The Lichenologist 49(1) 13ndash25 (2017) copy British Lichen Society 2017doi101017S0024282916000633

Two new species in the Micarea prasina group from Western Europe

Pieter P G VAN DEN BOOM A Maarten BRAND Brian J COPPINSand Emmanueumll SEacuteRUSIAUX

Abstract Detailed morphological and anatomical studies informed by molecular inferences withmtSSU as a marker revealed two new species ofMicarea inWestern Europe both belonging to the coregroup of the genus namely the M prasina group M herbarum from the Netherlands and Poland andMmeridionalis fromPortugal and ItalyMicarea herbarum looks like a small or depauperateM denigratabut clearly differs by the lack of gyrophoric acid while M meridionalis is distinguished by its granularthallus and the production of micareic acid

Key words lichen diversity lichenized Ascomycota molecular phylogeny taxonomy

Accepted for publication 8 August 2016

Introduction

Recently the study of the genus Micarea hasagain attracted the attention of lichenologistsThis is because the production of molecularsequences and phylogenetic reconstructions hasrevealed greater diversity in Micarea includingin Europe and Macaronesia (Czarnota ampGuzow-Krzemińska 2010 van den Boom ampErtz 2014 Guzow-Krzemińska et al 2016) andbecause a significant number of new speciescontinue to be described from all parts of theworld (van den Boom 2010 Seacuterusiaux et al2010 Svensson amp Thor 2011 Cacircceres et al2013 Aptroot amp Cacircceres 2014 Brand et al2014 Coacuterdova-Chaacutevez et al 2014 McCarthyamp Elix 2016) In Europe the genus wasinvestigated in a major revision (Coppins 1983)and was further studied at a smaller scale byCoppins (2009) for Great Britain and Irelandand Czarnota (2007) for Poland

The M prasina group is known to be veryvariable and was suspected of containing

additional species (Coppins 1983) Severalstudies dealing with collections within Europe(Czarnota amp Guzow-Krzemińska 2010Guzow-Krzemińska et al 2016) and outsideEurope (Barton ampLendemer 2014 Brand et al2014) have shown that it represents a complexassemblage of species that is yet to be properlydisentangled Based on morphological anato-mical chemical and molecular evidence ourstudy of the material belonging to that groupavailable to us from Europe yielded evidence oftwo undescribed species They are formallydescribed in this paper

Material and Methods

The material examined was collected by the authors inBelgium France Germany the Netherlands PortugalRomania andSpain (Canary IslandsTenerife) Anatomicalmeasurements were made on material mounted in diluteKOH for conidia and paraphysis width and in water for allother characters In each collection c 10 well-developedascospores representing the size and shape variationdetected conidia and paraphyses were measured with aprecision of 0middot1microm using camera lucida drawings Forthallus description we refer to Coppins (1983) for theuse of areolate- and goniocyst-type the latter being ldquoafinely granular thallus composed of discrete plusmn globularstructures mostly c 12ndash40microm diam these ecorticategranules consist of clustered algal cells intertwined andsurrounded by short-celled hyphae and never protected byan amorphous covering layerrdquo

Chemical compounds were studied using severalmethods response to UV light melting point determina-tion microcrystallization and thin-layer chromatography

P P G van den Boom Arafura 16 NL-5691 JA SonThe NetherlandsA M Brand Klipperwerf 5 NL-2317 DX LeidenThe NetherlandsB J Coppins Royal Botanic Garden Edinburgh 20AInverleith Row Edinburgh EH3 5LR UKE Seacuterusiaux (corresponding author) Evolution andConservation Biology Unit University of LiegravegeSart Tilman B22 B-4000 Liegravege Belgium EmailESerusiauxulgacbe

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(TLC) using solvent system A (toluene14-dioxaneaceticacid 180455) for all collections and C (tolueneacetic acid17030) when gyrophoric acid was suspected (Huneck ampYoshimura 1996 Orange et al 2010) in both cases visua-lization of spots was achieved with sulphuric acid sprayedover the plates followed by heating at 110 degC for c 5min

Well-preserved specimens lacking any visible symptomsof fungal infection were selected for DNA isolationExtraction of DNA and PCR amplification wereperformed following the protocol of Cubero et al (1999)We used the primers mrSSU1 and mrSSU3R (Zoller et al1999) for the production of mtSSU sequences Ampliconswere sequenced by Macrogenreg Sequence fragments wereassembled with Sequencher version 523 (Gene CodesCorporation Ann Arbor Michigan) Sequences were thensubjected to MEGABLAST searches to detect potentialcontamination They were included in a single matrixtogether with data extracted from GenBank published inAndersen amp Ekman (2005) Czarnota amp Guzow-Krzemińska (2010) Guzow-Krzemińska et al (2016) andvan den Boom amp Ertz (2014) We chose Byssolomaleucoblepharum and B subdiscordans as outgroup followingthe topology obtained for the Pilocarpaceae byMiadlikowska et al (2014) Accession data are included inTable 1 Sequences were aligned manually usingMacClade version 408 (MaddisonampMaddison 2005) andalignment was checked with the MAFTT software (Katohamp Standley 2013) Ambiguous regions were delimitedusing the online version of Gblocks v091b (Castresana2000) at httpmolevolcmimacsicescastresanaGblockshtml allowing for gap positions within the final blocks andcarefully checked manually The aligned matrix is availablefrom the corresponding author on request We inferredthe maximum likelihood tree and bootstrap supportvalues based on 1000 pseudoreplicates in the same runusing RAxML HPC2 version 728 (Stamatakis 2006Stamatakis et al 2008) with the GTRCAT model and thedefault settings as implemented on the CIPRES portal(Miller et al 2010) Phylogenetic trees were visualizedusing FigTree v123 (Rambaut 2009) Branch supportvalues were considered significant when ML bootstrap(BS)gt70 We included in our accessions for molecularinferences other specimens besides those belonging tothe two species described here as new for the followingreasons 1) we wished to assess the only phylogenetic treeavailable for the genus with further data (Andersen ampEkman 2005) 2) we suspect thatMicarea is more complexin other groups than the prasina group and we wished totest this hypothesis

Results

The data matrix includes 21 mtSSU sequencesnewly produced for the following speciesM adnata M byssacea M denigrataM doliiformis M herbarum M lignaria varlignaria M meridionalis M nowakiiM micrococca M prasina M pycnidiophoraM stipitata and M viridileprosa The single

most-likely tree (Fig 1) resolves two stronglysupported clades within Micarea Both speciesnewly described in this paper are resolvedwithin a single clade and more preciselywithin a strongly supported (BS = 98)subclade including all accessions of theM prasina group (Coppins 1983 2009Czarnota 2007 Czarnota amp Guzow-Krzemińska 2010 Guzow-Krzemińska et al2016) Within that subclade the resolution ispoor except for a strongly supported groupcomprising M byssacea M hedlundiiM micrococca A and B (sensu Czarnota ampGuzow-Krzemińska 2010) M viridileprosa andM xanthonicaOur new accessions of M nowakii from

Romania are identical to two accessions ofthe same species from Poland (Czarnota ampGuzow-Krzemińska 2010) In two furtheraccessions of the same species also fromPoland one is resolved as nearly identical toM herbarum sp nov and the other as sisterto accessions of the same speciesAll accessions of M prasina from Poland

(Czarnota amp Guzow-Krzemińska 2010) areresolved as a lineage (as lsquoprasina 1rsquo in Fig 1)separate to those from Belgium Franceand the USA (as lsquoprasina 2rsquo in Fig 2) thus twocryptic species might be involved and requirefurther studyWithin theM prasina group oursequence of M byssacea is nearly identical tothose referred to that species by Czarnota ampGuzow-Krzemińska (2010) our sequence ofM viridileprosa is identical to those of Czarnotaamp Guzow-Krzemińska (2010) and finallyour sequences of M micrococca belong toM micrococca ldquoArdquo of Czarnota amp Krzemińska(2010) While our sequences of M adnataM denigrata M doliiformis M lignaria varlignaria M nitschkeana and M pycnidiophoraare almost identical to those retrievedfrom GenBank this is not the case forM peliocarpa and M stipitata Indeed ouraccession of M peliocarpa from the Nether-lands is resolved with strong support asclosely related to the recently describedM usneae from Madeira (van den Boom ampErtz 2014) while the sequence fromNorway (Andersen amp Ekman 2005) isquite different (26 substitutions for themtSSU sequence) The same applies to our

14 THE LICHENOLOGIST Vol 49

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TABLE 1 Specimens and GenBank Accession numbers used in this study with their respective voucher information New sequences in bold

Name CountryCollector year collection number herbariumand DNA sample number where appropriate Publication

GenBankAcc no

Byssoloma leucoblepharum Portugal S Ekman - 3502 BG Andersen amp Ekman 2005 AY567778B subdiscordans USA T Toslashnsberg - 25968 BG Andersen amp Ekman 2005 AY567779Micarea adnata Norway H L Andersen - 48 BG Andersen amp Ekman 2005 AY567751M adnata France E Seacuterusiaux 2014 sn LG DNA 3438 KX459344M alabastrites Norway H L Andersen - 17 BG Andersen amp Ekman 2005 AY567764M assimilata Sweden Kanz amp C Printzen - sn BG Andersen amp Ekman 2005 AY567739M botryoides Norway H L Andersen - 79b BG Andersen amp Ekman 2005 AY567741M byssacea Norway H L Andersen - 34 BG (as M micrococca) Andersen amp Ekman 2005 AY567749M byssacea Estonia P Czarnota amp B Guzow-Krzemińska - GPN 4781 Czarnota amp Guzow-Krzemińska

2010EF453670

M byssacea Estonia P Czarnota amp B Guzow-Krzemińska - GPN 3956 Czarnota amp Guzow-Krzemińska2010

EF453690

M byssacea Poland P Czarnota amp B Guzow-Krzemińska - GPN 4751 Czarnota amp Guzow-Krzemińska2010

EF453664

M byssacea Germany P van den Boom 2014 50037 hb van den BoomLG DNA 3495

KX459345

M cinerea Norway T Toslashnsberg - 28572 BG Andersen amp Ekman 2005 AY567763M clavopycnidiata USA T Toslashnsberg - 27215 BG Andersen amp Ekman 2005 AY567747M coppinsii Norway T Toslashnsberg - 26075 BG Andersen amp Ekman 2005 AY567761M deminuta Not specified Z Palice amp Voriskova - 6745 hb Palice AY756446M denigrata Poland P Czarnota amp B Guzow-Krzemińska - GPN 4593 Czarnota amp Guzow-Krzemińska

2010EF453681

M denigrata Netherlands A M Brand 2014 63258 hb Brand LG DNA 3851 KX459346M denigrata Germany E Seacuterusiaux 2015 sn LG DNA 4381 KX459347M doliiformis United Kingdom A Orange 2006 sn A Orange LG NMW Seacuterusiaux et al 2010 GU138666M doliiformis Canary Isl Tenerife P van den Boom 2014 52014 hb van den Boom

LG DNA 4239KX459348

M elachista Sweden Koffman - 399 hb Koffman Andersen amp Ekman 2005 AY567755M elachista Poland P Czarnota amp B Guzow-Krzemińska - GPN 2986 Czarnota amp Guzow-Krzemińska

2010EF453680

M erratica Sweden U Arup - 99192 hb Arup Andersen amp Ekman 2005 AY567737M eximia Not specified Hermansson- 8866b UPS AY756447M flagellispora Australia Tasmania G Kantvilas- 6090 UPS AY756448M hedlundii Poland P Czarnota amp B Guzow-Krzemińska - GPN 3895 Czarnota amp Guzow-Krzemińska

2010EF453672

M hedlundii Poland P Czarnota amp B Guzow-Krzemińska - GPN 3915 Czarnota amp Guzow-Krzemińska2010

EF453667

2017New

Micarea

inWestern

Europe

mdashvan

denBoom

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TABLE 1 (continued )

Name CountryCollector year collection number herbariumand DNA sample number where appropriate Publication

GenBankAcc no

Micarea hedlundii Poland P Czarnota amp B Guzow-Krzemińska - GPN 4589 Czarnota amp Guzow-Krzemińska2010

EF453677

M herbarum sp nov Type Netherlands P amp G van den Boom 2015 52575 hb van den BoomLG DNA 4236

KX459349

M herbarum sp nov Netherlands A M Brand 2014 63193 hb Brand LG DNA 3852 KX459350M herbarum sp nov Poland P Czarnota amp B Guzow-Krzemińska - GPN 4634

(as M nowakii)Czarnota amp Guzow-Krzemińska2010

EF453692

M cf herbarum Poland P Czarnota amp B Guzow-Krzemińska - GPN 3464(as M nowakii)

Czarnota amp Guzow-Krzemińska2010

EF453665

M incrassata Not specified T Toslashnsberg - 17593 (BG) Andersen amp Ekman 2005 AY756449M lapillicola Czech Republic C Printzen - sn BG Andersen amp Ekman 2005 AY567735M leprosula Norway H L Andersen - 35 BG Andersen amp Ekman 2005 AY567762M lignaria var lignaria Norway H L Andersen - 18 BG Andersen amp Ekman 2005 AY567748M lignaria var lignaria France E Seacuterusiaux 2014 sn LG DNA 3435 KX459351M lignaria var lignaria Romania E Seacuterusiaux 2015 sn LG DNA 4375 KX459352M lithinella Norway H L Andersen - 80b BG Andersen amp Ekman 2005 AY567734M lynceola Czech Republic Z Palice 1996 - hb Palice Andersen amp Ekman 2005 AY567738M meridionalis sp nov Type Portugal P van den Boom 2015 - hb van den Boom

LG DNA 4279KX459353

M meridionalis sp nov Portugal P van den Boom 2015 - hb van den BoomLG DNA 4281

KX459354

M meridionalis sp nov Portugal P van den Boom 2015 - hb van den BoomLG DNA 4581

KX459355

M marginata Not specified S Bayerovaacute J Liska amp Z Palice- 5159 hb Palice AY756451M melaena Norway H L Andersen - 25 BG Andersen amp Ekman 2005 AY567743M micrococca Poland P Czarnota amp B Guzow-Krzemińska - GPN 3179 Czarnota amp Guzow-Krzemińska

2010EF453674

M micrococca Estonia P Czarnota amp B Guzow-Krzemińska - GPN 4782 Czarnota amp Guzow-Krzemińska2010

EF453676

M micrococca Poland P Czarnota amp B Guzow-Krzemińska - GPN 4179 Czarnota amp Guzow-Krzemińska2010

EF453691

M micrococca Poland P Czarnota amp B Guzow-Krzemińska - GPN 3632 Czarnota amp Guzow-Krzemińska2010

EF453668

M micrococca Poland P Czarnota amp B Guzow-Krzemińska - GPN 4553 Czarnota amp Guzow-Krzemińska2010

EF453683

M micrococca Poland P Czarnota amp B Guzow-Krzemińska - GPN 4059 Czarnota amp Guzow-Krzemińska2010

EF453663

M micrococca Poland P Czarnota amp B Guzow-Krzemińska - GPN 4456 Czarnota amp Guzow-Krzemińska2010

EF453662

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TABLE 1 (continued )

Name CountryCollector year collection number herbariumand DNA sample number where appropriate Publication

GenBankAcc no

Micarea micrococca Netherlands P amp B van den Boom 2014 50314 hb van den BoomLG DNA 3853

KX459356

M micrococca Netherlands P amp B van den Boom 2014 51244 hb van den BoomLG DNA 3855

KX459357

M micrococca Netherlands P amp B van den Boom 2015 52570 hb van den BoomLG DNA 4237

KX459358

M misella Norway H L Andersen - 73 BG Andersen amp Ekman 2005 AY567752M misella Poland P Czarnota amp B Guzow-Krzemińska - GPN 4593 Czarnota amp Guzow-Krzemińska

2010EF453687

M myriocarpa Norway H L Andersen - 37 BG Andersen amp Ekman 2005 AY567736M nitschkeana Czech Republic C Printzen - sn BG Andersen amp Ekman 2005 AY567758M nitschkeana Poland P Czarnota amp B Guzow-Krzemińska - GPN 3306 Czarnota amp Guzow-Krzemińska

2010EF453685

M nowakii Poland P Czarnota amp B Guzow-Krzemińska - GPN 4181 Czarnota amp Guzow-Krzemińska2010

EF453688

M nowakii Poland P Czarnota amp B Guzow-Krzemińska - GPN 4688 Czarnota amp Guzow-Krzemińska2010

EF453689

M nowakii Romania E Seacuterusiaux 2015 sn LG DNA 4380 KX459359M nowakii Romania E Seacuterusiaux 2015 sn LG DNA 4385 KX459360M paratropa Norway H L Andersen - 94 BG Andersen amp Ekman 2005 AY567740M peliocarpa Norway H L Andersen - 29 BG Andersen amp Ekman 2005 AY567760M peliocarpa Netherlands P van den Boom 2014 51318 hb van den Boom

LG DNA 3847KX459361

M prasina USA T Toslashnsberg - 30856 BG Czarnota amp Guzow-Krzemińska2010

AY756452

M prasina France E Seacuterusiaux 2014 sn LG DNA 3437 KX459362M prasina Belgium E Seacuterusiaux 2014 sn LG DNA 3609 KX459363M prasina Poland P Czarnota amp B Guzow-Krzemińska - GPN 3913 Czarnota amp Guzow-Krzemińska

2010EF453675

M prasina Poland P Czarnota amp B Guzow-Krzemińska - GPN 3914 Czarnota amp Guzow-Krzemińska2010

EF453669

M prasina Poland P Czarnota amp B Guzow-Krzemińska - GPN 4319 Czarnota amp Guzow-Krzemińska2010

EF453679

M prasina Poland P Czarnota amp B Guzow-Krzemińska - GPN 4489 Czarnota amp Guzow-Krzemińska2010

EF453678

M prasinella USA B McCune - 35337 BG Andersen amp Ekman 2005 AY567745M pycnidiophora USA T Toslashnsberg - 30881 BG Andersen amp Ekman 2005 AY567754M pycnidiophora Belgium E Seacuterusiaux 2014 sn LG DNA 3498 KX459364

2017New

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mdashvan

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TABLE 1 (continued )

Name CountryCollector year collection number herbariumand DNA sample number where appropriate Publication

GenBankAcc no

Micarea soralifera Poland M Kukwa 2014 12722 UGDA Guzow-Krzemińska et al 2016 KT119884M soralifera Poland M Kukwa 2014 12999 UGDA Guzow-Krzemińska et al 2016 KT119885M soralifera Type Poland M Kukwa 13001 amp A Łubek 2014 UGDA Guzow-Krzemińska et al 2016 KT119886M stipitata USA S Ekman - sn BG Andersen amp Ekman 2005 AY567756M stipitata Canary Is Tenerife E Seacuterusiaux 2014 sn LG DNA 3816 KX459365M subviridescens UK Scotland P Czarnota amp B Guzow-Krzemińska - GPN 3599 Czarnota amp Guzow-Krzemińska

2010EF453666

M synotheoides Norway H L Andersen - 47 BG Andersen amp Ekman 2005 AY567756M tomentosa Poland P Czarnota amp B Guzow-Krzemińska - GPN 3949 Czarnota amp Guzow-Krzemińska

2010EF453686

M turfosa Norway H L Andersen - 59 BG Andersen amp Ekman 2005 AY567742M usneae Portugal Madeira Pamp B van den Boom 2012 48057 BR van den Boom amp Ertz 2014 KF569511M viridileprosa Netherlands P amp B van den Boom 2013 50066 hb van den Boom

LG DNA 3493KX459366

M viridileprosa Poland P Czarnota amp B Guzow-Krzemińska - GPN 3436 Czarnota amp Guzow-Krzemińska2010

EF453671

M viridileprosa Poland P Czarnota amp B Guzow-Krzemińska - GPN 3869 Czarnota amp Guzow-Krzemińska2010

EF453673

M viridileprosa Poland P Czarnota amp B Guzow-Krzemińska - GPN 4518 Czarnota amp Guzow-Krzemińska2010

EF453684

M viridileprosa Poland P Czarnota amp B Guzow-Krzemińska - GPN 4527 Czarnota amp Guzow-Krzemińska2010

EF453682

M xanthonica USA T Tonsberg - 25674 BG Czarnota amp Guzow-Krzemińska2010

AY756454

Szczawinskia leucopoda USA T Toslashnsberg - 30044 BG Andersen amp Ekman 2005 AY567746

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100

95prasina 1

prasina 2

nowakii

herbarum

99

96

100

96

97

95100

100

100

100100

100

10090

100

98

8889

100

82

100 99

99

100

100

100

100

91

100

86

100

100

100

herbarumsoralifera

meridionalis

Micarea prasina group

subviridescens

xanthonica

hedlundiibyssacea

micrococca A

viridileprosa

micrococca B

tomentosa

adnatastipitata

stipitataelaschista

elaschistasynotheoides

pycnidiophoramisella

eximiabotryoides

turfosa

lynceola

flagellisporaclavopycnidiata

prasinellaSzczawinskia leucopodalithinella

lapillicolaparatropa

assimilata

doliiformisincrassata

marginatamyriocarpa

peliocarpapeliocarpa

usneaecoppinsiileprosula

alabastritescinerea

erratica deminuta

denigrata

nitschkeanamelaena

lignaria

Byssoloma subdiscordansByssoloma leucoblepharum

FIG 1 Most-likely phylogenetic tree for species of Micarea obtained from mtSSU sequences Branches in bold are those that obtained ML bootstrap supportgt70 and are indicated above branches New species are in bold

2017New

Micarea

inWestern

Europe

mdashvan

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sequences for M stipitata from Tenerife(Canary Islands) which differ by 22 substitu-tions from the sequence from the USAretrieved from GenBank (Andersen amp Ekman2005) Thus our limited sampling illuminatesthe rather poorly known diversity withinMicareaMorphological anatomical and chemical

results are included in the description of bothnew species

Taxonomy

Micarea herbarum Brand CoppinsSeacuterus amp van den Boom sp nov

MycoBank No MB 811051

Species inconspicuous with a very thin thalluscomprised of small greenish flattened or slightly convexareoles and often covered by a thin film of gelatinousgreen algal cells Apothecia abundant dark brown toblack 0middot15ndash0middot25mm diam immarginate Ascosporesellipsoid 6middot5ndash9middot7times2middot0ndash2middot6 microm (0ndash)1-septate PigmentSedifolia-grey K+ violet Mesopycnidia often abundantmesoconidia shortly bacilliform 3middot8ndash6middot1times1middot0ndash1middot2(ndash1middot3) micromNo secondary compounds detected

Type the Netherlands Noord-Brabant S of OirschotS rim of Oirschotse Heide Pinus-Quercus forest with manyfallen trunks of Quercus on wood of fallen trunk TDNgrid ref 513342 22 January 2015 PampB van den Boom52575 (LGmdashholotype hb vd Boomndashisotype)

(Fig 2AndashD)

Thallus very thin consisting of small greenishflattened or slightly convex areoles less than0middot1mm diam with crystals often partlycoated by a thin gelatinous film of greenalgal cells Photobiont micareoid 6ndash8microm diamthin-walled clustered in compact massesApothecia abundant subglobose immar-

ginate 0middot15 to 0middot25mm diam dark brownto black Hymenium c 30ndash40 microm highepihymenium with dark green-brown (K+violet) spots paraphyses sparse branchedc 1middot2ndash1middot4microm wide Hypothecium hyalineAsci 20ndash28times7ndash12microm Ascospores ellipsoid6middot5ndash9middot7times2middot0ndash2middot6microm (0ndash)1-septateMesopycnidia often abundant c 40ndash80 microm

top dark greenish grey (K+ violet) formedinside a thallus granule or outside of thelichenized thallus in the gelatinous matrixof free algal cells Mesoconidia shortly

bacilliform rarely obovoid 3middot8ndash6middot1times1middot0ndash1middot2(ndash1middot3) microm non-septate

Chemistry No chemical compoundspigment Sedifolia-grey (Meyer amp Printzen2000) K+ violet

Etymology The epithet chosen for thisspecies refers to a quite unusual habitat(decaying herbs) on which it has been foundseveral times

Habitat and distribution On soft and decayingwood on standing dead trunks on dead andwet stems of herbaceous plants or directly onsoil In the Netherlands accompanying specieson decaying wood include Absconditella spMicarea micrococca and Placynthiella dasaea andon soil Absconditella fossarum and Thelocarponlichenicola It is distributed throughout theNetherlands and has been detected in onecollection from Poland filed under the recentlydescribed M nowakii (Czarnota 2007) In thelatter collection it grows on decorticated woodwith M denigrata and M misella The speciesis very inconspicuous and can be easilyoverlooked because it is frequently coveredwithgelatinous algal cells No doubt it will be foundin other European countries

Notes In the monograph and revisionsof the genus in Europe (Coppins 1983 2009Czarnota 2007) this new species would keyout as Micarea denigrata if no chemical test forgyrophoric acid is performed Indeed it lookslike a small or depauperate M denigrata acommon and highly polymorphic speciesbut clearly differs by the lack of gyrophoricacid and the shorter mesoconidia inM denigrata (3middot0ndash4middot2(ndash5middot0)times1middot4ndash1middot8(ndash2middot0) micromfide Czarnota 2007) We interpret theconidia produced by our new species asmesoconidia as they originate in rather largepycnidia and they are regularly bacilliform(rarely obovoid) in shape and thus akin tothe mesoconidia produced by M denigrataNo validly published epithet reduced intosynonymy with this species in Coppins (1983)can be assigned to the new species Micareamisellamight also be confused with this speciesand differs mainly by its simple ascospores

20 THE LICHENOLOGIST Vol 49

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In addition M denigrata and M misella areresolved outside the M prasina clade in whichM herbarum is nested with strong supportPhylogenetic inferences frommtSSU sequencesposition M herbarum in an unsupported

clade within the strongly supported M prasinagroup together with both lineages referredto as M prasina and M nowakii a speciesdescribed from Poland (Czarnota 2007)M nowakii was segregated from M denigrata

A

B

C D

E

F

G H

FIG 2 Micarea herbarum and M meridionalis AndashD Micarea herbarum (holotype) A amp B habitus C amp Dascospores with green algal cells growing over the hymenium EndashH Micarea meridionalis (holotype) E amp F

habitus G amp H ascospores Scales A B E amp F = 0middot5mm C D G amp H = 20 microm In colour online

2017 New Micarea in Western Europemdashvan den Boom et al 21

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and M misella based on the production ofmicareic acid (vs gyrophoric acid or nosubstances for the other two respectively)Compared with M herbarum M nowakiihas slightly smaller ascospores (0(ndash1)-septate6middot0ndash8middot0(ndash8middot5)times2middot0ndash3middot0(ndash3middot2) microm fide Czarnota2007) and shorter and wider mesoconidia(3middot5ndash4middot0times1middot5ndash1middot8microm fide Czarnota 2007)The position of the two accessions of

M herbarum in our phylogenetic tree points toa relationship between M herbarum andM nowakii or even that M herbarum cannotbe distinguished fromM nowakii Among themtSSU sequences published by Czarnota ampGuzow-Krzemińska (2010) for M nowakiione accession (Poland P Czarnota 4634GPN) is resolved with strong support asclosely related to M herbarum We foundthat this collection represents M herbarum(no micareic acid detected by TLC) a furthercollection (Poland P Czarnota 3464 GPN)might represent either a further species in thatgroup or a variant ofM herbarum In fact thiscollection does not have apothecia but onlypycnidia no crystals of any lichen substancecould be detected by microcrystallizationand the phylogenetic tree resolved it as sisterwith all accessions referred to M herbarumMicarea melanobola (Nyl) Coppins is a

species so far known only from the typecollection in Finland collected in 1866(Coppins 1983) Although it has beenreduced into synonymy with M prasina byCzarnota (2007) we suspect this speciesto be close to M nowakii and thus toM herbarum Indeed M melanobola hasparaphyses with ldquoapices thickened withgreenish (K+ violet) pigment and up to1middot7 microm wide overtopping the tops of ascirdquo(Coppins 1983) a typical feature that seemsto be quite similar to the paraphyses ofM nowakii described with ldquoapices thickenedto 2ndash2middot5 microm and dull olive pigmentrdquo said toreact K+ violet (legend to fig 46 in Czarnota2007) The chemistry of the type ofM melanobola could not be studied for lackof material (Coppins 1983) The genuineidentity of the type material ofM melanobolashould therefore be re-evaluatedIt is worth mentioning that M herbarum is

the first species within the prasina group that

does not produce any secondary compoundsother than the pigment present in apotheciaThe biology of M herbarum sp nov is

remarkable as it very often grows over or withina gelatinous film of green algal cells Hyphaeconnected with the apothecia and pycnidiapenetrate into this layer Furthermorethese unknown algae can penetrate into thesubhymenium of the Micarea apothecia

Specimens examined The Netherlands Groningen1middot8km N van Sellingen E bank of Ruiten Aa N of bridgenear Rijsdam 52deg57middot8N 7deg8middot6E open area loamysoil recently scraped 1999 M Brand 39768 (hb Brand)Overijssel Hardenberg Rheezerveen Klimberg52deg34middot8N 6deg32middot9E dead wood in forest 2010 M Brand61133 (hb Brand) Noord-Holland Santpoort Duin enKruidberg tank ditch between Argus and Westerveld52deg26middot6N 4deg37middot4E open forest in dune area wood ofPopulus alba branches 2014 M Brand 63193 (hb Brand)Zuid-Holland Zoetermeer Sprinterpad N of Westerpark52deg3middot6N 4deg26middot7E rotting wood of dead Populus trunks2010 M Brand 60950 (hb Brand) GelderlandVierhouten Elspeetsche Heide W of bicycle trail52deg18middot6N 5deg48middot5E rotting wood in heathland 1999M Brand 39206 (hb Brand) Heerde Sprengen52deg23middot9N 6deg0middot2E wood of bridge in forest 1973M Brand 3254 (hb Brand) Garderen Speulderbos14m E of Dodenweg 52deg14middot65N 5deg41middot53E deadwood of fallen Fagus in forest 1998 M Brand 37726(hb Brand) Zeeland Tholen recently reclaimed areabetween Slaakdam and Haaftenpolder S side 51deg36middot3N4deg10middot8E twigs in open grass vegetation 1983 M Brand33842 (hb Brand) Noord-Brabant E of OirschotOirschotse Heide N side of secondary road to OirschotPinus forest and edge of Calluna heathland with someQuercus robur trees 51deg30N 5deg21E wood of fallen trunk2014 P amp B van den Boom 52533 (hb vd Boom)Limburg Weert Kolenhofweg 1km NE of Mildert51deg14middot6N 5deg48middot3E rotting wood of trunk in youngPinus plantation 2000 M Brand 41201 (hb Brand)Swalmen 2km NE Boschheide 51deg14middot4N 6deg3middot7Erecently partly scraped Calluna heathland 1992 M Brand29016 (hb Brand) E of Wanssum NE of WellerlooiDe Hamert open Pinus forest withCalluna heathland a fewscattered Pinus strobus trees and fallen trunks51deg32middot7N 6deg08middot6E dead wood of fallen P strobus 2015P amp B van den Boom 53197 (hb vd Boom)mdashPolandPojezierze Lubuskie Lakeland S of Motski Village52deg1442N 15deg2214E on wooden fence in well-lit placenear the edge of pine forest 2005 P Czarnota 4634 (GPN)

Micarea meridionalis van den BoomBrand Coppins amp Seacuterus sp nov

MycoBank No MB 811050

Thallus areolate areoles subglobose to irregularlyflattened 40ndash100microm diam pale to medium greenish

22 THE LICHENOLOGIST Vol 49

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or greenish grey to pale brownish apothecia abundant0middot10ndash0middot30(ndash0middot35) mm diam pale to dark brownish greyascospores ellipsoid 1(ndash3)-septate 8middot0ndash9middot4times3middot4ndash4middot0micrommesopycnidia often present 50ndash70micromdiam mesoconidia5middot8ndash6middot7times1middot0ndash1middot2microm bacilliform to fusiform Thallus andapothecia with micareic acid

Type Portugal Alentejo ESE of Odemira c 4 km E ofSanta Clara-a-Velha near storage lake (W side) Pinusforest 37deg30middot90N 8deg26middot66W 150m 16 February 2015on Pinus Pamp B van den Boom 52904 (LGmdashholotype hbBrand hb vd Boommdashisotypes)

(Fig 2EndashH)

Thallus plusmn effuse up to 4cm wide appearinggranular with ecorticate areoles 40ndash100micromdiam subglobose to irregularly flattenedcoalescing and forming a continuous crust orscattered patches upper surface usuallysmooth pale to medium greenish or greenishgrey to brownish grey to pale brown mattto slightly shiny up to 20micromthickmostly thinlycoated by gelatinous algae fine crystals presentrarely forming soredioid structures consisting ofloose goniocysts 10ndash14microm in diam Photobiontmicareoid algal cells globose 4ndash6microm diamthin-walled clustered in compact masses

Apothecia 0middot10ndash0middot30(ndash0middot35) mm diamabundant scattered sometimes tuberculateimmersed to semi-immersed in thallusgranules and thus seemingly marginatebecause of thallus parts present on the edge ofapothecium eventually becoming immargi-nate disc beige or pale to dark brownish greynever black slightly to moderately convexa few crystals sometimes seen in apotheciumsection Hymenium hyaline 40ndash50microm highwith spots pale greyish brown K+ violetN+ reddish excipulum in young apotheciahardly distinct up to 10microm wide paraphysesabundantly branched c 1middot2microm wide tips notor sometimes slightly widened up to 1middot5micromnot pigmented epithecium olive-greenishK+ violet N+ reddish hypothecium hyalineAsci slightly clavate 35ndash40times8ndash10microm8-spored Ascospores ellipsoid 1(ndash3)-septate8middot0ndash9middot4times3middot4ndash4middot0microm

Macropycnidia very rare c 50microm diamhyaline but top greyish macroconidia15ndash17times1middot1ndash1middot3microm 1-septate slightly curvedMesopycnidia often present immersed50ndash70microm diam hyaline mesoconidiabacilliform to fusiform 5middot8ndash6middot7times1middot0ndash1middot2microm

Chemistry Thallus and apothecia Kminus CminusKCminus Pminus micareic acid detected by TLC inthallus and apothecia pigment Sedifolia-grey(Meyer amp Printzen 2000) K+ violet

Etymology The epithet chosen for thisspecies refers to its southern distribution inEurope

Habitat and distribution Micarea meridionalisis known from several localities in westernPortugal in lowland and maritime areas in theAlentejo Estremadura and Lisboa provincesbetween 39deg40N and 37deg30N In theselocalities it is a corticolous species in ruderaland even dusty environments includingparklands and roadsides it has been foundon indigenous or planted tree species suchas Acacia longifolia Eucalyptus Pinus MorusNerium oleander andThuja OnAcacia longifoliaaccompanying species include Arthoniapruinata Cliostomum griffithii Coenogoniumtavaresiana Hyperphyscia adglutinata Lecanianaegelii and Lecanora lividocinerea Diploiciacanescens Waynea stoechadeana Candelariellareflexa and Physconia grisea are furtherassociated species found growing togetherTwo further collections have also been foundfrom CalabriaItaly here the species grows onPinus trees at sea level in unknown ecologicalconditions Further north it is known from theBotanical Garden of Rome in the city centreonAcanthosyris spinescens andQuercus pubescensand within the urban area of Rome on Quercussuber in a small remnant of semi-natural forest

Notes Micarea meridionalis is resolvedwithin the M prasina clade with strongsupport but its relationships within it areunclear It is easily distinguished by itsgranular thallus and the production ofmicareic acid The only otherMicarea speciesthat produce micareic acid are M nowakiiM prasina s str and M soralifera (Czarnota2007 Czarnota amp Guzow-Krzemińska 2010Guzow-Krzemińska et al 2016) In additionto the typical granular thallusMmeridionaliscan be distinguished from M nowakii by itslonger mesoconidia (3middot5ndash4middot0times1middot5ndash1middot8 micromfor M nowakii fide Czarnota (2007)) andfrom M prasina s str by its shorter

2017 New Micarea in Western Europemdashvan den Boom et al 23

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ascospores ((6ndash)8ndash12(ndash14) times (2middot5ndash)3middot0ndash4middot0(ndash5middot5) microm for M prasina s str fide Czarnota(2007)) Micarea soralifera has similargranular areoles to M meridionalis but isdistinguished by its distinct mostly discretesoralia Furthermore the ecology ofM meridionalis is much more ruderal andeutrophic than M nowakii M prasina s strand M soralifera all of which prefer moreacidic and nutrient-poor substrataNo validly published epithet reduced into

synonymy with M prasina in the monographand revisions of the genus in Europe(Coppins 1983 2009 Czarnota 2007) can beassigned to the new species

Specimens examined Portugal Estremadura E ofCaldas da Rainha road to Santa Catarina betweenCabeccedila Alta and Portela Pinus forest along road onPinus 2001 P amp B van den Boom 27727 27731 27736(hb vd Boom)Beira Nazareacute NE of town Pinus foreston Pinus 2003 M Brand 49616 (hb Brand) SetubalSerra da Arraacutebida 2 km SW of Aldeia near gate offormer chapel on old Morus 2003 M Brand 49832(hb Brand) ibid on Nerium oleander M Brand 49839(hb Brand) Alentejo NE of Cercal road N261 fromAlvalade to Satildeo Domingo mature Quercus suber treesalong field on Q suber 37deg55middot91N 8deg27middot93W 2015P amp B van den Boom 53059 (hb vd Boom) WNW ofCercal N of Vila Nova de Milfontes Praia da Ilhacoastal outcrops with shrubs including Acacia longifoliaon Acacia 37deg49middot79N 8deg47middot48W 2015 P amp B vanden Boom 52966 (hb vd Boom) ENE of Cercal c 1 kmE of the city along roadN262 rows of mature Eucalyptusglobulus trees on Eucalyptus 37deg48middot36N 8deg38middot32W2015 P amp B van den Boom 53089 (hb vd Boom)WNWofOdemira E of Almograve Longueira roadsideAcacia longifolia shrubs on Acacia 37deg39middot41N8deg46middot17W 2015 P amp B van den Boom 52664 (hb vdBoom) WNW of Odemira Cabo Sardatildeo Acacialongifolia shrubs in coastal area near lighthouse onAcacia 37deg35middot95N 8deg48middot95W 2015 P amp B van denBoom 52690 (hb vd Boom) SW of Odemira Boavistados Pinheiros botanical garden lsquoParque das Aacuteguasrsquomixed trees including Salix and Mimosaceae on astump 37deg34middot97N 8deg39middot62W 2015 P amp B van denBoom 52920 (hb vd Boom) SW of Odemira just S ofZambujeira 37deg31middot18N 8deg47middot11W coastal dune areawith Acacia longifolia on Acacia 2015 P amp B van denBoom 53032 (hb vd Boom) Lisboa Sintra park ontrunk of Thuja 2015 P amp B van den Boom 53940(hb vd Boom)mdashItaly Calabria Nicotera on Pinusbark 2000 D Puntillo (E) Lazio Rome Orto Botanicodi Roma alt 50m on Acanthosyris spinescens 1999S Ravera 3228 (RO) ibid alt 60m on Quercuspubescens 1999 G Brezzi [Ravera 3229] (RO) RomeParco di Monte Mario alt 137m on Quercus suber2000 G Brezzi [Ravera 3231] (RO)

We warmly thank Ido Cremasco and Laurent Gohy fortechnical assistance in the molecular laboratoryand herbarium at the University of Liegravege and PawełCzarnota Beta Guzow-Krzemińska Domenico Puntilloand Sonia Ravera for the loan of material andorproviding us with interesting data

REFERENCES

Andersen H L amp Ekman S (2005) Disintegration of theMicareaceae (lichenized Ascomycota) a molecularphylogeny based on mitochondrial rDNA sequencesMycological Research 109 21ndash30

Aptroot A amp Caacuteceres M E S (2014) New lichenspecies from termite nests in rainforest in BrazilianRondocircnia and adjacent Amazonas Lichenologist 46365ndash372

Barton J amp Lendemer J (2014)Micarea micrococca andM prasina the first assessment of two very similarspecies in eastern North America Bryologist 117223ndash231

Brand A M van den Boom P P G amp Seacuterusiaux E(2014) Unveiling a surprising diversity in the lichengenusMicarea (Pilocarpaceae) in Reacuteunion (Mascarenesarchipelago IndianOcean)Lichenologist 46 413ndash439

Caacuteceres M E S Mota D A de Jesus L S ampAptroot A (2013) The new lichen species Micareacorallothallina from Serra da Jiboacuteia an Atlanticrainforest enclave in Bahia NE Brazil Lichenologist45 371ndash373

Castresana J (2000) Selection of conserved blocks frommultiple alignments for their use in phylogeneticanalysis Molecular Biology and Evolution 17 540ndash552

Coppins B J (1983) A taxonomic study of the lichengenus Micarea in Europe Bulletin of the BritishMuseum (Natural History) Botany Series 11 17ndash214

Coppins B J (2009) Micarea In The Lichens of GreatBritain and Ireland (C W Smith A Aptroot B JCoppins A Fletcher O L Gilbert P W James ampP A Wolseley eds) 583ndash606 London BritishLichen Society

Coacuterdova-Chaacutevez O Aptroot A Castillo-Camposa GCaacuteceres M E S amp Peacuterez-Peacuterez R E (2014) Threenew lichen species from cloud forest inVeracuz Mexico Cryptogamie Mycologie 35 157ndash162

Cubero O F Crespo A Fatehi J amp Bridge P D(1999) DNA extraction and PCR amplificationmethod suitable for fresh herbarium-storedlichenized and other fungi Plant Systematics andEvolution 216 243ndash249

Czarnota P (2007) The lichen genus Micarea(Lecanorales Ascomycota) in Poland Polish BotanicalStudies 23 1ndash199

Czarnota P amp Guzow-Krzemińska B (2010) Aphylogenetic study of the Micarea prasina groupshows thatMicarea micrococca includes three distinctlineages Lichenologist 42 7ndash21

Guzow-Krzemińska B Czarnota P Łubek A ampKukwa M (2016) Micarea soralifera sp nova new sorediate species in the M prasina groupLichenologist 48 161ndash169

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httpswwwcambridgeorgcoreterms httpsdoiorg101017S0024282916000633Downloaded from httpswwwcambridgeorgcore IP address 876721120 on 23 Jan 2017 at 074300 subject to the Cambridge Core terms of use available at

Huneck S amp Yoshimura I (1996) Identification of LichenSubstances Berlin Heidelberg Springer-Verlag

Katoh K amp Standley D M (2013) MAFFT multiplesequence alignment software version 7 improve-ments in performance and usability MolecularBiology and Evolution 30 772ndash780

Maddison D amp Maddison W (2005) MacClade v408 Sunderland Massachusetts SinauerAssociates

McCarthy P M amp Elix J A (2016) A new species ofMicarea (lichenized Ascomycota Pilocarpaceae)from alpine Australia Telopea 19 31ndash35

Meyer B amp Printzen C (2000) Proposal for astandardized nomenclature and characterizationof insoluble lichen pigments Lichenologist 32571ndash583

Miadlikowska J Kauff F Houmlgnabba F Oliver J CMolnaacuter K Fraker E Gaya E Hafellner JHofstetter V Gueidan C et al (2014) A multi-gene phylogenetic synthesis for the class Lecanoro-mycetes (Ascomycota) 1307 fungi representing1139 infrageneric taxa 317 genera and 66 familiesMolecular Phylogenetics and Evolution 79 132ndash168

Miller M A Pfeiffer W amp Schwartz T (2010)Creating the CIPRES Science Gateway forinference of large phylogenetic trees In Proceedingsof the Gateway Computing Environments Workshop(GCE) 14 November 2010 New Orleans Louisianapp1ndash8

Orange A James P W amp White F J (2010)Microchemical Methods for the Identification of Lichens(2nd edition) London British Lichen Society

Rambaut A (2009) FigTree v123 Available fromhttptreebioeduksoftwarefigtree

Seacuterusiaux E Brand AMMotiejunaite J Orange AampCoppins B J (2010) Lecidea doliiformis belongs toMicarea Catillaria alba to Biatora and Biatoraligni-mollis occurs in Western Europe Bryologist 113333ndash344

Stamatakis A (2006) RAxML-VI-HPC maximumlikelihood-based phylogenetic analyses with thou-sands of taxa and mixed models Bioinformatics 222688ndash2690

Stamatakis A Hoover P amp Rougemont J (2008) Arapid bootstrap algorithm for the RAxML webservers Systematic Biology 57 758ndash771

Svensson M amp Thor G (2011)Micarea capitata a newbryophilous lichen from Sweden Lichenologist 43401ndash405

van den Boom P P G (2010) Lichens and lichenico-lous fungi fromLanzarote (Canary Islands) with thedescriptions of two new species CryptogamieMycologie 31 183ndash199

van den Boom P P G amp Ertz D (2014) A new speciesofMicarea (Pilocarpaceae) fromMadeira growing onUsnea Lichenologist 46 295ndash301

Zoller S Scheidegger C amp Sperisen C (1999) PCRprimers for the amplification of mitochondrialsmall subunit ribosomal DNA of lichen-formingascomycetes Lichenologist 31 511ndash516

2017 New Micarea in Western Europemdashvan den Boom et al 25

httpswwwcambridgeorgcoreterms httpsdoiorg101017S0024282916000633Downloaded from httpswwwcambridgeorgcore IP address 876721120 on 23 Jan 2017 at 074300 subject to the Cambridge Core terms of use available at