework with diverse datasets
From a practical perspective we advocate aprocess of iterative taxonomy (sensu Yeates et al2011) to circumscribe and refine species limitsusing multiple lines of evidence This iterativeprocess involves comparisons of morphologicaldata with a phylogenetic hypothesis (ie single-locus gene tree or concatenated multilocusphylogeny) to identify the least inclusive mono-phyletic clade in the topology characterized by atleast one unambiguously diagnostic morphologi-cal character (Miralles and Vences 2013) Thisphylogenetic tree-informed approach to assessingspecies boundaries represents a common practicein studies of lichen-forming fungi Previouslyunrecognized species-level clades with corre-sponding subtle or overlooked phenotypiccharacters have been commonly observed in bothcrustose lichens [eg Acarospraceae (Wedin et al2009) Graphidaceae (Rivas Plata and Lumbsch2011 Papong et al 2009) Lecanoraceae (Leavittet al 2011a) Lecideaceae (Ruprecht et al 2010)Mycoblastaceae (Spribille et al 2011) andTeloschistaceae (Vondraacutek et al 2009 Muggia
et al 2008)] and foliose and fruticose lichens [egLobariaceae (Moncada et al 2014 McDonaldet al 2003) Parmeliaceae (Leavitt et al 2013aWirtz et al 2012 Divakar et al 2005 Molinaet al 2004 Kroken and Taylor 2001) Peltigera-ceae (OrsquoBrien et al 2009 Goffinet et al 2003)Physciaceae (Elix et al 2009 Divakar et al2007) and Sphaerophoraceae (Houmlgnabba andWedin 2003)] As a specific example Luumlckinget al (2008) used a combination of medullarychemistry and underside pigmentation in speci-mens from the Heterodermia obscurata group inCosta Rica to corroborate monophyletic clades inan ITS gene tree as species-level lineages
While this iterative approach to speciesdelimitation and taxonomy has proven valuablefor understanding species boundaries anddescribing new taxa in some groups of lichenizedfungi a posteriori examination of morphologicaland chemical features has failed to reveal diag-nostic phenotypic characters in a number ofstudies (Muggia et al 2014 Leavitt et al 2011aOtaacutelora et al 2010 OrsquoBrien et al 2009) Fur-thermore a study of widespread species in thegenus Melanelixia (Parmeliaceae) indicated thatphenotypically cryptic lichen-forming fungalspecies-level lineages may be relatively ancientand diagnosable phenotypic differences may beabsent even millions of years after the initialdivergence (Leavitt et al 2012b) The latter studyhighlights the fact that species-level lineages maycommonly exist without any observable diag-nostic phenotypic characters calling into ques-tion the impetus for a universal application ofintegrative taxonomy
In keeping with the principle that as manylines of evidence as available should be com-bined to delimit species (Dayrat 2005) the for-malized integrative taxonomic approach (ITAXMiralles and Vences 2013) uses a mtDNA guidetree and observations from different types of datathat might provide conclusive evidence for theindependence of lineages and thus their identityas different species Miralles and Vences (2013)provide a non-exhaustive list of species delimi-tation criteria to be integrated in the ITAXapproach including (a) sympatric occurrencewithout admixture as revealed by consistent
2 The Dynamic Discipline of Species Delimitation hellip 31
differences in morphological or molecular char-acters at the same geographic location (b) strongdifferences in a behavioral morphological orgenetic character known to mediate prematingisolation (c) unviability or infertility of hybrids(d) lack of gene flow across a geographicalhybrid zone (e) congruent diagnostic differencesbetween sister lineages in various unlinkedmorphological character (f) absence of haplo-type sharing in several unlinked nuclear loci and(g) a combination of criteria endashf Speciesboundaries are based on seeking the least inclu-sive monophyletic group in the mtDNA treewhich fulfills at least one of the criteria listedabove Clearly the ITAX approach is sensitive tosample size and in order to reliably support thedistinctiveness of a given species it has beenrecommended that the sampling strategy includesat least five individuals per species (Miralles andVences 2013) A similar approach could beadopted for lichenized fungi using an ITS genetopology as guide tree rather than mtDNA
A total evidence approach including concat-enation has been a common approach for inte-grating information from different sourcesincluding independent genetic markers in phy-logenetic reconstructions (Kluge 1989 Wiens1998 de Queiroz et al 1995) Proponents forconcatenation of independent data in phyloge-netic analyses argue that when combining alldata the underlying signal of speciation mayemerge even if weakly contradictory signalis contained in the individual data partitions(Gatesy et al 1999) For example establishing apreliminary perspective of species boundariesfrom multilocus sequence data using concatena-tion may provide a reasonable starting point forscreening for cryptic species and species treeinference (Šlapeta et al 2006 Leavitt et al2011a Le Gac et al 2007 Leacheacute 2009)However concatenation and consensus methodsimply a risk of obtaining inflated support forincorrect relationships and information aboutvariance in gene coalescence is lost (Degnan andRosenberg 2006 2009 Kubatko and Degnan2007 Edwards 2009) In spite of the impetus forintegrating evidence from independent data
sources into empirical species delimitation stud-ies and taxonomy (Fujita et al 2012 Padial et al2009) most currently available species delimi-tation methods are unable to accommodate non-genetic data sources in a statistical framework
231 Selecting the Appropriate Data
In the face of increasing availability of geneticdata and associated bioinformatical approachesfor delimiting species researchers should care-fully consider what information is being sacri-ficed by the failure to consider non-genetic datain species delimitation studies and whetheraccuracy could be improved by the addition ofmultiple data types Morphological data havehistorically served as a proxy to identify repro-ductively isolated groups (ie ldquospeciesrdquo) (Ray1686 Fujita et al 2012) Current methods fordelimiting species using non-genetic data (egchemistry morphology and ecology) remainwoefully understudied For example morphol-ogy-based species circumscriptions are generallybased on one or more qualitative (or quantitative)morphological characters that do not appear tooverlap with other species However ascertain-ing that a given trait is truly fixed within apopulation with statistical confidence requiresunrealistic sample sizes even when allowing forsome level of polymorphism in the diagnosticcharacter (Wiens and Servedio 2000) Now var-ious combinations of datamdashfrom morphologygenetics geography and ecologymdashare acceptedas standard information for species delimitationstudies (Ruiz-Sanchez and Sosa 2010 Ross et al2010 Edwards and Knowles 2014)
Below we briefly discuss appropriate datasources for species delimitation studies of lichen-forming fungi However homoplastic characters(similar traits that are not derived from a com-mon ancestor) are common among many traitscommonly used to circumscribe fungal taxa thebiological significance of secondary metabolitevariation remains largely unknown (Lawrey1986) and ecological niches may be difficultto adequately characterize and model due to
32 SD Leavitt et al
microhabitat requirements and data resolutionTherefore we advocate a cautious approach toselecting appropriate and relevant data forassessing species boundaries
In spite of the potential challenges and limi-tations of using phenotypic data these traits haveprovided a plethora of valuable information forunderstanding species boundaries For examplein the Melanelixia fuliginosa group (Parmelia-ceae) a morphometric analysis using color isi-dia and marginal zone free of isidia ascharacters revealed a general pattern of differ-entiation between material formerly recognizedas subspecies fuliginosa and glabratula in spiteof the fact that considerable overlap betweengroups occurs in some characters individually(Arup and Berlin 2011) Furthermore the dis-tinction of the two groups was supported by aphylogenetic analysis of the ITS marker andecological differences with M fuliginosaoccurring predominantly on rock and M glab-ratula on bark (Arup and Berlin 2011) Thisstudy of the M fuliginosa group provides a fit-ting example of using multiple independentsuites of data including ecology morphologyand genetic information to establish a robusthypothesis of species boundaries
In lichen systematics phenotypic dataincluding thallus organization secondary metab-olites mode of reproduction ascoma-type andontogeny ascus and ascospore characters havehistorically played a prominent role (Printzen2009) Ascomatal characters have traditionallyheld a major role in higher-level classification(Printzen 2009) in contrast to species-levelclassification which also tends to include a widearray of vegetative and chemical charactersCommonly assessed morphological charactersinclude thallus form and size cortical features(eg maculae and pseudocyphellae) presenceformcolor of attachment structure (eg rhizines)and reproductive mode (ascomata vs vegetativediaspores) (Printzen 2009) Different morpho-logical types of vegetative diasporesmdashcorticatedisidia and ecorticated sorediamdashand their locationare commonly used to distinguish speciesAscomatal characters including morphologylocation (laminal vs marginal) position (sessile
vs immersed) presence of thalline margins colorof an apothecial disc and presence or color ofpruina are also commonly used in speciesdelimitations of lichen-forming fungi (Printzen2009) Other important characters may includethalline characters form color size and septationof ascospores size and form and structure of ascithe hamathecium type of epihymenium andhypothecium and the type of excipulum orperidium conidiomatal characters etc (Printzen2009)
Assessments of secondary metabolites hasplayed an important role in lichen taxonomybeginning with the introduction of simple spottests by Nylander (1866a b) The use of chem-istry in lichen taxonomy has been discussed indetail in numerous reviews (Lumbsch 1998a bRogers 1989 Brodo 1986 Egan 1986 Leuckert1985 Brodo 1978 Hawksworth 1976 Culber-son 1969 1970) and we refer readers to thesevaluable sources for a more comprehensive per-spective on lichen chemistry In short extrolites(secondary metabolites) belong to various clas-ses the most common and diverse include dep-sides depsidones chlorinated xanthones andanthraquinones (Lumbsch 2002 Culberson1969) The presence or absence of specificextrolites or their replacements by another sub-stance is widely used to distinguish speciesparticularly when correlated with differences ingeographic distributions However if morpho-logical or geographical differences betweenpopulations containing different extrolites are notapparent the taxonomic significance has beendisputed with some authors distinguishing themas species and others preferring to regard them aschemical races within a species In addition tosimply using the presence or absence of extro-lites Culberson and Culberson (1976) proposedto arrange lichen substances into chemosyn-dromes of closely related substances The pres-ence or absence of these chemosyndromes maypotentially be used as characters to delimit spe-cies regarding differences involving the samechemosyndromes as intraspecific variation anddistinct chemosyndromes as evidence for inter-specific populations (Lumbsch 1994 Gowan1986)
2 The Dynamic Discipline of Species Delimitation hellip 33
Due to the fact that species delimitationstudies often incorporate a substantial biogeo-graphical or ecological component ecologicalniche modeling plays an increasing role in phy-logenetic and taxonomic research Niche mod-eling can provide evidence for ecologicalisolation between populations based on eitherconserved or divergent ecological niches andtherefore can provide additional evidence sup-porting lineage independence between putativespecies The application of ecological nichemodeling has been applied to species delimita-tion studies in a number of cases (Ruiz-Sanchezand Sosa 2010 Leacheacute et al 2009 Raxworthyet al 2007 Rissler and Apodaca 2007) althoughit has not been explicitly applied to assess speciesboundaries in lichen-forming fungi By mappingthe spatial distribution of environmental suit-ability of climatic variables for candidate speciesthe application of ecological niche modeling canbe particularly important in cases where specieshave allopatric distributions (Raxworthy et al2007)
Ecological niche modeling utilizes knownassociations between a speciesrsquo occurrencelocalities and environmental variables to defineabiotic conditions within which populations canbe maintained (Guisan and Thuiller 2005) Themethodological approach for modeling is basedon four general properties (i) The current knownspeciesrsquo localities is the dependent variable (ii)the distribution is modeled as a map composed ofgrid cells at a specified resolution (iii) a range ofenvironmental variables (eg temperature pre-cipitation and solar exposure) are collected todescribe the characteristics of each cell and (iv)classifying the degree to which each cell is eithersuitable or unsuitable for each species under arange of models (Guisan and Thuiller 2005)
Ecological data also have the potential to playan important role in understanding speciesboundaries in lichen-forming fungi For exampleNash and Zavada (1977) demonstrated that Xant-hoparmelia populations with distinct chemistriesoccurring in the northern portion of the SonoranDesert exhibit habitat selection among differentrock substrates within a region with relativelyuniform climate and topography In another case
the parmelioid species Parmelia mayi is mor-phologically indistinguishable from P saxatilisbut can be separated by bioclimatic features andgenetic and chemical characters (Molina et al2011) McCune and Printzen(2011) assess distri-butions and climatic niches of species in theLecanora varia group inwesternUSAandprovidea model that uses continental influence and annualtemperature as themajor factors predicting speciesdistributions The distribution of Usnea hirta alichen commonly used in air quality biomonitor-ing research was modeled for a section of theWhite River National Forest in central Coloradobased on the presence of U hirta at 72 biomoni-toring reference sites distributed in the inter-mountain western United States (Shrestha et al2012) The best model for predicting U hirtadistribution included four variablesmdashsolar radia-tion average monthly precipitation and averagemonthly minimum and maximum temperatures(Shrestha et al 2012) These studies supportthe potential use of ecological niche modelingmethods in species delimitation studies of lichen-forming fungi
24 Conclusions What AboutTaxonomy
In most cases species circumscription and tax-onomy requires some degree of qualitativejudgment and individual interpretation Integrat-ing multiple types of data into an empiricalframework for delimiting species boundarieswhere species boundaries can be tested within ahypothetico-deductive framework with diversedatasets can provide robust hypotheses of speciesboundaries and taxonomic stability (Yeates et al2011) However it has long been known toevolutionary biologists that distinct species donot need to have diagnosable morphologicaldifferences (Mayr 1963) and increasing avail-ability of genetic data has allowed researchers toidentify species and to rigorously test speciesboundaries with a level of precision that wasunimaginable a decade ago While analyticaladvances in statistical species delimitation havebeen largely based on genetic data the utility of
34 SD Leavitt et al
these approaches to formal taxonomy remainselusive Due to these challenges an eclecticapproach to delimiting species and cautionagainst the reliance on any single dataset ormethod is required when delimiting species
While results from many recent studies maycontradict traditional species boundaries acrossmany groups of lichen-forming fungi we areoptimistic that this research represents substantialprogress toward a more accurate perspective onspecies boundaries and diversity in fungi As aresult of ongoing research of species boundariesin lichen-forming fungi the taxonomic value ofmany phenotypes is now better understood ourunderstanding of ecological evolutionary andbiogeographic patterns has improved and we canbegin to better understand patterns of symbiontinteractions in lichens Integrating new data(including novel morphological characters andgenetic data) will be essential to accurately rep-resent species-level diversity across all groups oflichenized fungi Hopefully an improved per-spective on lichen diversity also increases ourappreciation of these incredible symbiotic sys-tems While we are strong advocates for theapplication of independent data types in devel-oping an integrative taxonomy there is anincreasing need to formally recognize the exis-tence of phenotypically cryptic species-levellineages in lichen-forming fungi (see Hibbettet al 2011) In some cases a molecular taxon-omy may provide the most practical approach toconsistent treatment of mycobiont species withinlichen groups where diagnostic morphologicalcharacters are unidentifiable or practically notfeasible (Leavitt et al 2013c d)
These are exciting times for taxonomists andphylogeneticists A closer look at lichen taxon-omy with the inclusion of new data will help usto better understand the diversity of these fasci-nating organisms accurately interpret distribu-tion patterns and play a more important role inmeaningful conservation practices Howeversome level of uncertainty will accompany pro-gress In many taxonomic groups our traditionalapproach for species identification will likelyneed to be substantially modified The search forcorroborating morphological support for cryptic
species identified using molecular data willrequire meticulous and creative approaches toassess phenotypic variation in potentially unor-thodox ways We are hopeful that lichenologistswho traditionally have been eager to include newmethods such as chromatography in their rou-tine identifications will be amenable to includemolecular techniques to their routine examina-tion of specimens for identification and classifi-cation Although this may prove difficult toachieve by single individuals especially citizenscientists that traditionally play an important rolein lichen taxonomy the increasing numberof collaborative projects in the discipline (egLumbsch et al 2011 Crespo et al 2010Gueidan et al 2009) make us optimistic thatbroad-scale collaborative approaches will facili-tate the inclusion of molecular data in lichenresearch at all levels
Acknowledgments We are indebted to various col-leagues for valuable thought-provoking discussionnotably Matthew Nelsen (University of Chicago) AnaCrespo (Universidad Complutense de Madrid) PradeepDivakar (Universidad Complutense de Madrid) BeckettSterner (The Field Museum) and Joyce Havstad (TheField Museum) We also thank anonymous reviewers whoprovided valuable comments that improved this chapterSupport by the US National Science Foundation isgratefully acknowledged (ldquoHidden diversity in parmelioidlichensrdquo DEB-0949147)
References
Ahti T Hawksworth DL (2005) Xanthoparmelia steno-phylla the correct name for X somloeumlnsis one of themost widespread usnic acid containing species of thegenus The Lichenologist 37(4)363ndash366 doi101017S0024282905015197
Altermann S Leavitt SD Goward T Nelsen MPLumbsch HT (2014) How do you solve a problemlike Letharia A new look at cryptic species in lichen-forming fungi using Bayesian clustering and SNPSsfrom multilocus sequence data PLoS ONE 9(5)e97556 doi101371journalpone0097556
Amo de Paz G Cubas P Crespo A Elix JA Lumbsch HT(2012) Transoceanic dispersal and subsequent diver-sification on separate continents shaped diversity ofthe Xanthoparmelia pulla group (Ascomycota) PLoSONE 7(6)e39683 doi101371journalpone0039683
Arguumlello A Del Prado R Cubas P Crespo A (2007)Parmelina quercina (Parmeliaceae Lecanorales)
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Arup U Berlin ES (2011) A taxonomic study ofMelanelixia fuliginosa in Europe The Lichenologist43(02)89ndash97 doi101017S0024282910000678
Arup U Grube M (2000) Is Rhizoplaca (Lecanoraleslichenized Ascomycota) a monophyletic genus Can JBot 78(3)318ndash327 doi101139b00-006
Avise J Ball R (1990) Principles of genealogicalconcordance in species concepts and biological tax-onomy Oxf Surv Evol Biol 745ndash67
Baird NA Etter PD Atwood TS Currey MC Shiver ALLewis ZA Selker EU Cresko WA Johnson EA(2008) Rapid SNP discovery and genetic mappingusing sequenced RAD markers PLoS ONE 3(10)e3376 doi101371journalpone0003376
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Beaumont MA Nielsen R Robert C Hey J Gaggiotti OKnowles L Estoup A Panchal M Corander JHickerson M Sisson SA Fagundes N Chikhi LBeerli P Vitalis R Cornuet J-M Huelsenbeck J FollM Yang Z Rousset F Balding D Excoffier L (2010)In defence of model-based inference in phylogeogra-phy Mol Ecol 19(3)436ndash446 doi101111j1365-294X200904515x
Bickford D Lohman DJ Sodhi NS Ng PKL Meier RWinker K Ingram KK Das I (2007) Cryptic speciesas a window on diversity and conservation TrendsEcol Evol 22(3)148ndash155 doi101016jtree200611004
Bonan GB Shugart HH (1989) Environmental factors andecological processes in boreal forests Annu Rev EcolSyst 20(1989)1ndash28
Bond J Stockman A (2008) An integrative method fordelimiting cohesion species finding the population-species interface in a group of Californian trapdoorspiders with extreme genetic divergence and geo-graphic structuring Syst Biol 57(4)628ndash646 doi10108010635150802302443
Brodo IM (1978) Changing concepts regarding chemicaldiversity in lichens The Lichenologist 10(1)1ndash11doi101017S0024282978000031
Brodo IM (1986) Interpreting chemical variation inlichens for systematic purposes The Bryologist 89(2)132ndash138
Caley MJ Fisher R Mengersen K (2014) Global speciesrichness estimates have not converged Trends EcolEvol 29(4)187ndash188 doi101016jtree201402002
Camargo A Sites JW (2013) Species delimitation adecade after the Renaissance In Pavlinov I (ed) Thespecies problemmdashongoing issues InTech doi10577252664
Camargo A Morando M Avila LJ Sites JW (2012)Species delimitation with ABC and other coalescent-based methods a test of accuracy with simulations and
an empirical example with lizards of the Liolaemusdarwinii complex (Squamata Liolaemidae) Evolution66(9)2834ndash2849 doi101111j1558-5646201201640x
Carstens BC Dewey TA (2010) Species delimitationusing a combined coalescent and information-theoreticapproach an example from North American Myotisbats Syst Biol 59(4)400ndash414 doi101093sysbiosyq024
Carstens BC Pelletier TA Reid NM Satler JD (2013)How to fail at species delimitation Mol Ecol 22(17)4369ndash4383 doi101111mec12413
Corander J Marttinen P (2006) Bayesian identification ofadmixture events using multi-locus molecular mark-ers Mol Ecol 15(10)2833ndash2843 doi101111j1365-294X200602994x
Corander J Waldmann P Marttinen P Sillanpaa M(2004) BAPS 2 enhanced possibilities for the analysisof genetic population structure Bioinformatics 20(15)2363ndash2369 doi101093bioinformaticsbth250
Corander J Marttinen P Mantyniemi S (2006) Bayesianidentification of stock mixtures from molecular markerdata Fish Bull 104550ndash558
Corander J Marttinen P Siren J Tang J (2008) EnhancedBayesian modelling in BAPS software for learninggenetic structures of populations BMC Bioinf 9(1)539 doi1011861471-2105-9-539
Coyne JA Orr HA (2004) Speciation Sinauer AssociatesSunderland
Cracraft J (1983) Species concepts and speciation anal-ysis Curr Ornithol 1159ndash187
Crespo A Lumbsch HT (2010) Cryptic species in lichen-forming fungi IMA Fungus 1167ndash170 doi105598imafungus2010010209
Crespo A Peacuterez-Ortega S (2009) Cryptic species andspecies pairs in lichens a discussion on the relation-ship between molecular phylogenies and morpholog-ical characters Anales del Jardin Botanico de Madrid66(S1)71ndash81 doi103989ajbm2225
Crespo A Kauff F Divakar PK del Prado R Perez-Ortega S Amo de Paz G Ferencova Z Blanco ORoca-Valiente B Nunez-Zapata J Cubas P ArguelloA Elix JA Esslinger TL Hawksworth DL MillanesA Molina MC Wedin M Ahti T Aptroot A et al(2010) Phylogenetic generic classification of parmeli-oid lichens (Parmeliaceae Ascomycota) based onmolecular morphological and chemical evidenceTaxon 59(6)1735ndash1753
Culberson WL (1969) The use of chemistry in thesystematics of the lichens Taxon 18498ndash505
Culberson WL (1970) Chemosystematics and ecology oflichen-forming fungi Annu Rev Ecol Syst 1153ndash170
Culberson CF Culberson WL (1976) Chemosyndromicvariation in lichens Syst Bot 1325ndash339
Darwin C (1859) On the origin of species by means ofnatural selection or the preservation of favoured racesin the struggle for life J Murray London
Dayrat B (2005) Towards integrative taxonomy Biol JLinn Soc 85(3)407ndash415 doi101111j1095-8312200500503x
36 SD Leavitt et al
de Queiroz K (1998) The general lineage concept ofspecies species criteria and the process of speciationa conceptual unification and terminological recom-mendations In Howard DJ Berlocher SH (eds)Endless forms species and speciation Oxford Uni-versity Press Oxford pp 57ndash75
de Queiroz K (1999) The general lineage concept ofspecies and the defining properties of the speciescategory In Wilson RA (ed) Species new interdis-ciplinary essays MIT Press Cambridge pp 49ndash89
de Queiroz K (2007) Species concepts and speciesdelimitation Syst Biol 56(6)879ndash886 doi10108010635150701701083
de Queiroz A Donoghue MJ Kim J (1995) Separateversus combined analysis of phylogenetic evidenceAnnu Rev Ecol Syst 26(1)657ndash681 doi101146annureves26110195003301
Degnan J Rosenberg N (2006) Discordance of speciestrees with their most likely gene trees PLoS Genet 2(5)e68 doi101371journalpgen0020068
Degnan JH Rosenberg NA (2009) Gene tree discordancephylogenetic inference and the multispecies coales-cent Trends Ecol Evol 24(6)332ndash340 doihttpdxdoiorg101016jtree200901009
Del-Prado R Cubas P Lumbsch HT Divakar PK BlancoO de Paz GA Molina MC Crespo A (2010) Geneticdistances within and among species in monophyleticlineages of Parmeliaceae (Ascomycota) as a tool fortaxon delimitation Mol Phylogenet Evol 56(1)125ndash133 doi101016jympev201004014
Del-Prado R Divakar PK Crespo A (2011) Using geneticdistances in addition to ITS molecular phylogeny toidentify potential species in the Parmotrema reticul-atum complex a case study The Lichenologist 43(06)569ndash583 doi101017S0024282911000582
Del-Prado R Blanco O Lumbsch HT Divakar PK ElixJA Molina MC Crespo A (2013) Molecular phylog-eny and historical biogeography of the lichen-formingfungal genus Flavoparmelia (Ascomycota Parmelia-ceae) Taxon 62(5)928ndash939 doi101270562522
Dettman J Jacobson D Taylor J (2003a) A multilocusgenealogical approach to phylogenetic species recog-nition in the model eukaryote Neurospora Evolution57(12)2703ndash2720 doi101111j0014-38202003tb01514x
Dettman JR Jacobson DJ Turner E Pringle A TaylorJW (2003b) Reproductive isolation and phylogeneticdivergence in Neurospora comparing methods ofspecies recognition in a model eukaryote Evolution57(12)2721 doi10155403-074
Devkota S Cornejo C Werth S Chaudhary RP Sche-idegger C (2014) Characterization of microsatelliteloci in the Himalayan lichen fungus Lobaria pindar-ensis (Lobariaceae) Appl Plant Sci 2(5)1300101doi103732apps1300101
Divakar PK Molina MC Lumbsch HT Crespo A (2005)Parmelia barrenoae a new lichen species related toParmelia sulcata (Parmeliaceae) based on molecularand morphological data The Lichenologist 37(01)37ndash46 doi101017S0024282904014641
Divakar PK Amo De paz G del Prado R Esslinger TLCrespo A (2007) Upper cortex anatomy corroboratesphylogenetic hypothesis in species of Physconia(Ascomycota Lecanoromycetes) Mycol Res 111(11)1311ndash1320 doi101016jmycres200708009
Divakar PK Figueras G Hladun N Crespo A (2010)Molecular phylogenetic studies reveal an undescribedspecies within the North American concept of Mela-nelixia glabra (Parmeliaceae) Fungal Divers 42(1)47ndash55 doi101007s13225-010-0027-3
Donoghue MJ Gauthier A (2004) Implementing thephylocode Trends Ecol Evol 19(6)281ndash282 doi101016jtree200404004
Eaton DAR Ree RH (2013) Inferring phylogeny andintrogression using RADseq data an example fromflowering plants (Pedicularis Orobanchaceae) SystBiol 62(5)689ndash706 doi101093sysbiosyt032
Edwards SV (2009) Is a new and general theory ofmolecular systematics emerging Evolution 63(1)1ndash19 doi101111j1558-5646200800549x
Edwards DL Knowles LL (2014) Species detection andindividual assignment in species delimitation canintegrative data increase efficacy Proc R Soc B BiolSci 281(1777) doi101098rspb20132765
Egan RS (1986) Correlations and non-correlations ofchemical variation patterns with lichen morphologyand geography The Bryologist 8999ndash110
Elix JA Corush J Lumbsch HT (2009) Triterpenechemosyndromes and subtle morphological characterscharacterise lineages in the Physcia aipolia group inAustralia (Ascomycota) Syst Biodivers 7(04)479ndash487 doi101017S1477200009990223
Emerson KJ Merz CR Catchen JM Hohenlohe PACresko WA Bradshaw WE Holzapfel CM (2010)Resolving postglacial phylogeography using high-throughput sequencing Proc Natl Acad Sci 107(37)16196ndash16200 doi101073pnas1006538107
Ence DD Carstens BC (2011) SpedeSTEM a rapid andaccurate method for species delimitation Mol EcolResour 11(3)473ndash480 doi101111j1755-0998201002947x
EvannoGRegnaut SGoudet J (2005)Detecting thenumberof clusters of individuals using the software STRUC-TURE a simulation study Mol Ecol 14(8)2611ndash2620doi101111j1365-294X200502553x
Falush D Stephens M Pritchard JK (2003) Inference ofpopulation structure using multilocus genotype datalinked loci and correlated allele frequencies Genetics164(4)1567ndash1587 doi101111j1471-8286200701758x
Fan HH Kubatko LS (2011) Estimating species treesusing approximate Bayesian computation Mol Phylo-genet Evol 59(2)354ndash363 doi101016jympev201102019
Fernaacutendez-Mendoza F Printzen C (2013) Pleistoceneexpansion of the bipolar lichen Cetraria aculeata intothe Southern hemisphere Mol Ecol 22(7)1961ndash1983doi101111mec12210
Fernaacutendez-Mendoza F Domaschke S Garciacutea MA JordanP Martin MP Printzen C (2011) Population structure
2 The Dynamic Discipline of Species Delimitation hellip 37
of mycobionts and photobionts of the widespreadlichen Cetraria aculeata Mol Ecol 20(6)1208ndash1232doi101111j1365-294X201004993x
Fraley C Raftery A (2007) Model-based methods ofclassification Using the mclust software in chemo-metrics J Stat Softw 18i06 [Available online at httpwwwdoajorgdoajfunc=abstractampid=218544]
Fujisawa T Barraclough TG (2013) Delimiting speciesusing single-locus data and the generalized mixed yulecoalescent (GMYC) approach a revised method andevaluation on simulated datasets Syst Biol 62(5)707ndash724 doi101093sysbiosyt033
Fujita MK Leacheacute AD Burbrink FT McGuire JA MoritzC (2012) Coalescent-based species delimitation in anintegrative taxonomy Trends Ecol Evol 27(9)480ndash488 doi101016jtree201204012
Gatesy J OrsquoGrady P Baker RH (1999) Corroborationamong data sets in simultaneous analysis hiddensupport for phylogenetic relationships among higherlevel Artiodactyl taxa Cladistics 15(3)271ndash313doi101111j1096-00311999tb00268x
Gladieux P Ropars J Badouin H Branca A Aguileta Gde Vienne DM Rodriacuteguez de la Vega RC Branco SGiraud T (2014) Fungal evolutionary genomicsprovides insight into the mechanisms of adaptivedivergence in eukaryotes Mol Ecol 23(4)753ndash773doi101111mec12631
Goffinet B Miadlikowska J Goward T (2003) Phyloge-netic inferences based on nrDNA sequences supportfive morphospecies within the Peltigera didactylacomplex (Lichenized Ascomycota) The Bryologist106(3)349ndash364 doi10163901
Gowan SP (1986) Evolution of secondary natural productsin the genus Porpidia (Ascomycata Porpidiaceae) AmJ Bot 73606
Griffin PC Hoffmann AA (2014) Limited genetic diver-gence among Australian alpine Poa tussock grassescoupled with regional structuring points to ongoinggene flow and taxonomic challenges Ann Bot doi101093aobmcu017
Grube M Hawksworth DL (2007) Trouble with lichenthe re-evaluation and re-interpretation of thallus formand fruit body types in the molecular era Mycol Res111(9)1116ndash1132 doi101016jmycres200704008
Gueidan C Savi S Thues H Roux C Keller C Tibell LPrieto M Heimarsson S Breuss O Orange A FrobergL Wynns AA Navarro-Rosines P Krzewicka BPykaelae J Grube M Lutzoni F (2009) Genericclassification of the Verrucariaceae (Ascomycota)based on molecular and morphological evidencerecent progress and remaining challenges Taxon 58(1)184ndash208
Guillot G Mortier F Estoup A (2005) Geneland acomputer package for landscape genetics Mol EcolNotes 5(3)712ndash715 doi101111j1471-8286200501031x
Guillot G Renaud S Ledevin R Michaux J Claude J(2012) A unifying model for the analysis of pheno-typic genetic and geographic data Syst Biol 61(6)897ndash911 doi101093sysbiosys038
Guisan A Thuiller W (2005) Predicting species distribu-tion offering more than simple habitat models EcolLett 8(9)993ndash1009 doi101111j1461-0248200500792x
Hale ME (1990) A synopsis of the lichen genusXanthoparmelia (Vainio) Hale (AscomycotinaParmeliaceae) Smithsonian Institution Press Wash-ington DC
Hamilton CA Hendrixson BE Brewer MS Bond JE(2014) An evaluation of sampling effects on multipleDNA barcoding methods leads to an integrativeapproach for delimiting species a case study ofthe North American tarantula genus Aphonopelma(Araneae Mygalomorphae Theraphosidae) MolPhylogenet Evol 7179ndash93 doi101016jympev201311007
Hausdorf B (2011) Progress toward a general speciesconcept Evolution 65(4)923ndash931 doi101111j1558-5646201101231x
Hausdorf B Hennig C (2010) Species delimitation usingdominant and codominant multilocus markers SystBiol 59(5)491ndash503 doi101093sysbiosyq039
Hawksworth DL (1976) Lichen chemotaxonomy InBailey RH (ed) Lichenology progress and problemsAcademic London pp 139ndash184
Hebert PDN Cywinska A Ball SL deWaard JR (2003)Biological identifications through DNA barcodesProc R Soc Lond B Biol Sci 270(1512)313ndash321doi101098rspb20022218
Hebert PDN Stoeckle MY Zemlak TS Francis CM(2004) Identification of birds through DNA barcodesPLoS Biol 2(10)e312 doi101371journalpbio0020312
Heled J Drummond AJ (2010) Bayesian inference ofspecies trees from multilocus data Mol Biol Evol 27(3)570ndash580 doi101093molbevmsp274
Hey J (2006) On the failure of modern species conceptsTrends Ecol Evol 21(8)447ndash450 doi101016jtree200605011
Hibbett DS OhmanA Glotzer D NuhnMKirk P NilssonRH (2011) Progress in molecular and morphologicaltaxon discovery in Fungi and options for formalclassification on environmental sequences Fungal BiolRev 2538ndash47 doi101016jfbr201101001
Houmlgnabba F Wedin M (2003) Molecular phylogeny ofthe Sphaerophorus globosus species complex Cladis-tics 19(3)224ndash232 doi101111j1096-00312003tb00365x
Hudson RR Coyne JA (2002) Mathematical conse-quences of the genealogical species concept Evolu-tion 56(8)1557 doi101111j0014-38202002tb01467x
Huelsenbeck JP Andolfatto P Huelsenbeck ET (2011)Structurama Bayesian inference of population struc-ture Evol Bioinf Online 7(2011)55ndash59 doi104137EBOS6761
Jones G Oxelman B (2014) DISSECT an assignment-free Bayesian discovery method for species delimita-tion under the multispecies coalescent Bioinformaticsdoi101101003178
38 SD Leavitt et al
Kekkonen M Hebert PD (2014) DNA barcode-baseddelineation of putative species efficient start fortaxonomic workflows Mol Ecol Res doi1011111755-099812233
Kelly LJ Hollingsworth PM Coppins BJ Ellis CJHarrold P Tosh J Yahr R (2011) DNA barcoding oflichenized fungi demonstrates high identification suc-cess in a floristic context New Phytol 191(1)288ndash300doi101111j1469-8137201103677x
Kiss L (2012) Limits of nuclear ribosomal DNA internaltranscribed spacer (ITS) sequences as species barcodesfor fungi Proc Natl Acad Sci 109(27)E1811ndashE1811doi101073pnas1207143109
Kluge AG (1989) A concern for evidence and aphylogenetic hypothesis for relationships among Epi-crates (Boidae Serpentes) Syst Zool 38(1)7ndash25doi101093sysbio3817
Knowles LL Carstens BC (2007) Delimiting specieswithout monophyletic gene trees Syst Biol 56(6)887ndash895 doi10108010635150701701091
Kroken S Taylor JW (2001) A gene genealogical approachto recognize phylogenetic species boundaries in thelichenized fungus Letharia Mycologia 93(1)38ndash53
Kubatko LS Degnan JH (2007) Inconsistency of phylo-genetic estimates from concatenated data under coa-lescence Syst Biol 56(1)17ndash24 doi10108010635150601146041
Latch EK Dharmarajan G Glaubitz JC Rhodes OE Jr(2006) Relative performance of Bayesian clusteringsoftware for inferring population substructure andindividual assignments at low levels of populationdifferentiation Conserv Genet 7(2)295ndash302 doi101007s10592-005-9098-1
Lawrey JD (1986) Biological role of lichen substancesThe Bryologist 89(2)111ndash122
Le Gac M Hood ME Fournier E Giraud T (2007)Phylogenetic evidence of host-specific cryptic speciesin the anther smut fungus Evolution 61(1)15ndash26doi101111j1558-5646200700002x
Leacheacute AD (2009) Species tree discordance traces tophylogeographic clade boundaries in North Americanfence lizards (Sceloporus) Syst Biol 58(6)547ndash559doi101093sysbiosyp057
Leacheacute AD Fujita MK (2010) Bayesian species delim-itation in West African forest geckos (Hemidactylusfasciatus) Proc R Soc B Biol Sci 2773071ndash3077doi101098rspb20100662
Leacheacute AD Koo M Spencer C Papenfuss T Fisher RMcGuire J (2009) Quantifying ecological morpho-logical and genetic variation to delimit species in thecoast horned lizard species complex (Phrynosoma)Proc Natl Acad Sci USA 10612418ndash12423 doi101073pnas0906380106
Leacheacute AD Fujita MK Minin VN Bouckaert RR (2014)Species delimitation using genome-wide SNP dataSystematic Biology doi101093sysbiosyu018
Leavitt SD Fankhauser JD Leavitt DH Porter LDJohnson LA St Clair LL (2011a) Complex patterns ofspeciation in cosmopolitan ldquorock posyrdquo lichensmdashdiscovering and delimiting cryptic fungal species in
the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
Leavitt SD Esslinger TL Lumbsch HT (2012c) Neogene-dominated diversification in neotropical montanelichens dating divergence events in the lichen-form-ing fungal genus Oropogon (Parmeliaceae) Am J Bot99(11)1764ndash1777 doi103732ajb1200146
Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Lumbsch HT St Clair LL(2013c) DNA barcode identification of lichen-formingfungal species in the Rhizoplaca melanophthalmaspecies-complex (Lecanorales Lecanoraceae) includ-ing five new species MycoKeys 71ndash22 doi103897mycokeys74508
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Vondraacutek J Thorsten Lum-bsch H Clair LLS (2013d) Local representation ofglobal diversity in a cosmopolitan lichen-formingfungal species complex (Rhizoplaca Ascomycota)J Biogeogr 40(9)1792ndash1806 doi101111jbi12118
Leavitt SD Lumbsch HT Stenroos S St Clair LL(2013e) Pleistocene speciation in North American
2 The Dynamic Discipline of Species Delimitation hellip 39
lichenized fungi and the impact of alternative speciescircumscriptions and rates of molecular evolution ondivergence estimates PLoS ONE 8(12)e85240doi101371journalpone0085240
Leavitt SD Esslinger TL Hansen ES Divakar PK CrespoA Loomis BF Lumbsch HT (2014) DNA barcoding ofbrown Parmeliae (Parmeliaceae) species a molecularapproach for accurate specimen identification empha-sizing species in Greenland Organ Divers Evol 14(1)11ndash20 doi101007s13127-013-0147-1
Leliaert F Verbruggen H Vanormelingen P Steen FLopez-Bautista JM Zuccarello GC De Clerck O(2014) DNA-based species delimitation in algae Eur JPhycol 49(2) doi101080096702622014904524
Leuckert C (1985) Probleme der Flechten-Chemotaxon-omiemdashStoffkombinationen und ihre taxonomischeWertung Ber Deut Bot Ges 98401ndash408
Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
Lohtander K Kaumlllersjouml M Roland M Tehler A (2000)The family physciaceae in fennoscandia phylogenyinferred from its sequences Mycologia 92(4)728ndash735
Longton RE (1997) The role of bryophytes and lichens inpolar ecosystems In Woodin SJ Marquiss M (eds)Ecology of Arctic Environments Blackwell ScienceOxford pp 69ndash96 (Special publication No 13)
Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
Lumbsch HT (1994) Die Lecanora subfusca-Gruppe inAustralasien J Hattori Bot Lab 771ndash175
Lumbsch HT (1998a) Taxonomic use of metabolic data inlichen-forming fungi In Frisvad JC Bridge PDArora DK (eds) Chemical Fungal Taxonomy MarcelDekker New York pp 345ndash387
Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
Mayden RL (1997) A hierarchy of species concepts thedenouement in the saga of the species problem InClaridge MF Dawah HA Wilson MR (eds) Speciesthe units of biodiversity Chapman amp Hall Londonpp 381ndash424
Mayr E (1963) Animal species and evolution HarvardUniversity Press Cambridge
Mayr E (1970) Populations species and evolutionBelknap Press of Harvard University Press Cambridge
McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
McDonald T Miadlikowska J Lutzoni F (2003) Thelichen genus Sticta in the Great Smoky Mountains a
40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
McKay BD Mays HL Wu Y Li H Yao C-T Nishiumi IZou F (2013) An empirical comparison of character-based and coalescent-based approaches to speciesdelimitation in a young avian complex Mol Ecol 22(19)4943ndash4957 doi101111mec12446
Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
Mishler BD Brandon RN (1987) Individuality pluralismand the phylogenetic species concept Biol Philos 2397
Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
Nash TH Zavada M (1977) Population studies amongSonoran Desert species of Parmelia subg Xanthop-armelia (Parmeliaceae) Am J Bot 64(6)664ndash669
Nylander W (1866a) Circa novum in studio Lichenumcriterium chemicum Flora 49198ndash201
Nylander W (1866b) Hypochlorite of lime and hydrate ofpotash Two new criteria for the study of lichens(Tanslated and communicated by the Rev W ALeighton) Bot J Linn Soc 9358ndash365
OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
OrsquoNeill EM Schwartz R Bullock CT Williams JSShaffer HB Aguilar-Miguel X Parra-Olea G Weis-rock DW (2013) Parallel tagged amplicon sequencingreveals major lineages and phylogenetic structure inthe North American tiger salamander (Ambystomatigrinum) species complex Mol Ecol 22(1)111ndash129doi101111mec12049
Orock EA Leavitt SD Fonge BA St Clair LL LumbschHT (2012) DNA-based identification of lichen-form-ing fungi Can publicly available sequence databasesaid in lichen diversity inventories of Mount Cameroon(West Africa) The Lichenologist 44(6)833ndash839doi101017S0024282912000424
Otaacutelora MAG Martiacutenez I Aragoacuten G Molina MC (2010)Phylogeography and divergence date estimates of alichen species complex with a disjunct distributionpattern Am J Bot 97(2)216ndash223 doi103732ajb0900064
Padial J Castroviejo-Fisher S Kohler J Vila C ChaparroJ De la Riva I (2009) Deciphering the products ofevolution at the species level the need for anintegrative taxonomy Zoolog Scr 38(4)431ndash447doi101111j1463-6409200800381x
Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
Papong K Luumlcking R Thammathaworn A BoonpragobK (2009) Four new taxa of Chroodiscus (thelotremoidGraphidaceae) from Southeast Asia The Bryologist112(1)152ndash163 doi1016390007-2745-1121152
Park S-Y Choi J Kim JA Jeong M-H Kim S Lee Y-HHur J-S (2013a) Draft genome sequence of Cladoniamacilenta KoLRI003786 a lichen-forming fungusproducing biruloquinone Genome Announcements 1(5)e00695-13 doi101128genomeA00695-13
Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
Peacuterez-Ortega S Fernaacutendez-Mendoza F Raggio J VivasM Ascaso C Sancho LG Printzen C de los Riacuteos A(2012) Extreme phenotypic variation in Cetrariaaculeata (lichenized Ascomycota) adaptation or inci-dental modification Ann Bot 109(6)1133ndash1148doi101093aobmcs042
Pino-Bodas R Burgaz A Martiacuten M Lumbsch HT (2011)Phenotypical plasticity and homoplasy complicatespecies delimitation in the Cladonia gracilis group(Cladoniaceae Ascomycota) Organ Divers Evol 11(5)343ndash355 doi101007s13127-011-0062-2
Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
Pino-Bodas R Martiacuten M Burgaz A (2012b) Cladoniasubturgida and C iberica (Cladoniaceae) form asingle morphologically and chemically polymorphicspecies Mycol Prog 11(1)269ndash278 doi101007s11557-011-0746-1
Pino-Bodas R Ahti T Stenroos S Martiacuten MP BurgazAR (2013a) Multilocus approach to species recogni-tion in the Cladonia humilis complex (CladoniaceaeAscomycota) Am J Bot 100(4)664ndash678 doi103732ajb1200162
Pino-Bodas R Martiacuten AP Burgaz AR Lumbsch HT(2013b) Species delimitation in Cladonia (Ascomy-cota) a challenge to the DNA barcoding philosophyMol Ecol Resour 13(6)1058ndash1068 doi1011111755-099812086
Pons J Barraclough TG Gomez-Zurita J Cardoso ADuran DP Hazell S Kamoun S Sumlin WD VoglerAP (2006) Sequence-based species delimitation for theDNA taxonomy of undescribed insects Syst Biol 55(4)595ndash609 doi10108010635150600852011
Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
Pringle A Baker DM Platt JL Wares JP Latgeacute JPTaylor JW (2005) Cryptic speciation in the cosmo-politan and clonal human pathogenic fungus Asper-gillus fumigatus Evolution 59(9)1886ndash1899 doi10155404-2411
Printzen C (2009) Lichen systematics the role ofmorphological and molecular data to reconstructphylogenetic relationships Progress in botany vol71 Springer Berlin pp 233ndash275
Pritchard JK Stephens M Donnelly P (2000) Inference ofpopulation structure using multilocus genotype dataGenetics 155(2)945ndash959
Puillandre N Lambert A Brouillet S Achaz G (2012)ABGD automatic barcode gap discovery for primaryspecies delimitation Mol Ecol 21(8)1864ndash1877doi101111j1365-294X201105239x
Rannala B Yang Z (2003) Bayes estimation of speciesdivergence times and ancestral population sizes usingDNA sequences from multiple loci Genetics1641645ndash1656
Raxworthy CJ Ingram CM Rabibisoa N Pearson RG(2007) Applications of ecological niche modelingfor species delimitation a review and empiricalevaluation using day geckos (Phelsuma) fromMadagascar Syst Biol 56(6)907ndash923 doi10108010635150701775111
Ray J (1686) Historia planarum vol 1 Clark LondonReese Naeligsborg R Ekman S Tibell L (2007) Molecular
phylogeny of the genus Lecania (Ramalinaceaelichenized Ascomycota) Mycol Res 111(5)581ndash591doi101016jmycres200703001
Reid N Carstens B (2012) Phylogenetic estimation errorcan decrease the accuracy of species delimitation aBayesian implementation of the general mixed Yule-coalescent model BMC Evol Biol 12(1)196 doi1011861471-2148-12-196
Rissler L Apodaca J (2007) Adding more ecology intospecies delimitation ecological niche models andphylogeography help define cryptic species in theBlack Salamander (Aneides flavipunctatus) Syst Biol56924ndash942 doi10108010635150701703063
Rivas Plata E Luumlcking R (2013) High diversity ofGraphidaceae (lichenized Ascomycota Ostropales) inAmazonian Peruacute Fungal Divers 58(1)13ndash32 doi101007s13225-012-0172-y
Rivas Plata E Lumbsch HT (2011) Parallel evolution andphenotypic divergence in lichenized fungi a casestudy in the lichen-forming fungal family Graphida-ceae (Ascomycota Lecanoromycetes Ostropales)Mol Phylogenet Evol 61(1)45ndash63 doi101016jympev201104025
Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
Ross KG Gotzek D Ascunce MS Shoemaker DD (2010)Species delimitation a case study in a problematic anttaxon Syst Biol 59(2)162ndash184 doi101093sysbiosyp089
Rubin BER Ree RH Moreau CS (2012) Inferringphylogenies from RAD sequence data PLoS ONE 7(4)e33394 doi101371journalpone0033394
Rubinoff D (2006) Utility of mitochondrial DNA barcodesin species conservation Conserv Biol 20(4)1026ndash1033 doi101111j1523-1739200600372x
Ruiz-Sanchez E Sosa V (2010) Delimiting speciesboundaries within the Neotropical bamboo Otatea(Poaceae Bambusoideae) using molecular morpho-logical and ecological data Mol Phylogenet Evol 54(2)344ndash356 doi101016jympev200910035
Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
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Salicini I Ibaacutentildeez C Juste J (2011) Multilocus phylogenyand species delimitation within the Nattererrsquos batspecies complex in the Western Palearctic MolPhylogenet Evol 61(3)888ndash898 doi101016jympev201108010
Satler JD Carstens BC Hedin M (2013) Multilocusspecies delimitation in a complex of morphologicallyconserved trapdoor spiders (Mygalomorphae Antro-diaetidae Aliatypus) Syst Biol 62(6)805ndash823 doi101093sysbiosyt041
Schlick-Steiner BC Steiner FM Seifert B Stauffer CChristian E Crozier RH (2010) Integrative taxonomya multisource approach to exploring biodiversityAnnu Rev Entomol 55(1)421ndash438 doi101146annurev-ento-112408-085432
Schoch CL Seifert KA Huhndorf S Robert V SpougeJL Levesque CA Chen W Fungal Barcoding Con-sortium (2012) Nuclear ribosomal internal transcribedspacer (ITS) region as a universal DNA barcodemarker for Fungi Proc Natl Acad Sci 109(16)6241ndash6246 doi101073pnas1117018109
Seacuterusiaux E Villarreal AJC Wheeler T Goffinet B(2011) Recent origin active speciation and dispersalfor the lichen genus Nephroma (Peltigerales) inMacaronesia J Biogeogr 38(6)1138ndash1151 doi101111j1365-2699201002469x
Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
Simpson GG (1951) The species concept Evolution5285ndash298
Sites JW Marshall JC (2003) Delimiting species a renais-sance issue in systematic biology Trends Ecol Evol18462ndash470 doi101016S0169-5347(03)00184-8
Sites JW Marshall JC (2004) Operational criteria fordelimiting species Annu Rev Ecol Evol Syst 35(1)199ndash227 doi101146annurevecolsys35112202130128
Šlapeta J Loacutepez-Garciacutea P Moreira D (2006) Globaldispersal and ancient cryptic species in the smallestmarine eukaryotes Mol Biol Evol 23(1)23ndash29doi101093molbevmsj001
Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
Talavera G Dincă V Vila R (2013) Factors affectingspecies delimitations with the GMYC model insightsfrom a butterfly survey Methods Ecol Evol 4(12)1101ndash1110 doi1011112041-210X12107
Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
Tewksbury J Anderson JGT Bakker JD Billo TJDunwiddie PW Groom MJ Hampton SE HermanSG Levey DJ Machnicki NJ del Rio CM PowerME Rowell K Salomon AK Stacey L TrombulakSC Wheeler TA (2014) Natural historyrsquos place inscience and society BioScience doi101093bioscibiu032
Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
Werth S Cornejo C Scheidegger C (2013) Characteriza-tion of microsatellite loci in the lichen fungus Lobariapulmonaria (Lobariaceae) Appl Plant Sci 1(2)1200290 doi103732apps1200290
Westberg M Arup U Kaumlrnefelt I (2007) Phylogeneticstudies in the Candelariaceae (lichenized Ascomy-cota) based on nuclear ITS DNA sequence dataMycol Res 111(11)1277ndash1284 doi101017S0953756204002102
Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
2 The Dynamic Discipline of Species Delimitation hellip 43
(Lepidoptera Lycaenidae) Front Zoo 4(1)8 doi1011861742-9994-4-8
Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
integrative taxonomic framework bringing theseconceptual and methodological perspectivestogether (Hamilton et al 2014 Fujita et al 2012Salicini et al 2011 Yeates et al 2011 Padialet al 2010 Dayrat 2005 Will et al 2005 Wiensand Penkrot 2002) In practice any study linkingdifferent kinds of data to support hypotheses ofspecies boundaries including mapping morpho-logical characters onto a molecular phylogenycan be considered integrative Integrative meth-ods for species delimitation fall across a broadspectrum ranging from verbal and qualitativeassessments of data classes to quantitative meth-ods that allow different data types to contribute tostatistical species delimitation (Yeates et al2011) In most taxonomic studies utilizing bothmolecular and morphological data expert opinionis eventually used to some degree to evaluate thefinal status of a candidate species (eg Bond andStockman 2008) In this sense many studiesusing evidence for independent data sources fordelimiting species boundaries use an iterativeapproach (Yeates et al 2011) where speciesboundaries can be tested within a hypothetico-deductive framework with diverse datasets
From a practical perspective we advocate aprocess of iterative taxonomy (sensu Yeates et al2011) to circumscribe and refine species limitsusing multiple lines of evidence This iterativeprocess involves comparisons of morphologicaldata with a phylogenetic hypothesis (ie single-locus gene tree or concatenated multilocusphylogeny) to identify the least inclusive mono-phyletic clade in the topology characterized by atleast one unambiguously diagnostic morphologi-cal character (Miralles and Vences 2013) Thisphylogenetic tree-informed approach to assessingspecies boundaries represents a common practicein studies of lichen-forming fungi Previouslyunrecognized species-level clades with corre-sponding subtle or overlooked phenotypiccharacters have been commonly observed in bothcrustose lichens [eg Acarospraceae (Wedin et al2009) Graphidaceae (Rivas Plata and Lumbsch2011 Papong et al 2009) Lecanoraceae (Leavittet al 2011a) Lecideaceae (Ruprecht et al 2010)Mycoblastaceae (Spribille et al 2011) andTeloschistaceae (Vondraacutek et al 2009 Muggia
et al 2008)] and foliose and fruticose lichens [egLobariaceae (Moncada et al 2014 McDonaldet al 2003) Parmeliaceae (Leavitt et al 2013aWirtz et al 2012 Divakar et al 2005 Molinaet al 2004 Kroken and Taylor 2001) Peltigera-ceae (OrsquoBrien et al 2009 Goffinet et al 2003)Physciaceae (Elix et al 2009 Divakar et al2007) and Sphaerophoraceae (Houmlgnabba andWedin 2003)] As a specific example Luumlckinget al (2008) used a combination of medullarychemistry and underside pigmentation in speci-mens from the Heterodermia obscurata group inCosta Rica to corroborate monophyletic clades inan ITS gene tree as species-level lineages
While this iterative approach to speciesdelimitation and taxonomy has proven valuablefor understanding species boundaries anddescribing new taxa in some groups of lichenizedfungi a posteriori examination of morphologicaland chemical features has failed to reveal diag-nostic phenotypic characters in a number ofstudies (Muggia et al 2014 Leavitt et al 2011aOtaacutelora et al 2010 OrsquoBrien et al 2009) Fur-thermore a study of widespread species in thegenus Melanelixia (Parmeliaceae) indicated thatphenotypically cryptic lichen-forming fungalspecies-level lineages may be relatively ancientand diagnosable phenotypic differences may beabsent even millions of years after the initialdivergence (Leavitt et al 2012b) The latter studyhighlights the fact that species-level lineages maycommonly exist without any observable diag-nostic phenotypic characters calling into ques-tion the impetus for a universal application ofintegrative taxonomy
In keeping with the principle that as manylines of evidence as available should be com-bined to delimit species (Dayrat 2005) the for-malized integrative taxonomic approach (ITAXMiralles and Vences 2013) uses a mtDNA guidetree and observations from different types of datathat might provide conclusive evidence for theindependence of lineages and thus their identityas different species Miralles and Vences (2013)provide a non-exhaustive list of species delimi-tation criteria to be integrated in the ITAXapproach including (a) sympatric occurrencewithout admixture as revealed by consistent
2 The Dynamic Discipline of Species Delimitation hellip 31
differences in morphological or molecular char-acters at the same geographic location (b) strongdifferences in a behavioral morphological orgenetic character known to mediate prematingisolation (c) unviability or infertility of hybrids(d) lack of gene flow across a geographicalhybrid zone (e) congruent diagnostic differencesbetween sister lineages in various unlinkedmorphological character (f) absence of haplo-type sharing in several unlinked nuclear loci and(g) a combination of criteria endashf Speciesboundaries are based on seeking the least inclu-sive monophyletic group in the mtDNA treewhich fulfills at least one of the criteria listedabove Clearly the ITAX approach is sensitive tosample size and in order to reliably support thedistinctiveness of a given species it has beenrecommended that the sampling strategy includesat least five individuals per species (Miralles andVences 2013) A similar approach could beadopted for lichenized fungi using an ITS genetopology as guide tree rather than mtDNA
A total evidence approach including concat-enation has been a common approach for inte-grating information from different sourcesincluding independent genetic markers in phy-logenetic reconstructions (Kluge 1989 Wiens1998 de Queiroz et al 1995) Proponents forconcatenation of independent data in phyloge-netic analyses argue that when combining alldata the underlying signal of speciation mayemerge even if weakly contradictory signalis contained in the individual data partitions(Gatesy et al 1999) For example establishing apreliminary perspective of species boundariesfrom multilocus sequence data using concatena-tion may provide a reasonable starting point forscreening for cryptic species and species treeinference (Šlapeta et al 2006 Leavitt et al2011a Le Gac et al 2007 Leacheacute 2009)However concatenation and consensus methodsimply a risk of obtaining inflated support forincorrect relationships and information aboutvariance in gene coalescence is lost (Degnan andRosenberg 2006 2009 Kubatko and Degnan2007 Edwards 2009) In spite of the impetus forintegrating evidence from independent data
sources into empirical species delimitation stud-ies and taxonomy (Fujita et al 2012 Padial et al2009) most currently available species delimi-tation methods are unable to accommodate non-genetic data sources in a statistical framework
231 Selecting the Appropriate Data
In the face of increasing availability of geneticdata and associated bioinformatical approachesfor delimiting species researchers should care-fully consider what information is being sacri-ficed by the failure to consider non-genetic datain species delimitation studies and whetheraccuracy could be improved by the addition ofmultiple data types Morphological data havehistorically served as a proxy to identify repro-ductively isolated groups (ie ldquospeciesrdquo) (Ray1686 Fujita et al 2012) Current methods fordelimiting species using non-genetic data (egchemistry morphology and ecology) remainwoefully understudied For example morphol-ogy-based species circumscriptions are generallybased on one or more qualitative (or quantitative)morphological characters that do not appear tooverlap with other species However ascertain-ing that a given trait is truly fixed within apopulation with statistical confidence requiresunrealistic sample sizes even when allowing forsome level of polymorphism in the diagnosticcharacter (Wiens and Servedio 2000) Now var-ious combinations of datamdashfrom morphologygenetics geography and ecologymdashare acceptedas standard information for species delimitationstudies (Ruiz-Sanchez and Sosa 2010 Ross et al2010 Edwards and Knowles 2014)
Below we briefly discuss appropriate datasources for species delimitation studies of lichen-forming fungi However homoplastic characters(similar traits that are not derived from a com-mon ancestor) are common among many traitscommonly used to circumscribe fungal taxa thebiological significance of secondary metabolitevariation remains largely unknown (Lawrey1986) and ecological niches may be difficultto adequately characterize and model due to
32 SD Leavitt et al
microhabitat requirements and data resolutionTherefore we advocate a cautious approach toselecting appropriate and relevant data forassessing species boundaries
In spite of the potential challenges and limi-tations of using phenotypic data these traits haveprovided a plethora of valuable information forunderstanding species boundaries For examplein the Melanelixia fuliginosa group (Parmelia-ceae) a morphometric analysis using color isi-dia and marginal zone free of isidia ascharacters revealed a general pattern of differ-entiation between material formerly recognizedas subspecies fuliginosa and glabratula in spiteof the fact that considerable overlap betweengroups occurs in some characters individually(Arup and Berlin 2011) Furthermore the dis-tinction of the two groups was supported by aphylogenetic analysis of the ITS marker andecological differences with M fuliginosaoccurring predominantly on rock and M glab-ratula on bark (Arup and Berlin 2011) Thisstudy of the M fuliginosa group provides a fit-ting example of using multiple independentsuites of data including ecology morphologyand genetic information to establish a robusthypothesis of species boundaries
In lichen systematics phenotypic dataincluding thallus organization secondary metab-olites mode of reproduction ascoma-type andontogeny ascus and ascospore characters havehistorically played a prominent role (Printzen2009) Ascomatal characters have traditionallyheld a major role in higher-level classification(Printzen 2009) in contrast to species-levelclassification which also tends to include a widearray of vegetative and chemical charactersCommonly assessed morphological charactersinclude thallus form and size cortical features(eg maculae and pseudocyphellae) presenceformcolor of attachment structure (eg rhizines)and reproductive mode (ascomata vs vegetativediaspores) (Printzen 2009) Different morpho-logical types of vegetative diasporesmdashcorticatedisidia and ecorticated sorediamdashand their locationare commonly used to distinguish speciesAscomatal characters including morphologylocation (laminal vs marginal) position (sessile
vs immersed) presence of thalline margins colorof an apothecial disc and presence or color ofpruina are also commonly used in speciesdelimitations of lichen-forming fungi (Printzen2009) Other important characters may includethalline characters form color size and septationof ascospores size and form and structure of ascithe hamathecium type of epihymenium andhypothecium and the type of excipulum orperidium conidiomatal characters etc (Printzen2009)
Assessments of secondary metabolites hasplayed an important role in lichen taxonomybeginning with the introduction of simple spottests by Nylander (1866a b) The use of chem-istry in lichen taxonomy has been discussed indetail in numerous reviews (Lumbsch 1998a bRogers 1989 Brodo 1986 Egan 1986 Leuckert1985 Brodo 1978 Hawksworth 1976 Culber-son 1969 1970) and we refer readers to thesevaluable sources for a more comprehensive per-spective on lichen chemistry In short extrolites(secondary metabolites) belong to various clas-ses the most common and diverse include dep-sides depsidones chlorinated xanthones andanthraquinones (Lumbsch 2002 Culberson1969) The presence or absence of specificextrolites or their replacements by another sub-stance is widely used to distinguish speciesparticularly when correlated with differences ingeographic distributions However if morpho-logical or geographical differences betweenpopulations containing different extrolites are notapparent the taxonomic significance has beendisputed with some authors distinguishing themas species and others preferring to regard them aschemical races within a species In addition tosimply using the presence or absence of extro-lites Culberson and Culberson (1976) proposedto arrange lichen substances into chemosyn-dromes of closely related substances The pres-ence or absence of these chemosyndromes maypotentially be used as characters to delimit spe-cies regarding differences involving the samechemosyndromes as intraspecific variation anddistinct chemosyndromes as evidence for inter-specific populations (Lumbsch 1994 Gowan1986)
2 The Dynamic Discipline of Species Delimitation hellip 33
Due to the fact that species delimitationstudies often incorporate a substantial biogeo-graphical or ecological component ecologicalniche modeling plays an increasing role in phy-logenetic and taxonomic research Niche mod-eling can provide evidence for ecologicalisolation between populations based on eitherconserved or divergent ecological niches andtherefore can provide additional evidence sup-porting lineage independence between putativespecies The application of ecological nichemodeling has been applied to species delimita-tion studies in a number of cases (Ruiz-Sanchezand Sosa 2010 Leacheacute et al 2009 Raxworthyet al 2007 Rissler and Apodaca 2007) althoughit has not been explicitly applied to assess speciesboundaries in lichen-forming fungi By mappingthe spatial distribution of environmental suit-ability of climatic variables for candidate speciesthe application of ecological niche modeling canbe particularly important in cases where specieshave allopatric distributions (Raxworthy et al2007)
Ecological niche modeling utilizes knownassociations between a speciesrsquo occurrencelocalities and environmental variables to defineabiotic conditions within which populations canbe maintained (Guisan and Thuiller 2005) Themethodological approach for modeling is basedon four general properties (i) The current knownspeciesrsquo localities is the dependent variable (ii)the distribution is modeled as a map composed ofgrid cells at a specified resolution (iii) a range ofenvironmental variables (eg temperature pre-cipitation and solar exposure) are collected todescribe the characteristics of each cell and (iv)classifying the degree to which each cell is eithersuitable or unsuitable for each species under arange of models (Guisan and Thuiller 2005)
Ecological data also have the potential to playan important role in understanding speciesboundaries in lichen-forming fungi For exampleNash and Zavada (1977) demonstrated that Xant-hoparmelia populations with distinct chemistriesoccurring in the northern portion of the SonoranDesert exhibit habitat selection among differentrock substrates within a region with relativelyuniform climate and topography In another case
the parmelioid species Parmelia mayi is mor-phologically indistinguishable from P saxatilisbut can be separated by bioclimatic features andgenetic and chemical characters (Molina et al2011) McCune and Printzen(2011) assess distri-butions and climatic niches of species in theLecanora varia group inwesternUSAandprovidea model that uses continental influence and annualtemperature as themajor factors predicting speciesdistributions The distribution of Usnea hirta alichen commonly used in air quality biomonitor-ing research was modeled for a section of theWhite River National Forest in central Coloradobased on the presence of U hirta at 72 biomoni-toring reference sites distributed in the inter-mountain western United States (Shrestha et al2012) The best model for predicting U hirtadistribution included four variablesmdashsolar radia-tion average monthly precipitation and averagemonthly minimum and maximum temperatures(Shrestha et al 2012) These studies supportthe potential use of ecological niche modelingmethods in species delimitation studies of lichen-forming fungi
24 Conclusions What AboutTaxonomy
In most cases species circumscription and tax-onomy requires some degree of qualitativejudgment and individual interpretation Integrat-ing multiple types of data into an empiricalframework for delimiting species boundarieswhere species boundaries can be tested within ahypothetico-deductive framework with diversedatasets can provide robust hypotheses of speciesboundaries and taxonomic stability (Yeates et al2011) However it has long been known toevolutionary biologists that distinct species donot need to have diagnosable morphologicaldifferences (Mayr 1963) and increasing avail-ability of genetic data has allowed researchers toidentify species and to rigorously test speciesboundaries with a level of precision that wasunimaginable a decade ago While analyticaladvances in statistical species delimitation havebeen largely based on genetic data the utility of
34 SD Leavitt et al
these approaches to formal taxonomy remainselusive Due to these challenges an eclecticapproach to delimiting species and cautionagainst the reliance on any single dataset ormethod is required when delimiting species
While results from many recent studies maycontradict traditional species boundaries acrossmany groups of lichen-forming fungi we areoptimistic that this research represents substantialprogress toward a more accurate perspective onspecies boundaries and diversity in fungi As aresult of ongoing research of species boundariesin lichen-forming fungi the taxonomic value ofmany phenotypes is now better understood ourunderstanding of ecological evolutionary andbiogeographic patterns has improved and we canbegin to better understand patterns of symbiontinteractions in lichens Integrating new data(including novel morphological characters andgenetic data) will be essential to accurately rep-resent species-level diversity across all groups oflichenized fungi Hopefully an improved per-spective on lichen diversity also increases ourappreciation of these incredible symbiotic sys-tems While we are strong advocates for theapplication of independent data types in devel-oping an integrative taxonomy there is anincreasing need to formally recognize the exis-tence of phenotypically cryptic species-levellineages in lichen-forming fungi (see Hibbettet al 2011) In some cases a molecular taxon-omy may provide the most practical approach toconsistent treatment of mycobiont species withinlichen groups where diagnostic morphologicalcharacters are unidentifiable or practically notfeasible (Leavitt et al 2013c d)
These are exciting times for taxonomists andphylogeneticists A closer look at lichen taxon-omy with the inclusion of new data will help usto better understand the diversity of these fasci-nating organisms accurately interpret distribu-tion patterns and play a more important role inmeaningful conservation practices Howeversome level of uncertainty will accompany pro-gress In many taxonomic groups our traditionalapproach for species identification will likelyneed to be substantially modified The search forcorroborating morphological support for cryptic
species identified using molecular data willrequire meticulous and creative approaches toassess phenotypic variation in potentially unor-thodox ways We are hopeful that lichenologistswho traditionally have been eager to include newmethods such as chromatography in their rou-tine identifications will be amenable to includemolecular techniques to their routine examina-tion of specimens for identification and classifi-cation Although this may prove difficult toachieve by single individuals especially citizenscientists that traditionally play an important rolein lichen taxonomy the increasing numberof collaborative projects in the discipline (egLumbsch et al 2011 Crespo et al 2010Gueidan et al 2009) make us optimistic thatbroad-scale collaborative approaches will facili-tate the inclusion of molecular data in lichenresearch at all levels
Acknowledgments We are indebted to various col-leagues for valuable thought-provoking discussionnotably Matthew Nelsen (University of Chicago) AnaCrespo (Universidad Complutense de Madrid) PradeepDivakar (Universidad Complutense de Madrid) BeckettSterner (The Field Museum) and Joyce Havstad (TheField Museum) We also thank anonymous reviewers whoprovided valuable comments that improved this chapterSupport by the US National Science Foundation isgratefully acknowledged (ldquoHidden diversity in parmelioidlichensrdquo DEB-0949147)
References
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2 The Dynamic Discipline of Species Delimitation hellip 35
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Del-Prado R Divakar PK Crespo A (2011) Using geneticdistances in addition to ITS molecular phylogeny toidentify potential species in the Parmotrema reticul-atum complex a case study The Lichenologist 43(06)569ndash583 doi101017S0024282911000582
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Divakar PK Molina MC Lumbsch HT Crespo A (2005)Parmelia barrenoae a new lichen species related toParmelia sulcata (Parmeliaceae) based on molecularand morphological data The Lichenologist 37(01)37ndash46 doi101017S0024282904014641
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Hausdorf B Hennig C (2010) Species delimitation usingdominant and codominant multilocus markers SystBiol 59(5)491ndash503 doi101093sysbiosyq039
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Heled J Drummond AJ (2010) Bayesian inference ofspecies trees from multilocus data Mol Biol Evol 27(3)570ndash580 doi101093molbevmsp274
Hey J (2006) On the failure of modern species conceptsTrends Ecol Evol 21(8)447ndash450 doi101016jtree200605011
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the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
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Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
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Leavitt SD Lumbsch HT Stenroos S St Clair LL(2013e) Pleistocene speciation in North American
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Leavitt SD Esslinger TL Hansen ES Divakar PK CrespoA Loomis BF Lumbsch HT (2014) DNA barcoding ofbrown Parmeliae (Parmeliaceae) species a molecularapproach for accurate specimen identification empha-sizing species in Greenland Organ Divers Evol 14(1)11ndash20 doi101007s13127-013-0147-1
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Leuckert C (1985) Probleme der Flechten-Chemotaxon-omiemdashStoffkombinationen und ihre taxonomischeWertung Ber Deut Bot Ges 98401ndash408
Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
Lohtander K Kaumlllersjouml M Roland M Tehler A (2000)The family physciaceae in fennoscandia phylogenyinferred from its sequences Mycologia 92(4)728ndash735
Longton RE (1997) The role of bryophytes and lichens inpolar ecosystems In Woodin SJ Marquiss M (eds)Ecology of Arctic Environments Blackwell ScienceOxford pp 69ndash96 (Special publication No 13)
Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
Lumbsch HT (1994) Die Lecanora subfusca-Gruppe inAustralasien J Hattori Bot Lab 771ndash175
Lumbsch HT (1998a) Taxonomic use of metabolic data inlichen-forming fungi In Frisvad JC Bridge PDArora DK (eds) Chemical Fungal Taxonomy MarcelDekker New York pp 345ndash387
Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
Mayden RL (1997) A hierarchy of species concepts thedenouement in the saga of the species problem InClaridge MF Dawah HA Wilson MR (eds) Speciesthe units of biodiversity Chapman amp Hall Londonpp 381ndash424
Mayr E (1963) Animal species and evolution HarvardUniversity Press Cambridge
Mayr E (1970) Populations species and evolutionBelknap Press of Harvard University Press Cambridge
McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
McDonald T Miadlikowska J Lutzoni F (2003) Thelichen genus Sticta in the Great Smoky Mountains a
40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
McKay BD Mays HL Wu Y Li H Yao C-T Nishiumi IZou F (2013) An empirical comparison of character-based and coalescent-based approaches to speciesdelimitation in a young avian complex Mol Ecol 22(19)4943ndash4957 doi101111mec12446
Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
Mishler BD Brandon RN (1987) Individuality pluralismand the phylogenetic species concept Biol Philos 2397
Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
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Nylander W (1866a) Circa novum in studio Lichenumcriterium chemicum Flora 49198ndash201
Nylander W (1866b) Hypochlorite of lime and hydrate ofpotash Two new criteria for the study of lichens(Tanslated and communicated by the Rev W ALeighton) Bot J Linn Soc 9358ndash365
OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
OrsquoNeill EM Schwartz R Bullock CT Williams JSShaffer HB Aguilar-Miguel X Parra-Olea G Weis-rock DW (2013) Parallel tagged amplicon sequencingreveals major lineages and phylogenetic structure inthe North American tiger salamander (Ambystomatigrinum) species complex Mol Ecol 22(1)111ndash129doi101111mec12049
Orock EA Leavitt SD Fonge BA St Clair LL LumbschHT (2012) DNA-based identification of lichen-form-ing fungi Can publicly available sequence databasesaid in lichen diversity inventories of Mount Cameroon(West Africa) The Lichenologist 44(6)833ndash839doi101017S0024282912000424
Otaacutelora MAG Martiacutenez I Aragoacuten G Molina MC (2010)Phylogeography and divergence date estimates of alichen species complex with a disjunct distributionpattern Am J Bot 97(2)216ndash223 doi103732ajb0900064
Padial J Castroviejo-Fisher S Kohler J Vila C ChaparroJ De la Riva I (2009) Deciphering the products ofevolution at the species level the need for anintegrative taxonomy Zoolog Scr 38(4)431ndash447doi101111j1463-6409200800381x
Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
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Park S-Y Choi J Kim JA Jeong M-H Kim S Lee Y-HHur J-S (2013a) Draft genome sequence of Cladoniamacilenta KoLRI003786 a lichen-forming fungusproducing biruloquinone Genome Announcements 1(5)e00695-13 doi101128genomeA00695-13
Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
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Pino-Bodas R Burgaz A Martiacuten M Lumbsch HT (2011)Phenotypical plasticity and homoplasy complicatespecies delimitation in the Cladonia gracilis group(Cladoniaceae Ascomycota) Organ Divers Evol 11(5)343ndash355 doi101007s13127-011-0062-2
Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
Pino-Bodas R Martiacuten M Burgaz A (2012b) Cladoniasubturgida and C iberica (Cladoniaceae) form asingle morphologically and chemically polymorphicspecies Mycol Prog 11(1)269ndash278 doi101007s11557-011-0746-1
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Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
Pringle A Baker DM Platt JL Wares JP Latgeacute JPTaylor JW (2005) Cryptic speciation in the cosmo-politan and clonal human pathogenic fungus Asper-gillus fumigatus Evolution 59(9)1886ndash1899 doi10155404-2411
Printzen C (2009) Lichen systematics the role ofmorphological and molecular data to reconstructphylogenetic relationships Progress in botany vol71 Springer Berlin pp 233ndash275
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Puillandre N Lambert A Brouillet S Achaz G (2012)ABGD automatic barcode gap discovery for primaryspecies delimitation Mol Ecol 21(8)1864ndash1877doi101111j1365-294X201105239x
Rannala B Yang Z (2003) Bayes estimation of speciesdivergence times and ancestral population sizes usingDNA sequences from multiple loci Genetics1641645ndash1656
Raxworthy CJ Ingram CM Rabibisoa N Pearson RG(2007) Applications of ecological niche modelingfor species delimitation a review and empiricalevaluation using day geckos (Phelsuma) fromMadagascar Syst Biol 56(6)907ndash923 doi10108010635150701775111
Ray J (1686) Historia planarum vol 1 Clark LondonReese Naeligsborg R Ekman S Tibell L (2007) Molecular
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Reid N Carstens B (2012) Phylogenetic estimation errorcan decrease the accuracy of species delimitation aBayesian implementation of the general mixed Yule-coalescent model BMC Evol Biol 12(1)196 doi1011861471-2148-12-196
Rissler L Apodaca J (2007) Adding more ecology intospecies delimitation ecological niche models andphylogeography help define cryptic species in theBlack Salamander (Aneides flavipunctatus) Syst Biol56924ndash942 doi10108010635150701703063
Rivas Plata E Luumlcking R (2013) High diversity ofGraphidaceae (lichenized Ascomycota Ostropales) inAmazonian Peruacute Fungal Divers 58(1)13ndash32 doi101007s13225-012-0172-y
Rivas Plata E Lumbsch HT (2011) Parallel evolution andphenotypic divergence in lichenized fungi a casestudy in the lichen-forming fungal family Graphida-ceae (Ascomycota Lecanoromycetes Ostropales)Mol Phylogenet Evol 61(1)45ndash63 doi101016jympev201104025
Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
Ross KG Gotzek D Ascunce MS Shoemaker DD (2010)Species delimitation a case study in a problematic anttaxon Syst Biol 59(2)162ndash184 doi101093sysbiosyp089
Rubin BER Ree RH Moreau CS (2012) Inferringphylogenies from RAD sequence data PLoS ONE 7(4)e33394 doi101371journalpone0033394
Rubinoff D (2006) Utility of mitochondrial DNA barcodesin species conservation Conserv Biol 20(4)1026ndash1033 doi101111j1523-1739200600372x
Ruiz-Sanchez E Sosa V (2010) Delimiting speciesboundaries within the Neotropical bamboo Otatea(Poaceae Bambusoideae) using molecular morpho-logical and ecological data Mol Phylogenet Evol 54(2)344ndash356 doi101016jympev200910035
Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
morphological characters Antarctic Sci 22 (SpecialIssue 06)727ndash741 doi101017S0954102010000477
Salicini I Ibaacutentildeez C Juste J (2011) Multilocus phylogenyand species delimitation within the Nattererrsquos batspecies complex in the Western Palearctic MolPhylogenet Evol 61(3)888ndash898 doi101016jympev201108010
Satler JD Carstens BC Hedin M (2013) Multilocusspecies delimitation in a complex of morphologicallyconserved trapdoor spiders (Mygalomorphae Antro-diaetidae Aliatypus) Syst Biol 62(6)805ndash823 doi101093sysbiosyt041
Schlick-Steiner BC Steiner FM Seifert B Stauffer CChristian E Crozier RH (2010) Integrative taxonomya multisource approach to exploring biodiversityAnnu Rev Entomol 55(1)421ndash438 doi101146annurev-ento-112408-085432
Schoch CL Seifert KA Huhndorf S Robert V SpougeJL Levesque CA Chen W Fungal Barcoding Con-sortium (2012) Nuclear ribosomal internal transcribedspacer (ITS) region as a universal DNA barcodemarker for Fungi Proc Natl Acad Sci 109(16)6241ndash6246 doi101073pnas1117018109
Seacuterusiaux E Villarreal AJC Wheeler T Goffinet B(2011) Recent origin active speciation and dispersalfor the lichen genus Nephroma (Peltigerales) inMacaronesia J Biogeogr 38(6)1138ndash1151 doi101111j1365-2699201002469x
Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
Simpson GG (1951) The species concept Evolution5285ndash298
Sites JW Marshall JC (2003) Delimiting species a renais-sance issue in systematic biology Trends Ecol Evol18462ndash470 doi101016S0169-5347(03)00184-8
Sites JW Marshall JC (2004) Operational criteria fordelimiting species Annu Rev Ecol Evol Syst 35(1)199ndash227 doi101146annurevecolsys35112202130128
Šlapeta J Loacutepez-Garciacutea P Moreira D (2006) Globaldispersal and ancient cryptic species in the smallestmarine eukaryotes Mol Biol Evol 23(1)23ndash29doi101093molbevmsj001
Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
Talavera G Dincă V Vila R (2013) Factors affectingspecies delimitations with the GMYC model insightsfrom a butterfly survey Methods Ecol Evol 4(12)1101ndash1110 doi1011112041-210X12107
Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
Tewksbury J Anderson JGT Bakker JD Billo TJDunwiddie PW Groom MJ Hampton SE HermanSG Levey DJ Machnicki NJ del Rio CM PowerME Rowell K Salomon AK Stacey L TrombulakSC Wheeler TA (2014) Natural historyrsquos place inscience and society BioScience doi101093bioscibiu032
Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
Werth S Cornejo C Scheidegger C (2013) Characteriza-tion of microsatellite loci in the lichen fungus Lobariapulmonaria (Lobariaceae) Appl Plant Sci 1(2)1200290 doi103732apps1200290
Westberg M Arup U Kaumlrnefelt I (2007) Phylogeneticstudies in the Candelariaceae (lichenized Ascomy-cota) based on nuclear ITS DNA sequence dataMycol Res 111(11)1277ndash1284 doi101017S0953756204002102
Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
2 The Dynamic Discipline of Species Delimitation hellip 43
(Lepidoptera Lycaenidae) Front Zoo 4(1)8 doi1011861742-9994-4-8
Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
differences in morphological or molecular char-acters at the same geographic location (b) strongdifferences in a behavioral morphological orgenetic character known to mediate prematingisolation (c) unviability or infertility of hybrids(d) lack of gene flow across a geographicalhybrid zone (e) congruent diagnostic differencesbetween sister lineages in various unlinkedmorphological character (f) absence of haplo-type sharing in several unlinked nuclear loci and(g) a combination of criteria endashf Speciesboundaries are based on seeking the least inclu-sive monophyletic group in the mtDNA treewhich fulfills at least one of the criteria listedabove Clearly the ITAX approach is sensitive tosample size and in order to reliably support thedistinctiveness of a given species it has beenrecommended that the sampling strategy includesat least five individuals per species (Miralles andVences 2013) A similar approach could beadopted for lichenized fungi using an ITS genetopology as guide tree rather than mtDNA
A total evidence approach including concat-enation has been a common approach for inte-grating information from different sourcesincluding independent genetic markers in phy-logenetic reconstructions (Kluge 1989 Wiens1998 de Queiroz et al 1995) Proponents forconcatenation of independent data in phyloge-netic analyses argue that when combining alldata the underlying signal of speciation mayemerge even if weakly contradictory signalis contained in the individual data partitions(Gatesy et al 1999) For example establishing apreliminary perspective of species boundariesfrom multilocus sequence data using concatena-tion may provide a reasonable starting point forscreening for cryptic species and species treeinference (Šlapeta et al 2006 Leavitt et al2011a Le Gac et al 2007 Leacheacute 2009)However concatenation and consensus methodsimply a risk of obtaining inflated support forincorrect relationships and information aboutvariance in gene coalescence is lost (Degnan andRosenberg 2006 2009 Kubatko and Degnan2007 Edwards 2009) In spite of the impetus forintegrating evidence from independent data
sources into empirical species delimitation stud-ies and taxonomy (Fujita et al 2012 Padial et al2009) most currently available species delimi-tation methods are unable to accommodate non-genetic data sources in a statistical framework
231 Selecting the Appropriate Data
In the face of increasing availability of geneticdata and associated bioinformatical approachesfor delimiting species researchers should care-fully consider what information is being sacri-ficed by the failure to consider non-genetic datain species delimitation studies and whetheraccuracy could be improved by the addition ofmultiple data types Morphological data havehistorically served as a proxy to identify repro-ductively isolated groups (ie ldquospeciesrdquo) (Ray1686 Fujita et al 2012) Current methods fordelimiting species using non-genetic data (egchemistry morphology and ecology) remainwoefully understudied For example morphol-ogy-based species circumscriptions are generallybased on one or more qualitative (or quantitative)morphological characters that do not appear tooverlap with other species However ascertain-ing that a given trait is truly fixed within apopulation with statistical confidence requiresunrealistic sample sizes even when allowing forsome level of polymorphism in the diagnosticcharacter (Wiens and Servedio 2000) Now var-ious combinations of datamdashfrom morphologygenetics geography and ecologymdashare acceptedas standard information for species delimitationstudies (Ruiz-Sanchez and Sosa 2010 Ross et al2010 Edwards and Knowles 2014)
Below we briefly discuss appropriate datasources for species delimitation studies of lichen-forming fungi However homoplastic characters(similar traits that are not derived from a com-mon ancestor) are common among many traitscommonly used to circumscribe fungal taxa thebiological significance of secondary metabolitevariation remains largely unknown (Lawrey1986) and ecological niches may be difficultto adequately characterize and model due to
32 SD Leavitt et al
microhabitat requirements and data resolutionTherefore we advocate a cautious approach toselecting appropriate and relevant data forassessing species boundaries
In spite of the potential challenges and limi-tations of using phenotypic data these traits haveprovided a plethora of valuable information forunderstanding species boundaries For examplein the Melanelixia fuliginosa group (Parmelia-ceae) a morphometric analysis using color isi-dia and marginal zone free of isidia ascharacters revealed a general pattern of differ-entiation between material formerly recognizedas subspecies fuliginosa and glabratula in spiteof the fact that considerable overlap betweengroups occurs in some characters individually(Arup and Berlin 2011) Furthermore the dis-tinction of the two groups was supported by aphylogenetic analysis of the ITS marker andecological differences with M fuliginosaoccurring predominantly on rock and M glab-ratula on bark (Arup and Berlin 2011) Thisstudy of the M fuliginosa group provides a fit-ting example of using multiple independentsuites of data including ecology morphologyand genetic information to establish a robusthypothesis of species boundaries
In lichen systematics phenotypic dataincluding thallus organization secondary metab-olites mode of reproduction ascoma-type andontogeny ascus and ascospore characters havehistorically played a prominent role (Printzen2009) Ascomatal characters have traditionallyheld a major role in higher-level classification(Printzen 2009) in contrast to species-levelclassification which also tends to include a widearray of vegetative and chemical charactersCommonly assessed morphological charactersinclude thallus form and size cortical features(eg maculae and pseudocyphellae) presenceformcolor of attachment structure (eg rhizines)and reproductive mode (ascomata vs vegetativediaspores) (Printzen 2009) Different morpho-logical types of vegetative diasporesmdashcorticatedisidia and ecorticated sorediamdashand their locationare commonly used to distinguish speciesAscomatal characters including morphologylocation (laminal vs marginal) position (sessile
vs immersed) presence of thalline margins colorof an apothecial disc and presence or color ofpruina are also commonly used in speciesdelimitations of lichen-forming fungi (Printzen2009) Other important characters may includethalline characters form color size and septationof ascospores size and form and structure of ascithe hamathecium type of epihymenium andhypothecium and the type of excipulum orperidium conidiomatal characters etc (Printzen2009)
Assessments of secondary metabolites hasplayed an important role in lichen taxonomybeginning with the introduction of simple spottests by Nylander (1866a b) The use of chem-istry in lichen taxonomy has been discussed indetail in numerous reviews (Lumbsch 1998a bRogers 1989 Brodo 1986 Egan 1986 Leuckert1985 Brodo 1978 Hawksworth 1976 Culber-son 1969 1970) and we refer readers to thesevaluable sources for a more comprehensive per-spective on lichen chemistry In short extrolites(secondary metabolites) belong to various clas-ses the most common and diverse include dep-sides depsidones chlorinated xanthones andanthraquinones (Lumbsch 2002 Culberson1969) The presence or absence of specificextrolites or their replacements by another sub-stance is widely used to distinguish speciesparticularly when correlated with differences ingeographic distributions However if morpho-logical or geographical differences betweenpopulations containing different extrolites are notapparent the taxonomic significance has beendisputed with some authors distinguishing themas species and others preferring to regard them aschemical races within a species In addition tosimply using the presence or absence of extro-lites Culberson and Culberson (1976) proposedto arrange lichen substances into chemosyn-dromes of closely related substances The pres-ence or absence of these chemosyndromes maypotentially be used as characters to delimit spe-cies regarding differences involving the samechemosyndromes as intraspecific variation anddistinct chemosyndromes as evidence for inter-specific populations (Lumbsch 1994 Gowan1986)
2 The Dynamic Discipline of Species Delimitation hellip 33
Due to the fact that species delimitationstudies often incorporate a substantial biogeo-graphical or ecological component ecologicalniche modeling plays an increasing role in phy-logenetic and taxonomic research Niche mod-eling can provide evidence for ecologicalisolation between populations based on eitherconserved or divergent ecological niches andtherefore can provide additional evidence sup-porting lineage independence between putativespecies The application of ecological nichemodeling has been applied to species delimita-tion studies in a number of cases (Ruiz-Sanchezand Sosa 2010 Leacheacute et al 2009 Raxworthyet al 2007 Rissler and Apodaca 2007) althoughit has not been explicitly applied to assess speciesboundaries in lichen-forming fungi By mappingthe spatial distribution of environmental suit-ability of climatic variables for candidate speciesthe application of ecological niche modeling canbe particularly important in cases where specieshave allopatric distributions (Raxworthy et al2007)
Ecological niche modeling utilizes knownassociations between a speciesrsquo occurrencelocalities and environmental variables to defineabiotic conditions within which populations canbe maintained (Guisan and Thuiller 2005) Themethodological approach for modeling is basedon four general properties (i) The current knownspeciesrsquo localities is the dependent variable (ii)the distribution is modeled as a map composed ofgrid cells at a specified resolution (iii) a range ofenvironmental variables (eg temperature pre-cipitation and solar exposure) are collected todescribe the characteristics of each cell and (iv)classifying the degree to which each cell is eithersuitable or unsuitable for each species under arange of models (Guisan and Thuiller 2005)
Ecological data also have the potential to playan important role in understanding speciesboundaries in lichen-forming fungi For exampleNash and Zavada (1977) demonstrated that Xant-hoparmelia populations with distinct chemistriesoccurring in the northern portion of the SonoranDesert exhibit habitat selection among differentrock substrates within a region with relativelyuniform climate and topography In another case
the parmelioid species Parmelia mayi is mor-phologically indistinguishable from P saxatilisbut can be separated by bioclimatic features andgenetic and chemical characters (Molina et al2011) McCune and Printzen(2011) assess distri-butions and climatic niches of species in theLecanora varia group inwesternUSAandprovidea model that uses continental influence and annualtemperature as themajor factors predicting speciesdistributions The distribution of Usnea hirta alichen commonly used in air quality biomonitor-ing research was modeled for a section of theWhite River National Forest in central Coloradobased on the presence of U hirta at 72 biomoni-toring reference sites distributed in the inter-mountain western United States (Shrestha et al2012) The best model for predicting U hirtadistribution included four variablesmdashsolar radia-tion average monthly precipitation and averagemonthly minimum and maximum temperatures(Shrestha et al 2012) These studies supportthe potential use of ecological niche modelingmethods in species delimitation studies of lichen-forming fungi
24 Conclusions What AboutTaxonomy
In most cases species circumscription and tax-onomy requires some degree of qualitativejudgment and individual interpretation Integrat-ing multiple types of data into an empiricalframework for delimiting species boundarieswhere species boundaries can be tested within ahypothetico-deductive framework with diversedatasets can provide robust hypotheses of speciesboundaries and taxonomic stability (Yeates et al2011) However it has long been known toevolutionary biologists that distinct species donot need to have diagnosable morphologicaldifferences (Mayr 1963) and increasing avail-ability of genetic data has allowed researchers toidentify species and to rigorously test speciesboundaries with a level of precision that wasunimaginable a decade ago While analyticaladvances in statistical species delimitation havebeen largely based on genetic data the utility of
34 SD Leavitt et al
these approaches to formal taxonomy remainselusive Due to these challenges an eclecticapproach to delimiting species and cautionagainst the reliance on any single dataset ormethod is required when delimiting species
While results from many recent studies maycontradict traditional species boundaries acrossmany groups of lichen-forming fungi we areoptimistic that this research represents substantialprogress toward a more accurate perspective onspecies boundaries and diversity in fungi As aresult of ongoing research of species boundariesin lichen-forming fungi the taxonomic value ofmany phenotypes is now better understood ourunderstanding of ecological evolutionary andbiogeographic patterns has improved and we canbegin to better understand patterns of symbiontinteractions in lichens Integrating new data(including novel morphological characters andgenetic data) will be essential to accurately rep-resent species-level diversity across all groups oflichenized fungi Hopefully an improved per-spective on lichen diversity also increases ourappreciation of these incredible symbiotic sys-tems While we are strong advocates for theapplication of independent data types in devel-oping an integrative taxonomy there is anincreasing need to formally recognize the exis-tence of phenotypically cryptic species-levellineages in lichen-forming fungi (see Hibbettet al 2011) In some cases a molecular taxon-omy may provide the most practical approach toconsistent treatment of mycobiont species withinlichen groups where diagnostic morphologicalcharacters are unidentifiable or practically notfeasible (Leavitt et al 2013c d)
These are exciting times for taxonomists andphylogeneticists A closer look at lichen taxon-omy with the inclusion of new data will help usto better understand the diversity of these fasci-nating organisms accurately interpret distribu-tion patterns and play a more important role inmeaningful conservation practices Howeversome level of uncertainty will accompany pro-gress In many taxonomic groups our traditionalapproach for species identification will likelyneed to be substantially modified The search forcorroborating morphological support for cryptic
species identified using molecular data willrequire meticulous and creative approaches toassess phenotypic variation in potentially unor-thodox ways We are hopeful that lichenologistswho traditionally have been eager to include newmethods such as chromatography in their rou-tine identifications will be amenable to includemolecular techniques to their routine examina-tion of specimens for identification and classifi-cation Although this may prove difficult toachieve by single individuals especially citizenscientists that traditionally play an important rolein lichen taxonomy the increasing numberof collaborative projects in the discipline (egLumbsch et al 2011 Crespo et al 2010Gueidan et al 2009) make us optimistic thatbroad-scale collaborative approaches will facili-tate the inclusion of molecular data in lichenresearch at all levels
Acknowledgments We are indebted to various col-leagues for valuable thought-provoking discussionnotably Matthew Nelsen (University of Chicago) AnaCrespo (Universidad Complutense de Madrid) PradeepDivakar (Universidad Complutense de Madrid) BeckettSterner (The Field Museum) and Joyce Havstad (TheField Museum) We also thank anonymous reviewers whoprovided valuable comments that improved this chapterSupport by the US National Science Foundation isgratefully acknowledged (ldquoHidden diversity in parmelioidlichensrdquo DEB-0949147)
References
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Altermann S Leavitt SD Goward T Nelsen MPLumbsch HT (2014) How do you solve a problemlike Letharia A new look at cryptic species in lichen-forming fungi using Bayesian clustering and SNPSsfrom multilocus sequence data PLoS ONE 9(5)e97556 doi101371journalpone0097556
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2 The Dynamic Discipline of Species Delimitation hellip 35
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Culberson WL (1969) The use of chemistry in thesystematics of the lichens Taxon 18498ndash505
Culberson WL (1970) Chemosystematics and ecology oflichen-forming fungi Annu Rev Ecol Syst 1153ndash170
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Darwin C (1859) On the origin of species by means ofnatural selection or the preservation of favoured racesin the struggle for life J Murray London
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de Queiroz K (1999) The general lineage concept ofspecies and the defining properties of the speciescategory In Wilson RA (ed) Species new interdis-ciplinary essays MIT Press Cambridge pp 49ndash89
de Queiroz K (2007) Species concepts and speciesdelimitation Syst Biol 56(6)879ndash886 doi10108010635150701701083
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Degnan JH Rosenberg NA (2009) Gene tree discordancephylogenetic inference and the multispecies coales-cent Trends Ecol Evol 24(6)332ndash340 doihttpdxdoiorg101016jtree200901009
Del-Prado R Cubas P Lumbsch HT Divakar PK BlancoO de Paz GA Molina MC Crespo A (2010) Geneticdistances within and among species in monophyleticlineages of Parmeliaceae (Ascomycota) as a tool fortaxon delimitation Mol Phylogenet Evol 56(1)125ndash133 doi101016jympev201004014
Del-Prado R Divakar PK Crespo A (2011) Using geneticdistances in addition to ITS molecular phylogeny toidentify potential species in the Parmotrema reticul-atum complex a case study The Lichenologist 43(06)569ndash583 doi101017S0024282911000582
Del-Prado R Blanco O Lumbsch HT Divakar PK ElixJA Molina MC Crespo A (2013) Molecular phylog-eny and historical biogeography of the lichen-formingfungal genus Flavoparmelia (Ascomycota Parmelia-ceae) Taxon 62(5)928ndash939 doi101270562522
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Dettman JR Jacobson DJ Turner E Pringle A TaylorJW (2003b) Reproductive isolation and phylogeneticdivergence in Neurospora comparing methods ofspecies recognition in a model eukaryote Evolution57(12)2721 doi10155403-074
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Divakar PK Molina MC Lumbsch HT Crespo A (2005)Parmelia barrenoae a new lichen species related toParmelia sulcata (Parmeliaceae) based on molecularand morphological data The Lichenologist 37(01)37ndash46 doi101017S0024282904014641
Divakar PK Amo De paz G del Prado R Esslinger TLCrespo A (2007) Upper cortex anatomy corroboratesphylogenetic hypothesis in species of Physconia(Ascomycota Lecanoromycetes) Mycol Res 111(11)1311ndash1320 doi101016jmycres200708009
Divakar PK Figueras G Hladun N Crespo A (2010)Molecular phylogenetic studies reveal an undescribedspecies within the North American concept of Mela-nelixia glabra (Parmeliaceae) Fungal Divers 42(1)47ndash55 doi101007s13225-010-0027-3
Donoghue MJ Gauthier A (2004) Implementing thephylocode Trends Ecol Evol 19(6)281ndash282 doi101016jtree200404004
Eaton DAR Ree RH (2013) Inferring phylogeny andintrogression using RADseq data an example fromflowering plants (Pedicularis Orobanchaceae) SystBiol 62(5)689ndash706 doi101093sysbiosyt032
Edwards SV (2009) Is a new and general theory ofmolecular systematics emerging Evolution 63(1)1ndash19 doi101111j1558-5646200800549x
Edwards DL Knowles LL (2014) Species detection andindividual assignment in species delimitation canintegrative data increase efficacy Proc R Soc B BiolSci 281(1777) doi101098rspb20132765
Egan RS (1986) Correlations and non-correlations ofchemical variation patterns with lichen morphologyand geography The Bryologist 8999ndash110
Elix JA Corush J Lumbsch HT (2009) Triterpenechemosyndromes and subtle morphological characterscharacterise lineages in the Physcia aipolia group inAustralia (Ascomycota) Syst Biodivers 7(04)479ndash487 doi101017S1477200009990223
Emerson KJ Merz CR Catchen JM Hohenlohe PACresko WA Bradshaw WE Holzapfel CM (2010)Resolving postglacial phylogeography using high-throughput sequencing Proc Natl Acad Sci 107(37)16196ndash16200 doi101073pnas1006538107
Ence DD Carstens BC (2011) SpedeSTEM a rapid andaccurate method for species delimitation Mol EcolResour 11(3)473ndash480 doi101111j1755-0998201002947x
EvannoGRegnaut SGoudet J (2005)Detecting thenumberof clusters of individuals using the software STRUC-TURE a simulation study Mol Ecol 14(8)2611ndash2620doi101111j1365-294X200502553x
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Fan HH Kubatko LS (2011) Estimating species treesusing approximate Bayesian computation Mol Phylo-genet Evol 59(2)354ndash363 doi101016jympev201102019
Fernaacutendez-Mendoza F Printzen C (2013) Pleistoceneexpansion of the bipolar lichen Cetraria aculeata intothe Southern hemisphere Mol Ecol 22(7)1961ndash1983doi101111mec12210
Fernaacutendez-Mendoza F Domaschke S Garciacutea MA JordanP Martin MP Printzen C (2011) Population structure
2 The Dynamic Discipline of Species Delimitation hellip 37
of mycobionts and photobionts of the widespreadlichen Cetraria aculeata Mol Ecol 20(6)1208ndash1232doi101111j1365-294X201004993x
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Fujita MK Leacheacute AD Burbrink FT McGuire JA MoritzC (2012) Coalescent-based species delimitation in anintegrative taxonomy Trends Ecol Evol 27(9)480ndash488 doi101016jtree201204012
Gatesy J OrsquoGrady P Baker RH (1999) Corroborationamong data sets in simultaneous analysis hiddensupport for phylogenetic relationships among higherlevel Artiodactyl taxa Cladistics 15(3)271ndash313doi101111j1096-00311999tb00268x
Gladieux P Ropars J Badouin H Branca A Aguileta Gde Vienne DM Rodriacuteguez de la Vega RC Branco SGiraud T (2014) Fungal evolutionary genomicsprovides insight into the mechanisms of adaptivedivergence in eukaryotes Mol Ecol 23(4)753ndash773doi101111mec12631
Goffinet B Miadlikowska J Goward T (2003) Phyloge-netic inferences based on nrDNA sequences supportfive morphospecies within the Peltigera didactylacomplex (Lichenized Ascomycota) The Bryologist106(3)349ndash364 doi10163901
Gowan SP (1986) Evolution of secondary natural productsin the genus Porpidia (Ascomycata Porpidiaceae) AmJ Bot 73606
Griffin PC Hoffmann AA (2014) Limited genetic diver-gence among Australian alpine Poa tussock grassescoupled with regional structuring points to ongoinggene flow and taxonomic challenges Ann Bot doi101093aobmcu017
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Gueidan C Savi S Thues H Roux C Keller C Tibell LPrieto M Heimarsson S Breuss O Orange A FrobergL Wynns AA Navarro-Rosines P Krzewicka BPykaelae J Grube M Lutzoni F (2009) Genericclassification of the Verrucariaceae (Ascomycota)based on molecular and morphological evidencerecent progress and remaining challenges Taxon 58(1)184ndash208
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Guillot G Renaud S Ledevin R Michaux J Claude J(2012) A unifying model for the analysis of pheno-typic genetic and geographic data Syst Biol 61(6)897ndash911 doi101093sysbiosys038
Guisan A Thuiller W (2005) Predicting species distribu-tion offering more than simple habitat models EcolLett 8(9)993ndash1009 doi101111j1461-0248200500792x
Hale ME (1990) A synopsis of the lichen genusXanthoparmelia (Vainio) Hale (AscomycotinaParmeliaceae) Smithsonian Institution Press Wash-ington DC
Hamilton CA Hendrixson BE Brewer MS Bond JE(2014) An evaluation of sampling effects on multipleDNA barcoding methods leads to an integrativeapproach for delimiting species a case study ofthe North American tarantula genus Aphonopelma(Araneae Mygalomorphae Theraphosidae) MolPhylogenet Evol 7179ndash93 doi101016jympev201311007
Hausdorf B (2011) Progress toward a general speciesconcept Evolution 65(4)923ndash931 doi101111j1558-5646201101231x
Hausdorf B Hennig C (2010) Species delimitation usingdominant and codominant multilocus markers SystBiol 59(5)491ndash503 doi101093sysbiosyq039
Hawksworth DL (1976) Lichen chemotaxonomy InBailey RH (ed) Lichenology progress and problemsAcademic London pp 139ndash184
Hebert PDN Cywinska A Ball SL deWaard JR (2003)Biological identifications through DNA barcodesProc R Soc Lond B Biol Sci 270(1512)313ndash321doi101098rspb20022218
Hebert PDN Stoeckle MY Zemlak TS Francis CM(2004) Identification of birds through DNA barcodesPLoS Biol 2(10)e312 doi101371journalpbio0020312
Heled J Drummond AJ (2010) Bayesian inference ofspecies trees from multilocus data Mol Biol Evol 27(3)570ndash580 doi101093molbevmsp274
Hey J (2006) On the failure of modern species conceptsTrends Ecol Evol 21(8)447ndash450 doi101016jtree200605011
Hibbett DS OhmanA Glotzer D NuhnMKirk P NilssonRH (2011) Progress in molecular and morphologicaltaxon discovery in Fungi and options for formalclassification on environmental sequences Fungal BiolRev 2538ndash47 doi101016jfbr201101001
Houmlgnabba F Wedin M (2003) Molecular phylogeny ofthe Sphaerophorus globosus species complex Cladis-tics 19(3)224ndash232 doi101111j1096-00312003tb00365x
Hudson RR Coyne JA (2002) Mathematical conse-quences of the genealogical species concept Evolu-tion 56(8)1557 doi101111j0014-38202002tb01467x
Huelsenbeck JP Andolfatto P Huelsenbeck ET (2011)Structurama Bayesian inference of population struc-ture Evol Bioinf Online 7(2011)55ndash59 doi104137EBOS6761
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Kiss L (2012) Limits of nuclear ribosomal DNA internaltranscribed spacer (ITS) sequences as species barcodesfor fungi Proc Natl Acad Sci 109(27)E1811ndashE1811doi101073pnas1207143109
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Knowles LL Carstens BC (2007) Delimiting specieswithout monophyletic gene trees Syst Biol 56(6)887ndash895 doi10108010635150701701091
Kroken S Taylor JW (2001) A gene genealogical approachto recognize phylogenetic species boundaries in thelichenized fungus Letharia Mycologia 93(1)38ndash53
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Leacheacute AD (2009) Species tree discordance traces tophylogeographic clade boundaries in North Americanfence lizards (Sceloporus) Syst Biol 58(6)547ndash559doi101093sysbiosyp057
Leacheacute AD Fujita MK (2010) Bayesian species delim-itation in West African forest geckos (Hemidactylusfasciatus) Proc R Soc B Biol Sci 2773071ndash3077doi101098rspb20100662
Leacheacute AD Koo M Spencer C Papenfuss T Fisher RMcGuire J (2009) Quantifying ecological morpho-logical and genetic variation to delimit species in thecoast horned lizard species complex (Phrynosoma)Proc Natl Acad Sci USA 10612418ndash12423 doi101073pnas0906380106
Leacheacute AD Fujita MK Minin VN Bouckaert RR (2014)Species delimitation using genome-wide SNP dataSystematic Biology doi101093sysbiosyu018
Leavitt SD Fankhauser JD Leavitt DH Porter LDJohnson LA St Clair LL (2011a) Complex patterns ofspeciation in cosmopolitan ldquorock posyrdquo lichensmdashdiscovering and delimiting cryptic fungal species in
the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
Leavitt SD Esslinger TL Lumbsch HT (2012c) Neogene-dominated diversification in neotropical montanelichens dating divergence events in the lichen-form-ing fungal genus Oropogon (Parmeliaceae) Am J Bot99(11)1764ndash1777 doi103732ajb1200146
Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Lumbsch HT St Clair LL(2013c) DNA barcode identification of lichen-formingfungal species in the Rhizoplaca melanophthalmaspecies-complex (Lecanorales Lecanoraceae) includ-ing five new species MycoKeys 71ndash22 doi103897mycokeys74508
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Vondraacutek J Thorsten Lum-bsch H Clair LLS (2013d) Local representation ofglobal diversity in a cosmopolitan lichen-formingfungal species complex (Rhizoplaca Ascomycota)J Biogeogr 40(9)1792ndash1806 doi101111jbi12118
Leavitt SD Lumbsch HT Stenroos S St Clair LL(2013e) Pleistocene speciation in North American
2 The Dynamic Discipline of Species Delimitation hellip 39
lichenized fungi and the impact of alternative speciescircumscriptions and rates of molecular evolution ondivergence estimates PLoS ONE 8(12)e85240doi101371journalpone0085240
Leavitt SD Esslinger TL Hansen ES Divakar PK CrespoA Loomis BF Lumbsch HT (2014) DNA barcoding ofbrown Parmeliae (Parmeliaceae) species a molecularapproach for accurate specimen identification empha-sizing species in Greenland Organ Divers Evol 14(1)11ndash20 doi101007s13127-013-0147-1
Leliaert F Verbruggen H Vanormelingen P Steen FLopez-Bautista JM Zuccarello GC De Clerck O(2014) DNA-based species delimitation in algae Eur JPhycol 49(2) doi101080096702622014904524
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Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
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Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
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Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
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Mayr E (1970) Populations species and evolutionBelknap Press of Harvard University Press Cambridge
McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
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40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
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Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
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Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
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OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
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Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
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Park S-Y Choi J Kim JA Jeong M-H Kim S Lee Y-HHur J-S (2013a) Draft genome sequence of Cladoniamacilenta KoLRI003786 a lichen-forming fungusproducing biruloquinone Genome Announcements 1(5)e00695-13 doi101128genomeA00695-13
Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
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Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
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Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
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phylogeny of the genus Lecania (Ramalinaceaelichenized Ascomycota) Mycol Res 111(5)581ndash591doi101016jmycres200703001
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Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
Ross KG Gotzek D Ascunce MS Shoemaker DD (2010)Species delimitation a case study in a problematic anttaxon Syst Biol 59(2)162ndash184 doi101093sysbiosyp089
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Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
morphological characters Antarctic Sci 22 (SpecialIssue 06)727ndash741 doi101017S0954102010000477
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Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
Simpson GG (1951) The species concept Evolution5285ndash298
Sites JW Marshall JC (2003) Delimiting species a renais-sance issue in systematic biology Trends Ecol Evol18462ndash470 doi101016S0169-5347(03)00184-8
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Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
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Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
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Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
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Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
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Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
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Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
microhabitat requirements and data resolutionTherefore we advocate a cautious approach toselecting appropriate and relevant data forassessing species boundaries
In spite of the potential challenges and limi-tations of using phenotypic data these traits haveprovided a plethora of valuable information forunderstanding species boundaries For examplein the Melanelixia fuliginosa group (Parmelia-ceae) a morphometric analysis using color isi-dia and marginal zone free of isidia ascharacters revealed a general pattern of differ-entiation between material formerly recognizedas subspecies fuliginosa and glabratula in spiteof the fact that considerable overlap betweengroups occurs in some characters individually(Arup and Berlin 2011) Furthermore the dis-tinction of the two groups was supported by aphylogenetic analysis of the ITS marker andecological differences with M fuliginosaoccurring predominantly on rock and M glab-ratula on bark (Arup and Berlin 2011) Thisstudy of the M fuliginosa group provides a fit-ting example of using multiple independentsuites of data including ecology morphologyand genetic information to establish a robusthypothesis of species boundaries
In lichen systematics phenotypic dataincluding thallus organization secondary metab-olites mode of reproduction ascoma-type andontogeny ascus and ascospore characters havehistorically played a prominent role (Printzen2009) Ascomatal characters have traditionallyheld a major role in higher-level classification(Printzen 2009) in contrast to species-levelclassification which also tends to include a widearray of vegetative and chemical charactersCommonly assessed morphological charactersinclude thallus form and size cortical features(eg maculae and pseudocyphellae) presenceformcolor of attachment structure (eg rhizines)and reproductive mode (ascomata vs vegetativediaspores) (Printzen 2009) Different morpho-logical types of vegetative diasporesmdashcorticatedisidia and ecorticated sorediamdashand their locationare commonly used to distinguish speciesAscomatal characters including morphologylocation (laminal vs marginal) position (sessile
vs immersed) presence of thalline margins colorof an apothecial disc and presence or color ofpruina are also commonly used in speciesdelimitations of lichen-forming fungi (Printzen2009) Other important characters may includethalline characters form color size and septationof ascospores size and form and structure of ascithe hamathecium type of epihymenium andhypothecium and the type of excipulum orperidium conidiomatal characters etc (Printzen2009)
Assessments of secondary metabolites hasplayed an important role in lichen taxonomybeginning with the introduction of simple spottests by Nylander (1866a b) The use of chem-istry in lichen taxonomy has been discussed indetail in numerous reviews (Lumbsch 1998a bRogers 1989 Brodo 1986 Egan 1986 Leuckert1985 Brodo 1978 Hawksworth 1976 Culber-son 1969 1970) and we refer readers to thesevaluable sources for a more comprehensive per-spective on lichen chemistry In short extrolites(secondary metabolites) belong to various clas-ses the most common and diverse include dep-sides depsidones chlorinated xanthones andanthraquinones (Lumbsch 2002 Culberson1969) The presence or absence of specificextrolites or their replacements by another sub-stance is widely used to distinguish speciesparticularly when correlated with differences ingeographic distributions However if morpho-logical or geographical differences betweenpopulations containing different extrolites are notapparent the taxonomic significance has beendisputed with some authors distinguishing themas species and others preferring to regard them aschemical races within a species In addition tosimply using the presence or absence of extro-lites Culberson and Culberson (1976) proposedto arrange lichen substances into chemosyn-dromes of closely related substances The pres-ence or absence of these chemosyndromes maypotentially be used as characters to delimit spe-cies regarding differences involving the samechemosyndromes as intraspecific variation anddistinct chemosyndromes as evidence for inter-specific populations (Lumbsch 1994 Gowan1986)
2 The Dynamic Discipline of Species Delimitation hellip 33
Due to the fact that species delimitationstudies often incorporate a substantial biogeo-graphical or ecological component ecologicalniche modeling plays an increasing role in phy-logenetic and taxonomic research Niche mod-eling can provide evidence for ecologicalisolation between populations based on eitherconserved or divergent ecological niches andtherefore can provide additional evidence sup-porting lineage independence between putativespecies The application of ecological nichemodeling has been applied to species delimita-tion studies in a number of cases (Ruiz-Sanchezand Sosa 2010 Leacheacute et al 2009 Raxworthyet al 2007 Rissler and Apodaca 2007) althoughit has not been explicitly applied to assess speciesboundaries in lichen-forming fungi By mappingthe spatial distribution of environmental suit-ability of climatic variables for candidate speciesthe application of ecological niche modeling canbe particularly important in cases where specieshave allopatric distributions (Raxworthy et al2007)
Ecological niche modeling utilizes knownassociations between a speciesrsquo occurrencelocalities and environmental variables to defineabiotic conditions within which populations canbe maintained (Guisan and Thuiller 2005) Themethodological approach for modeling is basedon four general properties (i) The current knownspeciesrsquo localities is the dependent variable (ii)the distribution is modeled as a map composed ofgrid cells at a specified resolution (iii) a range ofenvironmental variables (eg temperature pre-cipitation and solar exposure) are collected todescribe the characteristics of each cell and (iv)classifying the degree to which each cell is eithersuitable or unsuitable for each species under arange of models (Guisan and Thuiller 2005)
Ecological data also have the potential to playan important role in understanding speciesboundaries in lichen-forming fungi For exampleNash and Zavada (1977) demonstrated that Xant-hoparmelia populations with distinct chemistriesoccurring in the northern portion of the SonoranDesert exhibit habitat selection among differentrock substrates within a region with relativelyuniform climate and topography In another case
the parmelioid species Parmelia mayi is mor-phologically indistinguishable from P saxatilisbut can be separated by bioclimatic features andgenetic and chemical characters (Molina et al2011) McCune and Printzen(2011) assess distri-butions and climatic niches of species in theLecanora varia group inwesternUSAandprovidea model that uses continental influence and annualtemperature as themajor factors predicting speciesdistributions The distribution of Usnea hirta alichen commonly used in air quality biomonitor-ing research was modeled for a section of theWhite River National Forest in central Coloradobased on the presence of U hirta at 72 biomoni-toring reference sites distributed in the inter-mountain western United States (Shrestha et al2012) The best model for predicting U hirtadistribution included four variablesmdashsolar radia-tion average monthly precipitation and averagemonthly minimum and maximum temperatures(Shrestha et al 2012) These studies supportthe potential use of ecological niche modelingmethods in species delimitation studies of lichen-forming fungi
24 Conclusions What AboutTaxonomy
In most cases species circumscription and tax-onomy requires some degree of qualitativejudgment and individual interpretation Integrat-ing multiple types of data into an empiricalframework for delimiting species boundarieswhere species boundaries can be tested within ahypothetico-deductive framework with diversedatasets can provide robust hypotheses of speciesboundaries and taxonomic stability (Yeates et al2011) However it has long been known toevolutionary biologists that distinct species donot need to have diagnosable morphologicaldifferences (Mayr 1963) and increasing avail-ability of genetic data has allowed researchers toidentify species and to rigorously test speciesboundaries with a level of precision that wasunimaginable a decade ago While analyticaladvances in statistical species delimitation havebeen largely based on genetic data the utility of
34 SD Leavitt et al
these approaches to formal taxonomy remainselusive Due to these challenges an eclecticapproach to delimiting species and cautionagainst the reliance on any single dataset ormethod is required when delimiting species
While results from many recent studies maycontradict traditional species boundaries acrossmany groups of lichen-forming fungi we areoptimistic that this research represents substantialprogress toward a more accurate perspective onspecies boundaries and diversity in fungi As aresult of ongoing research of species boundariesin lichen-forming fungi the taxonomic value ofmany phenotypes is now better understood ourunderstanding of ecological evolutionary andbiogeographic patterns has improved and we canbegin to better understand patterns of symbiontinteractions in lichens Integrating new data(including novel morphological characters andgenetic data) will be essential to accurately rep-resent species-level diversity across all groups oflichenized fungi Hopefully an improved per-spective on lichen diversity also increases ourappreciation of these incredible symbiotic sys-tems While we are strong advocates for theapplication of independent data types in devel-oping an integrative taxonomy there is anincreasing need to formally recognize the exis-tence of phenotypically cryptic species-levellineages in lichen-forming fungi (see Hibbettet al 2011) In some cases a molecular taxon-omy may provide the most practical approach toconsistent treatment of mycobiont species withinlichen groups where diagnostic morphologicalcharacters are unidentifiable or practically notfeasible (Leavitt et al 2013c d)
These are exciting times for taxonomists andphylogeneticists A closer look at lichen taxon-omy with the inclusion of new data will help usto better understand the diversity of these fasci-nating organisms accurately interpret distribu-tion patterns and play a more important role inmeaningful conservation practices Howeversome level of uncertainty will accompany pro-gress In many taxonomic groups our traditionalapproach for species identification will likelyneed to be substantially modified The search forcorroborating morphological support for cryptic
species identified using molecular data willrequire meticulous and creative approaches toassess phenotypic variation in potentially unor-thodox ways We are hopeful that lichenologistswho traditionally have been eager to include newmethods such as chromatography in their rou-tine identifications will be amenable to includemolecular techniques to their routine examina-tion of specimens for identification and classifi-cation Although this may prove difficult toachieve by single individuals especially citizenscientists that traditionally play an important rolein lichen taxonomy the increasing numberof collaborative projects in the discipline (egLumbsch et al 2011 Crespo et al 2010Gueidan et al 2009) make us optimistic thatbroad-scale collaborative approaches will facili-tate the inclusion of molecular data in lichenresearch at all levels
Acknowledgments We are indebted to various col-leagues for valuable thought-provoking discussionnotably Matthew Nelsen (University of Chicago) AnaCrespo (Universidad Complutense de Madrid) PradeepDivakar (Universidad Complutense de Madrid) BeckettSterner (The Field Museum) and Joyce Havstad (TheField Museum) We also thank anonymous reviewers whoprovided valuable comments that improved this chapterSupport by the US National Science Foundation isgratefully acknowledged (ldquoHidden diversity in parmelioidlichensrdquo DEB-0949147)
References
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Altermann S Leavitt SD Goward T Nelsen MPLumbsch HT (2014) How do you solve a problemlike Letharia A new look at cryptic species in lichen-forming fungi using Bayesian clustering and SNPSsfrom multilocus sequence data PLoS ONE 9(5)e97556 doi101371journalpone0097556
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2 The Dynamic Discipline of Species Delimitation hellip 35
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Corander J Marttinen P Mantyniemi S (2006) Bayesianidentification of stock mixtures from molecular markerdata Fish Bull 104550ndash558
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Crespo A Peacuterez-Ortega S (2009) Cryptic species andspecies pairs in lichens a discussion on the relation-ship between molecular phylogenies and morpholog-ical characters Anales del Jardin Botanico de Madrid66(S1)71ndash81 doi103989ajbm2225
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Culberson WL (1969) The use of chemistry in thesystematics of the lichens Taxon 18498ndash505
Culberson WL (1970) Chemosystematics and ecology oflichen-forming fungi Annu Rev Ecol Syst 1153ndash170
Culberson CF Culberson WL (1976) Chemosyndromicvariation in lichens Syst Bot 1325ndash339
Darwin C (1859) On the origin of species by means ofnatural selection or the preservation of favoured racesin the struggle for life J Murray London
Dayrat B (2005) Towards integrative taxonomy Biol JLinn Soc 85(3)407ndash415 doi101111j1095-8312200500503x
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de Queiroz K (1998) The general lineage concept ofspecies species criteria and the process of speciationa conceptual unification and terminological recom-mendations In Howard DJ Berlocher SH (eds)Endless forms species and speciation Oxford Uni-versity Press Oxford pp 57ndash75
de Queiroz K (1999) The general lineage concept ofspecies and the defining properties of the speciescategory In Wilson RA (ed) Species new interdis-ciplinary essays MIT Press Cambridge pp 49ndash89
de Queiroz K (2007) Species concepts and speciesdelimitation Syst Biol 56(6)879ndash886 doi10108010635150701701083
de Queiroz A Donoghue MJ Kim J (1995) Separateversus combined analysis of phylogenetic evidenceAnnu Rev Ecol Syst 26(1)657ndash681 doi101146annureves26110195003301
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Degnan JH Rosenberg NA (2009) Gene tree discordancephylogenetic inference and the multispecies coales-cent Trends Ecol Evol 24(6)332ndash340 doihttpdxdoiorg101016jtree200901009
Del-Prado R Cubas P Lumbsch HT Divakar PK BlancoO de Paz GA Molina MC Crespo A (2010) Geneticdistances within and among species in monophyleticlineages of Parmeliaceae (Ascomycota) as a tool fortaxon delimitation Mol Phylogenet Evol 56(1)125ndash133 doi101016jympev201004014
Del-Prado R Divakar PK Crespo A (2011) Using geneticdistances in addition to ITS molecular phylogeny toidentify potential species in the Parmotrema reticul-atum complex a case study The Lichenologist 43(06)569ndash583 doi101017S0024282911000582
Del-Prado R Blanco O Lumbsch HT Divakar PK ElixJA Molina MC Crespo A (2013) Molecular phylog-eny and historical biogeography of the lichen-formingfungal genus Flavoparmelia (Ascomycota Parmelia-ceae) Taxon 62(5)928ndash939 doi101270562522
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Dettman JR Jacobson DJ Turner E Pringle A TaylorJW (2003b) Reproductive isolation and phylogeneticdivergence in Neurospora comparing methods ofspecies recognition in a model eukaryote Evolution57(12)2721 doi10155403-074
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Divakar PK Molina MC Lumbsch HT Crespo A (2005)Parmelia barrenoae a new lichen species related toParmelia sulcata (Parmeliaceae) based on molecularand morphological data The Lichenologist 37(01)37ndash46 doi101017S0024282904014641
Divakar PK Amo De paz G del Prado R Esslinger TLCrespo A (2007) Upper cortex anatomy corroboratesphylogenetic hypothesis in species of Physconia(Ascomycota Lecanoromycetes) Mycol Res 111(11)1311ndash1320 doi101016jmycres200708009
Divakar PK Figueras G Hladun N Crespo A (2010)Molecular phylogenetic studies reveal an undescribedspecies within the North American concept of Mela-nelixia glabra (Parmeliaceae) Fungal Divers 42(1)47ndash55 doi101007s13225-010-0027-3
Donoghue MJ Gauthier A (2004) Implementing thephylocode Trends Ecol Evol 19(6)281ndash282 doi101016jtree200404004
Eaton DAR Ree RH (2013) Inferring phylogeny andintrogression using RADseq data an example fromflowering plants (Pedicularis Orobanchaceae) SystBiol 62(5)689ndash706 doi101093sysbiosyt032
Edwards SV (2009) Is a new and general theory ofmolecular systematics emerging Evolution 63(1)1ndash19 doi101111j1558-5646200800549x
Edwards DL Knowles LL (2014) Species detection andindividual assignment in species delimitation canintegrative data increase efficacy Proc R Soc B BiolSci 281(1777) doi101098rspb20132765
Egan RS (1986) Correlations and non-correlations ofchemical variation patterns with lichen morphologyand geography The Bryologist 8999ndash110
Elix JA Corush J Lumbsch HT (2009) Triterpenechemosyndromes and subtle morphological characterscharacterise lineages in the Physcia aipolia group inAustralia (Ascomycota) Syst Biodivers 7(04)479ndash487 doi101017S1477200009990223
Emerson KJ Merz CR Catchen JM Hohenlohe PACresko WA Bradshaw WE Holzapfel CM (2010)Resolving postglacial phylogeography using high-throughput sequencing Proc Natl Acad Sci 107(37)16196ndash16200 doi101073pnas1006538107
Ence DD Carstens BC (2011) SpedeSTEM a rapid andaccurate method for species delimitation Mol EcolResour 11(3)473ndash480 doi101111j1755-0998201002947x
EvannoGRegnaut SGoudet J (2005)Detecting thenumberof clusters of individuals using the software STRUC-TURE a simulation study Mol Ecol 14(8)2611ndash2620doi101111j1365-294X200502553x
Falush D Stephens M Pritchard JK (2003) Inference ofpopulation structure using multilocus genotype datalinked loci and correlated allele frequencies Genetics164(4)1567ndash1587 doi101111j1471-8286200701758x
Fan HH Kubatko LS (2011) Estimating species treesusing approximate Bayesian computation Mol Phylo-genet Evol 59(2)354ndash363 doi101016jympev201102019
Fernaacutendez-Mendoza F Printzen C (2013) Pleistoceneexpansion of the bipolar lichen Cetraria aculeata intothe Southern hemisphere Mol Ecol 22(7)1961ndash1983doi101111mec12210
Fernaacutendez-Mendoza F Domaschke S Garciacutea MA JordanP Martin MP Printzen C (2011) Population structure
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of mycobionts and photobionts of the widespreadlichen Cetraria aculeata Mol Ecol 20(6)1208ndash1232doi101111j1365-294X201004993x
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Fujisawa T Barraclough TG (2013) Delimiting speciesusing single-locus data and the generalized mixed yulecoalescent (GMYC) approach a revised method andevaluation on simulated datasets Syst Biol 62(5)707ndash724 doi101093sysbiosyt033
Fujita MK Leacheacute AD Burbrink FT McGuire JA MoritzC (2012) Coalescent-based species delimitation in anintegrative taxonomy Trends Ecol Evol 27(9)480ndash488 doi101016jtree201204012
Gatesy J OrsquoGrady P Baker RH (1999) Corroborationamong data sets in simultaneous analysis hiddensupport for phylogenetic relationships among higherlevel Artiodactyl taxa Cladistics 15(3)271ndash313doi101111j1096-00311999tb00268x
Gladieux P Ropars J Badouin H Branca A Aguileta Gde Vienne DM Rodriacuteguez de la Vega RC Branco SGiraud T (2014) Fungal evolutionary genomicsprovides insight into the mechanisms of adaptivedivergence in eukaryotes Mol Ecol 23(4)753ndash773doi101111mec12631
Goffinet B Miadlikowska J Goward T (2003) Phyloge-netic inferences based on nrDNA sequences supportfive morphospecies within the Peltigera didactylacomplex (Lichenized Ascomycota) The Bryologist106(3)349ndash364 doi10163901
Gowan SP (1986) Evolution of secondary natural productsin the genus Porpidia (Ascomycata Porpidiaceae) AmJ Bot 73606
Griffin PC Hoffmann AA (2014) Limited genetic diver-gence among Australian alpine Poa tussock grassescoupled with regional structuring points to ongoinggene flow and taxonomic challenges Ann Bot doi101093aobmcu017
Grube M Hawksworth DL (2007) Trouble with lichenthe re-evaluation and re-interpretation of thallus formand fruit body types in the molecular era Mycol Res111(9)1116ndash1132 doi101016jmycres200704008
Gueidan C Savi S Thues H Roux C Keller C Tibell LPrieto M Heimarsson S Breuss O Orange A FrobergL Wynns AA Navarro-Rosines P Krzewicka BPykaelae J Grube M Lutzoni F (2009) Genericclassification of the Verrucariaceae (Ascomycota)based on molecular and morphological evidencerecent progress and remaining challenges Taxon 58(1)184ndash208
Guillot G Mortier F Estoup A (2005) Geneland acomputer package for landscape genetics Mol EcolNotes 5(3)712ndash715 doi101111j1471-8286200501031x
Guillot G Renaud S Ledevin R Michaux J Claude J(2012) A unifying model for the analysis of pheno-typic genetic and geographic data Syst Biol 61(6)897ndash911 doi101093sysbiosys038
Guisan A Thuiller W (2005) Predicting species distribu-tion offering more than simple habitat models EcolLett 8(9)993ndash1009 doi101111j1461-0248200500792x
Hale ME (1990) A synopsis of the lichen genusXanthoparmelia (Vainio) Hale (AscomycotinaParmeliaceae) Smithsonian Institution Press Wash-ington DC
Hamilton CA Hendrixson BE Brewer MS Bond JE(2014) An evaluation of sampling effects on multipleDNA barcoding methods leads to an integrativeapproach for delimiting species a case study ofthe North American tarantula genus Aphonopelma(Araneae Mygalomorphae Theraphosidae) MolPhylogenet Evol 7179ndash93 doi101016jympev201311007
Hausdorf B (2011) Progress toward a general speciesconcept Evolution 65(4)923ndash931 doi101111j1558-5646201101231x
Hausdorf B Hennig C (2010) Species delimitation usingdominant and codominant multilocus markers SystBiol 59(5)491ndash503 doi101093sysbiosyq039
Hawksworth DL (1976) Lichen chemotaxonomy InBailey RH (ed) Lichenology progress and problemsAcademic London pp 139ndash184
Hebert PDN Cywinska A Ball SL deWaard JR (2003)Biological identifications through DNA barcodesProc R Soc Lond B Biol Sci 270(1512)313ndash321doi101098rspb20022218
Hebert PDN Stoeckle MY Zemlak TS Francis CM(2004) Identification of birds through DNA barcodesPLoS Biol 2(10)e312 doi101371journalpbio0020312
Heled J Drummond AJ (2010) Bayesian inference ofspecies trees from multilocus data Mol Biol Evol 27(3)570ndash580 doi101093molbevmsp274
Hey J (2006) On the failure of modern species conceptsTrends Ecol Evol 21(8)447ndash450 doi101016jtree200605011
Hibbett DS OhmanA Glotzer D NuhnMKirk P NilssonRH (2011) Progress in molecular and morphologicaltaxon discovery in Fungi and options for formalclassification on environmental sequences Fungal BiolRev 2538ndash47 doi101016jfbr201101001
Houmlgnabba F Wedin M (2003) Molecular phylogeny ofthe Sphaerophorus globosus species complex Cladis-tics 19(3)224ndash232 doi101111j1096-00312003tb00365x
Hudson RR Coyne JA (2002) Mathematical conse-quences of the genealogical species concept Evolu-tion 56(8)1557 doi101111j0014-38202002tb01467x
Huelsenbeck JP Andolfatto P Huelsenbeck ET (2011)Structurama Bayesian inference of population struc-ture Evol Bioinf Online 7(2011)55ndash59 doi104137EBOS6761
Jones G Oxelman B (2014) DISSECT an assignment-free Bayesian discovery method for species delimita-tion under the multispecies coalescent Bioinformaticsdoi101101003178
38 SD Leavitt et al
Kekkonen M Hebert PD (2014) DNA barcode-baseddelineation of putative species efficient start fortaxonomic workflows Mol Ecol Res doi1011111755-099812233
Kelly LJ Hollingsworth PM Coppins BJ Ellis CJHarrold P Tosh J Yahr R (2011) DNA barcoding oflichenized fungi demonstrates high identification suc-cess in a floristic context New Phytol 191(1)288ndash300doi101111j1469-8137201103677x
Kiss L (2012) Limits of nuclear ribosomal DNA internaltranscribed spacer (ITS) sequences as species barcodesfor fungi Proc Natl Acad Sci 109(27)E1811ndashE1811doi101073pnas1207143109
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Knowles LL Carstens BC (2007) Delimiting specieswithout monophyletic gene trees Syst Biol 56(6)887ndash895 doi10108010635150701701091
Kroken S Taylor JW (2001) A gene genealogical approachto recognize phylogenetic species boundaries in thelichenized fungus Letharia Mycologia 93(1)38ndash53
Kubatko LS Degnan JH (2007) Inconsistency of phylo-genetic estimates from concatenated data under coa-lescence Syst Biol 56(1)17ndash24 doi10108010635150601146041
Latch EK Dharmarajan G Glaubitz JC Rhodes OE Jr(2006) Relative performance of Bayesian clusteringsoftware for inferring population substructure andindividual assignments at low levels of populationdifferentiation Conserv Genet 7(2)295ndash302 doi101007s10592-005-9098-1
Lawrey JD (1986) Biological role of lichen substancesThe Bryologist 89(2)111ndash122
Le Gac M Hood ME Fournier E Giraud T (2007)Phylogenetic evidence of host-specific cryptic speciesin the anther smut fungus Evolution 61(1)15ndash26doi101111j1558-5646200700002x
Leacheacute AD (2009) Species tree discordance traces tophylogeographic clade boundaries in North Americanfence lizards (Sceloporus) Syst Biol 58(6)547ndash559doi101093sysbiosyp057
Leacheacute AD Fujita MK (2010) Bayesian species delim-itation in West African forest geckos (Hemidactylusfasciatus) Proc R Soc B Biol Sci 2773071ndash3077doi101098rspb20100662
Leacheacute AD Koo M Spencer C Papenfuss T Fisher RMcGuire J (2009) Quantifying ecological morpho-logical and genetic variation to delimit species in thecoast horned lizard species complex (Phrynosoma)Proc Natl Acad Sci USA 10612418ndash12423 doi101073pnas0906380106
Leacheacute AD Fujita MK Minin VN Bouckaert RR (2014)Species delimitation using genome-wide SNP dataSystematic Biology doi101093sysbiosyu018
Leavitt SD Fankhauser JD Leavitt DH Porter LDJohnson LA St Clair LL (2011a) Complex patterns ofspeciation in cosmopolitan ldquorock posyrdquo lichensmdashdiscovering and delimiting cryptic fungal species in
the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
Leavitt SD Esslinger TL Lumbsch HT (2012c) Neogene-dominated diversification in neotropical montanelichens dating divergence events in the lichen-form-ing fungal genus Oropogon (Parmeliaceae) Am J Bot99(11)1764ndash1777 doi103732ajb1200146
Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Lumbsch HT St Clair LL(2013c) DNA barcode identification of lichen-formingfungal species in the Rhizoplaca melanophthalmaspecies-complex (Lecanorales Lecanoraceae) includ-ing five new species MycoKeys 71ndash22 doi103897mycokeys74508
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Vondraacutek J Thorsten Lum-bsch H Clair LLS (2013d) Local representation ofglobal diversity in a cosmopolitan lichen-formingfungal species complex (Rhizoplaca Ascomycota)J Biogeogr 40(9)1792ndash1806 doi101111jbi12118
Leavitt SD Lumbsch HT Stenroos S St Clair LL(2013e) Pleistocene speciation in North American
2 The Dynamic Discipline of Species Delimitation hellip 39
lichenized fungi and the impact of alternative speciescircumscriptions and rates of molecular evolution ondivergence estimates PLoS ONE 8(12)e85240doi101371journalpone0085240
Leavitt SD Esslinger TL Hansen ES Divakar PK CrespoA Loomis BF Lumbsch HT (2014) DNA barcoding ofbrown Parmeliae (Parmeliaceae) species a molecularapproach for accurate specimen identification empha-sizing species in Greenland Organ Divers Evol 14(1)11ndash20 doi101007s13127-013-0147-1
Leliaert F Verbruggen H Vanormelingen P Steen FLopez-Bautista JM Zuccarello GC De Clerck O(2014) DNA-based species delimitation in algae Eur JPhycol 49(2) doi101080096702622014904524
Leuckert C (1985) Probleme der Flechten-Chemotaxon-omiemdashStoffkombinationen und ihre taxonomischeWertung Ber Deut Bot Ges 98401ndash408
Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
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Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
Lumbsch HT (1994) Die Lecanora subfusca-Gruppe inAustralasien J Hattori Bot Lab 771ndash175
Lumbsch HT (1998a) Taxonomic use of metabolic data inlichen-forming fungi In Frisvad JC Bridge PDArora DK (eds) Chemical Fungal Taxonomy MarcelDekker New York pp 345ndash387
Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
Mayden RL (1997) A hierarchy of species concepts thedenouement in the saga of the species problem InClaridge MF Dawah HA Wilson MR (eds) Speciesthe units of biodiversity Chapman amp Hall Londonpp 381ndash424
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Mayr E (1970) Populations species and evolutionBelknap Press of Harvard University Press Cambridge
McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
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40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
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Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
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Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
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Nylander W (1866a) Circa novum in studio Lichenumcriterium chemicum Flora 49198ndash201
Nylander W (1866b) Hypochlorite of lime and hydrate ofpotash Two new criteria for the study of lichens(Tanslated and communicated by the Rev W ALeighton) Bot J Linn Soc 9358ndash365
OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
OrsquoNeill EM Schwartz R Bullock CT Williams JSShaffer HB Aguilar-Miguel X Parra-Olea G Weis-rock DW (2013) Parallel tagged amplicon sequencingreveals major lineages and phylogenetic structure inthe North American tiger salamander (Ambystomatigrinum) species complex Mol Ecol 22(1)111ndash129doi101111mec12049
Orock EA Leavitt SD Fonge BA St Clair LL LumbschHT (2012) DNA-based identification of lichen-form-ing fungi Can publicly available sequence databasesaid in lichen diversity inventories of Mount Cameroon(West Africa) The Lichenologist 44(6)833ndash839doi101017S0024282912000424
Otaacutelora MAG Martiacutenez I Aragoacuten G Molina MC (2010)Phylogeography and divergence date estimates of alichen species complex with a disjunct distributionpattern Am J Bot 97(2)216ndash223 doi103732ajb0900064
Padial J Castroviejo-Fisher S Kohler J Vila C ChaparroJ De la Riva I (2009) Deciphering the products ofevolution at the species level the need for anintegrative taxonomy Zoolog Scr 38(4)431ndash447doi101111j1463-6409200800381x
Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
Papong K Luumlcking R Thammathaworn A BoonpragobK (2009) Four new taxa of Chroodiscus (thelotremoidGraphidaceae) from Southeast Asia The Bryologist112(1)152ndash163 doi1016390007-2745-1121152
Park S-Y Choi J Kim JA Jeong M-H Kim S Lee Y-HHur J-S (2013a) Draft genome sequence of Cladoniamacilenta KoLRI003786 a lichen-forming fungusproducing biruloquinone Genome Announcements 1(5)e00695-13 doi101128genomeA00695-13
Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
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Pino-Bodas R Burgaz A Martiacuten M Lumbsch HT (2011)Phenotypical plasticity and homoplasy complicatespecies delimitation in the Cladonia gracilis group(Cladoniaceae Ascomycota) Organ Divers Evol 11(5)343ndash355 doi101007s13127-011-0062-2
Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
Pino-Bodas R Martiacuten M Burgaz A (2012b) Cladoniasubturgida and C iberica (Cladoniaceae) form asingle morphologically and chemically polymorphicspecies Mycol Prog 11(1)269ndash278 doi101007s11557-011-0746-1
Pino-Bodas R Ahti T Stenroos S Martiacuten MP BurgazAR (2013a) Multilocus approach to species recogni-tion in the Cladonia humilis complex (CladoniaceaeAscomycota) Am J Bot 100(4)664ndash678 doi103732ajb1200162
Pino-Bodas R Martiacuten AP Burgaz AR Lumbsch HT(2013b) Species delimitation in Cladonia (Ascomy-cota) a challenge to the DNA barcoding philosophyMol Ecol Resour 13(6)1058ndash1068 doi1011111755-099812086
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Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
Pringle A Baker DM Platt JL Wares JP Latgeacute JPTaylor JW (2005) Cryptic speciation in the cosmo-politan and clonal human pathogenic fungus Asper-gillus fumigatus Evolution 59(9)1886ndash1899 doi10155404-2411
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Puillandre N Lambert A Brouillet S Achaz G (2012)ABGD automatic barcode gap discovery for primaryspecies delimitation Mol Ecol 21(8)1864ndash1877doi101111j1365-294X201105239x
Rannala B Yang Z (2003) Bayes estimation of speciesdivergence times and ancestral population sizes usingDNA sequences from multiple loci Genetics1641645ndash1656
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Ray J (1686) Historia planarum vol 1 Clark LondonReese Naeligsborg R Ekman S Tibell L (2007) Molecular
phylogeny of the genus Lecania (Ramalinaceaelichenized Ascomycota) Mycol Res 111(5)581ndash591doi101016jmycres200703001
Reid N Carstens B (2012) Phylogenetic estimation errorcan decrease the accuracy of species delimitation aBayesian implementation of the general mixed Yule-coalescent model BMC Evol Biol 12(1)196 doi1011861471-2148-12-196
Rissler L Apodaca J (2007) Adding more ecology intospecies delimitation ecological niche models andphylogeography help define cryptic species in theBlack Salamander (Aneides flavipunctatus) Syst Biol56924ndash942 doi10108010635150701703063
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Rivas Plata E Lumbsch HT (2011) Parallel evolution andphenotypic divergence in lichenized fungi a casestudy in the lichen-forming fungal family Graphida-ceae (Ascomycota Lecanoromycetes Ostropales)Mol Phylogenet Evol 61(1)45ndash63 doi101016jympev201104025
Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
Ross KG Gotzek D Ascunce MS Shoemaker DD (2010)Species delimitation a case study in a problematic anttaxon Syst Biol 59(2)162ndash184 doi101093sysbiosyp089
Rubin BER Ree RH Moreau CS (2012) Inferringphylogenies from RAD sequence data PLoS ONE 7(4)e33394 doi101371journalpone0033394
Rubinoff D (2006) Utility of mitochondrial DNA barcodesin species conservation Conserv Biol 20(4)1026ndash1033 doi101111j1523-1739200600372x
Ruiz-Sanchez E Sosa V (2010) Delimiting speciesboundaries within the Neotropical bamboo Otatea(Poaceae Bambusoideae) using molecular morpho-logical and ecological data Mol Phylogenet Evol 54(2)344ndash356 doi101016jympev200910035
Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
morphological characters Antarctic Sci 22 (SpecialIssue 06)727ndash741 doi101017S0954102010000477
Salicini I Ibaacutentildeez C Juste J (2011) Multilocus phylogenyand species delimitation within the Nattererrsquos batspecies complex in the Western Palearctic MolPhylogenet Evol 61(3)888ndash898 doi101016jympev201108010
Satler JD Carstens BC Hedin M (2013) Multilocusspecies delimitation in a complex of morphologicallyconserved trapdoor spiders (Mygalomorphae Antro-diaetidae Aliatypus) Syst Biol 62(6)805ndash823 doi101093sysbiosyt041
Schlick-Steiner BC Steiner FM Seifert B Stauffer CChristian E Crozier RH (2010) Integrative taxonomya multisource approach to exploring biodiversityAnnu Rev Entomol 55(1)421ndash438 doi101146annurev-ento-112408-085432
Schoch CL Seifert KA Huhndorf S Robert V SpougeJL Levesque CA Chen W Fungal Barcoding Con-sortium (2012) Nuclear ribosomal internal transcribedspacer (ITS) region as a universal DNA barcodemarker for Fungi Proc Natl Acad Sci 109(16)6241ndash6246 doi101073pnas1117018109
Seacuterusiaux E Villarreal AJC Wheeler T Goffinet B(2011) Recent origin active speciation and dispersalfor the lichen genus Nephroma (Peltigerales) inMacaronesia J Biogeogr 38(6)1138ndash1151 doi101111j1365-2699201002469x
Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
Simpson GG (1951) The species concept Evolution5285ndash298
Sites JW Marshall JC (2003) Delimiting species a renais-sance issue in systematic biology Trends Ecol Evol18462ndash470 doi101016S0169-5347(03)00184-8
Sites JW Marshall JC (2004) Operational criteria fordelimiting species Annu Rev Ecol Evol Syst 35(1)199ndash227 doi101146annurevecolsys35112202130128
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Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
Talavera G Dincă V Vila R (2013) Factors affectingspecies delimitations with the GMYC model insightsfrom a butterfly survey Methods Ecol Evol 4(12)1101ndash1110 doi1011112041-210X12107
Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
Tewksbury J Anderson JGT Bakker JD Billo TJDunwiddie PW Groom MJ Hampton SE HermanSG Levey DJ Machnicki NJ del Rio CM PowerME Rowell K Salomon AK Stacey L TrombulakSC Wheeler TA (2014) Natural historyrsquos place inscience and society BioScience doi101093bioscibiu032
Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
Werth S Cornejo C Scheidegger C (2013) Characteriza-tion of microsatellite loci in the lichen fungus Lobariapulmonaria (Lobariaceae) Appl Plant Sci 1(2)1200290 doi103732apps1200290
Westberg M Arup U Kaumlrnefelt I (2007) Phylogeneticstudies in the Candelariaceae (lichenized Ascomy-cota) based on nuclear ITS DNA sequence dataMycol Res 111(11)1277ndash1284 doi101017S0953756204002102
Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
2 The Dynamic Discipline of Species Delimitation hellip 43
(Lepidoptera Lycaenidae) Front Zoo 4(1)8 doi1011861742-9994-4-8
Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
Due to the fact that species delimitationstudies often incorporate a substantial biogeo-graphical or ecological component ecologicalniche modeling plays an increasing role in phy-logenetic and taxonomic research Niche mod-eling can provide evidence for ecologicalisolation between populations based on eitherconserved or divergent ecological niches andtherefore can provide additional evidence sup-porting lineage independence between putativespecies The application of ecological nichemodeling has been applied to species delimita-tion studies in a number of cases (Ruiz-Sanchezand Sosa 2010 Leacheacute et al 2009 Raxworthyet al 2007 Rissler and Apodaca 2007) althoughit has not been explicitly applied to assess speciesboundaries in lichen-forming fungi By mappingthe spatial distribution of environmental suit-ability of climatic variables for candidate speciesthe application of ecological niche modeling canbe particularly important in cases where specieshave allopatric distributions (Raxworthy et al2007)
Ecological niche modeling utilizes knownassociations between a speciesrsquo occurrencelocalities and environmental variables to defineabiotic conditions within which populations canbe maintained (Guisan and Thuiller 2005) Themethodological approach for modeling is basedon four general properties (i) The current knownspeciesrsquo localities is the dependent variable (ii)the distribution is modeled as a map composed ofgrid cells at a specified resolution (iii) a range ofenvironmental variables (eg temperature pre-cipitation and solar exposure) are collected todescribe the characteristics of each cell and (iv)classifying the degree to which each cell is eithersuitable or unsuitable for each species under arange of models (Guisan and Thuiller 2005)
Ecological data also have the potential to playan important role in understanding speciesboundaries in lichen-forming fungi For exampleNash and Zavada (1977) demonstrated that Xant-hoparmelia populations with distinct chemistriesoccurring in the northern portion of the SonoranDesert exhibit habitat selection among differentrock substrates within a region with relativelyuniform climate and topography In another case
the parmelioid species Parmelia mayi is mor-phologically indistinguishable from P saxatilisbut can be separated by bioclimatic features andgenetic and chemical characters (Molina et al2011) McCune and Printzen(2011) assess distri-butions and climatic niches of species in theLecanora varia group inwesternUSAandprovidea model that uses continental influence and annualtemperature as themajor factors predicting speciesdistributions The distribution of Usnea hirta alichen commonly used in air quality biomonitor-ing research was modeled for a section of theWhite River National Forest in central Coloradobased on the presence of U hirta at 72 biomoni-toring reference sites distributed in the inter-mountain western United States (Shrestha et al2012) The best model for predicting U hirtadistribution included four variablesmdashsolar radia-tion average monthly precipitation and averagemonthly minimum and maximum temperatures(Shrestha et al 2012) These studies supportthe potential use of ecological niche modelingmethods in species delimitation studies of lichen-forming fungi
24 Conclusions What AboutTaxonomy
In most cases species circumscription and tax-onomy requires some degree of qualitativejudgment and individual interpretation Integrat-ing multiple types of data into an empiricalframework for delimiting species boundarieswhere species boundaries can be tested within ahypothetico-deductive framework with diversedatasets can provide robust hypotheses of speciesboundaries and taxonomic stability (Yeates et al2011) However it has long been known toevolutionary biologists that distinct species donot need to have diagnosable morphologicaldifferences (Mayr 1963) and increasing avail-ability of genetic data has allowed researchers toidentify species and to rigorously test speciesboundaries with a level of precision that wasunimaginable a decade ago While analyticaladvances in statistical species delimitation havebeen largely based on genetic data the utility of
34 SD Leavitt et al
these approaches to formal taxonomy remainselusive Due to these challenges an eclecticapproach to delimiting species and cautionagainst the reliance on any single dataset ormethod is required when delimiting species
While results from many recent studies maycontradict traditional species boundaries acrossmany groups of lichen-forming fungi we areoptimistic that this research represents substantialprogress toward a more accurate perspective onspecies boundaries and diversity in fungi As aresult of ongoing research of species boundariesin lichen-forming fungi the taxonomic value ofmany phenotypes is now better understood ourunderstanding of ecological evolutionary andbiogeographic patterns has improved and we canbegin to better understand patterns of symbiontinteractions in lichens Integrating new data(including novel morphological characters andgenetic data) will be essential to accurately rep-resent species-level diversity across all groups oflichenized fungi Hopefully an improved per-spective on lichen diversity also increases ourappreciation of these incredible symbiotic sys-tems While we are strong advocates for theapplication of independent data types in devel-oping an integrative taxonomy there is anincreasing need to formally recognize the exis-tence of phenotypically cryptic species-levellineages in lichen-forming fungi (see Hibbettet al 2011) In some cases a molecular taxon-omy may provide the most practical approach toconsistent treatment of mycobiont species withinlichen groups where diagnostic morphologicalcharacters are unidentifiable or practically notfeasible (Leavitt et al 2013c d)
These are exciting times for taxonomists andphylogeneticists A closer look at lichen taxon-omy with the inclusion of new data will help usto better understand the diversity of these fasci-nating organisms accurately interpret distribu-tion patterns and play a more important role inmeaningful conservation practices Howeversome level of uncertainty will accompany pro-gress In many taxonomic groups our traditionalapproach for species identification will likelyneed to be substantially modified The search forcorroborating morphological support for cryptic
species identified using molecular data willrequire meticulous and creative approaches toassess phenotypic variation in potentially unor-thodox ways We are hopeful that lichenologistswho traditionally have been eager to include newmethods such as chromatography in their rou-tine identifications will be amenable to includemolecular techniques to their routine examina-tion of specimens for identification and classifi-cation Although this may prove difficult toachieve by single individuals especially citizenscientists that traditionally play an important rolein lichen taxonomy the increasing numberof collaborative projects in the discipline (egLumbsch et al 2011 Crespo et al 2010Gueidan et al 2009) make us optimistic thatbroad-scale collaborative approaches will facili-tate the inclusion of molecular data in lichenresearch at all levels
Acknowledgments We are indebted to various col-leagues for valuable thought-provoking discussionnotably Matthew Nelsen (University of Chicago) AnaCrespo (Universidad Complutense de Madrid) PradeepDivakar (Universidad Complutense de Madrid) BeckettSterner (The Field Museum) and Joyce Havstad (TheField Museum) We also thank anonymous reviewers whoprovided valuable comments that improved this chapterSupport by the US National Science Foundation isgratefully acknowledged (ldquoHidden diversity in parmelioidlichensrdquo DEB-0949147)
References
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Altermann S Leavitt SD Goward T Nelsen MPLumbsch HT (2014) How do you solve a problemlike Letharia A new look at cryptic species in lichen-forming fungi using Bayesian clustering and SNPSsfrom multilocus sequence data PLoS ONE 9(5)e97556 doi101371journalpone0097556
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de Queiroz K (2007) Species concepts and speciesdelimitation Syst Biol 56(6)879ndash886 doi10108010635150701701083
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Degnan JH Rosenberg NA (2009) Gene tree discordancephylogenetic inference and the multispecies coales-cent Trends Ecol Evol 24(6)332ndash340 doihttpdxdoiorg101016jtree200901009
Del-Prado R Cubas P Lumbsch HT Divakar PK BlancoO de Paz GA Molina MC Crespo A (2010) Geneticdistances within and among species in monophyleticlineages of Parmeliaceae (Ascomycota) as a tool fortaxon delimitation Mol Phylogenet Evol 56(1)125ndash133 doi101016jympev201004014
Del-Prado R Divakar PK Crespo A (2011) Using geneticdistances in addition to ITS molecular phylogeny toidentify potential species in the Parmotrema reticul-atum complex a case study The Lichenologist 43(06)569ndash583 doi101017S0024282911000582
Del-Prado R Blanco O Lumbsch HT Divakar PK ElixJA Molina MC Crespo A (2013) Molecular phylog-eny and historical biogeography of the lichen-formingfungal genus Flavoparmelia (Ascomycota Parmelia-ceae) Taxon 62(5)928ndash939 doi101270562522
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Divakar PK Molina MC Lumbsch HT Crespo A (2005)Parmelia barrenoae a new lichen species related toParmelia sulcata (Parmeliaceae) based on molecularand morphological data The Lichenologist 37(01)37ndash46 doi101017S0024282904014641
Divakar PK Amo De paz G del Prado R Esslinger TLCrespo A (2007) Upper cortex anatomy corroboratesphylogenetic hypothesis in species of Physconia(Ascomycota Lecanoromycetes) Mycol Res 111(11)1311ndash1320 doi101016jmycres200708009
Divakar PK Figueras G Hladun N Crespo A (2010)Molecular phylogenetic studies reveal an undescribedspecies within the North American concept of Mela-nelixia glabra (Parmeliaceae) Fungal Divers 42(1)47ndash55 doi101007s13225-010-0027-3
Donoghue MJ Gauthier A (2004) Implementing thephylocode Trends Ecol Evol 19(6)281ndash282 doi101016jtree200404004
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Edwards SV (2009) Is a new and general theory ofmolecular systematics emerging Evolution 63(1)1ndash19 doi101111j1558-5646200800549x
Edwards DL Knowles LL (2014) Species detection andindividual assignment in species delimitation canintegrative data increase efficacy Proc R Soc B BiolSci 281(1777) doi101098rspb20132765
Egan RS (1986) Correlations and non-correlations ofchemical variation patterns with lichen morphologyand geography The Bryologist 8999ndash110
Elix JA Corush J Lumbsch HT (2009) Triterpenechemosyndromes and subtle morphological characterscharacterise lineages in the Physcia aipolia group inAustralia (Ascomycota) Syst Biodivers 7(04)479ndash487 doi101017S1477200009990223
Emerson KJ Merz CR Catchen JM Hohenlohe PACresko WA Bradshaw WE Holzapfel CM (2010)Resolving postglacial phylogeography using high-throughput sequencing Proc Natl Acad Sci 107(37)16196ndash16200 doi101073pnas1006538107
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EvannoGRegnaut SGoudet J (2005)Detecting thenumberof clusters of individuals using the software STRUC-TURE a simulation study Mol Ecol 14(8)2611ndash2620doi101111j1365-294X200502553x
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Fan HH Kubatko LS (2011) Estimating species treesusing approximate Bayesian computation Mol Phylo-genet Evol 59(2)354ndash363 doi101016jympev201102019
Fernaacutendez-Mendoza F Printzen C (2013) Pleistoceneexpansion of the bipolar lichen Cetraria aculeata intothe Southern hemisphere Mol Ecol 22(7)1961ndash1983doi101111mec12210
Fernaacutendez-Mendoza F Domaschke S Garciacutea MA JordanP Martin MP Printzen C (2011) Population structure
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of mycobionts and photobionts of the widespreadlichen Cetraria aculeata Mol Ecol 20(6)1208ndash1232doi101111j1365-294X201004993x
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Fujita MK Leacheacute AD Burbrink FT McGuire JA MoritzC (2012) Coalescent-based species delimitation in anintegrative taxonomy Trends Ecol Evol 27(9)480ndash488 doi101016jtree201204012
Gatesy J OrsquoGrady P Baker RH (1999) Corroborationamong data sets in simultaneous analysis hiddensupport for phylogenetic relationships among higherlevel Artiodactyl taxa Cladistics 15(3)271ndash313doi101111j1096-00311999tb00268x
Gladieux P Ropars J Badouin H Branca A Aguileta Gde Vienne DM Rodriacuteguez de la Vega RC Branco SGiraud T (2014) Fungal evolutionary genomicsprovides insight into the mechanisms of adaptivedivergence in eukaryotes Mol Ecol 23(4)753ndash773doi101111mec12631
Goffinet B Miadlikowska J Goward T (2003) Phyloge-netic inferences based on nrDNA sequences supportfive morphospecies within the Peltigera didactylacomplex (Lichenized Ascomycota) The Bryologist106(3)349ndash364 doi10163901
Gowan SP (1986) Evolution of secondary natural productsin the genus Porpidia (Ascomycata Porpidiaceae) AmJ Bot 73606
Griffin PC Hoffmann AA (2014) Limited genetic diver-gence among Australian alpine Poa tussock grassescoupled with regional structuring points to ongoinggene flow and taxonomic challenges Ann Bot doi101093aobmcu017
Grube M Hawksworth DL (2007) Trouble with lichenthe re-evaluation and re-interpretation of thallus formand fruit body types in the molecular era Mycol Res111(9)1116ndash1132 doi101016jmycres200704008
Gueidan C Savi S Thues H Roux C Keller C Tibell LPrieto M Heimarsson S Breuss O Orange A FrobergL Wynns AA Navarro-Rosines P Krzewicka BPykaelae J Grube M Lutzoni F (2009) Genericclassification of the Verrucariaceae (Ascomycota)based on molecular and morphological evidencerecent progress and remaining challenges Taxon 58(1)184ndash208
Guillot G Mortier F Estoup A (2005) Geneland acomputer package for landscape genetics Mol EcolNotes 5(3)712ndash715 doi101111j1471-8286200501031x
Guillot G Renaud S Ledevin R Michaux J Claude J(2012) A unifying model for the analysis of pheno-typic genetic and geographic data Syst Biol 61(6)897ndash911 doi101093sysbiosys038
Guisan A Thuiller W (2005) Predicting species distribu-tion offering more than simple habitat models EcolLett 8(9)993ndash1009 doi101111j1461-0248200500792x
Hale ME (1990) A synopsis of the lichen genusXanthoparmelia (Vainio) Hale (AscomycotinaParmeliaceae) Smithsonian Institution Press Wash-ington DC
Hamilton CA Hendrixson BE Brewer MS Bond JE(2014) An evaluation of sampling effects on multipleDNA barcoding methods leads to an integrativeapproach for delimiting species a case study ofthe North American tarantula genus Aphonopelma(Araneae Mygalomorphae Theraphosidae) MolPhylogenet Evol 7179ndash93 doi101016jympev201311007
Hausdorf B (2011) Progress toward a general speciesconcept Evolution 65(4)923ndash931 doi101111j1558-5646201101231x
Hausdorf B Hennig C (2010) Species delimitation usingdominant and codominant multilocus markers SystBiol 59(5)491ndash503 doi101093sysbiosyq039
Hawksworth DL (1976) Lichen chemotaxonomy InBailey RH (ed) Lichenology progress and problemsAcademic London pp 139ndash184
Hebert PDN Cywinska A Ball SL deWaard JR (2003)Biological identifications through DNA barcodesProc R Soc Lond B Biol Sci 270(1512)313ndash321doi101098rspb20022218
Hebert PDN Stoeckle MY Zemlak TS Francis CM(2004) Identification of birds through DNA barcodesPLoS Biol 2(10)e312 doi101371journalpbio0020312
Heled J Drummond AJ (2010) Bayesian inference ofspecies trees from multilocus data Mol Biol Evol 27(3)570ndash580 doi101093molbevmsp274
Hey J (2006) On the failure of modern species conceptsTrends Ecol Evol 21(8)447ndash450 doi101016jtree200605011
Hibbett DS OhmanA Glotzer D NuhnMKirk P NilssonRH (2011) Progress in molecular and morphologicaltaxon discovery in Fungi and options for formalclassification on environmental sequences Fungal BiolRev 2538ndash47 doi101016jfbr201101001
Houmlgnabba F Wedin M (2003) Molecular phylogeny ofthe Sphaerophorus globosus species complex Cladis-tics 19(3)224ndash232 doi101111j1096-00312003tb00365x
Hudson RR Coyne JA (2002) Mathematical conse-quences of the genealogical species concept Evolu-tion 56(8)1557 doi101111j0014-38202002tb01467x
Huelsenbeck JP Andolfatto P Huelsenbeck ET (2011)Structurama Bayesian inference of population struc-ture Evol Bioinf Online 7(2011)55ndash59 doi104137EBOS6761
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Kekkonen M Hebert PD (2014) DNA barcode-baseddelineation of putative species efficient start fortaxonomic workflows Mol Ecol Res doi1011111755-099812233
Kelly LJ Hollingsworth PM Coppins BJ Ellis CJHarrold P Tosh J Yahr R (2011) DNA barcoding oflichenized fungi demonstrates high identification suc-cess in a floristic context New Phytol 191(1)288ndash300doi101111j1469-8137201103677x
Kiss L (2012) Limits of nuclear ribosomal DNA internaltranscribed spacer (ITS) sequences as species barcodesfor fungi Proc Natl Acad Sci 109(27)E1811ndashE1811doi101073pnas1207143109
Kluge AG (1989) A concern for evidence and aphylogenetic hypothesis for relationships among Epi-crates (Boidae Serpentes) Syst Zool 38(1)7ndash25doi101093sysbio3817
Knowles LL Carstens BC (2007) Delimiting specieswithout monophyletic gene trees Syst Biol 56(6)887ndash895 doi10108010635150701701091
Kroken S Taylor JW (2001) A gene genealogical approachto recognize phylogenetic species boundaries in thelichenized fungus Letharia Mycologia 93(1)38ndash53
Kubatko LS Degnan JH (2007) Inconsistency of phylo-genetic estimates from concatenated data under coa-lescence Syst Biol 56(1)17ndash24 doi10108010635150601146041
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Lawrey JD (1986) Biological role of lichen substancesThe Bryologist 89(2)111ndash122
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Leacheacute AD (2009) Species tree discordance traces tophylogeographic clade boundaries in North Americanfence lizards (Sceloporus) Syst Biol 58(6)547ndash559doi101093sysbiosyp057
Leacheacute AD Fujita MK (2010) Bayesian species delim-itation in West African forest geckos (Hemidactylusfasciatus) Proc R Soc B Biol Sci 2773071ndash3077doi101098rspb20100662
Leacheacute AD Koo M Spencer C Papenfuss T Fisher RMcGuire J (2009) Quantifying ecological morpho-logical and genetic variation to delimit species in thecoast horned lizard species complex (Phrynosoma)Proc Natl Acad Sci USA 10612418ndash12423 doi101073pnas0906380106
Leacheacute AD Fujita MK Minin VN Bouckaert RR (2014)Species delimitation using genome-wide SNP dataSystematic Biology doi101093sysbiosyu018
Leavitt SD Fankhauser JD Leavitt DH Porter LDJohnson LA St Clair LL (2011a) Complex patterns ofspeciation in cosmopolitan ldquorock posyrdquo lichensmdashdiscovering and delimiting cryptic fungal species in
the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
Leavitt SD Esslinger TL Lumbsch HT (2012c) Neogene-dominated diversification in neotropical montanelichens dating divergence events in the lichen-form-ing fungal genus Oropogon (Parmeliaceae) Am J Bot99(11)1764ndash1777 doi103732ajb1200146
Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Lumbsch HT St Clair LL(2013c) DNA barcode identification of lichen-formingfungal species in the Rhizoplaca melanophthalmaspecies-complex (Lecanorales Lecanoraceae) includ-ing five new species MycoKeys 71ndash22 doi103897mycokeys74508
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Vondraacutek J Thorsten Lum-bsch H Clair LLS (2013d) Local representation ofglobal diversity in a cosmopolitan lichen-formingfungal species complex (Rhizoplaca Ascomycota)J Biogeogr 40(9)1792ndash1806 doi101111jbi12118
Leavitt SD Lumbsch HT Stenroos S St Clair LL(2013e) Pleistocene speciation in North American
2 The Dynamic Discipline of Species Delimitation hellip 39
lichenized fungi and the impact of alternative speciescircumscriptions and rates of molecular evolution ondivergence estimates PLoS ONE 8(12)e85240doi101371journalpone0085240
Leavitt SD Esslinger TL Hansen ES Divakar PK CrespoA Loomis BF Lumbsch HT (2014) DNA barcoding ofbrown Parmeliae (Parmeliaceae) species a molecularapproach for accurate specimen identification empha-sizing species in Greenland Organ Divers Evol 14(1)11ndash20 doi101007s13127-013-0147-1
Leliaert F Verbruggen H Vanormelingen P Steen FLopez-Bautista JM Zuccarello GC De Clerck O(2014) DNA-based species delimitation in algae Eur JPhycol 49(2) doi101080096702622014904524
Leuckert C (1985) Probleme der Flechten-Chemotaxon-omiemdashStoffkombinationen und ihre taxonomischeWertung Ber Deut Bot Ges 98401ndash408
Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
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Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
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Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
Mayden RL (1997) A hierarchy of species concepts thedenouement in the saga of the species problem InClaridge MF Dawah HA Wilson MR (eds) Speciesthe units of biodiversity Chapman amp Hall Londonpp 381ndash424
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Mayr E (1970) Populations species and evolutionBelknap Press of Harvard University Press Cambridge
McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
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40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
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Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
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Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
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OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
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Orock EA Leavitt SD Fonge BA St Clair LL LumbschHT (2012) DNA-based identification of lichen-form-ing fungi Can publicly available sequence databasesaid in lichen diversity inventories of Mount Cameroon(West Africa) The Lichenologist 44(6)833ndash839doi101017S0024282912000424
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Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
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Park S-Y Choi J Kim JA Jeong M-H Kim S Lee Y-HHur J-S (2013a) Draft genome sequence of Cladoniamacilenta KoLRI003786 a lichen-forming fungusproducing biruloquinone Genome Announcements 1(5)e00695-13 doi101128genomeA00695-13
Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
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Pino-Bodas R Burgaz A Martiacuten M Lumbsch HT (2011)Phenotypical plasticity and homoplasy complicatespecies delimitation in the Cladonia gracilis group(Cladoniaceae Ascomycota) Organ Divers Evol 11(5)343ndash355 doi101007s13127-011-0062-2
Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
Pino-Bodas R Martiacuten M Burgaz A (2012b) Cladoniasubturgida and C iberica (Cladoniaceae) form asingle morphologically and chemically polymorphicspecies Mycol Prog 11(1)269ndash278 doi101007s11557-011-0746-1
Pino-Bodas R Ahti T Stenroos S Martiacuten MP BurgazAR (2013a) Multilocus approach to species recogni-tion in the Cladonia humilis complex (CladoniaceaeAscomycota) Am J Bot 100(4)664ndash678 doi103732ajb1200162
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Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
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phylogeny of the genus Lecania (Ramalinaceaelichenized Ascomycota) Mycol Res 111(5)581ndash591doi101016jmycres200703001
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Rissler L Apodaca J (2007) Adding more ecology intospecies delimitation ecological niche models andphylogeography help define cryptic species in theBlack Salamander (Aneides flavipunctatus) Syst Biol56924ndash942 doi10108010635150701703063
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Rivas Plata E Lumbsch HT (2011) Parallel evolution andphenotypic divergence in lichenized fungi a casestudy in the lichen-forming fungal family Graphida-ceae (Ascomycota Lecanoromycetes Ostropales)Mol Phylogenet Evol 61(1)45ndash63 doi101016jympev201104025
Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
Ross KG Gotzek D Ascunce MS Shoemaker DD (2010)Species delimitation a case study in a problematic anttaxon Syst Biol 59(2)162ndash184 doi101093sysbiosyp089
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Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
morphological characters Antarctic Sci 22 (SpecialIssue 06)727ndash741 doi101017S0954102010000477
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Seacuterusiaux E Villarreal AJC Wheeler T Goffinet B(2011) Recent origin active speciation and dispersalfor the lichen genus Nephroma (Peltigerales) inMacaronesia J Biogeogr 38(6)1138ndash1151 doi101111j1365-2699201002469x
Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
Simpson GG (1951) The species concept Evolution5285ndash298
Sites JW Marshall JC (2003) Delimiting species a renais-sance issue in systematic biology Trends Ecol Evol18462ndash470 doi101016S0169-5347(03)00184-8
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Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
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Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
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Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
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Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
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Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
these approaches to formal taxonomy remainselusive Due to these challenges an eclecticapproach to delimiting species and cautionagainst the reliance on any single dataset ormethod is required when delimiting species
While results from many recent studies maycontradict traditional species boundaries acrossmany groups of lichen-forming fungi we areoptimistic that this research represents substantialprogress toward a more accurate perspective onspecies boundaries and diversity in fungi As aresult of ongoing research of species boundariesin lichen-forming fungi the taxonomic value ofmany phenotypes is now better understood ourunderstanding of ecological evolutionary andbiogeographic patterns has improved and we canbegin to better understand patterns of symbiontinteractions in lichens Integrating new data(including novel morphological characters andgenetic data) will be essential to accurately rep-resent species-level diversity across all groups oflichenized fungi Hopefully an improved per-spective on lichen diversity also increases ourappreciation of these incredible symbiotic sys-tems While we are strong advocates for theapplication of independent data types in devel-oping an integrative taxonomy there is anincreasing need to formally recognize the exis-tence of phenotypically cryptic species-levellineages in lichen-forming fungi (see Hibbettet al 2011) In some cases a molecular taxon-omy may provide the most practical approach toconsistent treatment of mycobiont species withinlichen groups where diagnostic morphologicalcharacters are unidentifiable or practically notfeasible (Leavitt et al 2013c d)
These are exciting times for taxonomists andphylogeneticists A closer look at lichen taxon-omy with the inclusion of new data will help usto better understand the diversity of these fasci-nating organisms accurately interpret distribu-tion patterns and play a more important role inmeaningful conservation practices Howeversome level of uncertainty will accompany pro-gress In many taxonomic groups our traditionalapproach for species identification will likelyneed to be substantially modified The search forcorroborating morphological support for cryptic
species identified using molecular data willrequire meticulous and creative approaches toassess phenotypic variation in potentially unor-thodox ways We are hopeful that lichenologistswho traditionally have been eager to include newmethods such as chromatography in their rou-tine identifications will be amenable to includemolecular techniques to their routine examina-tion of specimens for identification and classifi-cation Although this may prove difficult toachieve by single individuals especially citizenscientists that traditionally play an important rolein lichen taxonomy the increasing numberof collaborative projects in the discipline (egLumbsch et al 2011 Crespo et al 2010Gueidan et al 2009) make us optimistic thatbroad-scale collaborative approaches will facili-tate the inclusion of molecular data in lichenresearch at all levels
Acknowledgments We are indebted to various col-leagues for valuable thought-provoking discussionnotably Matthew Nelsen (University of Chicago) AnaCrespo (Universidad Complutense de Madrid) PradeepDivakar (Universidad Complutense de Madrid) BeckettSterner (The Field Museum) and Joyce Havstad (TheField Museum) We also thank anonymous reviewers whoprovided valuable comments that improved this chapterSupport by the US National Science Foundation isgratefully acknowledged (ldquoHidden diversity in parmelioidlichensrdquo DEB-0949147)
References
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2 The Dynamic Discipline of Species Delimitation hellip 35
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Hey J (2006) On the failure of modern species conceptsTrends Ecol Evol 21(8)447ndash450 doi101016jtree200605011
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the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
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Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Lumbsch HT St Clair LL(2013c) DNA barcode identification of lichen-formingfungal species in the Rhizoplaca melanophthalmaspecies-complex (Lecanorales Lecanoraceae) includ-ing five new species MycoKeys 71ndash22 doi103897mycokeys74508
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Leliaert F Verbruggen H Vanormelingen P Steen FLopez-Bautista JM Zuccarello GC De Clerck O(2014) DNA-based species delimitation in algae Eur JPhycol 49(2) doi101080096702622014904524
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Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
Lohtander K Kaumlllersjouml M Roland M Tehler A (2000)The family physciaceae in fennoscandia phylogenyinferred from its sequences Mycologia 92(4)728ndash735
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Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
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Lumbsch HT (1998a) Taxonomic use of metabolic data inlichen-forming fungi In Frisvad JC Bridge PDArora DK (eds) Chemical Fungal Taxonomy MarcelDekker New York pp 345ndash387
Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
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Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
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40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
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Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
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Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
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Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
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Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
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Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
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Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
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42 SD Leavitt et al
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Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
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Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
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Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
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Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
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Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
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Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
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Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
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Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
includes four phylogenetically supported morphospe-cies Biol J Linn Soc 91(3)455ndash467 doi101111j1095-8312200700810x
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Bickford D Lohman DJ Sodhi NS Ng PKL Meier RWinker K Ingram KK Das I (2007) Cryptic speciesas a window on diversity and conservation TrendsEcol Evol 22(3)148ndash155 doi101016jtree200611004
Bonan GB Shugart HH (1989) Environmental factors andecological processes in boreal forests Annu Rev EcolSyst 20(1989)1ndash28
Bond J Stockman A (2008) An integrative method fordelimiting cohesion species finding the population-species interface in a group of Californian trapdoorspiders with extreme genetic divergence and geo-graphic structuring Syst Biol 57(4)628ndash646 doi10108010635150802302443
Brodo IM (1978) Changing concepts regarding chemicaldiversity in lichens The Lichenologist 10(1)1ndash11doi101017S0024282978000031
Brodo IM (1986) Interpreting chemical variation inlichens for systematic purposes The Bryologist 89(2)132ndash138
Caley MJ Fisher R Mengersen K (2014) Global speciesrichness estimates have not converged Trends EcolEvol 29(4)187ndash188 doi101016jtree201402002
Camargo A Sites JW (2013) Species delimitation adecade after the Renaissance In Pavlinov I (ed) Thespecies problemmdashongoing issues InTech doi10577252664
Camargo A Morando M Avila LJ Sites JW (2012)Species delimitation with ABC and other coalescent-based methods a test of accuracy with simulations and
an empirical example with lizards of the Liolaemusdarwinii complex (Squamata Liolaemidae) Evolution66(9)2834ndash2849 doi101111j1558-5646201201640x
Carstens BC Dewey TA (2010) Species delimitationusing a combined coalescent and information-theoreticapproach an example from North American Myotisbats Syst Biol 59(4)400ndash414 doi101093sysbiosyq024
Carstens BC Pelletier TA Reid NM Satler JD (2013)How to fail at species delimitation Mol Ecol 22(17)4369ndash4383 doi101111mec12413
Corander J Marttinen P (2006) Bayesian identification ofadmixture events using multi-locus molecular mark-ers Mol Ecol 15(10)2833ndash2843 doi101111j1365-294X200602994x
Corander J Waldmann P Marttinen P Sillanpaa M(2004) BAPS 2 enhanced possibilities for the analysisof genetic population structure Bioinformatics 20(15)2363ndash2369 doi101093bioinformaticsbth250
Corander J Marttinen P Mantyniemi S (2006) Bayesianidentification of stock mixtures from molecular markerdata Fish Bull 104550ndash558
Corander J Marttinen P Siren J Tang J (2008) EnhancedBayesian modelling in BAPS software for learninggenetic structures of populations BMC Bioinf 9(1)539 doi1011861471-2105-9-539
Coyne JA Orr HA (2004) Speciation Sinauer AssociatesSunderland
Cracraft J (1983) Species concepts and speciation anal-ysis Curr Ornithol 1159ndash187
Crespo A Lumbsch HT (2010) Cryptic species in lichen-forming fungi IMA Fungus 1167ndash170 doi105598imafungus2010010209
Crespo A Peacuterez-Ortega S (2009) Cryptic species andspecies pairs in lichens a discussion on the relation-ship between molecular phylogenies and morpholog-ical characters Anales del Jardin Botanico de Madrid66(S1)71ndash81 doi103989ajbm2225
Crespo A Kauff F Divakar PK del Prado R Perez-Ortega S Amo de Paz G Ferencova Z Blanco ORoca-Valiente B Nunez-Zapata J Cubas P ArguelloA Elix JA Esslinger TL Hawksworth DL MillanesA Molina MC Wedin M Ahti T Aptroot A et al(2010) Phylogenetic generic classification of parmeli-oid lichens (Parmeliaceae Ascomycota) based onmolecular morphological and chemical evidenceTaxon 59(6)1735ndash1753
Culberson WL (1969) The use of chemistry in thesystematics of the lichens Taxon 18498ndash505
Culberson WL (1970) Chemosystematics and ecology oflichen-forming fungi Annu Rev Ecol Syst 1153ndash170
Culberson CF Culberson WL (1976) Chemosyndromicvariation in lichens Syst Bot 1325ndash339
Darwin C (1859) On the origin of species by means ofnatural selection or the preservation of favoured racesin the struggle for life J Murray London
Dayrat B (2005) Towards integrative taxonomy Biol JLinn Soc 85(3)407ndash415 doi101111j1095-8312200500503x
36 SD Leavitt et al
de Queiroz K (1998) The general lineage concept ofspecies species criteria and the process of speciationa conceptual unification and terminological recom-mendations In Howard DJ Berlocher SH (eds)Endless forms species and speciation Oxford Uni-versity Press Oxford pp 57ndash75
de Queiroz K (1999) The general lineage concept ofspecies and the defining properties of the speciescategory In Wilson RA (ed) Species new interdis-ciplinary essays MIT Press Cambridge pp 49ndash89
de Queiroz K (2007) Species concepts and speciesdelimitation Syst Biol 56(6)879ndash886 doi10108010635150701701083
de Queiroz A Donoghue MJ Kim J (1995) Separateversus combined analysis of phylogenetic evidenceAnnu Rev Ecol Syst 26(1)657ndash681 doi101146annureves26110195003301
Degnan J Rosenberg N (2006) Discordance of speciestrees with their most likely gene trees PLoS Genet 2(5)e68 doi101371journalpgen0020068
Degnan JH Rosenberg NA (2009) Gene tree discordancephylogenetic inference and the multispecies coales-cent Trends Ecol Evol 24(6)332ndash340 doihttpdxdoiorg101016jtree200901009
Del-Prado R Cubas P Lumbsch HT Divakar PK BlancoO de Paz GA Molina MC Crespo A (2010) Geneticdistances within and among species in monophyleticlineages of Parmeliaceae (Ascomycota) as a tool fortaxon delimitation Mol Phylogenet Evol 56(1)125ndash133 doi101016jympev201004014
Del-Prado R Divakar PK Crespo A (2011) Using geneticdistances in addition to ITS molecular phylogeny toidentify potential species in the Parmotrema reticul-atum complex a case study The Lichenologist 43(06)569ndash583 doi101017S0024282911000582
Del-Prado R Blanco O Lumbsch HT Divakar PK ElixJA Molina MC Crespo A (2013) Molecular phylog-eny and historical biogeography of the lichen-formingfungal genus Flavoparmelia (Ascomycota Parmelia-ceae) Taxon 62(5)928ndash939 doi101270562522
Dettman J Jacobson D Taylor J (2003a) A multilocusgenealogical approach to phylogenetic species recog-nition in the model eukaryote Neurospora Evolution57(12)2703ndash2720 doi101111j0014-38202003tb01514x
Dettman JR Jacobson DJ Turner E Pringle A TaylorJW (2003b) Reproductive isolation and phylogeneticdivergence in Neurospora comparing methods ofspecies recognition in a model eukaryote Evolution57(12)2721 doi10155403-074
Devkota S Cornejo C Werth S Chaudhary RP Sche-idegger C (2014) Characterization of microsatelliteloci in the Himalayan lichen fungus Lobaria pindar-ensis (Lobariaceae) Appl Plant Sci 2(5)1300101doi103732apps1300101
Divakar PK Molina MC Lumbsch HT Crespo A (2005)Parmelia barrenoae a new lichen species related toParmelia sulcata (Parmeliaceae) based on molecularand morphological data The Lichenologist 37(01)37ndash46 doi101017S0024282904014641
Divakar PK Amo De paz G del Prado R Esslinger TLCrespo A (2007) Upper cortex anatomy corroboratesphylogenetic hypothesis in species of Physconia(Ascomycota Lecanoromycetes) Mycol Res 111(11)1311ndash1320 doi101016jmycres200708009
Divakar PK Figueras G Hladun N Crespo A (2010)Molecular phylogenetic studies reveal an undescribedspecies within the North American concept of Mela-nelixia glabra (Parmeliaceae) Fungal Divers 42(1)47ndash55 doi101007s13225-010-0027-3
Donoghue MJ Gauthier A (2004) Implementing thephylocode Trends Ecol Evol 19(6)281ndash282 doi101016jtree200404004
Eaton DAR Ree RH (2013) Inferring phylogeny andintrogression using RADseq data an example fromflowering plants (Pedicularis Orobanchaceae) SystBiol 62(5)689ndash706 doi101093sysbiosyt032
Edwards SV (2009) Is a new and general theory ofmolecular systematics emerging Evolution 63(1)1ndash19 doi101111j1558-5646200800549x
Edwards DL Knowles LL (2014) Species detection andindividual assignment in species delimitation canintegrative data increase efficacy Proc R Soc B BiolSci 281(1777) doi101098rspb20132765
Egan RS (1986) Correlations and non-correlations ofchemical variation patterns with lichen morphologyand geography The Bryologist 8999ndash110
Elix JA Corush J Lumbsch HT (2009) Triterpenechemosyndromes and subtle morphological characterscharacterise lineages in the Physcia aipolia group inAustralia (Ascomycota) Syst Biodivers 7(04)479ndash487 doi101017S1477200009990223
Emerson KJ Merz CR Catchen JM Hohenlohe PACresko WA Bradshaw WE Holzapfel CM (2010)Resolving postglacial phylogeography using high-throughput sequencing Proc Natl Acad Sci 107(37)16196ndash16200 doi101073pnas1006538107
Ence DD Carstens BC (2011) SpedeSTEM a rapid andaccurate method for species delimitation Mol EcolResour 11(3)473ndash480 doi101111j1755-0998201002947x
EvannoGRegnaut SGoudet J (2005)Detecting thenumberof clusters of individuals using the software STRUC-TURE a simulation study Mol Ecol 14(8)2611ndash2620doi101111j1365-294X200502553x
Falush D Stephens M Pritchard JK (2003) Inference ofpopulation structure using multilocus genotype datalinked loci and correlated allele frequencies Genetics164(4)1567ndash1587 doi101111j1471-8286200701758x
Fan HH Kubatko LS (2011) Estimating species treesusing approximate Bayesian computation Mol Phylo-genet Evol 59(2)354ndash363 doi101016jympev201102019
Fernaacutendez-Mendoza F Printzen C (2013) Pleistoceneexpansion of the bipolar lichen Cetraria aculeata intothe Southern hemisphere Mol Ecol 22(7)1961ndash1983doi101111mec12210
Fernaacutendez-Mendoza F Domaschke S Garciacutea MA JordanP Martin MP Printzen C (2011) Population structure
2 The Dynamic Discipline of Species Delimitation hellip 37
of mycobionts and photobionts of the widespreadlichen Cetraria aculeata Mol Ecol 20(6)1208ndash1232doi101111j1365-294X201004993x
Fraley C Raftery A (2007) Model-based methods ofclassification Using the mclust software in chemo-metrics J Stat Softw 18i06 [Available online at httpwwwdoajorgdoajfunc=abstractampid=218544]
Fujisawa T Barraclough TG (2013) Delimiting speciesusing single-locus data and the generalized mixed yulecoalescent (GMYC) approach a revised method andevaluation on simulated datasets Syst Biol 62(5)707ndash724 doi101093sysbiosyt033
Fujita MK Leacheacute AD Burbrink FT McGuire JA MoritzC (2012) Coalescent-based species delimitation in anintegrative taxonomy Trends Ecol Evol 27(9)480ndash488 doi101016jtree201204012
Gatesy J OrsquoGrady P Baker RH (1999) Corroborationamong data sets in simultaneous analysis hiddensupport for phylogenetic relationships among higherlevel Artiodactyl taxa Cladistics 15(3)271ndash313doi101111j1096-00311999tb00268x
Gladieux P Ropars J Badouin H Branca A Aguileta Gde Vienne DM Rodriacuteguez de la Vega RC Branco SGiraud T (2014) Fungal evolutionary genomicsprovides insight into the mechanisms of adaptivedivergence in eukaryotes Mol Ecol 23(4)753ndash773doi101111mec12631
Goffinet B Miadlikowska J Goward T (2003) Phyloge-netic inferences based on nrDNA sequences supportfive morphospecies within the Peltigera didactylacomplex (Lichenized Ascomycota) The Bryologist106(3)349ndash364 doi10163901
Gowan SP (1986) Evolution of secondary natural productsin the genus Porpidia (Ascomycata Porpidiaceae) AmJ Bot 73606
Griffin PC Hoffmann AA (2014) Limited genetic diver-gence among Australian alpine Poa tussock grassescoupled with regional structuring points to ongoinggene flow and taxonomic challenges Ann Bot doi101093aobmcu017
Grube M Hawksworth DL (2007) Trouble with lichenthe re-evaluation and re-interpretation of thallus formand fruit body types in the molecular era Mycol Res111(9)1116ndash1132 doi101016jmycres200704008
Gueidan C Savi S Thues H Roux C Keller C Tibell LPrieto M Heimarsson S Breuss O Orange A FrobergL Wynns AA Navarro-Rosines P Krzewicka BPykaelae J Grube M Lutzoni F (2009) Genericclassification of the Verrucariaceae (Ascomycota)based on molecular and morphological evidencerecent progress and remaining challenges Taxon 58(1)184ndash208
Guillot G Mortier F Estoup A (2005) Geneland acomputer package for landscape genetics Mol EcolNotes 5(3)712ndash715 doi101111j1471-8286200501031x
Guillot G Renaud S Ledevin R Michaux J Claude J(2012) A unifying model for the analysis of pheno-typic genetic and geographic data Syst Biol 61(6)897ndash911 doi101093sysbiosys038
Guisan A Thuiller W (2005) Predicting species distribu-tion offering more than simple habitat models EcolLett 8(9)993ndash1009 doi101111j1461-0248200500792x
Hale ME (1990) A synopsis of the lichen genusXanthoparmelia (Vainio) Hale (AscomycotinaParmeliaceae) Smithsonian Institution Press Wash-ington DC
Hamilton CA Hendrixson BE Brewer MS Bond JE(2014) An evaluation of sampling effects on multipleDNA barcoding methods leads to an integrativeapproach for delimiting species a case study ofthe North American tarantula genus Aphonopelma(Araneae Mygalomorphae Theraphosidae) MolPhylogenet Evol 7179ndash93 doi101016jympev201311007
Hausdorf B (2011) Progress toward a general speciesconcept Evolution 65(4)923ndash931 doi101111j1558-5646201101231x
Hausdorf B Hennig C (2010) Species delimitation usingdominant and codominant multilocus markers SystBiol 59(5)491ndash503 doi101093sysbiosyq039
Hawksworth DL (1976) Lichen chemotaxonomy InBailey RH (ed) Lichenology progress and problemsAcademic London pp 139ndash184
Hebert PDN Cywinska A Ball SL deWaard JR (2003)Biological identifications through DNA barcodesProc R Soc Lond B Biol Sci 270(1512)313ndash321doi101098rspb20022218
Hebert PDN Stoeckle MY Zemlak TS Francis CM(2004) Identification of birds through DNA barcodesPLoS Biol 2(10)e312 doi101371journalpbio0020312
Heled J Drummond AJ (2010) Bayesian inference ofspecies trees from multilocus data Mol Biol Evol 27(3)570ndash580 doi101093molbevmsp274
Hey J (2006) On the failure of modern species conceptsTrends Ecol Evol 21(8)447ndash450 doi101016jtree200605011
Hibbett DS OhmanA Glotzer D NuhnMKirk P NilssonRH (2011) Progress in molecular and morphologicaltaxon discovery in Fungi and options for formalclassification on environmental sequences Fungal BiolRev 2538ndash47 doi101016jfbr201101001
Houmlgnabba F Wedin M (2003) Molecular phylogeny ofthe Sphaerophorus globosus species complex Cladis-tics 19(3)224ndash232 doi101111j1096-00312003tb00365x
Hudson RR Coyne JA (2002) Mathematical conse-quences of the genealogical species concept Evolu-tion 56(8)1557 doi101111j0014-38202002tb01467x
Huelsenbeck JP Andolfatto P Huelsenbeck ET (2011)Structurama Bayesian inference of population struc-ture Evol Bioinf Online 7(2011)55ndash59 doi104137EBOS6761
Jones G Oxelman B (2014) DISSECT an assignment-free Bayesian discovery method for species delimita-tion under the multispecies coalescent Bioinformaticsdoi101101003178
38 SD Leavitt et al
Kekkonen M Hebert PD (2014) DNA barcode-baseddelineation of putative species efficient start fortaxonomic workflows Mol Ecol Res doi1011111755-099812233
Kelly LJ Hollingsworth PM Coppins BJ Ellis CJHarrold P Tosh J Yahr R (2011) DNA barcoding oflichenized fungi demonstrates high identification suc-cess in a floristic context New Phytol 191(1)288ndash300doi101111j1469-8137201103677x
Kiss L (2012) Limits of nuclear ribosomal DNA internaltranscribed spacer (ITS) sequences as species barcodesfor fungi Proc Natl Acad Sci 109(27)E1811ndashE1811doi101073pnas1207143109
Kluge AG (1989) A concern for evidence and aphylogenetic hypothesis for relationships among Epi-crates (Boidae Serpentes) Syst Zool 38(1)7ndash25doi101093sysbio3817
Knowles LL Carstens BC (2007) Delimiting specieswithout monophyletic gene trees Syst Biol 56(6)887ndash895 doi10108010635150701701091
Kroken S Taylor JW (2001) A gene genealogical approachto recognize phylogenetic species boundaries in thelichenized fungus Letharia Mycologia 93(1)38ndash53
Kubatko LS Degnan JH (2007) Inconsistency of phylo-genetic estimates from concatenated data under coa-lescence Syst Biol 56(1)17ndash24 doi10108010635150601146041
Latch EK Dharmarajan G Glaubitz JC Rhodes OE Jr(2006) Relative performance of Bayesian clusteringsoftware for inferring population substructure andindividual assignments at low levels of populationdifferentiation Conserv Genet 7(2)295ndash302 doi101007s10592-005-9098-1
Lawrey JD (1986) Biological role of lichen substancesThe Bryologist 89(2)111ndash122
Le Gac M Hood ME Fournier E Giraud T (2007)Phylogenetic evidence of host-specific cryptic speciesin the anther smut fungus Evolution 61(1)15ndash26doi101111j1558-5646200700002x
Leacheacute AD (2009) Species tree discordance traces tophylogeographic clade boundaries in North Americanfence lizards (Sceloporus) Syst Biol 58(6)547ndash559doi101093sysbiosyp057
Leacheacute AD Fujita MK (2010) Bayesian species delim-itation in West African forest geckos (Hemidactylusfasciatus) Proc R Soc B Biol Sci 2773071ndash3077doi101098rspb20100662
Leacheacute AD Koo M Spencer C Papenfuss T Fisher RMcGuire J (2009) Quantifying ecological morpho-logical and genetic variation to delimit species in thecoast horned lizard species complex (Phrynosoma)Proc Natl Acad Sci USA 10612418ndash12423 doi101073pnas0906380106
Leacheacute AD Fujita MK Minin VN Bouckaert RR (2014)Species delimitation using genome-wide SNP dataSystematic Biology doi101093sysbiosyu018
Leavitt SD Fankhauser JD Leavitt DH Porter LDJohnson LA St Clair LL (2011a) Complex patterns ofspeciation in cosmopolitan ldquorock posyrdquo lichensmdashdiscovering and delimiting cryptic fungal species in
the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
Leavitt SD Esslinger TL Lumbsch HT (2012c) Neogene-dominated diversification in neotropical montanelichens dating divergence events in the lichen-form-ing fungal genus Oropogon (Parmeliaceae) Am J Bot99(11)1764ndash1777 doi103732ajb1200146
Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Lumbsch HT St Clair LL(2013c) DNA barcode identification of lichen-formingfungal species in the Rhizoplaca melanophthalmaspecies-complex (Lecanorales Lecanoraceae) includ-ing five new species MycoKeys 71ndash22 doi103897mycokeys74508
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Vondraacutek J Thorsten Lum-bsch H Clair LLS (2013d) Local representation ofglobal diversity in a cosmopolitan lichen-formingfungal species complex (Rhizoplaca Ascomycota)J Biogeogr 40(9)1792ndash1806 doi101111jbi12118
Leavitt SD Lumbsch HT Stenroos S St Clair LL(2013e) Pleistocene speciation in North American
2 The Dynamic Discipline of Species Delimitation hellip 39
lichenized fungi and the impact of alternative speciescircumscriptions and rates of molecular evolution ondivergence estimates PLoS ONE 8(12)e85240doi101371journalpone0085240
Leavitt SD Esslinger TL Hansen ES Divakar PK CrespoA Loomis BF Lumbsch HT (2014) DNA barcoding ofbrown Parmeliae (Parmeliaceae) species a molecularapproach for accurate specimen identification empha-sizing species in Greenland Organ Divers Evol 14(1)11ndash20 doi101007s13127-013-0147-1
Leliaert F Verbruggen H Vanormelingen P Steen FLopez-Bautista JM Zuccarello GC De Clerck O(2014) DNA-based species delimitation in algae Eur JPhycol 49(2) doi101080096702622014904524
Leuckert C (1985) Probleme der Flechten-Chemotaxon-omiemdashStoffkombinationen und ihre taxonomischeWertung Ber Deut Bot Ges 98401ndash408
Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
Lohtander K Kaumlllersjouml M Roland M Tehler A (2000)The family physciaceae in fennoscandia phylogenyinferred from its sequences Mycologia 92(4)728ndash735
Longton RE (1997) The role of bryophytes and lichens inpolar ecosystems In Woodin SJ Marquiss M (eds)Ecology of Arctic Environments Blackwell ScienceOxford pp 69ndash96 (Special publication No 13)
Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
Lumbsch HT (1994) Die Lecanora subfusca-Gruppe inAustralasien J Hattori Bot Lab 771ndash175
Lumbsch HT (1998a) Taxonomic use of metabolic data inlichen-forming fungi In Frisvad JC Bridge PDArora DK (eds) Chemical Fungal Taxonomy MarcelDekker New York pp 345ndash387
Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
Mayden RL (1997) A hierarchy of species concepts thedenouement in the saga of the species problem InClaridge MF Dawah HA Wilson MR (eds) Speciesthe units of biodiversity Chapman amp Hall Londonpp 381ndash424
Mayr E (1963) Animal species and evolution HarvardUniversity Press Cambridge
Mayr E (1970) Populations species and evolutionBelknap Press of Harvard University Press Cambridge
McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
McDonald T Miadlikowska J Lutzoni F (2003) Thelichen genus Sticta in the Great Smoky Mountains a
40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
McKay BD Mays HL Wu Y Li H Yao C-T Nishiumi IZou F (2013) An empirical comparison of character-based and coalescent-based approaches to speciesdelimitation in a young avian complex Mol Ecol 22(19)4943ndash4957 doi101111mec12446
Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
Mishler BD Brandon RN (1987) Individuality pluralismand the phylogenetic species concept Biol Philos 2397
Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
Nash TH Zavada M (1977) Population studies amongSonoran Desert species of Parmelia subg Xanthop-armelia (Parmeliaceae) Am J Bot 64(6)664ndash669
Nylander W (1866a) Circa novum in studio Lichenumcriterium chemicum Flora 49198ndash201
Nylander W (1866b) Hypochlorite of lime and hydrate ofpotash Two new criteria for the study of lichens(Tanslated and communicated by the Rev W ALeighton) Bot J Linn Soc 9358ndash365
OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
OrsquoNeill EM Schwartz R Bullock CT Williams JSShaffer HB Aguilar-Miguel X Parra-Olea G Weis-rock DW (2013) Parallel tagged amplicon sequencingreveals major lineages and phylogenetic structure inthe North American tiger salamander (Ambystomatigrinum) species complex Mol Ecol 22(1)111ndash129doi101111mec12049
Orock EA Leavitt SD Fonge BA St Clair LL LumbschHT (2012) DNA-based identification of lichen-form-ing fungi Can publicly available sequence databasesaid in lichen diversity inventories of Mount Cameroon(West Africa) The Lichenologist 44(6)833ndash839doi101017S0024282912000424
Otaacutelora MAG Martiacutenez I Aragoacuten G Molina MC (2010)Phylogeography and divergence date estimates of alichen species complex with a disjunct distributionpattern Am J Bot 97(2)216ndash223 doi103732ajb0900064
Padial J Castroviejo-Fisher S Kohler J Vila C ChaparroJ De la Riva I (2009) Deciphering the products ofevolution at the species level the need for anintegrative taxonomy Zoolog Scr 38(4)431ndash447doi101111j1463-6409200800381x
Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
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Park S-Y Choi J Kim JA Jeong M-H Kim S Lee Y-HHur J-S (2013a) Draft genome sequence of Cladoniamacilenta KoLRI003786 a lichen-forming fungusproducing biruloquinone Genome Announcements 1(5)e00695-13 doi101128genomeA00695-13
Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
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Pino-Bodas R Burgaz A Martiacuten M Lumbsch HT (2011)Phenotypical plasticity and homoplasy complicatespecies delimitation in the Cladonia gracilis group(Cladoniaceae Ascomycota) Organ Divers Evol 11(5)343ndash355 doi101007s13127-011-0062-2
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Pino-Bodas R Martiacuten M Burgaz A (2012b) Cladoniasubturgida and C iberica (Cladoniaceae) form asingle morphologically and chemically polymorphicspecies Mycol Prog 11(1)269ndash278 doi101007s11557-011-0746-1
Pino-Bodas R Ahti T Stenroos S Martiacuten MP BurgazAR (2013a) Multilocus approach to species recogni-tion in the Cladonia humilis complex (CladoniaceaeAscomycota) Am J Bot 100(4)664ndash678 doi103732ajb1200162
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Puillandre N Lambert A Brouillet S Achaz G (2012)ABGD automatic barcode gap discovery for primaryspecies delimitation Mol Ecol 21(8)1864ndash1877doi101111j1365-294X201105239x
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Raxworthy CJ Ingram CM Rabibisoa N Pearson RG(2007) Applications of ecological niche modelingfor species delimitation a review and empiricalevaluation using day geckos (Phelsuma) fromMadagascar Syst Biol 56(6)907ndash923 doi10108010635150701775111
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Rubinoff D (2006) Utility of mitochondrial DNA barcodesin species conservation Conserv Biol 20(4)1026ndash1033 doi101111j1523-1739200600372x
Ruiz-Sanchez E Sosa V (2010) Delimiting speciesboundaries within the Neotropical bamboo Otatea(Poaceae Bambusoideae) using molecular morpho-logical and ecological data Mol Phylogenet Evol 54(2)344ndash356 doi101016jympev200910035
Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
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Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
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Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
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Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
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Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
2 The Dynamic Discipline of Species Delimitation hellip 43
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Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
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Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
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Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
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Del-Prado R Cubas P Lumbsch HT Divakar PK BlancoO de Paz GA Molina MC Crespo A (2010) Geneticdistances within and among species in monophyleticlineages of Parmeliaceae (Ascomycota) as a tool fortaxon delimitation Mol Phylogenet Evol 56(1)125ndash133 doi101016jympev201004014
Del-Prado R Divakar PK Crespo A (2011) Using geneticdistances in addition to ITS molecular phylogeny toidentify potential species in the Parmotrema reticul-atum complex a case study The Lichenologist 43(06)569ndash583 doi101017S0024282911000582
Del-Prado R Blanco O Lumbsch HT Divakar PK ElixJA Molina MC Crespo A (2013) Molecular phylog-eny and historical biogeography of the lichen-formingfungal genus Flavoparmelia (Ascomycota Parmelia-ceae) Taxon 62(5)928ndash939 doi101270562522
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Dettman JR Jacobson DJ Turner E Pringle A TaylorJW (2003b) Reproductive isolation and phylogeneticdivergence in Neurospora comparing methods ofspecies recognition in a model eukaryote Evolution57(12)2721 doi10155403-074
Devkota S Cornejo C Werth S Chaudhary RP Sche-idegger C (2014) Characterization of microsatelliteloci in the Himalayan lichen fungus Lobaria pindar-ensis (Lobariaceae) Appl Plant Sci 2(5)1300101doi103732apps1300101
Divakar PK Molina MC Lumbsch HT Crespo A (2005)Parmelia barrenoae a new lichen species related toParmelia sulcata (Parmeliaceae) based on molecularand morphological data The Lichenologist 37(01)37ndash46 doi101017S0024282904014641
Divakar PK Amo De paz G del Prado R Esslinger TLCrespo A (2007) Upper cortex anatomy corroboratesphylogenetic hypothesis in species of Physconia(Ascomycota Lecanoromycetes) Mycol Res 111(11)1311ndash1320 doi101016jmycres200708009
Divakar PK Figueras G Hladun N Crespo A (2010)Molecular phylogenetic studies reveal an undescribedspecies within the North American concept of Mela-nelixia glabra (Parmeliaceae) Fungal Divers 42(1)47ndash55 doi101007s13225-010-0027-3
Donoghue MJ Gauthier A (2004) Implementing thephylocode Trends Ecol Evol 19(6)281ndash282 doi101016jtree200404004
Eaton DAR Ree RH (2013) Inferring phylogeny andintrogression using RADseq data an example fromflowering plants (Pedicularis Orobanchaceae) SystBiol 62(5)689ndash706 doi101093sysbiosyt032
Edwards SV (2009) Is a new and general theory ofmolecular systematics emerging Evolution 63(1)1ndash19 doi101111j1558-5646200800549x
Edwards DL Knowles LL (2014) Species detection andindividual assignment in species delimitation canintegrative data increase efficacy Proc R Soc B BiolSci 281(1777) doi101098rspb20132765
Egan RS (1986) Correlations and non-correlations ofchemical variation patterns with lichen morphologyand geography The Bryologist 8999ndash110
Elix JA Corush J Lumbsch HT (2009) Triterpenechemosyndromes and subtle morphological characterscharacterise lineages in the Physcia aipolia group inAustralia (Ascomycota) Syst Biodivers 7(04)479ndash487 doi101017S1477200009990223
Emerson KJ Merz CR Catchen JM Hohenlohe PACresko WA Bradshaw WE Holzapfel CM (2010)Resolving postglacial phylogeography using high-throughput sequencing Proc Natl Acad Sci 107(37)16196ndash16200 doi101073pnas1006538107
Ence DD Carstens BC (2011) SpedeSTEM a rapid andaccurate method for species delimitation Mol EcolResour 11(3)473ndash480 doi101111j1755-0998201002947x
EvannoGRegnaut SGoudet J (2005)Detecting thenumberof clusters of individuals using the software STRUC-TURE a simulation study Mol Ecol 14(8)2611ndash2620doi101111j1365-294X200502553x
Falush D Stephens M Pritchard JK (2003) Inference ofpopulation structure using multilocus genotype datalinked loci and correlated allele frequencies Genetics164(4)1567ndash1587 doi101111j1471-8286200701758x
Fan HH Kubatko LS (2011) Estimating species treesusing approximate Bayesian computation Mol Phylo-genet Evol 59(2)354ndash363 doi101016jympev201102019
Fernaacutendez-Mendoza F Printzen C (2013) Pleistoceneexpansion of the bipolar lichen Cetraria aculeata intothe Southern hemisphere Mol Ecol 22(7)1961ndash1983doi101111mec12210
Fernaacutendez-Mendoza F Domaschke S Garciacutea MA JordanP Martin MP Printzen C (2011) Population structure
2 The Dynamic Discipline of Species Delimitation hellip 37
of mycobionts and photobionts of the widespreadlichen Cetraria aculeata Mol Ecol 20(6)1208ndash1232doi101111j1365-294X201004993x
Fraley C Raftery A (2007) Model-based methods ofclassification Using the mclust software in chemo-metrics J Stat Softw 18i06 [Available online at httpwwwdoajorgdoajfunc=abstractampid=218544]
Fujisawa T Barraclough TG (2013) Delimiting speciesusing single-locus data and the generalized mixed yulecoalescent (GMYC) approach a revised method andevaluation on simulated datasets Syst Biol 62(5)707ndash724 doi101093sysbiosyt033
Fujita MK Leacheacute AD Burbrink FT McGuire JA MoritzC (2012) Coalescent-based species delimitation in anintegrative taxonomy Trends Ecol Evol 27(9)480ndash488 doi101016jtree201204012
Gatesy J OrsquoGrady P Baker RH (1999) Corroborationamong data sets in simultaneous analysis hiddensupport for phylogenetic relationships among higherlevel Artiodactyl taxa Cladistics 15(3)271ndash313doi101111j1096-00311999tb00268x
Gladieux P Ropars J Badouin H Branca A Aguileta Gde Vienne DM Rodriacuteguez de la Vega RC Branco SGiraud T (2014) Fungal evolutionary genomicsprovides insight into the mechanisms of adaptivedivergence in eukaryotes Mol Ecol 23(4)753ndash773doi101111mec12631
Goffinet B Miadlikowska J Goward T (2003) Phyloge-netic inferences based on nrDNA sequences supportfive morphospecies within the Peltigera didactylacomplex (Lichenized Ascomycota) The Bryologist106(3)349ndash364 doi10163901
Gowan SP (1986) Evolution of secondary natural productsin the genus Porpidia (Ascomycata Porpidiaceae) AmJ Bot 73606
Griffin PC Hoffmann AA (2014) Limited genetic diver-gence among Australian alpine Poa tussock grassescoupled with regional structuring points to ongoinggene flow and taxonomic challenges Ann Bot doi101093aobmcu017
Grube M Hawksworth DL (2007) Trouble with lichenthe re-evaluation and re-interpretation of thallus formand fruit body types in the molecular era Mycol Res111(9)1116ndash1132 doi101016jmycres200704008
Gueidan C Savi S Thues H Roux C Keller C Tibell LPrieto M Heimarsson S Breuss O Orange A FrobergL Wynns AA Navarro-Rosines P Krzewicka BPykaelae J Grube M Lutzoni F (2009) Genericclassification of the Verrucariaceae (Ascomycota)based on molecular and morphological evidencerecent progress and remaining challenges Taxon 58(1)184ndash208
Guillot G Mortier F Estoup A (2005) Geneland acomputer package for landscape genetics Mol EcolNotes 5(3)712ndash715 doi101111j1471-8286200501031x
Guillot G Renaud S Ledevin R Michaux J Claude J(2012) A unifying model for the analysis of pheno-typic genetic and geographic data Syst Biol 61(6)897ndash911 doi101093sysbiosys038
Guisan A Thuiller W (2005) Predicting species distribu-tion offering more than simple habitat models EcolLett 8(9)993ndash1009 doi101111j1461-0248200500792x
Hale ME (1990) A synopsis of the lichen genusXanthoparmelia (Vainio) Hale (AscomycotinaParmeliaceae) Smithsonian Institution Press Wash-ington DC
Hamilton CA Hendrixson BE Brewer MS Bond JE(2014) An evaluation of sampling effects on multipleDNA barcoding methods leads to an integrativeapproach for delimiting species a case study ofthe North American tarantula genus Aphonopelma(Araneae Mygalomorphae Theraphosidae) MolPhylogenet Evol 7179ndash93 doi101016jympev201311007
Hausdorf B (2011) Progress toward a general speciesconcept Evolution 65(4)923ndash931 doi101111j1558-5646201101231x
Hausdorf B Hennig C (2010) Species delimitation usingdominant and codominant multilocus markers SystBiol 59(5)491ndash503 doi101093sysbiosyq039
Hawksworth DL (1976) Lichen chemotaxonomy InBailey RH (ed) Lichenology progress and problemsAcademic London pp 139ndash184
Hebert PDN Cywinska A Ball SL deWaard JR (2003)Biological identifications through DNA barcodesProc R Soc Lond B Biol Sci 270(1512)313ndash321doi101098rspb20022218
Hebert PDN Stoeckle MY Zemlak TS Francis CM(2004) Identification of birds through DNA barcodesPLoS Biol 2(10)e312 doi101371journalpbio0020312
Heled J Drummond AJ (2010) Bayesian inference ofspecies trees from multilocus data Mol Biol Evol 27(3)570ndash580 doi101093molbevmsp274
Hey J (2006) On the failure of modern species conceptsTrends Ecol Evol 21(8)447ndash450 doi101016jtree200605011
Hibbett DS OhmanA Glotzer D NuhnMKirk P NilssonRH (2011) Progress in molecular and morphologicaltaxon discovery in Fungi and options for formalclassification on environmental sequences Fungal BiolRev 2538ndash47 doi101016jfbr201101001
Houmlgnabba F Wedin M (2003) Molecular phylogeny ofthe Sphaerophorus globosus species complex Cladis-tics 19(3)224ndash232 doi101111j1096-00312003tb00365x
Hudson RR Coyne JA (2002) Mathematical conse-quences of the genealogical species concept Evolu-tion 56(8)1557 doi101111j0014-38202002tb01467x
Huelsenbeck JP Andolfatto P Huelsenbeck ET (2011)Structurama Bayesian inference of population struc-ture Evol Bioinf Online 7(2011)55ndash59 doi104137EBOS6761
Jones G Oxelman B (2014) DISSECT an assignment-free Bayesian discovery method for species delimita-tion under the multispecies coalescent Bioinformaticsdoi101101003178
38 SD Leavitt et al
Kekkonen M Hebert PD (2014) DNA barcode-baseddelineation of putative species efficient start fortaxonomic workflows Mol Ecol Res doi1011111755-099812233
Kelly LJ Hollingsworth PM Coppins BJ Ellis CJHarrold P Tosh J Yahr R (2011) DNA barcoding oflichenized fungi demonstrates high identification suc-cess in a floristic context New Phytol 191(1)288ndash300doi101111j1469-8137201103677x
Kiss L (2012) Limits of nuclear ribosomal DNA internaltranscribed spacer (ITS) sequences as species barcodesfor fungi Proc Natl Acad Sci 109(27)E1811ndashE1811doi101073pnas1207143109
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Knowles LL Carstens BC (2007) Delimiting specieswithout monophyletic gene trees Syst Biol 56(6)887ndash895 doi10108010635150701701091
Kroken S Taylor JW (2001) A gene genealogical approachto recognize phylogenetic species boundaries in thelichenized fungus Letharia Mycologia 93(1)38ndash53
Kubatko LS Degnan JH (2007) Inconsistency of phylo-genetic estimates from concatenated data under coa-lescence Syst Biol 56(1)17ndash24 doi10108010635150601146041
Latch EK Dharmarajan G Glaubitz JC Rhodes OE Jr(2006) Relative performance of Bayesian clusteringsoftware for inferring population substructure andindividual assignments at low levels of populationdifferentiation Conserv Genet 7(2)295ndash302 doi101007s10592-005-9098-1
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Le Gac M Hood ME Fournier E Giraud T (2007)Phylogenetic evidence of host-specific cryptic speciesin the anther smut fungus Evolution 61(1)15ndash26doi101111j1558-5646200700002x
Leacheacute AD (2009) Species tree discordance traces tophylogeographic clade boundaries in North Americanfence lizards (Sceloporus) Syst Biol 58(6)547ndash559doi101093sysbiosyp057
Leacheacute AD Fujita MK (2010) Bayesian species delim-itation in West African forest geckos (Hemidactylusfasciatus) Proc R Soc B Biol Sci 2773071ndash3077doi101098rspb20100662
Leacheacute AD Koo M Spencer C Papenfuss T Fisher RMcGuire J (2009) Quantifying ecological morpho-logical and genetic variation to delimit species in thecoast horned lizard species complex (Phrynosoma)Proc Natl Acad Sci USA 10612418ndash12423 doi101073pnas0906380106
Leacheacute AD Fujita MK Minin VN Bouckaert RR (2014)Species delimitation using genome-wide SNP dataSystematic Biology doi101093sysbiosyu018
Leavitt SD Fankhauser JD Leavitt DH Porter LDJohnson LA St Clair LL (2011a) Complex patterns ofspeciation in cosmopolitan ldquorock posyrdquo lichensmdashdiscovering and delimiting cryptic fungal species in
the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
Leavitt SD Esslinger TL Lumbsch HT (2012c) Neogene-dominated diversification in neotropical montanelichens dating divergence events in the lichen-form-ing fungal genus Oropogon (Parmeliaceae) Am J Bot99(11)1764ndash1777 doi103732ajb1200146
Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Lumbsch HT St Clair LL(2013c) DNA barcode identification of lichen-formingfungal species in the Rhizoplaca melanophthalmaspecies-complex (Lecanorales Lecanoraceae) includ-ing five new species MycoKeys 71ndash22 doi103897mycokeys74508
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Vondraacutek J Thorsten Lum-bsch H Clair LLS (2013d) Local representation ofglobal diversity in a cosmopolitan lichen-formingfungal species complex (Rhizoplaca Ascomycota)J Biogeogr 40(9)1792ndash1806 doi101111jbi12118
Leavitt SD Lumbsch HT Stenroos S St Clair LL(2013e) Pleistocene speciation in North American
2 The Dynamic Discipline of Species Delimitation hellip 39
lichenized fungi and the impact of alternative speciescircumscriptions and rates of molecular evolution ondivergence estimates PLoS ONE 8(12)e85240doi101371journalpone0085240
Leavitt SD Esslinger TL Hansen ES Divakar PK CrespoA Loomis BF Lumbsch HT (2014) DNA barcoding ofbrown Parmeliae (Parmeliaceae) species a molecularapproach for accurate specimen identification empha-sizing species in Greenland Organ Divers Evol 14(1)11ndash20 doi101007s13127-013-0147-1
Leliaert F Verbruggen H Vanormelingen P Steen FLopez-Bautista JM Zuccarello GC De Clerck O(2014) DNA-based species delimitation in algae Eur JPhycol 49(2) doi101080096702622014904524
Leuckert C (1985) Probleme der Flechten-Chemotaxon-omiemdashStoffkombinationen und ihre taxonomischeWertung Ber Deut Bot Ges 98401ndash408
Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
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Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
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Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
Mayden RL (1997) A hierarchy of species concepts thedenouement in the saga of the species problem InClaridge MF Dawah HA Wilson MR (eds) Speciesthe units of biodiversity Chapman amp Hall Londonpp 381ndash424
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Mayr E (1970) Populations species and evolutionBelknap Press of Harvard University Press Cambridge
McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
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40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
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Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
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Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
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Nylander W (1866a) Circa novum in studio Lichenumcriterium chemicum Flora 49198ndash201
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OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
OrsquoNeill EM Schwartz R Bullock CT Williams JSShaffer HB Aguilar-Miguel X Parra-Olea G Weis-rock DW (2013) Parallel tagged amplicon sequencingreveals major lineages and phylogenetic structure inthe North American tiger salamander (Ambystomatigrinum) species complex Mol Ecol 22(1)111ndash129doi101111mec12049
Orock EA Leavitt SD Fonge BA St Clair LL LumbschHT (2012) DNA-based identification of lichen-form-ing fungi Can publicly available sequence databasesaid in lichen diversity inventories of Mount Cameroon(West Africa) The Lichenologist 44(6)833ndash839doi101017S0024282912000424
Otaacutelora MAG Martiacutenez I Aragoacuten G Molina MC (2010)Phylogeography and divergence date estimates of alichen species complex with a disjunct distributionpattern Am J Bot 97(2)216ndash223 doi103732ajb0900064
Padial J Castroviejo-Fisher S Kohler J Vila C ChaparroJ De la Riva I (2009) Deciphering the products ofevolution at the species level the need for anintegrative taxonomy Zoolog Scr 38(4)431ndash447doi101111j1463-6409200800381x
Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
Papong K Luumlcking R Thammathaworn A BoonpragobK (2009) Four new taxa of Chroodiscus (thelotremoidGraphidaceae) from Southeast Asia The Bryologist112(1)152ndash163 doi1016390007-2745-1121152
Park S-Y Choi J Kim JA Jeong M-H Kim S Lee Y-HHur J-S (2013a) Draft genome sequence of Cladoniamacilenta KoLRI003786 a lichen-forming fungusproducing biruloquinone Genome Announcements 1(5)e00695-13 doi101128genomeA00695-13
Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
Peacuterez-Ortega S Fernaacutendez-Mendoza F Raggio J VivasM Ascaso C Sancho LG Printzen C de los Riacuteos A(2012) Extreme phenotypic variation in Cetrariaaculeata (lichenized Ascomycota) adaptation or inci-dental modification Ann Bot 109(6)1133ndash1148doi101093aobmcs042
Pino-Bodas R Burgaz A Martiacuten M Lumbsch HT (2011)Phenotypical plasticity and homoplasy complicatespecies delimitation in the Cladonia gracilis group(Cladoniaceae Ascomycota) Organ Divers Evol 11(5)343ndash355 doi101007s13127-011-0062-2
Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
Pino-Bodas R Martiacuten M Burgaz A (2012b) Cladoniasubturgida and C iberica (Cladoniaceae) form asingle morphologically and chemically polymorphicspecies Mycol Prog 11(1)269ndash278 doi101007s11557-011-0746-1
Pino-Bodas R Ahti T Stenroos S Martiacuten MP BurgazAR (2013a) Multilocus approach to species recogni-tion in the Cladonia humilis complex (CladoniaceaeAscomycota) Am J Bot 100(4)664ndash678 doi103732ajb1200162
Pino-Bodas R Martiacuten AP Burgaz AR Lumbsch HT(2013b) Species delimitation in Cladonia (Ascomy-cota) a challenge to the DNA barcoding philosophyMol Ecol Resour 13(6)1058ndash1068 doi1011111755-099812086
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Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
Pringle A Baker DM Platt JL Wares JP Latgeacute JPTaylor JW (2005) Cryptic speciation in the cosmo-politan and clonal human pathogenic fungus Asper-gillus fumigatus Evolution 59(9)1886ndash1899 doi10155404-2411
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Puillandre N Lambert A Brouillet S Achaz G (2012)ABGD automatic barcode gap discovery for primaryspecies delimitation Mol Ecol 21(8)1864ndash1877doi101111j1365-294X201105239x
Rannala B Yang Z (2003) Bayes estimation of speciesdivergence times and ancestral population sizes usingDNA sequences from multiple loci Genetics1641645ndash1656
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phylogeny of the genus Lecania (Ramalinaceaelichenized Ascomycota) Mycol Res 111(5)581ndash591doi101016jmycres200703001
Reid N Carstens B (2012) Phylogenetic estimation errorcan decrease the accuracy of species delimitation aBayesian implementation of the general mixed Yule-coalescent model BMC Evol Biol 12(1)196 doi1011861471-2148-12-196
Rissler L Apodaca J (2007) Adding more ecology intospecies delimitation ecological niche models andphylogeography help define cryptic species in theBlack Salamander (Aneides flavipunctatus) Syst Biol56924ndash942 doi10108010635150701703063
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Rivas Plata E Lumbsch HT (2011) Parallel evolution andphenotypic divergence in lichenized fungi a casestudy in the lichen-forming fungal family Graphida-ceae (Ascomycota Lecanoromycetes Ostropales)Mol Phylogenet Evol 61(1)45ndash63 doi101016jympev201104025
Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
Ross KG Gotzek D Ascunce MS Shoemaker DD (2010)Species delimitation a case study in a problematic anttaxon Syst Biol 59(2)162ndash184 doi101093sysbiosyp089
Rubin BER Ree RH Moreau CS (2012) Inferringphylogenies from RAD sequence data PLoS ONE 7(4)e33394 doi101371journalpone0033394
Rubinoff D (2006) Utility of mitochondrial DNA barcodesin species conservation Conserv Biol 20(4)1026ndash1033 doi101111j1523-1739200600372x
Ruiz-Sanchez E Sosa V (2010) Delimiting speciesboundaries within the Neotropical bamboo Otatea(Poaceae Bambusoideae) using molecular morpho-logical and ecological data Mol Phylogenet Evol 54(2)344ndash356 doi101016jympev200910035
Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
morphological characters Antarctic Sci 22 (SpecialIssue 06)727ndash741 doi101017S0954102010000477
Salicini I Ibaacutentildeez C Juste J (2011) Multilocus phylogenyand species delimitation within the Nattererrsquos batspecies complex in the Western Palearctic MolPhylogenet Evol 61(3)888ndash898 doi101016jympev201108010
Satler JD Carstens BC Hedin M (2013) Multilocusspecies delimitation in a complex of morphologicallyconserved trapdoor spiders (Mygalomorphae Antro-diaetidae Aliatypus) Syst Biol 62(6)805ndash823 doi101093sysbiosyt041
Schlick-Steiner BC Steiner FM Seifert B Stauffer CChristian E Crozier RH (2010) Integrative taxonomya multisource approach to exploring biodiversityAnnu Rev Entomol 55(1)421ndash438 doi101146annurev-ento-112408-085432
Schoch CL Seifert KA Huhndorf S Robert V SpougeJL Levesque CA Chen W Fungal Barcoding Con-sortium (2012) Nuclear ribosomal internal transcribedspacer (ITS) region as a universal DNA barcodemarker for Fungi Proc Natl Acad Sci 109(16)6241ndash6246 doi101073pnas1117018109
Seacuterusiaux E Villarreal AJC Wheeler T Goffinet B(2011) Recent origin active speciation and dispersalfor the lichen genus Nephroma (Peltigerales) inMacaronesia J Biogeogr 38(6)1138ndash1151 doi101111j1365-2699201002469x
Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
Simpson GG (1951) The species concept Evolution5285ndash298
Sites JW Marshall JC (2003) Delimiting species a renais-sance issue in systematic biology Trends Ecol Evol18462ndash470 doi101016S0169-5347(03)00184-8
Sites JW Marshall JC (2004) Operational criteria fordelimiting species Annu Rev Ecol Evol Syst 35(1)199ndash227 doi101146annurevecolsys35112202130128
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Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
Talavera G Dincă V Vila R (2013) Factors affectingspecies delimitations with the GMYC model insightsfrom a butterfly survey Methods Ecol Evol 4(12)1101ndash1110 doi1011112041-210X12107
Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
Tewksbury J Anderson JGT Bakker JD Billo TJDunwiddie PW Groom MJ Hampton SE HermanSG Levey DJ Machnicki NJ del Rio CM PowerME Rowell K Salomon AK Stacey L TrombulakSC Wheeler TA (2014) Natural historyrsquos place inscience and society BioScience doi101093bioscibiu032
Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
Werth S Cornejo C Scheidegger C (2013) Characteriza-tion of microsatellite loci in the lichen fungus Lobariapulmonaria (Lobariaceae) Appl Plant Sci 1(2)1200290 doi103732apps1200290
Westberg M Arup U Kaumlrnefelt I (2007) Phylogeneticstudies in the Candelariaceae (lichenized Ascomy-cota) based on nuclear ITS DNA sequence dataMycol Res 111(11)1277ndash1284 doi101017S0953756204002102
Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
2 The Dynamic Discipline of Species Delimitation hellip 43
(Lepidoptera Lycaenidae) Front Zoo 4(1)8 doi1011861742-9994-4-8
Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
of mycobionts and photobionts of the widespreadlichen Cetraria aculeata Mol Ecol 20(6)1208ndash1232doi101111j1365-294X201004993x
Fraley C Raftery A (2007) Model-based methods ofclassification Using the mclust software in chemo-metrics J Stat Softw 18i06 [Available online at httpwwwdoajorgdoajfunc=abstractampid=218544]
Fujisawa T Barraclough TG (2013) Delimiting speciesusing single-locus data and the generalized mixed yulecoalescent (GMYC) approach a revised method andevaluation on simulated datasets Syst Biol 62(5)707ndash724 doi101093sysbiosyt033
Fujita MK Leacheacute AD Burbrink FT McGuire JA MoritzC (2012) Coalescent-based species delimitation in anintegrative taxonomy Trends Ecol Evol 27(9)480ndash488 doi101016jtree201204012
Gatesy J OrsquoGrady P Baker RH (1999) Corroborationamong data sets in simultaneous analysis hiddensupport for phylogenetic relationships among higherlevel Artiodactyl taxa Cladistics 15(3)271ndash313doi101111j1096-00311999tb00268x
Gladieux P Ropars J Badouin H Branca A Aguileta Gde Vienne DM Rodriacuteguez de la Vega RC Branco SGiraud T (2014) Fungal evolutionary genomicsprovides insight into the mechanisms of adaptivedivergence in eukaryotes Mol Ecol 23(4)753ndash773doi101111mec12631
Goffinet B Miadlikowska J Goward T (2003) Phyloge-netic inferences based on nrDNA sequences supportfive morphospecies within the Peltigera didactylacomplex (Lichenized Ascomycota) The Bryologist106(3)349ndash364 doi10163901
Gowan SP (1986) Evolution of secondary natural productsin the genus Porpidia (Ascomycata Porpidiaceae) AmJ Bot 73606
Griffin PC Hoffmann AA (2014) Limited genetic diver-gence among Australian alpine Poa tussock grassescoupled with regional structuring points to ongoinggene flow and taxonomic challenges Ann Bot doi101093aobmcu017
Grube M Hawksworth DL (2007) Trouble with lichenthe re-evaluation and re-interpretation of thallus formand fruit body types in the molecular era Mycol Res111(9)1116ndash1132 doi101016jmycres200704008
Gueidan C Savi S Thues H Roux C Keller C Tibell LPrieto M Heimarsson S Breuss O Orange A FrobergL Wynns AA Navarro-Rosines P Krzewicka BPykaelae J Grube M Lutzoni F (2009) Genericclassification of the Verrucariaceae (Ascomycota)based on molecular and morphological evidencerecent progress and remaining challenges Taxon 58(1)184ndash208
Guillot G Mortier F Estoup A (2005) Geneland acomputer package for landscape genetics Mol EcolNotes 5(3)712ndash715 doi101111j1471-8286200501031x
Guillot G Renaud S Ledevin R Michaux J Claude J(2012) A unifying model for the analysis of pheno-typic genetic and geographic data Syst Biol 61(6)897ndash911 doi101093sysbiosys038
Guisan A Thuiller W (2005) Predicting species distribu-tion offering more than simple habitat models EcolLett 8(9)993ndash1009 doi101111j1461-0248200500792x
Hale ME (1990) A synopsis of the lichen genusXanthoparmelia (Vainio) Hale (AscomycotinaParmeliaceae) Smithsonian Institution Press Wash-ington DC
Hamilton CA Hendrixson BE Brewer MS Bond JE(2014) An evaluation of sampling effects on multipleDNA barcoding methods leads to an integrativeapproach for delimiting species a case study ofthe North American tarantula genus Aphonopelma(Araneae Mygalomorphae Theraphosidae) MolPhylogenet Evol 7179ndash93 doi101016jympev201311007
Hausdorf B (2011) Progress toward a general speciesconcept Evolution 65(4)923ndash931 doi101111j1558-5646201101231x
Hausdorf B Hennig C (2010) Species delimitation usingdominant and codominant multilocus markers SystBiol 59(5)491ndash503 doi101093sysbiosyq039
Hawksworth DL (1976) Lichen chemotaxonomy InBailey RH (ed) Lichenology progress and problemsAcademic London pp 139ndash184
Hebert PDN Cywinska A Ball SL deWaard JR (2003)Biological identifications through DNA barcodesProc R Soc Lond B Biol Sci 270(1512)313ndash321doi101098rspb20022218
Hebert PDN Stoeckle MY Zemlak TS Francis CM(2004) Identification of birds through DNA barcodesPLoS Biol 2(10)e312 doi101371journalpbio0020312
Heled J Drummond AJ (2010) Bayesian inference ofspecies trees from multilocus data Mol Biol Evol 27(3)570ndash580 doi101093molbevmsp274
Hey J (2006) On the failure of modern species conceptsTrends Ecol Evol 21(8)447ndash450 doi101016jtree200605011
Hibbett DS OhmanA Glotzer D NuhnMKirk P NilssonRH (2011) Progress in molecular and morphologicaltaxon discovery in Fungi and options for formalclassification on environmental sequences Fungal BiolRev 2538ndash47 doi101016jfbr201101001
Houmlgnabba F Wedin M (2003) Molecular phylogeny ofthe Sphaerophorus globosus species complex Cladis-tics 19(3)224ndash232 doi101111j1096-00312003tb00365x
Hudson RR Coyne JA (2002) Mathematical conse-quences of the genealogical species concept Evolu-tion 56(8)1557 doi101111j0014-38202002tb01467x
Huelsenbeck JP Andolfatto P Huelsenbeck ET (2011)Structurama Bayesian inference of population struc-ture Evol Bioinf Online 7(2011)55ndash59 doi104137EBOS6761
Jones G Oxelman B (2014) DISSECT an assignment-free Bayesian discovery method for species delimita-tion under the multispecies coalescent Bioinformaticsdoi101101003178
38 SD Leavitt et al
Kekkonen M Hebert PD (2014) DNA barcode-baseddelineation of putative species efficient start fortaxonomic workflows Mol Ecol Res doi1011111755-099812233
Kelly LJ Hollingsworth PM Coppins BJ Ellis CJHarrold P Tosh J Yahr R (2011) DNA barcoding oflichenized fungi demonstrates high identification suc-cess in a floristic context New Phytol 191(1)288ndash300doi101111j1469-8137201103677x
Kiss L (2012) Limits of nuclear ribosomal DNA internaltranscribed spacer (ITS) sequences as species barcodesfor fungi Proc Natl Acad Sci 109(27)E1811ndashE1811doi101073pnas1207143109
Kluge AG (1989) A concern for evidence and aphylogenetic hypothesis for relationships among Epi-crates (Boidae Serpentes) Syst Zool 38(1)7ndash25doi101093sysbio3817
Knowles LL Carstens BC (2007) Delimiting specieswithout monophyletic gene trees Syst Biol 56(6)887ndash895 doi10108010635150701701091
Kroken S Taylor JW (2001) A gene genealogical approachto recognize phylogenetic species boundaries in thelichenized fungus Letharia Mycologia 93(1)38ndash53
Kubatko LS Degnan JH (2007) Inconsistency of phylo-genetic estimates from concatenated data under coa-lescence Syst Biol 56(1)17ndash24 doi10108010635150601146041
Latch EK Dharmarajan G Glaubitz JC Rhodes OE Jr(2006) Relative performance of Bayesian clusteringsoftware for inferring population substructure andindividual assignments at low levels of populationdifferentiation Conserv Genet 7(2)295ndash302 doi101007s10592-005-9098-1
Lawrey JD (1986) Biological role of lichen substancesThe Bryologist 89(2)111ndash122
Le Gac M Hood ME Fournier E Giraud T (2007)Phylogenetic evidence of host-specific cryptic speciesin the anther smut fungus Evolution 61(1)15ndash26doi101111j1558-5646200700002x
Leacheacute AD (2009) Species tree discordance traces tophylogeographic clade boundaries in North Americanfence lizards (Sceloporus) Syst Biol 58(6)547ndash559doi101093sysbiosyp057
Leacheacute AD Fujita MK (2010) Bayesian species delim-itation in West African forest geckos (Hemidactylusfasciatus) Proc R Soc B Biol Sci 2773071ndash3077doi101098rspb20100662
Leacheacute AD Koo M Spencer C Papenfuss T Fisher RMcGuire J (2009) Quantifying ecological morpho-logical and genetic variation to delimit species in thecoast horned lizard species complex (Phrynosoma)Proc Natl Acad Sci USA 10612418ndash12423 doi101073pnas0906380106
Leacheacute AD Fujita MK Minin VN Bouckaert RR (2014)Species delimitation using genome-wide SNP dataSystematic Biology doi101093sysbiosyu018
Leavitt SD Fankhauser JD Leavitt DH Porter LDJohnson LA St Clair LL (2011a) Complex patterns ofspeciation in cosmopolitan ldquorock posyrdquo lichensmdashdiscovering and delimiting cryptic fungal species in
the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
Leavitt SD Esslinger TL Lumbsch HT (2012c) Neogene-dominated diversification in neotropical montanelichens dating divergence events in the lichen-form-ing fungal genus Oropogon (Parmeliaceae) Am J Bot99(11)1764ndash1777 doi103732ajb1200146
Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Lumbsch HT St Clair LL(2013c) DNA barcode identification of lichen-formingfungal species in the Rhizoplaca melanophthalmaspecies-complex (Lecanorales Lecanoraceae) includ-ing five new species MycoKeys 71ndash22 doi103897mycokeys74508
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Vondraacutek J Thorsten Lum-bsch H Clair LLS (2013d) Local representation ofglobal diversity in a cosmopolitan lichen-formingfungal species complex (Rhizoplaca Ascomycota)J Biogeogr 40(9)1792ndash1806 doi101111jbi12118
Leavitt SD Lumbsch HT Stenroos S St Clair LL(2013e) Pleistocene speciation in North American
2 The Dynamic Discipline of Species Delimitation hellip 39
lichenized fungi and the impact of alternative speciescircumscriptions and rates of molecular evolution ondivergence estimates PLoS ONE 8(12)e85240doi101371journalpone0085240
Leavitt SD Esslinger TL Hansen ES Divakar PK CrespoA Loomis BF Lumbsch HT (2014) DNA barcoding ofbrown Parmeliae (Parmeliaceae) species a molecularapproach for accurate specimen identification empha-sizing species in Greenland Organ Divers Evol 14(1)11ndash20 doi101007s13127-013-0147-1
Leliaert F Verbruggen H Vanormelingen P Steen FLopez-Bautista JM Zuccarello GC De Clerck O(2014) DNA-based species delimitation in algae Eur JPhycol 49(2) doi101080096702622014904524
Leuckert C (1985) Probleme der Flechten-Chemotaxon-omiemdashStoffkombinationen und ihre taxonomischeWertung Ber Deut Bot Ges 98401ndash408
Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
Lohtander K Kaumlllersjouml M Roland M Tehler A (2000)The family physciaceae in fennoscandia phylogenyinferred from its sequences Mycologia 92(4)728ndash735
Longton RE (1997) The role of bryophytes and lichens inpolar ecosystems In Woodin SJ Marquiss M (eds)Ecology of Arctic Environments Blackwell ScienceOxford pp 69ndash96 (Special publication No 13)
Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
Lumbsch HT (1994) Die Lecanora subfusca-Gruppe inAustralasien J Hattori Bot Lab 771ndash175
Lumbsch HT (1998a) Taxonomic use of metabolic data inlichen-forming fungi In Frisvad JC Bridge PDArora DK (eds) Chemical Fungal Taxonomy MarcelDekker New York pp 345ndash387
Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
Mayden RL (1997) A hierarchy of species concepts thedenouement in the saga of the species problem InClaridge MF Dawah HA Wilson MR (eds) Speciesthe units of biodiversity Chapman amp Hall Londonpp 381ndash424
Mayr E (1963) Animal species and evolution HarvardUniversity Press Cambridge
Mayr E (1970) Populations species and evolutionBelknap Press of Harvard University Press Cambridge
McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
McDonald T Miadlikowska J Lutzoni F (2003) Thelichen genus Sticta in the Great Smoky Mountains a
40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
McKay BD Mays HL Wu Y Li H Yao C-T Nishiumi IZou F (2013) An empirical comparison of character-based and coalescent-based approaches to speciesdelimitation in a young avian complex Mol Ecol 22(19)4943ndash4957 doi101111mec12446
Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
Mishler BD Brandon RN (1987) Individuality pluralismand the phylogenetic species concept Biol Philos 2397
Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
Nash TH Zavada M (1977) Population studies amongSonoran Desert species of Parmelia subg Xanthop-armelia (Parmeliaceae) Am J Bot 64(6)664ndash669
Nylander W (1866a) Circa novum in studio Lichenumcriterium chemicum Flora 49198ndash201
Nylander W (1866b) Hypochlorite of lime and hydrate ofpotash Two new criteria for the study of lichens(Tanslated and communicated by the Rev W ALeighton) Bot J Linn Soc 9358ndash365
OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
OrsquoNeill EM Schwartz R Bullock CT Williams JSShaffer HB Aguilar-Miguel X Parra-Olea G Weis-rock DW (2013) Parallel tagged amplicon sequencingreveals major lineages and phylogenetic structure inthe North American tiger salamander (Ambystomatigrinum) species complex Mol Ecol 22(1)111ndash129doi101111mec12049
Orock EA Leavitt SD Fonge BA St Clair LL LumbschHT (2012) DNA-based identification of lichen-form-ing fungi Can publicly available sequence databasesaid in lichen diversity inventories of Mount Cameroon(West Africa) The Lichenologist 44(6)833ndash839doi101017S0024282912000424
Otaacutelora MAG Martiacutenez I Aragoacuten G Molina MC (2010)Phylogeography and divergence date estimates of alichen species complex with a disjunct distributionpattern Am J Bot 97(2)216ndash223 doi103732ajb0900064
Padial J Castroviejo-Fisher S Kohler J Vila C ChaparroJ De la Riva I (2009) Deciphering the products ofevolution at the species level the need for anintegrative taxonomy Zoolog Scr 38(4)431ndash447doi101111j1463-6409200800381x
Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
Papong K Luumlcking R Thammathaworn A BoonpragobK (2009) Four new taxa of Chroodiscus (thelotremoidGraphidaceae) from Southeast Asia The Bryologist112(1)152ndash163 doi1016390007-2745-1121152
Park S-Y Choi J Kim JA Jeong M-H Kim S Lee Y-HHur J-S (2013a) Draft genome sequence of Cladoniamacilenta KoLRI003786 a lichen-forming fungusproducing biruloquinone Genome Announcements 1(5)e00695-13 doi101128genomeA00695-13
Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
Peacuterez-Ortega S Fernaacutendez-Mendoza F Raggio J VivasM Ascaso C Sancho LG Printzen C de los Riacuteos A(2012) Extreme phenotypic variation in Cetrariaaculeata (lichenized Ascomycota) adaptation or inci-dental modification Ann Bot 109(6)1133ndash1148doi101093aobmcs042
Pino-Bodas R Burgaz A Martiacuten M Lumbsch HT (2011)Phenotypical plasticity and homoplasy complicatespecies delimitation in the Cladonia gracilis group(Cladoniaceae Ascomycota) Organ Divers Evol 11(5)343ndash355 doi101007s13127-011-0062-2
Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
Pino-Bodas R Martiacuten M Burgaz A (2012b) Cladoniasubturgida and C iberica (Cladoniaceae) form asingle morphologically and chemically polymorphicspecies Mycol Prog 11(1)269ndash278 doi101007s11557-011-0746-1
Pino-Bodas R Ahti T Stenroos S Martiacuten MP BurgazAR (2013a) Multilocus approach to species recogni-tion in the Cladonia humilis complex (CladoniaceaeAscomycota) Am J Bot 100(4)664ndash678 doi103732ajb1200162
Pino-Bodas R Martiacuten AP Burgaz AR Lumbsch HT(2013b) Species delimitation in Cladonia (Ascomy-cota) a challenge to the DNA barcoding philosophyMol Ecol Resour 13(6)1058ndash1068 doi1011111755-099812086
Pons J Barraclough TG Gomez-Zurita J Cardoso ADuran DP Hazell S Kamoun S Sumlin WD VoglerAP (2006) Sequence-based species delimitation for theDNA taxonomy of undescribed insects Syst Biol 55(4)595ndash609 doi10108010635150600852011
Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
Pringle A Baker DM Platt JL Wares JP Latgeacute JPTaylor JW (2005) Cryptic speciation in the cosmo-politan and clonal human pathogenic fungus Asper-gillus fumigatus Evolution 59(9)1886ndash1899 doi10155404-2411
Printzen C (2009) Lichen systematics the role ofmorphological and molecular data to reconstructphylogenetic relationships Progress in botany vol71 Springer Berlin pp 233ndash275
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Puillandre N Lambert A Brouillet S Achaz G (2012)ABGD automatic barcode gap discovery for primaryspecies delimitation Mol Ecol 21(8)1864ndash1877doi101111j1365-294X201105239x
Rannala B Yang Z (2003) Bayes estimation of speciesdivergence times and ancestral population sizes usingDNA sequences from multiple loci Genetics1641645ndash1656
Raxworthy CJ Ingram CM Rabibisoa N Pearson RG(2007) Applications of ecological niche modelingfor species delimitation a review and empiricalevaluation using day geckos (Phelsuma) fromMadagascar Syst Biol 56(6)907ndash923 doi10108010635150701775111
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phylogeny of the genus Lecania (Ramalinaceaelichenized Ascomycota) Mycol Res 111(5)581ndash591doi101016jmycres200703001
Reid N Carstens B (2012) Phylogenetic estimation errorcan decrease the accuracy of species delimitation aBayesian implementation of the general mixed Yule-coalescent model BMC Evol Biol 12(1)196 doi1011861471-2148-12-196
Rissler L Apodaca J (2007) Adding more ecology intospecies delimitation ecological niche models andphylogeography help define cryptic species in theBlack Salamander (Aneides flavipunctatus) Syst Biol56924ndash942 doi10108010635150701703063
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Rivas Plata E Lumbsch HT (2011) Parallel evolution andphenotypic divergence in lichenized fungi a casestudy in the lichen-forming fungal family Graphida-ceae (Ascomycota Lecanoromycetes Ostropales)Mol Phylogenet Evol 61(1)45ndash63 doi101016jympev201104025
Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
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Rubin BER Ree RH Moreau CS (2012) Inferringphylogenies from RAD sequence data PLoS ONE 7(4)e33394 doi101371journalpone0033394
Rubinoff D (2006) Utility of mitochondrial DNA barcodesin species conservation Conserv Biol 20(4)1026ndash1033 doi101111j1523-1739200600372x
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Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
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Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
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Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
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Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
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Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
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Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
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Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
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Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
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Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
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44 SD Leavitt et al
Kekkonen M Hebert PD (2014) DNA barcode-baseddelineation of putative species efficient start fortaxonomic workflows Mol Ecol Res doi1011111755-099812233
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Kroken S Taylor JW (2001) A gene genealogical approachto recognize phylogenetic species boundaries in thelichenized fungus Letharia Mycologia 93(1)38ndash53
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Le Gac M Hood ME Fournier E Giraud T (2007)Phylogenetic evidence of host-specific cryptic speciesin the anther smut fungus Evolution 61(1)15ndash26doi101111j1558-5646200700002x
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Leacheacute AD Fujita MK (2010) Bayesian species delim-itation in West African forest geckos (Hemidactylusfasciatus) Proc R Soc B Biol Sci 2773071ndash3077doi101098rspb20100662
Leacheacute AD Koo M Spencer C Papenfuss T Fisher RMcGuire J (2009) Quantifying ecological morpho-logical and genetic variation to delimit species in thecoast horned lizard species complex (Phrynosoma)Proc Natl Acad Sci USA 10612418ndash12423 doi101073pnas0906380106
Leacheacute AD Fujita MK Minin VN Bouckaert RR (2014)Species delimitation using genome-wide SNP dataSystematic Biology doi101093sysbiosyu018
Leavitt SD Fankhauser JD Leavitt DH Porter LDJohnson LA St Clair LL (2011a) Complex patterns ofspeciation in cosmopolitan ldquorock posyrdquo lichensmdashdiscovering and delimiting cryptic fungal species in
the lichen-forming Rhizoplaca melanophthalma spe-cies-complex (Lecanoraceae Ascomycota) MolPhylogenet Evolu 59(3)587ndash602 doi101016jympev201103020
Leavitt SD Johnson L St Clair LL (2011b) Speciesdelimitation and evolution in morphologically andchemically diverse communities of the lichen-forminggenus Xanthoparmelia (Parmeliaceae Ascomycota) inwestern North America Am J Bot 98 (2)175ndash188doi103732ajb1000230
Leavitt SD Johnson LA Goward T St Clair LL (2011c)Species delimitation in taxonomically difficult lichen-forming fungi an example from morphologically andchemically diverse Xanthoparmelia (Parmeliaceae) inNorth America Mol Phylogen Evol 60(3)317ndash332doi101016jympev201105012
Leavitt S Esslinger T Divakar P Lumbsch H (2012a)Miocene and Pliocene dominated diversification of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) and Pleistocene population expan-sions BMC Evol Biol 12(1)176 doi1011861471-2148-12-176
Leavitt SD Esslinger TL Divakar PK Lumbsch HT(2012b) Miocene divergence phenotypically crypticlineages and contrasting distribution patterns incommon lichen-forming fungi (Ascomycota Parmeli-aceae) Biol J Linn Soc 1007920ndash937 doi101111j1095-8312201201978x
Leavitt SD Esslinger TL Lumbsch HT (2012c) Neogene-dominated diversification in neotropical montanelichens dating divergence events in the lichen-form-ing fungal genus Oropogon (Parmeliaceae) Am J Bot99(11)1764ndash1777 doi103732ajb1200146
Leavitt SD Esslinger TL Nelsen MP Lumbsch HT(2013a) Further species diversity in NeotropicalOropogon (Lecanoromycetes Parmeliaceae) in Cen-tral America The Lichenologist 45(04)553ndash564doi101017S0024282913000212
Leavitt SD Esslinger TL Spribille T Divakar PKLumbsch HT (2013b) Multilocus phylogeny of thelichen-forming fungal genus Melanohalea (Parmelia-ceae Ascomycota) insights on diversity distributionsand a comparison of species tree and concatenatedtopologies Mol Phylogenet Evol 66(2013)138ndash152doi101016jympev201209013
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Lumbsch HT St Clair LL(2013c) DNA barcode identification of lichen-formingfungal species in the Rhizoplaca melanophthalmaspecies-complex (Lecanorales Lecanoraceae) includ-ing five new species MycoKeys 71ndash22 doi103897mycokeys74508
Leavitt SD Fernaacutendez-Mendoza F Peacuterez-Ortega SSohrabi M Divakar PK Vondraacutek J Thorsten Lum-bsch H Clair LLS (2013d) Local representation ofglobal diversity in a cosmopolitan lichen-formingfungal species complex (Rhizoplaca Ascomycota)J Biogeogr 40(9)1792ndash1806 doi101111jbi12118
Leavitt SD Lumbsch HT Stenroos S St Clair LL(2013e) Pleistocene speciation in North American
2 The Dynamic Discipline of Species Delimitation hellip 39
lichenized fungi and the impact of alternative speciescircumscriptions and rates of molecular evolution ondivergence estimates PLoS ONE 8(12)e85240doi101371journalpone0085240
Leavitt SD Esslinger TL Hansen ES Divakar PK CrespoA Loomis BF Lumbsch HT (2014) DNA barcoding ofbrown Parmeliae (Parmeliaceae) species a molecularapproach for accurate specimen identification empha-sizing species in Greenland Organ Divers Evol 14(1)11ndash20 doi101007s13127-013-0147-1
Leliaert F Verbruggen H Vanormelingen P Steen FLopez-Bautista JM Zuccarello GC De Clerck O(2014) DNA-based species delimitation in algae Eur JPhycol 49(2) doi101080096702622014904524
Leuckert C (1985) Probleme der Flechten-Chemotaxon-omiemdashStoffkombinationen und ihre taxonomischeWertung Ber Deut Bot Ges 98401ndash408
Lewis ZA Shiver AL Stiffler N Miller MR Johnson EASelker EU (2007) High-density detection of restric-tion-site-associated DNA markers for rapid mappingof mutated loci in Neurospora Genetics 177(2)1163ndash1171 doi101534genetics107078147
Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
Lohtander K Kaumlllersjouml M Roland M Tehler A (2000)The family physciaceae in fennoscandia phylogenyinferred from its sequences Mycologia 92(4)728ndash735
Longton RE (1997) The role of bryophytes and lichens inpolar ecosystems In Woodin SJ Marquiss M (eds)Ecology of Arctic Environments Blackwell ScienceOxford pp 69ndash96 (Special publication No 13)
Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
Lumbsch HT (1994) Die Lecanora subfusca-Gruppe inAustralasien J Hattori Bot Lab 771ndash175
Lumbsch HT (1998a) Taxonomic use of metabolic data inlichen-forming fungi In Frisvad JC Bridge PDArora DK (eds) Chemical Fungal Taxonomy MarcelDekker New York pp 345ndash387
Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
Mayden RL (1997) A hierarchy of species concepts thedenouement in the saga of the species problem InClaridge MF Dawah HA Wilson MR (eds) Speciesthe units of biodiversity Chapman amp Hall Londonpp 381ndash424
Mayr E (1963) Animal species and evolution HarvardUniversity Press Cambridge
Mayr E (1970) Populations species and evolutionBelknap Press of Harvard University Press Cambridge
McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
McDonald T Miadlikowska J Lutzoni F (2003) Thelichen genus Sticta in the Great Smoky Mountains a
40 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
McKay BD Mays HL Wu Y Li H Yao C-T Nishiumi IZou F (2013) An empirical comparison of character-based and coalescent-based approaches to speciesdelimitation in a young avian complex Mol Ecol 22(19)4943ndash4957 doi101111mec12446
Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
Mishler BD Brandon RN (1987) Individuality pluralismand the phylogenetic species concept Biol Philos 2397
Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
Nash TH Zavada M (1977) Population studies amongSonoran Desert species of Parmelia subg Xanthop-armelia (Parmeliaceae) Am J Bot 64(6)664ndash669
Nylander W (1866a) Circa novum in studio Lichenumcriterium chemicum Flora 49198ndash201
Nylander W (1866b) Hypochlorite of lime and hydrate ofpotash Two new criteria for the study of lichens(Tanslated and communicated by the Rev W ALeighton) Bot J Linn Soc 9358ndash365
OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
OrsquoNeill EM Schwartz R Bullock CT Williams JSShaffer HB Aguilar-Miguel X Parra-Olea G Weis-rock DW (2013) Parallel tagged amplicon sequencingreveals major lineages and phylogenetic structure inthe North American tiger salamander (Ambystomatigrinum) species complex Mol Ecol 22(1)111ndash129doi101111mec12049
Orock EA Leavitt SD Fonge BA St Clair LL LumbschHT (2012) DNA-based identification of lichen-form-ing fungi Can publicly available sequence databasesaid in lichen diversity inventories of Mount Cameroon(West Africa) The Lichenologist 44(6)833ndash839doi101017S0024282912000424
Otaacutelora MAG Martiacutenez I Aragoacuten G Molina MC (2010)Phylogeography and divergence date estimates of alichen species complex with a disjunct distributionpattern Am J Bot 97(2)216ndash223 doi103732ajb0900064
Padial J Castroviejo-Fisher S Kohler J Vila C ChaparroJ De la Riva I (2009) Deciphering the products ofevolution at the species level the need for anintegrative taxonomy Zoolog Scr 38(4)431ndash447doi101111j1463-6409200800381x
Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
Papong K Luumlcking R Thammathaworn A BoonpragobK (2009) Four new taxa of Chroodiscus (thelotremoidGraphidaceae) from Southeast Asia The Bryologist112(1)152ndash163 doi1016390007-2745-1121152
Park S-Y Choi J Kim JA Jeong M-H Kim S Lee Y-HHur J-S (2013a) Draft genome sequence of Cladoniamacilenta KoLRI003786 a lichen-forming fungusproducing biruloquinone Genome Announcements 1(5)e00695-13 doi101128genomeA00695-13
Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
Peacuterez-Ortega S Fernaacutendez-Mendoza F Raggio J VivasM Ascaso C Sancho LG Printzen C de los Riacuteos A(2012) Extreme phenotypic variation in Cetrariaaculeata (lichenized Ascomycota) adaptation or inci-dental modification Ann Bot 109(6)1133ndash1148doi101093aobmcs042
Pino-Bodas R Burgaz A Martiacuten M Lumbsch HT (2011)Phenotypical plasticity and homoplasy complicatespecies delimitation in the Cladonia gracilis group(Cladoniaceae Ascomycota) Organ Divers Evol 11(5)343ndash355 doi101007s13127-011-0062-2
Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
Pino-Bodas R Martiacuten M Burgaz A (2012b) Cladoniasubturgida and C iberica (Cladoniaceae) form asingle morphologically and chemically polymorphicspecies Mycol Prog 11(1)269ndash278 doi101007s11557-011-0746-1
Pino-Bodas R Ahti T Stenroos S Martiacuten MP BurgazAR (2013a) Multilocus approach to species recogni-tion in the Cladonia humilis complex (CladoniaceaeAscomycota) Am J Bot 100(4)664ndash678 doi103732ajb1200162
Pino-Bodas R Martiacuten AP Burgaz AR Lumbsch HT(2013b) Species delimitation in Cladonia (Ascomy-cota) a challenge to the DNA barcoding philosophyMol Ecol Resour 13(6)1058ndash1068 doi1011111755-099812086
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Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
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Rubin BER Ree RH Moreau CS (2012) Inferringphylogenies from RAD sequence data PLoS ONE 7(4)e33394 doi101371journalpone0033394
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Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
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Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
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Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
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2 The Dynamic Discipline of Species Delimitation hellip 43
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Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
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Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
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Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
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lichenized fungi and the impact of alternative speciescircumscriptions and rates of molecular evolution ondivergence estimates PLoS ONE 8(12)e85240doi101371journalpone0085240
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Lindblom L Soslashchting U (2008) Taxonomic revision ofXanthomendoza borealis and Xanthoria mawsonii(Lecanoromycetes Ascomycota) The Lichenologist40(05)399ndash409 doi101017S0024282908007937
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Luumlcking R (2012) Predicting species richness in tropicallichenized fungi with lsquomodularrsquo combinations ofcharacter states Biodiver Conserv 21(9)2341ndash2360doi101007s10531-011-0217-7
Luumlcking R del Prado R Lumbsch HT Will-Wolf SAptroot A Sipman HJM Umana L Chaves JL (2008)Phytogenetic patterns of morphological and chemicalcharacters and reproductive mode in the Heterodermiaobscurata group in Costa Rica (Ascomycota Physci-aceae) Syst Biodivers 6(1)31ndash41 doi101017S1477200007002629
Lumbsch HT (1994) Die Lecanora subfusca-Gruppe inAustralasien J Hattori Bot Lab 771ndash175
Lumbsch HT (1998a) Taxonomic use of metabolic data inlichen-forming fungi In Frisvad JC Bridge PDArora DK (eds) Chemical Fungal Taxonomy MarcelDekker New York pp 345ndash387
Lumbsch HT (1998b) The use of metabolic data inlichenology at the species and subspecific levels TheLichenologist 30(4ndash5)357ndash367 doi101017S0024282992000380
Lumbsch HT (2002) Analysis of phenolic products inlichens for identification and taxonomy Protocols inlichenology Culturing biochemistry ecophysiologyand use in biomonitoring Springer Berlin
Lumbsch HT Leavitt SD (2011) Goodbye morphologyA paradigm shift in the delimitation of species in
lichenized fungi Fungal Divers 50(1)59ndash72 doi101007s13225-011-0123-z
Lumbsch HT Ahti T Altermann S Amo de Paz GAptroot A Arup U Barcenas Pentildea A Bawingan PABenatti MN Betancourt L Bjoumlrk CR Boonpragob KBrand M Bungartz F Caceres MES Candan MChaves JL Clerc P Common R Coppins BJ CrespoA Dal Forno M Divakar PK Duya MV Elix JAElvebakk A Fankhauser J Farkas E Ferraro LIFischer E Galloway DJ Gaya E Giralt M Goward TGrube M Hafellner J Hernandez JE Herrera-CamposMA Kalb K Kaumlrnefelt I Kantvilas G Killmann DKirika P Knudesn K Komposch H Kondratyuk SLawrey JD Mangold A Marcelli MP McCune BPMichlig A Miranda Gonzalez R Moncada B Naika-tini A Nelsen MP Oslashvstedal DO Palice Z Papong KParnmen S Peacuterez-Ortega S Printzen C Rico VJRivas Plata E Robayo J Rosabal D Ruprecht USalazar Allen N Sancho L Santos de Jesus L SantosVieira T Schultz M Seaward MRD Seacuterusiaux ESchmitt I Sipman HJM Sohrabi M Soslashchting USoslashgaard MZ Sparrius LB Spielmann A Spribille TSutjaritturakan J Thammathaworn A Thell A ThorG Thuumls H Timdal E Truong C Tuumlrk R UmantildeaTenorio L Upreti D van den Boom P Vivas RebueltaM Wedin M Will-Wolf S Wirth V Wirtz N Yahr RYeshitela K Ziemmeck F Wheeler T Luumlcking R(2011) One hundred new species of lichenized fungi asignature of undiscovered global diversity Phytotaxa181ndash127
Mark K Saag L Saag A Thell A Randlane T (2012)Testing morphology-based delimitation of Vulpicidajuniperinus and V tubulosus (Parmeliaceae) usingthree molecular markers The Lichenologist 44(06)757ndash772 doi101017S0024282912000448
Martiacuten MP LaGreca S Lumbsch HT (2003) Molecularphylogeny of Diploschistes inferred from ITSsequence data The Lichenologist 35(01)27ndash32doi101006lich20020427
Masters BC Fan V Ross HA (2011) Species delimitationmdasha geneious plugin for the exploration of speciesboundaries Mol Ecol Res 11(1)154ndash157 doi101111j1755-0998201002896x
Mayden RL (1997) A hierarchy of species concepts thedenouement in the saga of the species problem InClaridge MF Dawah HA Wilson MR (eds) Speciesthe units of biodiversity Chapman amp Hall Londonpp 381ndash424
Mayr E (1963) Animal species and evolution HarvardUniversity Press Cambridge
Mayr E (1970) Populations species and evolutionBelknap Press of Harvard University Press Cambridge
McCune B (2000) Lichen communities as indicators offorest health The Bryologist 103(2)353ndash356 doi1016390007-2745(2000)103[0353LCAIOF]20CO2
McCune B Printzen C (2011) Distribution and climaticniches of the Lecanora varia group in western USABibliotheca Lichenologica 106225ndash234
McDonald T Miadlikowska J Lutzoni F (2003) Thelichen genus Sticta in the Great Smoky Mountains a
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phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
McKay BD Mays HL Wu Y Li H Yao C-T Nishiumi IZou F (2013) An empirical comparison of character-based and coalescent-based approaches to speciesdelimitation in a young avian complex Mol Ecol 22(19)4943ndash4957 doi101111mec12446
Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
Mishler BD Brandon RN (1987) Individuality pluralismand the phylogenetic species concept Biol Philos 2397
Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
Nash TH Zavada M (1977) Population studies amongSonoran Desert species of Parmelia subg Xanthop-armelia (Parmeliaceae) Am J Bot 64(6)664ndash669
Nylander W (1866a) Circa novum in studio Lichenumcriterium chemicum Flora 49198ndash201
Nylander W (1866b) Hypochlorite of lime and hydrate ofpotash Two new criteria for the study of lichens(Tanslated and communicated by the Rev W ALeighton) Bot J Linn Soc 9358ndash365
OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
OrsquoNeill EM Schwartz R Bullock CT Williams JSShaffer HB Aguilar-Miguel X Parra-Olea G Weis-rock DW (2013) Parallel tagged amplicon sequencingreveals major lineages and phylogenetic structure inthe North American tiger salamander (Ambystomatigrinum) species complex Mol Ecol 22(1)111ndash129doi101111mec12049
Orock EA Leavitt SD Fonge BA St Clair LL LumbschHT (2012) DNA-based identification of lichen-form-ing fungi Can publicly available sequence databasesaid in lichen diversity inventories of Mount Cameroon(West Africa) The Lichenologist 44(6)833ndash839doi101017S0024282912000424
Otaacutelora MAG Martiacutenez I Aragoacuten G Molina MC (2010)Phylogeography and divergence date estimates of alichen species complex with a disjunct distributionpattern Am J Bot 97(2)216ndash223 doi103732ajb0900064
Padial J Castroviejo-Fisher S Kohler J Vila C ChaparroJ De la Riva I (2009) Deciphering the products ofevolution at the species level the need for anintegrative taxonomy Zoolog Scr 38(4)431ndash447doi101111j1463-6409200800381x
Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
Papong K Luumlcking R Thammathaworn A BoonpragobK (2009) Four new taxa of Chroodiscus (thelotremoidGraphidaceae) from Southeast Asia The Bryologist112(1)152ndash163 doi1016390007-2745-1121152
Park S-Y Choi J Kim JA Jeong M-H Kim S Lee Y-HHur J-S (2013a) Draft genome sequence of Cladoniamacilenta KoLRI003786 a lichen-forming fungusproducing biruloquinone Genome Announcements 1(5)e00695-13 doi101128genomeA00695-13
Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
Peacuterez-Ortega S Fernaacutendez-Mendoza F Raggio J VivasM Ascaso C Sancho LG Printzen C de los Riacuteos A(2012) Extreme phenotypic variation in Cetrariaaculeata (lichenized Ascomycota) adaptation or inci-dental modification Ann Bot 109(6)1133ndash1148doi101093aobmcs042
Pino-Bodas R Burgaz A Martiacuten M Lumbsch HT (2011)Phenotypical plasticity and homoplasy complicatespecies delimitation in the Cladonia gracilis group(Cladoniaceae Ascomycota) Organ Divers Evol 11(5)343ndash355 doi101007s13127-011-0062-2
Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
Pino-Bodas R Martiacuten M Burgaz A (2012b) Cladoniasubturgida and C iberica (Cladoniaceae) form asingle morphologically and chemically polymorphicspecies Mycol Prog 11(1)269ndash278 doi101007s11557-011-0746-1
Pino-Bodas R Ahti T Stenroos S Martiacuten MP BurgazAR (2013a) Multilocus approach to species recogni-tion in the Cladonia humilis complex (CladoniaceaeAscomycota) Am J Bot 100(4)664ndash678 doi103732ajb1200162
Pino-Bodas R Martiacuten AP Burgaz AR Lumbsch HT(2013b) Species delimitation in Cladonia (Ascomy-cota) a challenge to the DNA barcoding philosophyMol Ecol Resour 13(6)1058ndash1068 doi1011111755-099812086
Pons J Barraclough TG Gomez-Zurita J Cardoso ADuran DP Hazell S Kamoun S Sumlin WD VoglerAP (2006) Sequence-based species delimitation for theDNA taxonomy of undescribed insects Syst Biol 55(4)595ndash609 doi10108010635150600852011
Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
Pringle A Baker DM Platt JL Wares JP Latgeacute JPTaylor JW (2005) Cryptic speciation in the cosmo-politan and clonal human pathogenic fungus Asper-gillus fumigatus Evolution 59(9)1886ndash1899 doi10155404-2411
Printzen C (2009) Lichen systematics the role ofmorphological and molecular data to reconstructphylogenetic relationships Progress in botany vol71 Springer Berlin pp 233ndash275
Pritchard JK Stephens M Donnelly P (2000) Inference ofpopulation structure using multilocus genotype dataGenetics 155(2)945ndash959
Puillandre N Lambert A Brouillet S Achaz G (2012)ABGD automatic barcode gap discovery for primaryspecies delimitation Mol Ecol 21(8)1864ndash1877doi101111j1365-294X201105239x
Rannala B Yang Z (2003) Bayes estimation of speciesdivergence times and ancestral population sizes usingDNA sequences from multiple loci Genetics1641645ndash1656
Raxworthy CJ Ingram CM Rabibisoa N Pearson RG(2007) Applications of ecological niche modelingfor species delimitation a review and empiricalevaluation using day geckos (Phelsuma) fromMadagascar Syst Biol 56(6)907ndash923 doi10108010635150701775111
Ray J (1686) Historia planarum vol 1 Clark LondonReese Naeligsborg R Ekman S Tibell L (2007) Molecular
phylogeny of the genus Lecania (Ramalinaceaelichenized Ascomycota) Mycol Res 111(5)581ndash591doi101016jmycres200703001
Reid N Carstens B (2012) Phylogenetic estimation errorcan decrease the accuracy of species delimitation aBayesian implementation of the general mixed Yule-coalescent model BMC Evol Biol 12(1)196 doi1011861471-2148-12-196
Rissler L Apodaca J (2007) Adding more ecology intospecies delimitation ecological niche models andphylogeography help define cryptic species in theBlack Salamander (Aneides flavipunctatus) Syst Biol56924ndash942 doi10108010635150701703063
Rivas Plata E Luumlcking R (2013) High diversity ofGraphidaceae (lichenized Ascomycota Ostropales) inAmazonian Peruacute Fungal Divers 58(1)13ndash32 doi101007s13225-012-0172-y
Rivas Plata E Lumbsch HT (2011) Parallel evolution andphenotypic divergence in lichenized fungi a casestudy in the lichen-forming fungal family Graphida-ceae (Ascomycota Lecanoromycetes Ostropales)Mol Phylogenet Evol 61(1)45ndash63 doi101016jympev201104025
Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
Ross KG Gotzek D Ascunce MS Shoemaker DD (2010)Species delimitation a case study in a problematic anttaxon Syst Biol 59(2)162ndash184 doi101093sysbiosyp089
Rubin BER Ree RH Moreau CS (2012) Inferringphylogenies from RAD sequence data PLoS ONE 7(4)e33394 doi101371journalpone0033394
Rubinoff D (2006) Utility of mitochondrial DNA barcodesin species conservation Conserv Biol 20(4)1026ndash1033 doi101111j1523-1739200600372x
Ruiz-Sanchez E Sosa V (2010) Delimiting speciesboundaries within the Neotropical bamboo Otatea(Poaceae Bambusoideae) using molecular morpho-logical and ecological data Mol Phylogenet Evol 54(2)344ndash356 doi101016jympev200910035
Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
morphological characters Antarctic Sci 22 (SpecialIssue 06)727ndash741 doi101017S0954102010000477
Salicini I Ibaacutentildeez C Juste J (2011) Multilocus phylogenyand species delimitation within the Nattererrsquos batspecies complex in the Western Palearctic MolPhylogenet Evol 61(3)888ndash898 doi101016jympev201108010
Satler JD Carstens BC Hedin M (2013) Multilocusspecies delimitation in a complex of morphologicallyconserved trapdoor spiders (Mygalomorphae Antro-diaetidae Aliatypus) Syst Biol 62(6)805ndash823 doi101093sysbiosyt041
Schlick-Steiner BC Steiner FM Seifert B Stauffer CChristian E Crozier RH (2010) Integrative taxonomya multisource approach to exploring biodiversityAnnu Rev Entomol 55(1)421ndash438 doi101146annurev-ento-112408-085432
Schoch CL Seifert KA Huhndorf S Robert V SpougeJL Levesque CA Chen W Fungal Barcoding Con-sortium (2012) Nuclear ribosomal internal transcribedspacer (ITS) region as a universal DNA barcodemarker for Fungi Proc Natl Acad Sci 109(16)6241ndash6246 doi101073pnas1117018109
Seacuterusiaux E Villarreal AJC Wheeler T Goffinet B(2011) Recent origin active speciation and dispersalfor the lichen genus Nephroma (Peltigerales) inMacaronesia J Biogeogr 38(6)1138ndash1151 doi101111j1365-2699201002469x
Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
Simpson GG (1951) The species concept Evolution5285ndash298
Sites JW Marshall JC (2003) Delimiting species a renais-sance issue in systematic biology Trends Ecol Evol18462ndash470 doi101016S0169-5347(03)00184-8
Sites JW Marshall JC (2004) Operational criteria fordelimiting species Annu Rev Ecol Evol Syst 35(1)199ndash227 doi101146annurevecolsys35112202130128
Šlapeta J Loacutepez-Garciacutea P Moreira D (2006) Globaldispersal and ancient cryptic species in the smallestmarine eukaryotes Mol Biol Evol 23(1)23ndash29doi101093molbevmsj001
Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
Talavera G Dincă V Vila R (2013) Factors affectingspecies delimitations with the GMYC model insightsfrom a butterfly survey Methods Ecol Evol 4(12)1101ndash1110 doi1011112041-210X12107
Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
Tewksbury J Anderson JGT Bakker JD Billo TJDunwiddie PW Groom MJ Hampton SE HermanSG Levey DJ Machnicki NJ del Rio CM PowerME Rowell K Salomon AK Stacey L TrombulakSC Wheeler TA (2014) Natural historyrsquos place inscience and society BioScience doi101093bioscibiu032
Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
Werth S Cornejo C Scheidegger C (2013) Characteriza-tion of microsatellite loci in the lichen fungus Lobariapulmonaria (Lobariaceae) Appl Plant Sci 1(2)1200290 doi103732apps1200290
Westberg M Arup U Kaumlrnefelt I (2007) Phylogeneticstudies in the Candelariaceae (lichenized Ascomy-cota) based on nuclear ITS DNA sequence dataMycol Res 111(11)1277ndash1284 doi101017S0953756204002102
Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
2 The Dynamic Discipline of Species Delimitation hellip 43
(Lepidoptera Lycaenidae) Front Zoo 4(1)8 doi1011861742-9994-4-8
Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
phylogenetic study of morphological chemical andmolecular data The Bryologist 106(1)61ndash79 doi1016390007-2745(2003)106[0061TLGSIT]20CO2
McKay BD Mays HL Wu Y Li H Yao C-T Nishiumi IZou F (2013) An empirical comparison of character-based and coalescent-based approaches to speciesdelimitation in a young avian complex Mol Ecol 22(19)4943ndash4957 doi101111mec12446
Miller MR Dunham JP Amores A Cresko WA JohnsonEA (2007) Rapid and cost-effective polymorphismidentification and genotyping using restriction siteassociated DNA (RAD) markers Genome Res 17(2)240ndash248 doi101101gr5681207
Miralles A Vences M (2013) New metrics for compar-ison of taxonomies reveal striking discrepanciesamong species delimitation methods in Madascincuslizards PLoS ONE 8(7)e68242 doi101371journalpone0068242
Mishler BD Brandon RN (1987) Individuality pluralismand the phylogenetic species concept Biol Philos 2397
Molina M Crespo A Blanco O Lumbsch HT Hawks-worth DL (2004) Phylogenetic relationships andspecies concepts in Parmelia s str (Parmeliaceae)inferred from nuclear ITS rDNA and β-tubulinsequences The Lichenologist 36(01)37ndash54 doi101017S0024282904013933
Molina M Del-Prado R Divakar P Saacutenchez-Mata DCrespo A (2011) Another example of cryptic diversityin lichen-forming fungi the new species Parmeliamayi (Ascomycota Parmeliaceae) Organ Divers Evol11(5)331ndash342 doi101007s13127-011-0060-4
Monaghan MT Wild R Elliot M Fujisawa T Balke MInward DJG Lees DC Ranaivosolo R Eggleton PBarraclough TG Vogler AP (2009) Acceleratedspecies inventory on Madagascar using coalescent-based models of species delineation Syst Biol 58(3)298ndash311 doi101093sysbiosyp027
Moncada B Reidy B Luumlcking R (2014) A phylogeneticrevision of Hawaiian Pseudocyphellaria (lichenizedAscomycota Lobariaceae) reveals eight new speciesand a high degree of inferred endemism The Bryol-ogist 117(2)119ndash160 doi httpdxdoiorg1016390007-2745-1172119
Moreau CS (2009) Inferring ant evolution in the age ofmolecular data Myrmecological New 12201
Muggia L Grube M Tretiach M (2008) A combinedmolecular and morphological approach to speciesdelimitation in black-fruited endolithic Caloplacahigh genetic and low morphological diversity MycolRes 112(1)36ndash49 doi101016jmycres200702001
Muggia L Peacuterez-Ortega S Fryday A Spribille T GrubeM (2014) Global assessment of genetic variation andphenotypic plasticity in the lichen-forming speciesTephromela atra Fungal Divers 64(1)233ndash251doi101007s13225-013-0271-4
Nash TH Zavada M (1977) Population studies amongSonoran Desert species of Parmelia subg Xanthop-armelia (Parmeliaceae) Am J Bot 64(6)664ndash669
Nylander W (1866a) Circa novum in studio Lichenumcriterium chemicum Flora 49198ndash201
Nylander W (1866b) Hypochlorite of lime and hydrate ofpotash Two new criteria for the study of lichens(Tanslated and communicated by the Rev W ALeighton) Bot J Linn Soc 9358ndash365
OrsquoBrien HE Miadlikowska J Lutzoni F (2009) Assessingreproductive isolation in highly diverse communitiesof the lichen-forming fugnal genus Peltigera Evolu-tion 63(8)2076ndash2086 doi101111j1558-5646200900685x
OrsquoMeara BC (2010) New heuristic methods for jointspecies delimitation and species tree inference SystBiol 59(1)59ndash73 doi101093sysbiosyp077
OrsquoNeill EM Schwartz R Bullock CT Williams JSShaffer HB Aguilar-Miguel X Parra-Olea G Weis-rock DW (2013) Parallel tagged amplicon sequencingreveals major lineages and phylogenetic structure inthe North American tiger salamander (Ambystomatigrinum) species complex Mol Ecol 22(1)111ndash129doi101111mec12049
Orock EA Leavitt SD Fonge BA St Clair LL LumbschHT (2012) DNA-based identification of lichen-form-ing fungi Can publicly available sequence databasesaid in lichen diversity inventories of Mount Cameroon(West Africa) The Lichenologist 44(6)833ndash839doi101017S0024282912000424
Otaacutelora MAG Martiacutenez I Aragoacuten G Molina MC (2010)Phylogeography and divergence date estimates of alichen species complex with a disjunct distributionpattern Am J Bot 97(2)216ndash223 doi103732ajb0900064
Padial J Castroviejo-Fisher S Kohler J Vila C ChaparroJ De la Riva I (2009) Deciphering the products ofevolution at the species level the need for anintegrative taxonomy Zoolog Scr 38(4)431ndash447doi101111j1463-6409200800381x
Padial J Miralles A De la Riva I Vences M (2010) Theintegrative future of taxonomy Front Zoo 7(1)16doi1011861742-9994-7-16
Papong K Luumlcking R Thammathaworn A BoonpragobK (2009) Four new taxa of Chroodiscus (thelotremoidGraphidaceae) from Southeast Asia The Bryologist112(1)152ndash163 doi1016390007-2745-1121152
Park S-Y Choi J Kim JA Jeong M-H Kim S Lee Y-HHur J-S (2013a) Draft genome sequence of Cladoniamacilenta KoLRI003786 a lichen-forming fungusproducing biruloquinone Genome Announcements 1(5)e00695-13 doi101128genomeA00695-13
Park S-Y Choi J Kim JA Yu N-H Kim S KondratyukSY Lee Y-H Hur J-S (2013b) Draft genome sequenceof lichen-forming fungus Caloplaca flavorubescensStrain KoLRI002931 Genome Announcements 1(4)e00678-13 doi101128genomeA00678-13
Park S-Y Choi J Lee G-W Kim JA Oh S-O Jeong M-HYu N-H Kim S Lee Y-H Hur J-S (2014) Draftgenome sequence of lichen-forming fungus Cladoniametacorallifera Strain KoLRI002260 GenomeAnnouncements 2(1)e01065-13 doi101128genomeA01065-13
Parnmen S Rangsiruji A Mongkolsuk P Boonpragob KNutakki A Lumbsch HT (2012) Using phylogenetic
2 The Dynamic Discipline of Species Delimitation hellip 41
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
Peacuterez-Ortega S Fernaacutendez-Mendoza F Raggio J VivasM Ascaso C Sancho LG Printzen C de los Riacuteos A(2012) Extreme phenotypic variation in Cetrariaaculeata (lichenized Ascomycota) adaptation or inci-dental modification Ann Bot 109(6)1133ndash1148doi101093aobmcs042
Pino-Bodas R Burgaz A Martiacuten M Lumbsch HT (2011)Phenotypical plasticity and homoplasy complicatespecies delimitation in the Cladonia gracilis group(Cladoniaceae Ascomycota) Organ Divers Evol 11(5)343ndash355 doi101007s13127-011-0062-2
Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
Pino-Bodas R Martiacuten M Burgaz A (2012b) Cladoniasubturgida and C iberica (Cladoniaceae) form asingle morphologically and chemically polymorphicspecies Mycol Prog 11(1)269ndash278 doi101007s11557-011-0746-1
Pino-Bodas R Ahti T Stenroos S Martiacuten MP BurgazAR (2013a) Multilocus approach to species recogni-tion in the Cladonia humilis complex (CladoniaceaeAscomycota) Am J Bot 100(4)664ndash678 doi103732ajb1200162
Pino-Bodas R Martiacuten AP Burgaz AR Lumbsch HT(2013b) Species delimitation in Cladonia (Ascomy-cota) a challenge to the DNA barcoding philosophyMol Ecol Resour 13(6)1058ndash1068 doi1011111755-099812086
Pons J Barraclough TG Gomez-Zurita J Cardoso ADuran DP Hazell S Kamoun S Sumlin WD VoglerAP (2006) Sequence-based species delimitation for theDNA taxonomy of undescribed insects Syst Biol 55(4)595ndash609 doi10108010635150600852011
Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
Pringle A Baker DM Platt JL Wares JP Latgeacute JPTaylor JW (2005) Cryptic speciation in the cosmo-politan and clonal human pathogenic fungus Asper-gillus fumigatus Evolution 59(9)1886ndash1899 doi10155404-2411
Printzen C (2009) Lichen systematics the role ofmorphological and molecular data to reconstructphylogenetic relationships Progress in botany vol71 Springer Berlin pp 233ndash275
Pritchard JK Stephens M Donnelly P (2000) Inference ofpopulation structure using multilocus genotype dataGenetics 155(2)945ndash959
Puillandre N Lambert A Brouillet S Achaz G (2012)ABGD automatic barcode gap discovery for primaryspecies delimitation Mol Ecol 21(8)1864ndash1877doi101111j1365-294X201105239x
Rannala B Yang Z (2003) Bayes estimation of speciesdivergence times and ancestral population sizes usingDNA sequences from multiple loci Genetics1641645ndash1656
Raxworthy CJ Ingram CM Rabibisoa N Pearson RG(2007) Applications of ecological niche modelingfor species delimitation a review and empiricalevaluation using day geckos (Phelsuma) fromMadagascar Syst Biol 56(6)907ndash923 doi10108010635150701775111
Ray J (1686) Historia planarum vol 1 Clark LondonReese Naeligsborg R Ekman S Tibell L (2007) Molecular
phylogeny of the genus Lecania (Ramalinaceaelichenized Ascomycota) Mycol Res 111(5)581ndash591doi101016jmycres200703001
Reid N Carstens B (2012) Phylogenetic estimation errorcan decrease the accuracy of species delimitation aBayesian implementation of the general mixed Yule-coalescent model BMC Evol Biol 12(1)196 doi1011861471-2148-12-196
Rissler L Apodaca J (2007) Adding more ecology intospecies delimitation ecological niche models andphylogeography help define cryptic species in theBlack Salamander (Aneides flavipunctatus) Syst Biol56924ndash942 doi10108010635150701703063
Rivas Plata E Luumlcking R (2013) High diversity ofGraphidaceae (lichenized Ascomycota Ostropales) inAmazonian Peruacute Fungal Divers 58(1)13ndash32 doi101007s13225-012-0172-y
Rivas Plata E Lumbsch HT (2011) Parallel evolution andphenotypic divergence in lichenized fungi a casestudy in the lichen-forming fungal family Graphida-ceae (Ascomycota Lecanoromycetes Ostropales)Mol Phylogenet Evol 61(1)45ndash63 doi101016jympev201104025
Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
Ross KG Gotzek D Ascunce MS Shoemaker DD (2010)Species delimitation a case study in a problematic anttaxon Syst Biol 59(2)162ndash184 doi101093sysbiosyp089
Rubin BER Ree RH Moreau CS (2012) Inferringphylogenies from RAD sequence data PLoS ONE 7(4)e33394 doi101371journalpone0033394
Rubinoff D (2006) Utility of mitochondrial DNA barcodesin species conservation Conserv Biol 20(4)1026ndash1033 doi101111j1523-1739200600372x
Ruiz-Sanchez E Sosa V (2010) Delimiting speciesboundaries within the Neotropical bamboo Otatea(Poaceae Bambusoideae) using molecular morpho-logical and ecological data Mol Phylogenet Evol 54(2)344ndash356 doi101016jympev200910035
Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
morphological characters Antarctic Sci 22 (SpecialIssue 06)727ndash741 doi101017S0954102010000477
Salicini I Ibaacutentildeez C Juste J (2011) Multilocus phylogenyand species delimitation within the Nattererrsquos batspecies complex in the Western Palearctic MolPhylogenet Evol 61(3)888ndash898 doi101016jympev201108010
Satler JD Carstens BC Hedin M (2013) Multilocusspecies delimitation in a complex of morphologicallyconserved trapdoor spiders (Mygalomorphae Antro-diaetidae Aliatypus) Syst Biol 62(6)805ndash823 doi101093sysbiosyt041
Schlick-Steiner BC Steiner FM Seifert B Stauffer CChristian E Crozier RH (2010) Integrative taxonomya multisource approach to exploring biodiversityAnnu Rev Entomol 55(1)421ndash438 doi101146annurev-ento-112408-085432
Schoch CL Seifert KA Huhndorf S Robert V SpougeJL Levesque CA Chen W Fungal Barcoding Con-sortium (2012) Nuclear ribosomal internal transcribedspacer (ITS) region as a universal DNA barcodemarker for Fungi Proc Natl Acad Sci 109(16)6241ndash6246 doi101073pnas1117018109
Seacuterusiaux E Villarreal AJC Wheeler T Goffinet B(2011) Recent origin active speciation and dispersalfor the lichen genus Nephroma (Peltigerales) inMacaronesia J Biogeogr 38(6)1138ndash1151 doi101111j1365-2699201002469x
Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
Simpson GG (1951) The species concept Evolution5285ndash298
Sites JW Marshall JC (2003) Delimiting species a renais-sance issue in systematic biology Trends Ecol Evol18462ndash470 doi101016S0169-5347(03)00184-8
Sites JW Marshall JC (2004) Operational criteria fordelimiting species Annu Rev Ecol Evol Syst 35(1)199ndash227 doi101146annurevecolsys35112202130128
Šlapeta J Loacutepez-Garciacutea P Moreira D (2006) Globaldispersal and ancient cryptic species in the smallestmarine eukaryotes Mol Biol Evol 23(1)23ndash29doi101093molbevmsj001
Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
Talavera G Dincă V Vila R (2013) Factors affectingspecies delimitations with the GMYC model insightsfrom a butterfly survey Methods Ecol Evol 4(12)1101ndash1110 doi1011112041-210X12107
Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
Tewksbury J Anderson JGT Bakker JD Billo TJDunwiddie PW Groom MJ Hampton SE HermanSG Levey DJ Machnicki NJ del Rio CM PowerME Rowell K Salomon AK Stacey L TrombulakSC Wheeler TA (2014) Natural historyrsquos place inscience and society BioScience doi101093bioscibiu032
Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
Werth S Cornejo C Scheidegger C (2013) Characteriza-tion of microsatellite loci in the lichen fungus Lobariapulmonaria (Lobariaceae) Appl Plant Sci 1(2)1200290 doi103732apps1200290
Westberg M Arup U Kaumlrnefelt I (2007) Phylogeneticstudies in the Candelariaceae (lichenized Ascomy-cota) based on nuclear ITS DNA sequence dataMycol Res 111(11)1277ndash1284 doi101017S0953756204002102
Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
2 The Dynamic Discipline of Species Delimitation hellip 43
(Lepidoptera Lycaenidae) Front Zoo 4(1)8 doi1011861742-9994-4-8
Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
and coalescent methods to understand the speciesdiversity in the Cladia aggregata complex (Ascomy-cota Lecanorales) PLoS ONE 7(12)e52245 doi101371journalpone0052245
Peacuterez-Ortega S Fernaacutendez-Mendoza F Raggio J VivasM Ascaso C Sancho LG Printzen C de los Riacuteos A(2012) Extreme phenotypic variation in Cetrariaaculeata (lichenized Ascomycota) adaptation or inci-dental modification Ann Bot 109(6)1133ndash1148doi101093aobmcs042
Pino-Bodas R Burgaz A Martiacuten M Lumbsch HT (2011)Phenotypical plasticity and homoplasy complicatespecies delimitation in the Cladonia gracilis group(Cladoniaceae Ascomycota) Organ Divers Evol 11(5)343ndash355 doi101007s13127-011-0062-2
Pino-Bodas R Burgaz AR Martin MP Lumbsch HT(2012a) Species delimitations in the Cladonia cariosagroup (Cladoniaceae Ascomycota) The Lichenologist44(01)121ndash135 doi101017S002428291100065X
Pino-Bodas R Martiacuten M Burgaz A (2012b) Cladoniasubturgida and C iberica (Cladoniaceae) form asingle morphologically and chemically polymorphicspecies Mycol Prog 11(1)269ndash278 doi101007s11557-011-0746-1
Pino-Bodas R Ahti T Stenroos S Martiacuten MP BurgazAR (2013a) Multilocus approach to species recogni-tion in the Cladonia humilis complex (CladoniaceaeAscomycota) Am J Bot 100(4)664ndash678 doi103732ajb1200162
Pino-Bodas R Martiacuten AP Burgaz AR Lumbsch HT(2013b) Species delimitation in Cladonia (Ascomy-cota) a challenge to the DNA barcoding philosophyMol Ecol Resour 13(6)1058ndash1068 doi1011111755-099812086
Pons J Barraclough TG Gomez-Zurita J Cardoso ADuran DP Hazell S Kamoun S Sumlin WD VoglerAP (2006) Sequence-based species delimitation for theDNA taxonomy of undescribed insects Syst Biol 55(4)595ndash609 doi10108010635150600852011
Porada P Weber B Elbert W Poumlschl U Kleidon A(2014) Estimating impacts of lichens and bryophyteson global biogeochemical cycles Global BiogeochemCycles 28(2)71ndash85 doi1010022013GB004705
Pringle A Baker DM Platt JL Wares JP Latgeacute JPTaylor JW (2005) Cryptic speciation in the cosmo-politan and clonal human pathogenic fungus Asper-gillus fumigatus Evolution 59(9)1886ndash1899 doi10155404-2411
Printzen C (2009) Lichen systematics the role ofmorphological and molecular data to reconstructphylogenetic relationships Progress in botany vol71 Springer Berlin pp 233ndash275
Pritchard JK Stephens M Donnelly P (2000) Inference ofpopulation structure using multilocus genotype dataGenetics 155(2)945ndash959
Puillandre N Lambert A Brouillet S Achaz G (2012)ABGD automatic barcode gap discovery for primaryspecies delimitation Mol Ecol 21(8)1864ndash1877doi101111j1365-294X201105239x
Rannala B Yang Z (2003) Bayes estimation of speciesdivergence times and ancestral population sizes usingDNA sequences from multiple loci Genetics1641645ndash1656
Raxworthy CJ Ingram CM Rabibisoa N Pearson RG(2007) Applications of ecological niche modelingfor species delimitation a review and empiricalevaluation using day geckos (Phelsuma) fromMadagascar Syst Biol 56(6)907ndash923 doi10108010635150701775111
Ray J (1686) Historia planarum vol 1 Clark LondonReese Naeligsborg R Ekman S Tibell L (2007) Molecular
phylogeny of the genus Lecania (Ramalinaceaelichenized Ascomycota) Mycol Res 111(5)581ndash591doi101016jmycres200703001
Reid N Carstens B (2012) Phylogenetic estimation errorcan decrease the accuracy of species delimitation aBayesian implementation of the general mixed Yule-coalescent model BMC Evol Biol 12(1)196 doi1011861471-2148-12-196
Rissler L Apodaca J (2007) Adding more ecology intospecies delimitation ecological niche models andphylogeography help define cryptic species in theBlack Salamander (Aneides flavipunctatus) Syst Biol56924ndash942 doi10108010635150701703063
Rivas Plata E Luumlcking R (2013) High diversity ofGraphidaceae (lichenized Ascomycota Ostropales) inAmazonian Peruacute Fungal Divers 58(1)13ndash32 doi101007s13225-012-0172-y
Rivas Plata E Lumbsch HT (2011) Parallel evolution andphenotypic divergence in lichenized fungi a casestudy in the lichen-forming fungal family Graphida-ceae (Ascomycota Lecanoromycetes Ostropales)Mol Phylogenet Evol 61(1)45ndash63 doi101016jympev201104025
Rogers RW (1989) Chemical variation and the speciesconcept in lichenized ascomycetes Bot J Linn Soc101229ndash239
Ross HA (2014) The incidence of species-level paraphylyin animals a re-assessment Mol Phylogenet Evol7610ndash17 doi101016jympev201402021
Ross KG Gotzek D Ascunce MS Shoemaker DD (2010)Species delimitation a case study in a problematic anttaxon Syst Biol 59(2)162ndash184 doi101093sysbiosyp089
Rubin BER Ree RH Moreau CS (2012) Inferringphylogenies from RAD sequence data PLoS ONE 7(4)e33394 doi101371journalpone0033394
Rubinoff D (2006) Utility of mitochondrial DNA barcodesin species conservation Conserv Biol 20(4)1026ndash1033 doi101111j1523-1739200600372x
Ruiz-Sanchez E Sosa V (2010) Delimiting speciesboundaries within the Neotropical bamboo Otatea(Poaceae Bambusoideae) using molecular morpho-logical and ecological data Mol Phylogenet Evol 54(2)344ndash356 doi101016jympev200910035
Ruprecht U Lumbsch HT Brunauer G Green TGA TuumlrkR (2010) Diversity of Lecidea (Lecideaceae Asco-mycota) species revealed by molecular data and
42 SD Leavitt et al
morphological characters Antarctic Sci 22 (SpecialIssue 06)727ndash741 doi101017S0954102010000477
Salicini I Ibaacutentildeez C Juste J (2011) Multilocus phylogenyand species delimitation within the Nattererrsquos batspecies complex in the Western Palearctic MolPhylogenet Evol 61(3)888ndash898 doi101016jympev201108010
Satler JD Carstens BC Hedin M (2013) Multilocusspecies delimitation in a complex of morphologicallyconserved trapdoor spiders (Mygalomorphae Antro-diaetidae Aliatypus) Syst Biol 62(6)805ndash823 doi101093sysbiosyt041
Schlick-Steiner BC Steiner FM Seifert B Stauffer CChristian E Crozier RH (2010) Integrative taxonomya multisource approach to exploring biodiversityAnnu Rev Entomol 55(1)421ndash438 doi101146annurev-ento-112408-085432
Schoch CL Seifert KA Huhndorf S Robert V SpougeJL Levesque CA Chen W Fungal Barcoding Con-sortium (2012) Nuclear ribosomal internal transcribedspacer (ITS) region as a universal DNA barcodemarker for Fungi Proc Natl Acad Sci 109(16)6241ndash6246 doi101073pnas1117018109
Seacuterusiaux E Villarreal AJC Wheeler T Goffinet B(2011) Recent origin active speciation and dispersalfor the lichen genus Nephroma (Peltigerales) inMacaronesia J Biogeogr 38(6)1138ndash1151 doi101111j1365-2699201002469x
Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
Simpson GG (1951) The species concept Evolution5285ndash298
Sites JW Marshall JC (2003) Delimiting species a renais-sance issue in systematic biology Trends Ecol Evol18462ndash470 doi101016S0169-5347(03)00184-8
Sites JW Marshall JC (2004) Operational criteria fordelimiting species Annu Rev Ecol Evol Syst 35(1)199ndash227 doi101146annurevecolsys35112202130128
Šlapeta J Loacutepez-Garciacutea P Moreira D (2006) Globaldispersal and ancient cryptic species in the smallestmarine eukaryotes Mol Biol Evol 23(1)23ndash29doi101093molbevmsj001
Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
Talavera G Dincă V Vila R (2013) Factors affectingspecies delimitations with the GMYC model insightsfrom a butterfly survey Methods Ecol Evol 4(12)1101ndash1110 doi1011112041-210X12107
Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
Tewksbury J Anderson JGT Bakker JD Billo TJDunwiddie PW Groom MJ Hampton SE HermanSG Levey DJ Machnicki NJ del Rio CM PowerME Rowell K Salomon AK Stacey L TrombulakSC Wheeler TA (2014) Natural historyrsquos place inscience and society BioScience doi101093bioscibiu032
Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
Werth S Cornejo C Scheidegger C (2013) Characteriza-tion of microsatellite loci in the lichen fungus Lobariapulmonaria (Lobariaceae) Appl Plant Sci 1(2)1200290 doi103732apps1200290
Westberg M Arup U Kaumlrnefelt I (2007) Phylogeneticstudies in the Candelariaceae (lichenized Ascomy-cota) based on nuclear ITS DNA sequence dataMycol Res 111(11)1277ndash1284 doi101017S0953756204002102
Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
2 The Dynamic Discipline of Species Delimitation hellip 43
(Lepidoptera Lycaenidae) Front Zoo 4(1)8 doi1011861742-9994-4-8
Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
morphological characters Antarctic Sci 22 (SpecialIssue 06)727ndash741 doi101017S0954102010000477
Salicini I Ibaacutentildeez C Juste J (2011) Multilocus phylogenyand species delimitation within the Nattererrsquos batspecies complex in the Western Palearctic MolPhylogenet Evol 61(3)888ndash898 doi101016jympev201108010
Satler JD Carstens BC Hedin M (2013) Multilocusspecies delimitation in a complex of morphologicallyconserved trapdoor spiders (Mygalomorphae Antro-diaetidae Aliatypus) Syst Biol 62(6)805ndash823 doi101093sysbiosyt041
Schlick-Steiner BC Steiner FM Seifert B Stauffer CChristian E Crozier RH (2010) Integrative taxonomya multisource approach to exploring biodiversityAnnu Rev Entomol 55(1)421ndash438 doi101146annurev-ento-112408-085432
Schoch CL Seifert KA Huhndorf S Robert V SpougeJL Levesque CA Chen W Fungal Barcoding Con-sortium (2012) Nuclear ribosomal internal transcribedspacer (ITS) region as a universal DNA barcodemarker for Fungi Proc Natl Acad Sci 109(16)6241ndash6246 doi101073pnas1117018109
Seacuterusiaux E Villarreal AJC Wheeler T Goffinet B(2011) Recent origin active speciation and dispersalfor the lichen genus Nephroma (Peltigerales) inMacaronesia J Biogeogr 38(6)1138ndash1151 doi101111j1365-2699201002469x
Shrestha G Peterson SL St Clair LL (2012) Predictingthe distribution of the air pollution sensitive lichenspecies Usnea hirta The Lichenologist 44(04)511ndash521 doi101017S0024282912000060
Simpson GG (1951) The species concept Evolution5285ndash298
Sites JW Marshall JC (2003) Delimiting species a renais-sance issue in systematic biology Trends Ecol Evol18462ndash470 doi101016S0169-5347(03)00184-8
Sites JW Marshall JC (2004) Operational criteria fordelimiting species Annu Rev Ecol Evol Syst 35(1)199ndash227 doi101146annurevecolsys35112202130128
Šlapeta J Loacutepez-Garciacutea P Moreira D (2006) Globaldispersal and ancient cryptic species in the smallestmarine eukaryotes Mol Biol Evol 23(1)23ndash29doi101093molbevmsj001
Spribille T Klug B Mayrhofer H (2011) A phylogeneticanalysis of the boreal lichen Mycoblastus sanguina-rius (Mycoblastaceae lichenized Ascomycota) revealscryptic clades correlated with fatty acid profiles MolPhylogenet Evol 59(3)603ndash614 doi101016jympev201103021
Stenroos SK DePriest PT (1998) SSU rDNA phylogenyof cladoniiform lichens Am J Bot 85(11)1548ndash1559
Talavera G Dincă V Vila R (2013) Factors affectingspecies delimitations with the GMYC model insightsfrom a butterfly survey Methods Ecol Evol 4(12)1101ndash1110 doi1011112041-210X12107
Taylor JW Jacobson DJ Kroken S Kasuga T GeiserDM Hibbett DS Fisher MC (2000) Phylogeneticspecies recognition and species concepts in fungi
Fungal Genet Biol 31(1)21ndash32 doi101006fgbi20001228
Tewksbury J Anderson JGT Bakker JD Billo TJDunwiddie PW Groom MJ Hampton SE HermanSG Levey DJ Machnicki NJ del Rio CM PowerME Rowell K Salomon AK Stacey L TrombulakSC Wheeler TA (2014) Natural historyrsquos place inscience and society BioScience doi101093bioscibiu032
Thell A Houmlgnabba F Elix JA Feuerer T Kaumlrnefelt IMyllys L Randlane T Saag A Stenroos S Ahti TSeaward MRD (2009) Phylogeny of the cetrarioidcore (Parmeliaceae) based on five genetic markersThe Lichenologist 41(05)489ndash511 doi101017S0024282909990090
Velmala S Myllys L Halonen P Goward T Ahti T(2009) Molecular data show that Bryoria fremontiiand B tortuosa (Parmeliaceae) are conspecific TheLichenologist 41(03)231ndash242 doi101017S0024282909008573
Vondraacutek J Řiacuteha P Arup U Soslashchting U (2009) Thetaxonomy of the Caloplaca citrina group (Teloschist-aceae) in the Black Sea region with contributions tothe cryptic species concept in lichenology TheLichenologist 41(06)571ndash604 doi101017S0024282909008317
Wagner CE Keller I Wittwer S Selz OM Mwaiko SGreuter L Sivasundar A Seehausen O (2013)Genome-wide RAD sequence data provide unprece-dented resolution of species boundaries and relation-ships in the Lake Victoria cichlid adaptive radiationMol Ecol 22(3)787ndash798 doi101111mec12023
Wang Y-Y Liu B Zhang X-Y Zhou Q-M Zhang T LiH Yu Y-F Zhang X-L Hao X-Y Wang M Wang LWei J-C (2014) Genome characteristics reveal theimpact of lichenization on lichen-forming fungusEndocarpon pusillum Hedwig (Verrucariales Asco-mycota) BMC Genom 15(1)34 doi1011861471-2164-15-34
Wedin M Westberg M Crewe AT Tehler A Purvis OW(2009) Species delimitation and evolution of metalbioaccumulation in the lichenized Acarospora smar-agdula (Ascomycota Fungi) complex Cladistics 25(2)161ndash172 doi101111j1096-0031200900240x
Weisrock DW Rasoloarison RM Fiorentino I RalisonJM Goodman SM Kappeler PM Yoder AD (2010)Delimiting species without nuclear monophyly inMadagascarrsquos mouse lemurs PLoS ONE 5(3)e9883doi101371journalpone0009883
Werth S Cornejo C Scheidegger C (2013) Characteriza-tion of microsatellite loci in the lichen fungus Lobariapulmonaria (Lobariaceae) Appl Plant Sci 1(2)1200290 doi103732apps1200290
Westberg M Arup U Kaumlrnefelt I (2007) Phylogeneticstudies in the Candelariaceae (lichenized Ascomy-cota) based on nuclear ITS DNA sequence dataMycol Res 111(11)1277ndash1284 doi101017S0953756204002102
Wiemers M Fiedler K (2007) Does the DNA barcodinggap existmdashA case study in blue butterflies
2 The Dynamic Discipline of Species Delimitation hellip 43
(Lepidoptera Lycaenidae) Front Zoo 4(1)8 doi1011861742-9994-4-8
Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al
(Lepidoptera Lycaenidae) Front Zoo 4(1)8 doi1011861742-9994-4-8
Wiens JJ (1998) Combining data sets with differentphylogenetic histories Syst Biol 4568ndash581 doi101080106351598260581
Wiens JJ Penkrot T (2002) Delimiting species usingDNA and morphological variation and discordantspecies limits in spiny lizards (Sceloporus) Syst Biol5169ndash91 doi101080106351502753475880
Wiens JJ Servedio MR (2000) Species delimitation insystematics inferring diagnostic differences betweenspecies Proc R Soc Lond B Biol Sci 267(1444)631ndash636 doi101098rspb20001049
Will KW Mishler BD Wheeler QD (2005) The perils ofDNA barcoding and the need for integrative taxon-omy Syst Biol 54(5)844ndash851 doi10108010635150500354878
Wirtz N Printzen C Lumbsch HT (2012) Using haplo-type networks estimation of gene flow and phenotypic
characters to understand species delimitation in fungiof a predominantly Antarctic Usnea group (Ascomy-cota Parmeliaceae) Organ Divers Evol 12(1)17ndash37doi101007s13127-011-0066-y
Yang Z Rannala B (2010) Bayesian species delimitationusing multilocus sequence data Proc Natl Acad Sci107(20)9264ndash9269 doi101073pnas0913022107
Yeates DK Seago A Nelson L Cameron SL JosephLEO Trueman JWH (2011) Integrative taxonomy oriterative taxonomy Syst Entomol 36(2)209ndash217doi101111j1365-3113201000558x
Zhang C Zhang D-X Zhu T Yang Z (2011) Evaluationof a Bayesian coalescent method of species delimita-tion Syst Biol 60(6)747ndash761 doi101093sysbiosyr071
Zhang J Kapli P Pavlidis P Stamatakis A (2013) Ageneral species delimitation method with applicationsto phylogenetic placements Bioinformatics 29(22)2869ndash2876 doi101093bioinformaticsbtt499
44 SD Leavitt et al