Major clades of parmelioid lichens (Parmeliaceae, Ascomycota) and the evolution of their morphological and chemical diversity

Molecular Phylogenetics and Evolution 39 (2006) 52–69www.elsevier.com/locate/ympev

Major clades of parmelioid lichens (Parmeliaceae, Ascomycota) and the evolution of their morphological and chemical diversity

Oscar Blanco a, Ana Crespo a, Richard H. Ree b, H. Thorsten Lumbsch b,¤

a Departamento de Biología Vegetal II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid 28040, Spainb Department of Botany, The Field Museum, 1400 S. Lake Shore Drive, Chicago, IL 60605, USA

Received 31 March 2005; revised 11 November 2005; accepted 28 December 2005Available online 14 February 2006

Abstract

Parmelioid lichens comprise about 1500 species and have a worldwide distribution. Numerous species are widely distributed and wellknown, including important bioindicators for atmospheric pollution. The phylogeny and classiWcation of parmelioid lichens has been amatter of debate for several decades. Previous studies using molecular data have helped to establish hypotheses of the phylogeny of cer-tain clades within this group. In this study, we infer the phylogeny of major clades of parmelioid lichens using DNA sequence data fromtwo nuclear loci and one mitochondrial locus from 145 specimens (117 species) that represent the morphological and chemical diversity inthese taxa. Parmelioid lichens are not monophyletic; however, a core group is strongly supported as monophyletic, excluding Arctoparm-elia and Melanelia s. str., and including Parmeliopsis and Parmelaria. Within this group, seven well-supported clades are found, but therelationships among them remain unresolved. Stochastic mapping on a MC/MCMC tree sampling was employed to infer the evolution oftwo morphological and two chemical traits believed to be important for the evolutionary success of these lichens, and have also been usedas major characters for classiWcation. The results suggest that these characters have been gained and lost multiple times during the diver-siWcation of parmelioid lichens.© 2006 Elsevier Inc. All rights reserved.

Keywords: Parmeliaceae; Ascomycota; Lichens; Stochastic mapping; Character evolution

1. Introduction The morphology and physiological eVects of these pores

In the course of co-evolution between ascomycete fungiand their photosynthetic partners in lichen symbioses,numerous morphological structures and secondary metab-olites have evolved that are believed to have an importantadaptive role for the success of these symbiotic organisms(Lange, 1992; Rikkinen, 1995; Sanders, 2001). The morpho-logical traits occur mostly in foliose lichens and includecortical layers composed of fungal hyphae that protectphotosynthetic partners from high insolation (Büdel andScheidegger, 1996; Jahns, 1973, 1988), and pores of diVerentkinds and sizes that facilitate gas exchange through the cor-tex—analogues to stoma in bryophytes and kormophytes.

* Corresponding author. Fax: +1 312 665 7158.E-mail address: [email protected] (H.T. Lumbsch).

1055-7903/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2005.12.015

have been studied intensely during the last decades (Belt-man, 1978; Green et al., 1981, 1985; Hale, 1973, 1981; Lum-bsch and Kothe, 1992; Sancho et al., 2000; Yoshimura andHurutani, 1987), but their evolutionary history is currentlypoorly understood.

In addition to morphological adaptations, lichens arewell known for the number and quantity of secondarymetabolites they produce (Culberson, 1969; Huneck andYoshimura, 1996). Of particular interest is the adaptivevalue of cortical substances, including UV absorbent com-pounds or pigments that screen visible light and UV(BeGora and Fahselt, 2001; Bjerke et al., 2002; Rikkinen,1995; Solhaug and Gauslaa, 1996; Solhaug et al., 2003).These adaptive morphological structures and secondarymetabolites were frequently used as taxonomic characterswithout any clear concept about their evolutionary origins.While alternative hypotheses about the evolutionary origin

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O. Blanco et al. / Molecular Phylogenetics and Evolution 39 (2006) 52–69 53

of lichen symbioses exist (Gargas et al., 1995; Lutzoni et al.,2001) and a high plasticity in the life-style of some ostropa-lean fungi has been demonstrated (Wedin et al., 2004), theevolution of adaptive morphological and chemical charac-ters in lichens has been examined only in few studies. Theseinclude the reconstruction of the evolution of polyspory(Reeb et al., 2004) and the evolution of tri-partite symbioses(Miadlikowska and Lutzoni, 2004). This lack of attention issurprising, given the importance of these characters inlichen symbiosis and their frequent use in taxonomy. In thisstudy, we have used parmelioid lichens (Parmeliaceae)(DePriest, 1999), a group of morphologically complexlichen-forming fungi (Henssen and Jahns, 1974), as a modelto study the evolution of these traits focusing in two mor-phological and two chemical characters.

Parmeliaceae is one of the largest families of lichen-forming fungi, and has a worldwide distribution. It includesseveral common and well-known species, including taxasuch as Parmelia sulcata, Flavoparmelia caperata, andPunctelia subrudecta that are frequently used as bioindica-tors for atmospheric pollution (Crespo et al., 2004; Nimiset al., 2002). In general, the family is characterized morpho-logically by a certain type of ascoma ontogeny and thepresence of an ascomatal structure called a cupulate exciple(Henssen and Jahns, 1974). Some authors prefer to segre-tate some taxa from Parmeliaceae at the family level, suchas Alectoriaceae or Hypogymniaceae (Elix, 1979; Erikssonand Hawksworth, 1998; Kärnefelt and Thell, 1992; Krog,1982; Poelt, 1973), but segregation of these families is notsupported by molecular evidence (Mattsson and Wedin,1999; Mattsson et al., 2004; Thell et al., 2004; Wedin et al.,1999).

Traditionally, two large genera, Parmelia and Cetraria,were distinguished within the core group of the family,and these correspond more or less to the parmelioid(DePriest, 1999) and cetrarioid group (Kärnefelt et al.,1992; Randlane and Saag, 1993; Randlane et al., 1997),respectively. Parmelioid lichens encompass approximately1500 taxa (Hale and DePriest, 1999), which were formerlyclassiWed within Parmelia sensu lato. This group includesspecies which are mainly foliose, mostly rhizinate lichenswith laminal apothecia and simple, hyaline ascospores(Hale, 1987), and have their centre of diversity in oceanic-temperate, tropical, and subtropical ecosystems. Genericconcepts in lichen-forming fungi have changed dramati-cally since the late 1960s (Rambold and Triebel, 1999),with the Parmeliaceae being a prominent example(DePriest, 1999; Hale, 1984; Nimis, 1998). In crustosegroups, ascomatal characters (e.g., ascus-type, ascoma andascospore development) and hamathecial features haveoften been utilized to circumscribe segregate genera, butthese characters have not been as widely used in parmeli-oid lichens, which frequently occur as sterile species. Forthis reason, morphological and chemical characters havemore often been employed to segregate genera in thisgroup, though acceptance of the new genera has not beenuniform (e.g., Clauzade and Roux, 1985; Eriksson and

Hawksworth, 1986; Llimona and Hladun, 2001; Purviset al., 1992). Based on Hale’s concept, DePriest (1999)gave an overview of the historic development of genericconcepts in parmelioid lichens and included 36 genera inthis group (Table 1). In recent years, some of these segre-gates have been included within other genera based onmorphological and/or molecular evidence, e.g., Rimeliellawithin Canomaculina (Elix, 1997); Chondropsis, Parap-armelia, and Neofuscelia within Xanthoparmelia (Blancoet al., 2004a; Elix, 2003; Hawksworth and Crespo, 2002);and Canomaculina, Concamerella, and Rimelia withinParmotrema (Blanco et al., 2005). Other groups have beenfound to be heterogeneous such as Melanelia (Blancoet al., 2004b) (Table 1). Crespo et al. (2001) demonstratedthe monophyly of a group of parmelioid lichens usingmitochondrial rDNA, a result corroborated by Mattssonet al. (2004), who used four diVerent loci in a higher-levelphylogenetic study. However, the relationships amongmajor clades within parmelioids remain poorly under-stood, and the evolution of morphological and chemicalcharacters within parmelioid lichens has not yet beenstudied.

We chose to study two morphological and two chemi-cal traits that are usually interpreted as being adaptivefor these symbiotic fungi (Hale, 1981; Rikkinen, 1995).The morphological characters are two types of perfora-tions of the thallus surface that allow gas exchange of thephotosynthetic partner: (a) pores in the epicortex and (b)a cortical structure that reaches the algal layer, called apseudocyphella (Fig. 1). The epicortex is a polysaccha-ride layer about 0.6 �m thick, lying over the cortex of sev-eral, unrelated macrolichens. This layer is analogous tothe cuticle in higher plants. Pores are 15–40 �m in diam.,with a density of 100–400 pores per mm¡2, and may berestricted to the epicortex or include cortical layers, butnot the medulla (Beltman, 1978; Hale, 1973, 1981).Pseudocyphellae are ontogenetically derived from poresin the cortex, have a diameter of 200–2000 �m and a den-sity of 1–2 per mm¡2. The cortex is dissolved or partiallyreduced in these structures and medullary hyphae areusually involved (Feuerer and Marth, 1997; Hale, 1973,1981). The type of perforation is usually believed to be animportant character at the generic level and was evenregarded as important at the subfamilial rank (Elix, 1993;Hale, 1981; Henssen, 1992).

The two chemical characters investigated are the pres-ence of cortical substances that are believed to play animportant ecological role in screening UV and visible light:the pigment usnic acid absorbs both UV and visible light,and atranorin, a colourless depside of the �-orcinol type,has a strong UV absorption (Huneck and Yoshimura,1996). All four characters described above have beenregarded as key characters distinguishing genera andgeneric groups within parmelioid lichens (Elix, 1993).

Sequences of two nuclear and single mitochondrialribosomal DNA loci were used for phylogeny reconstruc-tion. Most sequences were used in studies focused on

54 O. Blanco et al. / Molecular Phylogenetics and Evolution 39 (2006) 52–69

certain generic groups within parmelioid lichens (Blancoet al., 2004a,b, 2005). Here, they are used with 20 newsequences to infer higher-level phylogenetic relationshipswithin this group in Bayesian and maximum parsimony

frameworks. On the basis of the Bayesian tree sampling,histories of morphological and chemical character evolu-tion are mapped and used to evaluate previous hypothesesdeveloped on the basis of classical comparative methods.

Table 1Genera of Parmelioid lichens as accepted by DePriest (1999)

Changes in classiWcations since DePriest (1999) Number of species Genera included in this study

Allantoparmelia 3 ¡Almbornia 2 ¡Arctoparmelia 5 +Bulborrhizina 1 ¡Bulbothrix 52 +Canomaculina ParmotremaCanoparmelia 49 +Cetrariastrum 5 ¡Concamerella ParmotremaEverniastrum 40 +Flavoparmelia 35 +Flavpunctelia 7 +Hypotrachyna ca. 190 +Karoowia 19 +Melanelia 8 +Melanelia s. lat. Melanelixia 11 +Melanelia s. lat. Melanohalea 19 +Myelochroa 28 +Namakwa 2 ¡Neofuscelia Xanthoparmelia +Omphalora 1 ¡Paraparmelia Xanthoparmelia +Parmelia ca. 45 +Parmelina ca. 15 +Parmelinella 5 +Parmelinopsis 25 +Parmotrema ca. 350 +Parmotremopsis 2 ¡Placoparmelia 1 ¡Pleurosticta 3 +Pseudoparmelia 4 ¡Psiloparmelia 12 ¡Punctelia 34 +Relicina 54 +Relicinopsis 5 ¡Rimelia Parmotrema +Xanthomaculina 2 +Xanthoparmelia ca. 650 +

Fig. 1. SEM picture of the morphology of perforations in the upper surface of parmelioid lichens. (A) Pored epicortex in Parmotrema cetratum. (B)Pseudocyphella in Melanohalea exasperata.

A B

60µm 60µm


2. Materials and methods

2.1. Taxon sampling

We sampled 109 species of parmelioid lichens, includingthe major clades and representatives of the morphologicaland chemical diversity within this group. The sampleincludes 20 out of 31 parmelioid genera based on theDePriest (1999) classiWcation, considering that some generawere reduced to synonymy (Blanco et al., 2004a, 2005), andthe two newly described genera Melanelixia and Melanoha-lea (Blanco et al., 2004b). Samples of eight other Parmelia-ceae were also included. Pseudevernia furfuracea was usedas outgroup since it has been shown that it does not belongto parmelioid lichens (Mattsson et al., 2004). The data setincludes 406 sequences, the majority of which are from pre-vious publications by us (Blanco et al., 2004a,b, 2005;Crespo et al., 2001), and 20 sequences newly generated forthis study. GenBank accession numbers, locality, andvoucher information are given in Table 2.

2.2. Molecular methods

Samples prepared from freshly collected and frozen her-barium specimens were ground with sterile glass pestles.Total genomic DNA was extracted using the DNeasy PlantMini Kit (Qiagen) according to the manufacturer’s instruc-tions with slight modiWcations described in Crespo et al.(2001). Dilutions of the total DNA were used for PCRampliWcations of nuclear ITS and LSU rRNA loci, and themitochondrial SSU rRNA. Fungal nu ITS rDNA wasampliWed using the primers ITS1F (Gardes and Bruns,1993) and ITS4 (White et al., 1990); nu LSU rDNA wasampliWed using the primers LR0R (Rehner and Samuels,1994) and LR5 (Vilgalys and Hester, 1990), and mt SSUrDNA was ampliWed using the primers mrSSU1 andmrSSU3R (Zoller et al., 1999), NMS1 and NMS2 (Li et al.,1994), and MSU1 and MSU7 (Zhou and Stanosz, 2001).AmpliWcations were performed in 50 �L volumes contain-ing a reaction mixture of 10 �L diluted DNA, 5 �L of 10£DNA polymerase buVer (Biotools) (containing MgCl22 mM, 10 mM Tris–HCl, pH 8.0, 50 mM KCl, 1 mM EDTA,and 0.1% Triton X-100), 1 �L of dinucleotide triphosphate(dNTPs), containing 10 mM of each base, 2.5 �L of eachprimer (10 �M), 1.25�L of DNA polymerase (1 U/�L), and27.5 �L dH2O.

AmpliWcations for nu ITS and LSU rDNA were carriedout in an automatic thermocycler (Techne Progene) andperformed using the following programs: initial denatur-ation at 94 °C for 5 min, and 30 cycles of: 94 °C for 1 min,54–60 °C (ITS rDNA) and 60 °C (LSU rDNA) for 1 min,72 °C for 1.5 min, and a Wnal extension at 72 °C for 5 min.PCR ampliWcation for mitochondrial rDNA was carriedout in a Hybaid OmniGene thermocycler using the follow-ing program: initial denaturation at 94 °C for 5 min and 35cycles of: 94 °C for 1 min, 57–58 °C for 1 min, and 72 °C for1.5 min, and a Wnal extension at 72 °C for 5 min.

The PCR products were subsequently cleaned using theDNA PuriWcation Column kit (Biotools) according to themanufacturer’s instructions. The cleaned PCR productswere sequenced with the same primers used in the ampliW-cations. The ABI Prism Dye Terminator Cycle SequencingReady reaction kit (Applied Biosystems) was used and thefollowing settings were carried out: denaturation for 3 minat 94 °C and 25 cycles at: 96 °C for 10 s, 50 °C for 5 s, and60 °C for 4 min. Sequencing reactions were electrophoresedon a 3730 DNA analyser (Applied Biosystems). Sequencefragments obtained were assembled with SeqMan 4.03(DNAStar) and manually adjusted.

2.3. Sequence alignments

The nu ITS and mt SSU rDNA data sets contained highlyvariable sequence portions in the alignment. Because stan-dard alignment programs such as Clustal (Thompson et al.,1994) become less reliable when highly variable sequences areinvolved, we used an alignment procedure that employs a lin-ear Hidden Markov Model (HMM) for the alignment, asimplemented in the software SAM (Hughey and Krogh,1996). Sequences of 145 specimens (Table 2) were alignedseparately for the three regions. Ninety-nine base pairs in themt SSU, 61bp in the nu ITS and 188 in the nu LSU data setthat could not be aligned with statistical conWdence wereexcluded from the phylogenetic analysis.

2.4. Phylogenetic analyses

For phylogenetic analysis, we used a Bayesian approachthat allows eYcient analysis of complex nucleotide substitu-tion models in a parametric statistical framework (Huelsen-beck et al., 2001; Larget and Simon, 1999) and includesestimation of uncertainty (Huelsenbeck et al., 2000). Poster-ior probabilities and bootstrap support values from maxi-mum parsimony or maximum likelihood analyses have beendemonstrated to diVer, and these diVerences have been inter-preted in diVerent ways (Alfaro et al., 2003; Simmons et al.,2004; Suzuki et al., 2002; Wilcox et al., 2002). Bayesian sup-port values appear to be overestimates in certain cases, espe-cially when short branches are involved. In contrast,bootstrap values are commonly underestimates and can beviewed as helpful lower bounds of support values (Douadyet al., 2003), but are more sensitive to long-branch attraction.We also performed maximum parsimony analyses, includingnonparametric bootstrapping. Here, we adopt a conservativeperspective and consider well-supported clades to be thosethat have a posterior probability of at least 0.95 as well asbootstrap support equal to or above 70%.

2.4.1. Bayesian analysesThe Bayesian (B/MCMC) analyses were performed using

MrBayes 3.0 (Huelsenbeck and Ronquist, 2001). Posteriorprobabilities were approximated by sampling trees using aMarkov chain Monte Carlo (MCMC) method. For all datasets the general time reversible model of nucleotide substitu-

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volution 39 (2006) 52–69Table 2Species and specimens of Parmeliaceae analysed

Pseudocyphellae Atranorin Usnic acid

No No YesNo Yes NoNo Yes NoNo Yes NoNo Yes NoNo Yes NoNo No YesNo No YesNo No YesNo No YesNo No YesYes Yes NoYes Yes NoNo Yes NoNo Yes NoNo Yes NoNo Yes NoNo Yes NoNo Yes NoNo Yes NoNo Yes NoNo No YesNo Yes NoNo No YesYes No NoYes No NoNo No NoNo No NoNo No NoNo No NoNo No NoNo No NoNo No NoNo No NoNo No NoNo No NoNo No NoNo No NoNo No NoYes No NoYes No NoYes No NoYes No NoYes No NoYes No NoYes No No

Group Species Voucher Locality mt SSU nu ITS nu LSU Pored

Parmelioid (DePriest, 1999)

Arctoparmelia centrifuga MAF 6879 Sweden AF351156 AY581054 AY578917 YesBulbothrix meizospora GPGC 02-000786 India AY611127 AY611068 AY607780 YesB. setschwanensis MAF 10212 China — AY611069 AY607781 YesCanoparmelia crozalsiana MAF 7658 Spain AY586594 AY586571 AY584831 YesEverniastrum cirrhatum Trest 149 Costa Rica AY611128 AY611070 AY607782 YesE. nepalense GPGC 02-000924 India AY611129 AY611071 AY607783 YesFlavoparmelia baltimorensis 1 MAF 7660 USA AY586583 AY586559 AY584832 YesF. baltimorensis 2 MAF 10174 USA AY586584 AY586560 AY584833 YesF. caperata 1 MAF 6045 Spain AF351163 AY581059 AY578922 YesF. caperata 2 MAF 10175 China AY586585 AY586561 AY584834 YesF. soredians MAF 10176 Spain AY586586 AY586562 AY584835 YesFlavopunctelia Xaventior 1 MAF 6046 Spain AF351164 AY581060 AY578923 NoF. Xaventior 2 DCH-19 China AY586587 AY586563 — NoHypotrachyna adducta MAF 10206 China AY785277 AY785270 AY785263 YesH. ciliata MAF 10185 China AY785280 AY785273 AY785266 YesH. costaricensis MAF 10211 Costa Rica AY785276 AY785269 AY785262 YesH. endochlora MAF 10178 Great Britain AY611130 AY611072 AY607784 YesH. immaculata MAF 7462 Australia AY611131 AY611073 AY607785 YesH. inWrma MAF 10210 China AY785278 AY785271 AY785264 YesH. laevigata MAF 10177 Great Britain AY611132 AY611074 AY607786 YesH. revoluta MAF 6047 Spain AF351166 AY611075 AY607787 YesH. sinuosa MAF 10179 Great Britain AY611133 AY611076 AY607788 YesH. taylorensis MAF 9921 Great Britain AY582298 AY581061 AY578924 YesKaroowia saxeti Aptroot 53350 Taiwan AY582299 AY581063 AY578926 YesMelanelia disjuncta Mayrhofer 13743 Austria AY611134 AY611077 AY607789 NoMelanelia stygia Haikonen 20365 Finland AY611154 AY611097 AY607809 NoMelanelixia fuliginosa 1 MAF 7640 Spain AY611141 AY611084 AY607796 YesM. fuliginosa 2 MAF 10222 Spain AY611142 AY611085 AY607797 YesM. fuliginosa 3 MAF 10219 Spain AY611143 AY611086 AY607798 YesM. fuliginosa 4 MAF 10223 Spain AY611146 AY611089 AY607801 YesM. fuliginosa 5 MAF 10229 Spain AY611145 AY611088 AY607800 YesM. glabra 1 MAF 7634 Spain AY582300 AY581064 AY578927 YesM. glabra 2 MAF 10228 Spain AY611144 AY611087 AY607799 YesM. subargentifera 1 MAF 6049 Spain AY611155 AY611098 AY607810 YesM. subargentifera 2 MAF 9909 Spain AY582301 AY581065 AY578928 YesM. subaurifera 1 MAF 10221 Spain AY611152 AY611096 AY607807 YesM. subaurifera 2 MAF 10217 Spain AY611157 AY611100 AY607812 YesM. subaurifera 3 MAF 10216 Spain AY611158 AY611101 AY607813 YesM. subaurifera 4 MAF 10215 Great Britain AY611156 AY611099 AY607811 YesM. elegantula 1 MAF 10226 Spain AY611136 AY611079 AY607791 NoM. elegantula 2 MAF 10218 Spain AY611135 AY611078 AY607790 NoM. elegantula 3 MAF 10231 Spain AY611151 AY611094 AY607806 NoM. elegantula 4 MAF 10224 Spain AY61137 AY611080 AY607792 NoM. exasperata 1 MAF 7636 Spain AY611140 AY611083 AY607795 NoM. exasperata 2 MAF 10214 Spain AY611138 AY611081 AY607793 NoM. aV. exasperata 1 MAF 10227 Spain AY611139 AY611082 AY607794 No

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57M. aV. exasperata 2 MAF 10225 Spain AY611149 AY611092 AY607804 No Yes No NoM. aV. exasperata 3 MAF 10230 Spain AY611153 AY611095 AY607808 No Yes No No

Yes No NoYes No NoYes No NoNo Yes NoNo Yes NoYes Yes NoYes Yes NoYes Yes NoYes Yes NoYes Yes NoNo Yes NoNo Yes NoNo Yes NoNo Yes NoNo Yes NoNo Yes NoNo Yes NoNo Yes NoNo Yes NoNo Yes NoNo Yes NoNo Yes NoNo Yes NoNo Yes NoNo Yes NoNo Yes NoNo Yes NoNo Yes NoNo Yes NoNo Yes NoNo Yes NoNo Yes NoNo Yes NoNo Yes NoNo No NoYes Yes NoYes Yes NoYes Yes NoYes Yes NoYes Yes NoYes Yes NoNo No YesNo No YesNo No YesNo No NoNo No YesNo No Yes

(continued on next page)

M. exasperatula MAF 10213 Spain AY611147 AY611090 AY607802 NoM. olivacea Vitikainen 16196 Finland AY611148 AY611091 AY607803 NoM. septentrionalis Ahti 60893 Finland AY611150 AY611093 AY607805 NoMyelochroa irrugans MAF 10207 China AY611160 AY611103 AY607815 YesM. metarevoluta MAF 10208 China AY611159 AY611102 AY607814 YesParmelia pinnatiWda MAF 7272 Russia AY611161 AY036988 — NoP. saxatilis MAF 6804 Sweden AF351172 AF350027 AY578947 NoP. serrana MAF 9756 Spain AY582319 AY295109 AY578948 NoP. squarrosa MAF 7281 Japan AY611162 AY036975 AY607816 NoP. sulcata MAF 6054 Great Britain AY582320 AY581083 AY578949 NoParmelina pastillifera MAF 6058 Spain AY611163 AY611104 AY607817 YesP. quercina MAF 6057 Spain AY611164 AY611105 AY607818 YesP. tiliacea MAF 6056 Spain AF351173 AY581084 AY578950 YesParmelinella wallichiana LWG 20-77171 India AY611165 AY611106 AY607819 YesParmelinopsis horrescens MAF 9913 Spain AY582321 AY581085 AY578951 YesP. minarum 1 MAF 7639 Spain AY582322 AY581086 AY578952 YesP. minarum 2 MAF 10220 China AY611168 AY611110 — YesP. neodamaziana MAF 10182 Australia AY611166 AY611107 AY607820 YesP. subfatiscens MAF 6878 Australia AF351174 AY611108 AY607821 YesParmotrema cetratum Osorio 9424 Uruguay AY586598 AY586576 AY584847 YesP. crinitum MAF 6061 Portugal AY586589 AY586565 AY584837 YesP. Wstulatum Osorio 9423 Uruguay AY582297 AY581057 AY578920 YesP. haitiense MAF 7657 Australia AY582295 AY581055 AY578918 YesP. hypoleucinum MAF 7636 Spain AY586590 AY586567 AY584839 YesP. perforatum Cole 7983 USA AY586591 AY586568 AY584840 YesP. perlatum MAF 6965 Portugal AY586580 AY586566 AY584838 YesP. pilosum MAF 7656 Uruguay AY582296 AY581056 AY578919 YesP. pseudoreticulatum MAF 7650 Spain AY586600 AY586578 AY584849 YesP. reticulatum 1 MAF 6067 Portugal AF351184 AY586579 AY584850 YesP. reticulatum 2 MAF 10164 China AY586599 AY586577 AY584848 YesP. robustum MAF 10166 Portugal AY586592 AY586569 AY584841 YesP. subcaperatum HO 324283 Australia AY586581 AY586557 AY584829 YesP. subtinctorium GPGC 02-000696 India AY586582 AY586558 AY584830 YesP. tinctorum MAF 10163 Australia AY586593 AY586570 AY584842 YesPleurosticta acetabulum MAF 9914 Spain AY582323 AY581087 AY578953 YesPunctelia borreri MAF 9919 Portugal AY582324 AY581088 AY578954 NoP. pseudocoralloidea MAF 6922 Australia AY586595 AY586572 AY584843 NoP. rudecta 1 MAF 7661 USA AY586596 AY586573 AY584844 NoP. rudecta 2 MAF 10162 USA AY586597 AY586574 AY584845 NoP. subXava Elix 42705 Australia AF351183 AY586575 AY584846 NoP. subrudecta MAF 9918 Portugal AY582325 AY581089 AY578955 NoRelicina subnigra MAF 10184 Australia AY785281 AY785274 AY785267 YesR. sydneyensis MAF 10183 Australia AY785282 AY785275 AY785268 YesXanthoparmelia angustiphylla MAF 6768 USA AY582328 AY581092 AY578958 YesX. atticoides MAF 6744 USA AY582302 AY581066 AY578929 YesX. brachinaensis Elix 30651 Australia — AY581062 AY578925 YesX. conspersa MAF 6793 Spain AF351186 AY581096 AY578962 Yes

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Table 2 (continued)

Group Sp eudocyphellae Atranorin Usnic acid

X. No YesX. No YesX. No NoX. No NoX. No YesX. No NoX. No NoX. No YesX. No YesX. No YesX. Yes NoX. Yes NoX. No NoX. No NoX. No YesX. No YesX. Yes NoX. Yes NoX. No YesX. No NoX. No NoX. No YesX. No NoX.X. No NoX. No YesX. Yes NoX. No YesX. No Yes

X. No Yes

X. No NoX. No YesX. No NoX. Yes NoX. No YesX. No YesX. No YesX. No YesX. No Yes

ecies Voucher Locality mt SSU nu ITS nu LSU Pored Ps

crespoae 1 MAF 7524 Australia AY582332 AY581097 AY578963 Yes No crespoae 2 MAF 7440 Australia AY582333 AY581098 AY578964 Yes No delisei MAF 7659 Spain AY582304 AY581068 AY578931 Yes No aV. delisei MAF 7432 Australia AY582303 AY581067 AY578930 Yes No digitiformis MAF 7525 Australia AY582334 AY581099 AY578965 Yes No glabrans MAF 7665 Australia AY582305 AY581069 AY578932 Yes No aV. glabrans MAF 9912 Spain AY582308 AY581072 AY578935 Yes No hueana GZU 46511 Namibia AY582326 AY581090 AY578956 Yes No isidiovagans MAF 9956 Spain AY582330 AY581094 AY578960 Yes No lineola MAF 9955 USA AY582338 — AY578970 Yes No lithophila MAF 6900 Australia AF351171 AY581077 AY578941 Yes No lithophiloides MAF 7471 Australia AY582314 AY581078 AY578942 Yes No loxodes 1 MAF 7072 Spain AY582313 AY581076 AY578940 Yes No loxodes 2 MAF 6206 Spain AY582306 AY581070 AY578933 Yes No mougeotii 1 MAF 6802 Spain AY582335 AY037006 AY578966 Yes No mougeotii 2 MAF 9916 Spain AY582336 AY581100 AY578967 Yes No murina MAF 9915 Australia AY582315 AY581079 AY578943 Yes No norcapnodes MAF 7532 Australia AY582316 AY581080 AY578944 Yes No notata Elix 42648 Australia AY582337 AY581101 AY578968 Yes No pokornyi 1 MAF 6052 Spain AY582307 AY037005 AY578934 Yes No pokornyi 2 MAF 9908 Spain AY582312 AY581075 AY578939 Yes No protomatrae MAF 6216 Spain AY582339 AY581104 AY578972 Yes No pulla MAF 6794 Spain AF351169 AY581071 — Yes No pulla — Poland — — AJ421433 pulloides MAF 6784 Spain AY582309 AY037004 AY578936 Yes No reptans Elix 42635 Australia — AY581102 AY578969 Yes No scotophylla Elix 30650 Australia AY582317 AY581081 AY578945 Yes No semiviridis Elix 30294 Australia AF351158 AY581058 AY578921 Yes No stenophylla MAF 9917 Spain AY582329 AY581093 AY578959 Yes No

subdiZuens MAF 9910 Spain AY582340 AY581105 AY578973 Yes No

subincerta MAF 7494 Australia AY582310 AY581073 AY578937 Yes No sublaevis MAF 7460 Spain AY582341 AY581106 AY578974 Yes No subprolixa MAF 7667 Australia AY582311 AY581074 AY578938 Yes No subspodochroa MAF 7463 Australia AY582318 AY581082 AY578946 Yes No sub verrucigera MAF 10209 Spain AY582327 AY581091 AY578957 Yes No tegeta MAF 7523 Australia AY582342 AY581107 AY578975 Yes No tinctina 1 MAF 6070 Spain AY582343 AY581108 AY578976 Yes No tinctina 2 BCN-13861 Spain AY582344 AY581109 AY578977 Yes No tinctina 3 BCN-13862 Spain AY582345 AY581110 AY578978 Yes No


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tion (Rodríguez et al., 1990) including estimation of invariantsites and assuming a discrete gamma distribution with six ratecategories (GTR+I+G) was used and parameters were cal-culated for each partition separately as proposed by Nylanderet al. (2004). In the separate single-partition analyses, MrBA-YES was run with eight simultaneous chains for all data sets.In the combined analysis, 12 simultaneous chains were used.

MrBAYES was run for 106 generations on each data setand 2£106 generations for the combined analysis. Treeswere sampled every 100 generations yielding 10,000 and20,000 trees, respectively. The Wrst 1000 trees were discardedas the “burn in” of the chain. We plotted the log-likelihoodscores of sample points against generation time usingTRACER 1.0 (http://evolve.zoo.ox.ac.uk/software.html?idD tracer) to conWrm probable chain stationarityafter the Wrst 100,000 generations. For the remaining trees ineach analysis, a majority-rule consensus tree with averagebranch lengths was calculated using the sumt option ofMrBayes. Phylogenetic trees were visualized using the pro-gram Treeview (Page, 1996). Congruence between the datasets was assessed by comparing bootstrap support of cladesabove 70% for each locus. We used this approach instead ofcomparing posterior probabilities to avoid the potentialproblem of overestimated support for short branches inBayesian analyses.

2.4.2. Maximum parsimony analysesThe maximum parsimony (MP) analyses were per-

formed employing PAUP* 4.0 (SwoVord, 2003) using theheuristic search option with 200 random sequence addi-tions, TBR branch swapping and MulTrees option ineVect, equally weighted characters and gaps treated asmissing data. Nonparametric bootstrap support (Felsen-stein, 1985) for each clade was estimated based on 2000replications, using the heuristic search option with 200random sequence additions and the MulTrees option ineVect. To assess homoplasy levels, consistency index (CI),retention index (RI), and rescaled consistency (RC) index(Farris, 1989) were calculated from each parsimonysearch in PAUP*.

2.5. Character evolution

To evaluate the evolution of morphological and chemicalcharacters in a Bayesian framework, we have used stochas-tic mapping and Bayesian Metropolis-coupled MarkovChain Monte Carlo (MC/MCMC) tree sampling. Stochasticmapping was originally developed by Nielsen (2002) to inferthe phylogenetic locations of nucleotide substitutions andthe method was extended to morphological characters byHuelsenbeck et al. (2003). Stochastic mapping estimates thehistory of character changes on a phylogeny using simula-tions that employ an explicit transformation model, thusavoiding the tendency of parsimony to underestimate theactual number of changes that occurred (Felsenstein, 1978).It takes as input character-state data for extant species and aphylogenetic tree of those species, and uses a model parame-

http://evolve.zoo.ox.ac.uk/software.html?id=tracer








terized by a matrix of instantaneous rates of change betweenstates (Q), a vector of stationary frequencies for characterstates (�), and tree length, measured as the expected amountof change in the character of interest. Characters were codedas follows: epicortex nonpored (0) or pored (1); pseudocyp-hellae absent (0) or present (1); usnic acid absent (0) or pres-ent (1); and atranorin absent (0) or present (1) (Table 2).Presence and absence of atranorin and usnic acid was studiedusing standardized TLC and HPLC methods (Lumbsch,2002). Presence or absence of pores/fenestrations andpseudocyphellae was examined using SEM. Small pieces ofthalli ca. 5mm in diam. were cut from samples, air-dried,Wxed to a metallic stub, and sputtered with gold-palladium ina vacuum. A Jeol (JSM 6400) electron microscope was usedfor the analysis.

Uncertainty in the phylogeny was accommodated bymapping each character over 19,000 trees sampled in thecombined analysis. For each character, a mapping resultingin the observed states at terminal nodes was performed foreach tree. The simulations were run with branch lengthsscaled to yield a total tree length of 1 and the bias parameter�0 was drawn from a uniform prior distribution. Each map-ping was recorded as two sets of taxon bipartitions (with abipartition corresponding to a clade subtended by a charac-ter-state change): one set for gains of state 1, and one forlosses. Recording mappings in this manner facilitates thesummary of results across trees that may diVer in topology.Stochastic mapping was carried out using a software library,stmap, written by RHR and available from http://www.phylodiversity.net/rree/software.html.

3. Results

3.1. Phylogenetic analyses

Ambiguously aligned regions and major insertions, rep-resenting spliceosomal and group I introns in the nuclear

ribosomal DNA (Bhattacharya et al., 2000; Cubero et al.,2000; Gargas et al., 1995), were excluded from all analyses(99 bp for mt SSU, 188 for the nu LSU, and 61 for the nuITS). We produced a matrix of 2237 unambiguouslyaligned nucleotide position characters in the combinedanalysis, including 645 of the mt SSU, 1038 of the nu LSU,and 554 of the nu ITS rDNA. Five hundred and forty-sixcharacters were variable in the mt SSU, 850 in the nu LSU,and 493 in the ITS rDNA data set. Congruence in phyloge-netic signal between the three loci was high overall, with themajority of clades supported by one single-gene analysisnot being contradicted in the others. Areas of conXict, inwhich strongly supported clades were contradicted inanother analysis, were restricted to a very few terminalnodes: (a) in the mt SSU tree Hypotrachyna adducta andH. ciliata clustered together with 93% bootstrap support,while in the nu ITS tree H. adducta is sister group ofH. inWrma (100%); (b) Parmotrema haitiense grouped withP. pilosum in the mt SSU tree (82%), whereas P. haitiense issister to P. subtinctorum in the nu ITS tree (100%); (c) Mel-anohalea exasperata is monophyletic in the nu ITS tree(93%), while this species is paraphyletic in the mt SSU tree.The combined alignment is available in Treebase (S1400).(http://www.treebase.org/treebase).

Summary statistics for the data sets analysed undermaximum parsimony and B/MCMC are given in Table 3.The amount of homoplasy diVers between the gene parti-tions: the mt SSU rDNA exhibits the lowest amount ofhomoplasy, while the highest amount of homoplasy ispresent in the nu ITS rDNA gene partition. Base composi-tion varies considerably between the mitochondrial andnuclear data sets. The nu LSU rDNA was the most GCrich (51.9%), while the mitochondrial data set had a muchlower GC content (39.4%). The percentage of invariablesites also diVers between the data sets, being lowest in themt SSU rDNA (13.5%). The gamma shape parameteralpha is similar between the gene partitions. The nu ITS,

Table 3Comparison of performance of data partitions under parsimony and in a Bayesian framework

Nu LSU Nu ITS Mt SSU Combined

No of parsimony informative sites 164 231 141 536No. steps 1054 2172 655 3959CI 0.298 0.227 0.394 0.268RI 0.648 0.677 0.822 0.696RC 0.193 0.153 0.324 0.187No. nodes with bootstrap 7 70% 33 59 28 76Mean likelihood ¡6593 (§3.907) ¡10603 (§1.118) ¡4438 (§2.567) ¡21656 (§2.530)�Amean/all 0.228 (§0.003) 0.224 (§0.006) 0.327 (§0.003) 0.251 (§0.003)�Cmean/all 0.225 (§0.003) 0.287 (§0.005) 0.155 (§0.003) 0.227 (§0.003)�Gmean/all 0.294 (§0.003) 0.227 (§0.005) 0.239 (§0.004) 0.264 (§0.004)�Tmean/all 0.254 (§0.003) 0.263 (§0.003) 0.280 (§0.004) 0.258 (§0.003)r (AC)mean/all 1.445 (§0.211) 1.061 (§0.133) 0.953 (§0.120) 1.080 (§0.008)r (AG)mean/all 4.944 (§0.849) 3.818 (§0.324) 2.936 (§0.377) 3.192 (§0.208)r (AT)mean/all 0.769 (§0.093) 2.359 (§0.257) 1.353 (§0.172) 1.611 (§0.113)r (CG)mean/all 0.644 (§0.073) 0.776 (§0.078) 0.553(§0.088) 0.600 (§0.038)r (CT)mean/all 9.900 (§0.188) 9.769 (§0.098) 3.374 (§0.480) 8.756 (§0.676)�mean/all 0.500 (§0.0002) 0.570 (§0.0018) 0.501 (§0.0002) 0.578 (§0.007)P(invar) mean/all 0.219 (§0.013) 0.324 (§0.041) 0.135 (§0.014) 0.181 (§0.003)No. of nodes with PP 7 0.95 48 75 42 85

http://www.phylodiversity.net/rree/software.html



http://www.treebase.org/treebase

http://www.treebase.org/treebase


Fig. 2. Phylogenetic relationships of parmelioid lichens inferred from a combined analysis of nuclear ITS, LSU and mitochondrial SSU rDNA sequences.50% majority-rule consensus tree of 19,000 trees sampled using a Bayesian MC/MCMC analysis. Branches with posterior probabilities above 0.94 and alsobootstrap support under parsimony equal or above 70% are indicated in bold.

0.1

Xanthoparmelia lithophiloidesXanthoparmelia scotophylla

Xanthoparmelia subspodochroaXanthoparmelia lithophila

Xanthoparmelia norcapnodesXanthoparmelia crespoae 1Xanthoparmelia crespoae 2

Xanthoparmelia murinaXanthoparmelia digitiformisXanthoparmelia tinctina 2Xanthoparmelia tinctina 3Xanthoparmelia tinctina 1Xanthoparmelia semiviridisXanthoparmelia reptans

Xanthoparmelia vicentei 2Xanthoparmelia vicentei 1

Xanthoparmelia isidiovagansXanthoparmelia conspersaXanthoparmelia atticoides

Xanthoparmelia lineolaXanthoparmelia sublaevis

Xanthoparmelia stenophylla Xanthoparmelia subdiffluensXanthoparmelia protomatraeXanthoparmelia angustiphylla

Xanthoparmelia brachinaensisXanthoparmelia notata

Xanthioarmelia subverrucigeraXanthoparmelia verrucigera

Xanthoparmelia tranvaalensisXanthoparmelia deliseiXanthoparmelia loxodes 2

Xanthoparemelia aff. glabransXanthoparmelia puloidesXanthoparmelia pokornyi 1Xanthoparmelia pulla Xanthoparmelia pokornyi 2

Xanthoparmelia loxodes 1Xanthoparmelia aff. delisei

Xanthoparmelia subprolixaXanthoparmelia subincerta

Xanthoparmelia glabransXanthoparmelia mougeotii 1Xanthoparmelia mougeotii 2Xanthoparmelia tegeta

Xanthoparmelia hueanaKaroowia saxeti

Parmotrema crinitumParmotrema perlatum

Parmotrema robustumParmotrema pilosum

Parmotrema fistulatumParmotrema tinctorum

Parmotrema reticulatum 2Parmotrema reticulatum 1

Parmotrema pseudoreticulatumParmotrema cetratum

Parmotrema haitiense Parmotrema subtinctorium

Parmotrema subcaperatumParmelaria subthomsonii

Parmotrema hypoleucinumParmotrema perforatum

Canoparmelia crozalsianaFlavoparmelia caperata 1

Flavoparmelia caperata 2Flavoparmelia baltimorensis 1

Flavoparmelia baltimorensis 2Flavoparmelia soredians

Punctelia rudecta 1Punctelia rudecta 2

Punctelia subrudectaPunctelia borreri

Punctelia pseudocoralloideaPunctelia subflava

Flavopunctelia flaventior 1Flavopunctelia flaventior 2

Melanelixia subaurifera 1Melanelixia subaurifera 3

Melanelixia subaurifera 2Melanelixia subaurifera 4

Melanelixia fuliginosa 2Melanelixia fuliginosa 5

Melanelixia fuliginosa 3Melanelixia fuliginosa 4Melanelixia fuliginosa 1Melanelixia subargentifera 1Melanelixia subargentifera 2

Melanelixia glabra 2Melanelixia glabra 1

Parmelia pinnatifidaParmelia saxatilis

Parmelia serranaParmelia squarrosaParmelia sulcata

Pleurosticta acetabulumMelanohalea exasperata 2Melanohalea exasperata 1

Melanohalea elegantula 4Melanohalea exasperatula

Melanohalea elegantula 2Melanohalea elegantula 3Melanohalea elegantula 1

Melanohalea aff. exasperata 1Melanohalea aff. exasperata 3

Melanohalea aff. exasperata 2Melanohalea olivacea

Melanohalea septentrionalisMelanelia disjuncta Parmelinopsis minarum 1

Parmelinopsis minarum 2Parmelinopsis subfatiscens

Parmelinopsis horrescensParmelinopsis neodamaziana

Hypotrachyna revolutaHypotrachyna immaculata

Everniastrum cirrhatumEverniastrum nepalense

Hypotachyna endochloraHypotrachyna laevigata

Hypotrachyna taylorensisHypotrachyna sinuosa

Hypotrachyna adductaHypotrachyna infirma

Hypotrachyna ciliataHypotrachyna costaricensis

Bulbothrix meizosporaBulbothrix setschwanensis

Parmelinella wallichianaParmelina pastilliferaParmelina tiliaceaParmelina quercina

Myelochroa metarevolutaMyelochroa irrugans

Parmeliopsis ambiguaParmeliopsis hyperopta

Relicina subnigraRelicina sydneyensis

Cetaria aculeataVulpicida pinastri

Melanelia stygiaPannoparmelia angustata

Arctoparmelia centrifugaBrodoa atrofusca

Pseudevernia furfuracea

Parmelioid lichens

Parmelina-clade

Hyp

otra

chyn

a-cl

ade

Melanohalea-clade

Parmelia-clade

Melanelixia-clade

Parmotrema-clade

Xanthoparmelia-clade


having the highest level of homoplasy, provides the high-est level of highly supported internodes (58 with MP, 75with B/MCMC). In this regard, the contribution of mtSSU is about half of the ITS (28 with MP and 42 with B/MCMC).

Monophyly of a core group of parmelioid lichens isstrongly supported (Fig. 2), but parmelioid lichens as cir-cumscribed by DePriest (1999) are polyphyletic. Arctop-armelia and Melanelia s. str. are outside this well-supportedclade, and two genera not accepted as parmelioid inDePriest (1999), Parmeliopsis and Parmelaria, fall within it.In all analyses, the backbone of the phylogeny of parmeli-oid lichens lacks support. However, several well-supported

Table 5Frequencies of gains and losses of a pored epicortex in the phylogeny ofparmelioid lichens from stochastic mapping simulations

Changes are shown up to a cumulative frequency of 0.95.

Gains !losses

0 1 2 3 4 5 6 7 8 9 10

0 0.02 0.05 0.06 0.011 0.01 0.012 0.01 0.03 0.033 0.02 0.024 0.01 0.04 0.01 0.015 0.07 0.03 0.016 0.39 0.027 0.098 0.01

Table 6Frequencies of gains and losses of pseudocyphellae in the phylogeny ofparmelioid lichens from stochastic mapping simulations


Gains !losses

0 1 2 3 4 5 6 7

0 0.07 0.46 0.031 0.03 0.13 0.012 0.04 0.013 0.044 0.01 0.035 0.01 0.026 0.037 0.038 0.01

clades are resolved (Fig. 2), and seven of these are discussedbelow. The majority of these well-supported clades arecharacterized by a unique combination of some diagnosticcharacters (Table 4).

3.1.1. Xanthoparmelia-cladeThis clade corresponds to the genera Xanthoparmelia

and Karoowia in the circumscription accepted by Blancoet al. (2004a). The genera include species that have cell wall

Table 7Frequencies of gains and losses of usnic acid in the phylogeny of parmeli-oid lichens from stochastic mapping simulations


Gains !losses

0 1 2 3 4 5 6 7 8 9 10 11

0123 0.10 0.024 0.01 0.58 0.025 0.004 0.01 0.03 0.0046 0.04 0.07 0.017 0.01 0.018 0.019 0.01

10111213 0.0114 0.0115 0.004

Table 8Frequencies of gains and losses of atranorin in the phylogeny of parmeli-oid lichens from stochastic mapping simulations


Gains !losses

0 1 2 3 4 5 6 7 8 9 10 11

01 0.27 0.05 0.012 0.24 0.05 0.013 0.07 0.05 0.014 0.05 0.05 0.015 0.01 0.03 0.016 0.02 0.017 0.01

Table 4Morphological, chemical and geographical characters of main clades of parmelioid lichens

Cell wall polysaccharide Pores/fenestrations Pseudocyphellae Centre of distribution Ecology

Hypotrachyna-clade Isolichenan Present Absent Subcosmopolitan TropicalMelanelixia-clade Not determined Present Absent Northern Hemisphere TemperateMelanohalea-clade Not determined Absent On verrucae or isidia

tips, circular to slightly elliptic

Northern Hemisphere Temperate

Parmelia-clade Isolichenan Absent EYgurate, linear Both Hemispheres Temperate to borealParmelina-clade Isolichenan Present Absent Both Hemispheres TemperateParmotrema-clade Unknown type Present/Absent Punctiform or absent Southern Hemisphere Tropical, subtropical

to temperateXanthoparmelia-clade Xanthoparmelia-type

lichenanPresent Absent Southern Hemisphere Subtropical, semi-arid


polysaccharides with Xanthoparmelia-type lichenan. Mostspecies occur in the Southern Hemisphere in arid or semi-arid subtropical areas, and to some extend into temperateregions. The species in this clade lack pseudocyphellae,have a pored epicortex, and show a considerable variationin cortical chemistry. Species lacking phenolic corticalmetabolites are placed here, as well as species containingusnic acid or atranorin.

3.1.2. Parmotrema-cladeThis clade includes the large genus Parmotrema and

some smaller genera, including Flavoparmelia, Flavopunct-elia, Punctelia, Parmelaria, and Canoparmelia crozalsiana.Parmotrema in the classiWcation of Blanco et al. (2005)includes species that have a cell wall polysaccharide ofunknown type. Its centre of distribution is in the SouthernHemisphere. It is especially diverse in tropical and

Fig. 3. The four most probable distinct mappings of pored epicortex history on the phylogeny of parmelioid lichens. Thick branches represent the presenceof a pored epicortex. Each mapping is shown on a majority-rule consensus of the trees on which individual histories were simulated: i.e., the set of treesover which simulations of character history yielded an identical set of transformations. Identical sets were determined on the basis of the taxon biparti-tions deWning the branches on which gains and losses occurred, and their probabilities are derived from frequencies of occurrence in the stochastic map-ping analysis (see text).


subtropical areas, with some species extending into tem-perate regions. In this clade some species have a poredepicortex, or pseudocyphellae, and may contain atranorinor usnic acid as cortical pigment.

3.1.3. Melanelixia-cladeThis recently described genus (Blanco et al., 2004b) has a

cell wall polysaccharide that has not yet been determined.This genus is distributed in the Northern Hemisphere,mainly in temperate regions. It is characterized by having apored or fenestrate epicortex, lacking pseudocyphellae.Neither atranorin nor usnic acid are present as corticalcompounds, but species in this group do contain melanoidsubstances that are responsible for their brown colour.

3.1.4. Parmelia-cladeThis clade represents the monophyletic Parmelia s. str.

with isolichenan as a cell wall polysaccharide. It has aworldwide distribution. The species have linear to eYguratepseudocyphellae, lacking pores, and contain atranorin as acortical substance.

3.1.5. Melanohalea-cladeThis clade corresponds to the recently described genus

Melanohalea (Blanco et al., 2004b). Its cell wall polysaccha-ride is not determined. It occurs in the Northern Hemi-sphere mainly in temperate regions, and includes specieslacking a pored epicortex, but having pseudocyphellae. Thisgenus, as Melanelixia, has a brown coloration that is due tomelanoid compounds and its species also lack atranorinand usnic acid.

3.1.6. Hypotrachyna-cladeThis clade includes species of the genera Bulbothrix,

Everniastrum, Hypotrachyna, Parmelinella, and Parmelin-opsis. The genus Hypotrachyna is not monophyletic in ouranalysis. All these genera have isolichenan as a polysaccha-ride cell wall. They are currently poorly known and havetheir centre of speciation in the tropical and subtropicalregions of both hemispheres. All species in this clade have apored epicortex and lack pseudocyphellae. They may con-tain atranorin, usnic acid or lichenoxanthone as corticalsubstances.

3.1.7. Parmelina-cladeThe Parmelina clade includes species of Myelochroa, a

group widely distributed in the Northern Hemisphere,and Parmelina, which is cosmopolitan, but occurs mainlyin the Southern Hemisphere. The species in this clade haveisolichenan as polysaccharide cell walls, a pored epicortex,lack pseudocyphellae, contain atranorin and lack usnicacid as cortical compound.


Tables 5–8 summarize the posterior probabilitiesof gains and losses in each of the examined characterover the set of trees sampled by MCMC. Figs. 3–6 showthe four most probable distinct mappings of each char-acter, where a distinct mapping refers to a unique set oftaxon bipartitions corresponding to the clades sub-tended by changes in character state (see Section 2). Allcharacters exhibit considerable variability in the

Fig. 4. The two most probable distinct mappings of pseudocyphellae history on the phylogeny of parmelioid lichens. Thick branches represent the presenceof pseudocyphellae. Each mapping is shown on a majority-rule consensus tree as in Fig. 3.


distribution of gains and losses, indicating the phyloge-netic uncertainty associated with the inference ofcharacter evolution.

Fig. 5. The most probable distinct mapping of usnic acid history on thephylogeny of parmelioid lichens. Thick branches represent the presence ofusnic acid. The mapping is shown on a majority-rule consensus tree as inFig. 3.

3.2.1. Pored epicortexThe character mapping suggests that a pored epicortex

has been gained 0–10 times and lost 0–8 times over thetree (Table 5, Fig. 3). The three most probable gain–losspairs account for about 55% of the posterior probabilitydensity and suggest no gains and 5–7 losses of the poredepicortex. The proportion of stochastic mappings includ-ing more losses than gains is 0.68.

3.2.2. PseudocyphellaeThis trait has evolved 1–7 times and been lost 0–8 times

(Table 6, Fig. 4). Fifty-nine percent of the posterior prob-ability density is represented by two combinations ofgains and losses suggesting 0–1 losses and 5–6 gains of thepseudocyphellae. The proportion of stochastic mappingsincluding more gains than losses is 0.78.

3.2.3. Usnic acidFor this character the character mapping analysis sug-

gests 1 and 5–11 gains and 3–9 and 13–15 losses (Table 7,Fig. 5). The most probable numbers of gains and losses (9and 4, respectively) accounts for 58% of the posteriorprobability density. The proportion of stochastic map-pings including more gains than losses is 0.66.

3.2.4. AtranorinFive to 11 gains and 1–7 losses for atranorin are

inferred from character mapping (Table 8, Fig. 6). Fifty-one percent of the posterior probability density is repre-sented by the two most probable gain–loss pairs that sug-gest 8–9 gains and 1–2 losses. The proportion of

Fig. 6. The two most probable distinct mappings of atranorin history on the phylogeny of parmelioid lichens. Thick branches represent the presence ofatranorin. Each mapping is shown on a majority-rule consensus tree as in Fig. 3.


stochastic mappings including more gains than losses is0.91.

4. Discussion

Although this is a phylogenetic study that samplesbroadly both taxonomically and morphologically parme-lioid lichens, and includes most currently accepted genera,the species studied represent less than 10% of the totalspecies diversity in the lineage. Further, some geographicregions that are centres of distribution of parmelioidlichens, such as southern Africa and south-eastern Asiaremain poorly sampled. Therefore, the phylogeneticrelationships presented here should be regarded aspreliminary.

The nuclear and mitochondrial gene partitions sup-ported the same overall topology and no substantial inter-genomic conXict was found for the major clades. Thecombined three-region data set provided the most robustsupport of the parmelioid lichen phylogeny overall, indicat-ing that multi-locus data sets will be necessary to furtherresolve the phylogenetic relationships in this lineage offungi.

Characteristic diVerences in bootstrap and Bayesiansupport values are well documented (e.g., Alfaro et al.,2003; Douady et al., 2003; Suzuki et al., 2002; Wilcoxet al., 2002). Our results conWrm the common pattern thatthe Bayesian support is always higher than bootstrap val-ues. Both metrics appear to converge to some extent inthe combined analyses. The increase in stronglysupported nodes in the combined analyses gives hopethat with the addition of further gene partitions, thebackbone of the parmelioid lichens phylogeny can beresolved.

4.1. Major clades and intergeneric relationships within parmelioid lichens

Although the backbone is not resolved, we can never-theless draw some conclusions about the phylogeny ofparmelioid lichens. The monophyly of a core group ofparmelioid lichens is strongly supported. This groupincludes Parmelaria, a cetrariod genus (Randlane et al.,1997), and Parmeliopsis, a genus not included in the clas-siWcation of parmelioid lichens by DePriest (1999). Othergenera, formely considered as parmelioid, fell outside thismonophyletic group, e.g., Arctoparmelia and Melanelias. str.

Parmelaria, a genus of two species with laminal tosubmarginal apothecia, is very close to Parmotremain thallus morphology (Culberson, 1962). Our molecularanalyses support this relationship (Blanco et al., 2005)with Parmotrema. Parmeliopsis has not beenconsidered as parmelioid (DePriest, 1999), although itWts well morphologically, since it has a special type ofconidiophores (Glück, 1899), which do not occur in otherParmeliaceae. In agreement with previous molecular

studies (Crespo et al., 2001; Mattsson et al., 2004) ourdata suggest that Parmeliopsis belongs to parmelioidlichens.

Arctoparmelia, a genus morphologically similar toXanthoparmelia (Elix, 1993), falls outside of the core ofparmelioid group. However, Arctoparmelia diVerschemically from other parmelioid lichens in havingCetraria-type lichenan as cell wall polysaccharide(Elix, 1993). Our data conWrm previous molecular analy-ses in which no relationship between Arctoparmeliaand Xanthoparmelia or other investigated parmelioidgenera has been found (Blanco et al., 2004a; Thell et al.,2004).

Melanelia s. str. is also outside of the core of parmelioidlichens. Our results corroborate previous studies (Blancoet al., 2004b; Mattsson et al., 2004; Thell et al., 2004) whereMelanelia stygia is distantly related to Melanelixia andMelanohalea, and more closely related with cetrarioidlichens. Thell (1995) transferred the Cetraria hepatizongroup to Melanelia based on morphological and chemicalcharacters demonstrating relationships with cetrarioidlichens. However, Melanelia is still polyphyletic (Blancoet al., 2004b; Mattsson et al., 2004), since M. stygia and M.disjuncta do not cluster together. M. disjuncta has beenshown as closely related to Pleurosticta acetabulum (Matts-son et al., 2004). Our results do not show this relationship.Additional studies are necessary to clarify the status of M.disjuncta.

Within the monophyletic core group of parmelioid group,seven well-supported clades are recovered by the moleculardata, and these are also deWned by a combination of mor-phological, chemical, and geographical characters (Table 3).These clades correspond to genera or generic groups andsupport recent generic rearrangements, such as the enlarge-ment of Parmotrema (Blanco et al., 2005) and Xanthoparm-elia (Blanco et al., 2004a), and the segregation ofMelanelixia and Melanohalea from Melanelia (Blanco et al.,2004b). Two of these clades, Parmotrema and Xanthoparm-elia, were also shown by Thell et al. (2004). The resultsfurther suggest that additional studies are necessary to clarifythe monophyly of other currently accepted genera, such asKaroowia, Canoparmelia, and Parmelaria.

Generic concepts in the Hypotrachyna-clade clearly needrevision, since Hypotrachyna itself is polyphyletic. Largertaxon sampling is needed in some groups, such as the Parm-elia- and Parmelina-clades, and more taxa and characterswill be required to discover the relationships of other gen-era, such as Parmeliopsis, Pleurosticta, and Relicina.


Morphological characters, such as the type of corticalperforations and cortical chemistry, have generally beenregarded as key characters for classiWcation in parmelioidlichens. The presence of diVerent types of cortical perfora-tions was even believed to be important at subfamilial rank(Elix, 1993; Hale, 1981; Henssen, 1992). However, neither


those species having pseudocyphellae nor those having apored epicortex form a monophyletic group, and corticalchemistry does not appear to be important at the supragen-eric level, showing variability within some genera (e.g.,Xanthoparmelia, Hypotrachyna). It thus appears that thetaxonomic value of these characters has been overestimatedin classiWcations of parmelioid lichens.

Phylogenetic inferences of character evolution suggestthat the pored epicortex is a plesiomorphic state within par-melioid lichens and has been lost several times independentlywithin the group. However, some character mappings involv-ing numerous gains from a poreless ancestor are also present,albeit at low probability, in the 0.95% conWdence intervalestimated by stochastic mapping.

Pseudocyphellae appear to have been gained moreoften than lost in the phylogeny of parmelioid lichens,evolving independently in the Punctelia-, Flavopunctelia-,Parmelia-, and Melanohalea-clades and also in Melaneliadisjuncta.

The presence of usnic acid shows less phylogenetic conser-vation than the two morphological characters examined.This compound appears to have been lost and gained severaltimes, supporting recent views (Blanco et al., 2004a; Elix,2003) that the taxonomic value of this character has beenoverestimated in previous classiWcations.

Similarly, atranorin also exhibits considerable homoplasy,with a clear trend toward more gains than losses. The distri-bution of this character suggests that atranorin is of limitedtaxonomic value at generic or suprageneric level in parmeli-oid lichens. We currently have no knowledge about the pro-cesses that regulate the presence or absence of secondarymetabolites in lichen-forming fungi, but further investigationof genes involved in the production of secondary metabolites,such as polyketide synthases (Grube and Blaha, 2003; Sch-mitt et al., 2005) are likely to improve our understanding ofthe evolution of such characters.

Acknowledgments

This project has been supported by the Spanish Minis-try of Science and Technology (CGL2004-1848/BOS)from the Ministry of Educación and Science to A.C. and astart up fund of the Field Museum to H.T.L. Sequencingwas carried out at the Unidad de Genómica (ParqueCientíWco de Madrid) by M. Isabel García and SEM facil-ities were provided by the CAI de Microscopía Electró-nica Luis Bru of the UCM by Eugenio Baldonedo. We areindebted to various colleagues for sending fresh materialof several species, oVering some sequences and also fortheir contributions and critical comments, notably to J.A.Elix, P.K. Divakar, and M.C. Molina.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at doi:10.1016/j.ympev.2005.12.015.

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Documents

Major clades of parmelioid lichens (Parmeliaceae, Ascomycota) and the evolution of their morphological and chemical diversity