Taxonomic divergence of the green naked-stipe members of the genusMicroglossum (Helotiales)Viktor Kučera a Pavel Lizoň a and Michal Tomšovskyacuteb

aPlant Biology and Biodiversity Center Institute of Botany Slovak Academy of Sciences Duacutebravskaacute cesta 9 SK-845 23 Bratislava SlovakiabFaculty of Forestry and Wood Technology Mendel University in Brno Zemědělskaacute 3 CZndash613 00 Brno Czech Republic

ABSTRACTFour new species of the Ascomycete genus Microglossum are recognized based on morphologicalcharacters and DNA sequences of nuc rDNA (ITS region and 28S gene) and the second largestsubunit of RNA polymerase II (RPB2) They differ from Microglossum nudipes by the color of theascocarps and the sizes and shapes of ascospores asci and paraphyses A lectotype is proposedand an emended description is provided for M nudipes Descriptions of new species Microglossumclavatum M truncatum M pretense and M tenebrosum are provided Other closely related speciesin the group of green earth tongues include Microglossum viride M rickii and M griseoviride Anidentification key to green Microglossum species is presented

ARTICLE HISTORYReceived 13 December 2016Accepted 16 December 2016

KEYWORDSBoudier earth tonguesLeotiomycetes new taxasystematics

INTRODUCTION

Because of their unusual appearance earth tongues haveinterested mycologists for a long time Previous molecularanalyses (Sandnes 2006 Wang et al 2006 Ohenoja 2010)revealed that these fungi do not have a monophyleticorigin The class Geoglossomycetes was establishedrecently by Schoch et al (2009) as a sister taxon toLeotiomycetes Currently Geoglossomycetes includeseven more-or-less well-delimitated genera (Hustad et al2011 Hustad et al 2013) but the number of extant speciesis uncertainMicroglossum does not seem to belong withinGeoglossomycetes (Schoch et al 2009 Hustad et al 2013)and is still considered a member of Helotiales in the poly-phyletic class Leotiomycetes (Wang et al 2006)

Microglossum was described by Gillet (1879) for thegreen species Geoglossum viride Pers and the olivac-eous G olivaceum Pers Geoglossum viride was pub-lished by Persoon (1796) and mentioned but withoutdescription and thus not validly published 2 y before(Persoon 1794) Later Durand (1908) selectedMicroglossum viride (Pers Fr) Gillet as the lectotypeof Microglossum The generic name Microglossum hasbeen generally used for green earth tongues since thattime Saccardo (1884) based his Microglossum (nomillegit Art 531) on Geoglossum hookeri Cooke

Species of Microglossum are usually colorful fungiwith an earth tongue appearance with a sterile stipe

and fertile hymenium occurring in undisturbed grass-lands About 36 species are reported worldwide (wwwmycobankorg wwwindexfungorumorg)

Since 1917 the nameMicroglossum nudipes Boud wasapplied to all collections of Microglossum with a greennaked stipe Our DNA sequencing analyses employingthe nuc rDNA internal transcribed spacer (ITS1-58S-ITS2 = ITS) and the 28S and RPB2 genes indicated thatseven taxa could be differentiated in the material westudied These assumptions were supported by macro-morphological characters especially color of the ascomataand to a certain extent by micromorphology

MATERIALS AND METHODS

Morphological studiesmdashMacromorphological characterswere observed in fresh material Micromorphologicalstructures were studied in dried material using anAxioScope A1 compound microscope (Carl ZeissGoumlttingen Germany) at a magnification of 1000times with anoil immersion lens Fragments of material were examinedin 5 KOH tap water Melzerrsquos reagent and a solution ofCongo Red in ammonia Measurements were taken fromdry material treated in tap water directly under themicroscope using the eyepiece micrometer Dimensionsof micromorphological characters were estimated as anaverage (av) plusmn 1 standard deviation from 30measurements for each taxon with 10th and 90th

CONTACT Pavel Lizoň pavellizonsavbaskColor versions of one or more of the figures in the article can be found online at wwwtandfonlinecomumyc

Supplemental data for this article can be accessed on the publisherrsquos Web site

MYCOLOGIA2017 VOL 109 NO 1 46ndash54httpdxdoiorg1010800027551420161274620

copy 2017 The Mycological Society of America

percentiles in parentheses and Q value of ascosporespresent lengthwidth ratio Acronyms for herbaria followIndex herbariorum (Thiers continuously updated) Alldescriptions are based on dried studied collectionsSeveral collections were examined microscopically also inliving condition (indicated by an exclamation mark) thedata from these fresh collections are included inparentheses in the descriptions For the list of studiedMicroglossum specimens see SUPPLEMENTARY TABLE2 The pH reaction of the substratewasmeasured directly inthe field using HANNA HI 99121

Molecular studiesmdashSixty two recently collectedspecimens of Microglossum were selected for molecularanalyses DNA was isolated from dried fungal materialusing the PowerSoil DNA isolation kit (Mo Bio CarlsbadCalifornia USA) DNA fragments encompassing the ITSregion and the D1ndashD2 domains of the 28S gene wereamplified using the following primer combinationsITS5LR6 or ITS1ITS4 and LR0RLR6 in cases ofdifficult amplification (White et al 1990 Moncalvo et al2000) The polymerase chain reaction (PCR) profile forprimers ITS5LR6 was as follows touchdown PCRinitiated with a 2-min denaturation at 94 C annealingtemperature for first amplification cycle 60 Csubsequently incrementally reduced by 1 C per cycle forthe next 9 cycles followed by 36 amplification cycles eachconsisting of 30 s denaturation at 94 C 30 s annealing at50 C and 1 min extension at 72 C concluding with 10min incubation at 72 C PCR regimes using other primercombinations followed the protocols of Kučera et al(2014b) Amplicons of the domain 6ndash7 region of thesecond largest subunit of RNA polymerase II (RPB2)were obtained using primers fRPB2-5F (Liu et al 1999)and RPB2-P7R (Hansen et al 2005) following protocolsoutlined by Hansen et al (2005)

Gene fragments were amplified in a Mastercycler_epthermocycler (Eppendorf Hamburg Germany)Amplicons were custom-purified and sequenced atMacrogen (Seoul Korea) Sequences were deposited inthe European Molecular Biology Laboratory (EMBL)Nucleotide Sequence Database (Kl513002ndashKJ513011KX382834ndashKX382893)

Two data sets were subjected to phylogenetic analyses(i) one including ITS of all specimens and (ii) a combinedITS-28S-RPB2 data set of selected representatives of eachlineage of the M nudipes complex (SUPPLEMENTARYTABLE 1) Sequences were aligned usingMAFFT version7 utilizing the Q-INS-i option (Katoh and Toh 2008) TheITS data set was supplemented with sequences of Mcyanobasis Iglesias amp Arauzo M griseoviride V KučeraLizoň amp M Tomšovskyacute M olivaceum (Pers) Gillet M

rufescens (Greacutelet) Bon M rufum (Schwein) UnderwandM viride obtained from GenBank The ITS sequenceof Leotia lubrica (Scop) Pers strain ZW-GEO54-Clark(GenBank no AY789349) was selected as an outgroupThe combined ITS-28S-RPB2 data set was 2546 bp longwhereas intron sites (132ndash178 bp in some species dividedin two sections) occurring in some 28S sequences wereexcluded from the analyses Microglossum griseoviride(SAV F-9920) andM viride (SAV F-10249) were selectedas outgroups for combined analysis

Maximum likelihood (ML) and Bayesian inference(BI) analyses were used to estimate phylogenetic relation-ships of both data sets The combined data set was parti-tioned into three subsets of nucleotide sites ITS 28S andRPB2 because of different models relevant to each gene orregion The likelihood settings from the best-fit model forITS (TiM2+I+G) and the combined data set (TiM2ef+I+G = ITS TIM1+I+G = 28S TrN+G = RPB2) wereselected by the Akaike information criterion injModelTest2 (Darriba et al 2012) BI analysis was con-ducted with MrBayes 326 (Ronquist et al 2012) Thecombined data set was partitioned into three subsets ofnucleotide sites ITS 28S and RPB2 We ran four chainsfor 10 million generations The burn-in value (1 milliongenerations) was estimated using Tracer 16 (Rambautet al 2014) Sampling frequency was set to every 1000thgeneration ML analysis was performed with RAxML-HPC 8 (Stamatakis 2014) with a GTRCAT model ofevolution Nodal support was determined by nonpara-metric bootstrapping (BS) with rapid bootstrappingoption setting number of replicates automatically

RESULTS

Morphological studymdashAnalysis of macro- andmicromorphological characters of the studied collectionsshowed that combinations of characters are unique for eachof the taxa described below Characteristic forMicroglossum nudipes is a combination of olivaceous-green hymenium large asci and ascospores with thinapically branched paraphyses Blue-green or gray-greenascocarps a flattened stipe and sometimes apicallythickened paraphyses are distinguishing characters for Mpratense V Kučera Tomšovskyacute amp Lizoň Microglossumtruncatum V Kučera Tomšovskyacute amp Lizoň has a brown-green or brown-colored hymenium and truncate ascocarpsat least when young and paraphyses that are branched inthe middle and basal parts Microglossum tenebrosum VKučera Tomšovskyacute Lizoň amp F Hampe has dark green(dark blue-green) ascocarps and short ascospores lt15 μmwhereas M clavatum V Kučera Tomšovskyacute amp Lizoň hasclavate often flattened ascocarps a green hymenium shortstipe and relatively large ascospores lt20 (ndash25) μm

MYCOLOGIA 47

Microglossum parvisporumV Kučera Lizoňamp Tomšovskyacutehas the smallest ascospores in this complex of green naked-stipe species

Phylogenetic analysesmdashThe aligned ITS data set wascomposed of 513 positions and included 325 conserved159 variable and 36 unique sites as determined by MEGA606 (Tamura et al 2013) The combined ITS-28S-RPB2data set had 2546 positions including 2139 conserved 398variable and 333 unique sites The sequence data sets aredeposited in TreeBASE (Submission ID 19742)

The phylogenetic trees (FIGS 1 2) revealed that theMnudipes complex was composed of M fuscorubens BoudM parvisporum and four new species M clavatum Mpratense M tenebrosum andM truncatum Microglossumnudipes sensu stricto still seems to be variable and could bean aggregate of more species so we use the indication Mnudipes aff in this paper The M nudipes complex is

proximal to M rufum M rufescens M olivaceum andM cyanobasis The previously reported sequenced speci-men M cf nudipes (SAV 10024) (Kučera et al 2014b) fellwithinM pratense Microglossum griseoviride andM virideform a basal clade

TAXONOMY

Microglossum Gillet Champignons de France Discom125 1879

= Helote Hazsl Magyar Tud Akad Eacutertes 11(19)81881

= Ochroglossum S Imai Sci Rep Yokohama NatlUniv Sect 2 46 1955

Type species Microglossum viride (Pers Fr) GilletChampignons de France Discom 125 1879

Ascocarps fleshy erect stipitate clavate ascigerousportion only in upper part with the general form and

Figure 1 Phylogenetic tree of the internal transcribed spacer (ITS) region conducted by Bayesian analysis (for legends to collectionnumbers see SUPPLEMENTARY TABLE 1) Numbers at branches indicate maximum likelihood bootstrap proportion (left) andBayesian posterior probability (right) values The type specimens are highlighted in bold The asterisk () indicates different topologyin the two analyses Bar = number of expected substitutions per position

48 V KUČERA ET AL NEW MICROGLOSSUM TAXA

habit resembling species of the genus Geoglossum cla-vate or tongue-shaped somewhat compressed alwayscolorful (green brown or reddish brown olivaceousyellow clay) never dark brown or black Asci clavate-cylindrical opening by an amyloid pore Ascospores 8biseriate hyaline smooth ellipsoidal fusiform orcylindrical to oblong aseptate or septate paraphysesstraight slightly curved or circinate at the apex

Two basic groups of green earth tongues can bedistinguished easily (i) a group characterized by scalystipes including M viride M rickii Imai and M gri-seoviride (Kučera et al 2014b) and (ii) a group withnaked smooth stipe including M nudipes and M oli-vaceum both in a broad sense

KEY TO GREEN MICROGLOSSUM SPECIES

1 Stipe covered with scales ascocarp green yellowish-green grayish-green apex of paraphyses with a ldquocaprdquoof pigment 2

1prime Stipe naked green pinkish-green olive-greenblue-green apex of paraphyses lacking a ldquocaprdquo ofpigment 4

2 Ascocarp lt15 cm tall asci lt80 μm (65ndash75 times 9ndash10μm) long ascospores 10ndash14 times 4ndash5 μm known onlyfrom Brasilia M rickiib

2prime Ascocarps lt8 cm tall asci gt80 μm long 3

3 Ascocarp yellow-green growing in wet places oftenwith liverworts asci 106ndash134 times 95ndash12 μm ascos-pores 18ndash22 times 5ndash7 μm M viridea

3prime Ascocarp gray-green growing on soil in the forestasci 105ndash139 times 8ndash10 μm ascospores 16ndash20 times 4ndash5μm M griseoviridea

4 Stipe constituting 12ndash34 of the mature ascocarpdominant color of the hymenium buff pink olivestipe is light brown usually mixed with other colorsascospores 13ndash15 times 4 μm M rufescensc

4prime Stipe equal to or shorter in length than hyme-nium ascocarps without pink and olivecolor 5

Figure 2 Phylogenetic tree for a combined analysis of the ITS region and 28S and RPB2 genes conducted by Bayesian analysis (forlegends to collection numbers see SUPPLEMENTARY TABLE 1) Numbers at branches indicate maximum likelihood bootstrapproportion (left) and Bayesian posterior probability (right) values Type specimens are highlighted in bold Bar = number ofexpected substitutions per position

MYCOLOGIA 49

5 Ascospores le16 um long growing in open areas oncalcareous bedrock 6

5prime Ascospores gt16 um long growing in mesophilousmeadows or forests 7

6 Ascocarps white-green when old with violac-eous color asci lt80 μm long M parvisporuma

6prime Ascocarps dark green when old dark greenwith dark violet tint asci 80ndash95 μm long M tenebrosum

7 Asci gt105 μm long growing in theforests 8

7prime Asci lt105 μm long growing on mesophilousmeadows pastures 9

8 Ascocarp green hymenium with brownish tintstipe blue-green paraphyses branched also inupper 13 and in the middle 108prime Ascocarp with brown color present also on thestipe paraphyses tips swollen lt5 μm branched inbasal 13 M nudipes aff

9 Hymenium concolorous with sterile ascospores135ndash165 times 4ndash5 μm M pratense

9prime Hymenium usually brownish-green or brownstipe blue-green ascospores 15ndash18 times 4ndash5μm M truncatum

10 Ascocarp flattened hymenium club-shapedusually longer than the stipe paraphyses branchedat the base and in the middle M clavatum10prime Hymeniummace-shaped as long as stipe para-physes branched both at basal and apical part M nudipesd

In the sense of aKučera et al 2014a 2014b bImai 1942cMoingeon 2004 dBoudier 1917

Microglossum nudipes Boud Bull Soc Mycol France3316 1917

Typification FRANCE Vienne Savigneacute Dec 1913A Greacutelet (lectotype designated here ex herb BoudierPC 0139818) MBT 373899

Description of the lectotype Ascocarps (10ndash)168ndash313(ndash38) mm high tongue-like or club-shaped stipitateHymenium (5ndash)78ndash152(ndash18) times 1ndash38(ndash6) mm mace-shaped cylindrical clavate truncate or lanceolate long-itudinally grooved sometimes twisted and almost lacu-nose or compressed on the side glabrous nakedsubolivaceous dark green or grayish-green Hymeniumconstituting approximately the upper 12 of the ascocarpStipe (5ndash)86ndash166(ndash20) times 05ndash4 mm cylindrical some-times flexuous naked blue-green or concolorous withhymenium not paler at the base flesh yellow-green (20dry ascocarps examined) Asci (80ndash)914ndash1103(ndash130) times(6ndash)71ndash95(ndash11) μm 8-spored clavate rounded at the

apex narrowly tapered towards the base biseriate aboveuniseriate below pore dark blue in Melzerrsquos reagentAscospores (12ndash)166ndash205(ndash23) times (4ndash)42ndash52 μm Qvalue = 24ndash55 (av = 39) usually slightly curved orsigmoid ends obtuse or tapering hyaline without clearlyvisible lipid bodies true septa not observed Paraphysesfiliform 1ndash2 μmwide straight branched at the base someof the paraphyses branched in apical part apical cellsfiliform (1ndash)11ndash25(ndash3) μm or rarely slightly swollenlt35 μm

Habitat Growing among the mosses underBuxus sp

Distribution FranceAdditional specimens examined See

SUPPLEMENTARY TABLE 2Notes Microglossum nudipes was described by Boudier

(1917) from Savigneacute (Vienne France) in December 1913where it was as noted by him first collected by D Arnouldand consequently by L Greacutelet The description presentedboth in Latin and French has important data but unfortu-nately lacks details to allow it to be connected unequivo-cally to a type specimen There are four specimens in PClabeled Microglossum nudipes all collected by Greacutelet twodated 1913 two collected later in 1938 Arnould is notmentioned as collector on any label The collections from1938 are not conspecific with those from 1913 and accord-ing to our observations represent M rufescens MoreoverBoudierrsquos description does not fully correspondwith any ofthe specimens As noted by Van Brummelen (1985)Boudierrsquos microscopic measurements were often exagger-ated in his descriptions as a result of an error in theconstruction of hismeasuring scale and his sporemeasure-ments after 1885 were usually about 10 too large Whenmeasurement of asci and spore size in his descriptions areadjusted by lowering them by 20 allowing for shrinkageby drying and Boudierrsquos inaccurate scale they roughlycorrespond with our observations of PC 0139818 Thuswe emended the description of Microglossum nudipes toinclude these details DNA studies using Boudierrsquos originalmaterial were not done because of the age of the specimenUnfortunately no specimen among recently collectedmaterial corresponds with the lectotype of M nudipesand the second collection gathered from the type localityin 1913 Our visit to the original locality in France in 2015to recollect the species was unsuccessful

Microglossum truncatum V Kučera Tomšovskyacute ampLizoň sp nov FIG 3a bMycoBank MB817350

Typification SLOVAKIA Biele Karpaty Mts NovaacuteBošaacuteca ca 17 km SE from the church Španie settle-ment Natural Monument Blažejovaacute (48deg52prime333PrimeN17deg49prime034PrimeE alt 415 m) in a meadow 7 Nov 2013

50 V KUČERA ET AL NEW MICROGLOSSUM TAXA

V Kučera V Kautman and I Kautmanovaacute (holotypeSAV F-11280) DNA sequences from the holotypeITS = KX382861 28S = KX382861 RPB2 = KX382875

Etymology From truncatus (Latin) ending abruptlyascocarps truncate when young

Ascocarps (19ndash)254ndash443(ndash55) [(20ndash)302ndash555(ndash60)]mm high tongue-like or club-shaped stipitateHymenium (8ndash)222ndash285(ndash30) times (1ndash)32ndash41(ndash7) [(10ndash)253ndash323(ndash44) times (1ndash)31ndash52(ndash8)] mm cylindrical club-shaped truncate or lanceolate only slightly grooved ver-tically glabrous naked brownish-green or light brown orgreen-brown dark green or brownish-green when dryHymenium usually constituting more than the upper 12of the ascocarp Stipe (10ndash)152ndash201(ndash27) times 1ndash22(ndash3)[(11ndash)165ndash236(ndash29) times 12ndash31(ndash4)] mm cylindricalblue-green not paler at the base (15 ascocarps examined)Asci (75ndash)851ndash987(ndash105) times (8ndash)84ndash96(ndash10) [(95ndash)1206ndash1353(ndash150) times (8ndash)101ndash115(12)] μm 8-sporedclavate rounded at the apex narrowly tapered towardsthe base biseriate above uniseriate below pore bluing in

Melzerrsquos reagent Ascospores (14ndash)154ndash183(ndash20) times 4ndash47(ndash5) [(17ndash)182ndash198(ndash21) times 4ndash5(55)] μm Q value = 33ndash48 (av = 38) hyaline ellipsoidal to oblong aseptateParaphyses filiform straight branched in basal partsome of the paraphyses branched in apical part apicalcells filiform 1ndash2 μm or only slightly swollen (lt3 μm)

Habitat On soil among grass in mesophilous mea-dows with soil pH 57ndash61

Distribution France Russia Slovakia SwedenAdditional specimens examined See

SUPPLEMENTARY TABLE 2Notes Ascocarps of Microglossum truncatum are

brown or brownish-green in the hymenium whereasthe stipe is blue-green and oval in cross-section Themost similar species is M nudipes aff which differs bya brown coloration on the stipe and is usually dumb-bell-shaped in cross-section Microglossum truncatummay be macroscopically confused with M parvisporumbut it clearly differs in the size of asci (85ndash98 times 8ndash9 μmvs 66ndash79 times 6 μm) and ascospores (15ndash18 times 4ndash5 μm vs

Figure 3 Microglossum species with green naked stipes A M truncatum SAV F-11261 (M Krivuš) B M truncatum SAV F-11280 (VKučera) C M pratense SAV-11020 (P Marstad) D M pratense SAV-10024 (V Kučera) E F M tenebrosum SAV F-11278 (F Hampe) GM nudipes aff SAV F-11051 (T Knutsson) H M nudipes aff SAV F-11285 (V Kaounas) I M nudipes aff SAV F-11271 (U Pera) Bars =25 cm (A) and 5 cm (BndashI)

MYCOLOGIA 51

11ndash14 times 3ndash4 μm) and acidity of the substrate (pH 57ndash61 vs pH 635)

Microglossum pratense V Kučera Tomšovskyacute ampLizoň sp nov FIG 3c dMycoBank MB808093

Typification SLOVAKIA Stolickeacute vrchy MtsMuraacutenska Huta village Prednaacute Hora recreation area for-mer ski slope ca 2 km SSW from the village (48deg45prime3124PrimeN 20deg06prime2698PrimeE alt 789 m) in grass 13 Oct 2010 VKautman and V Kučera (holotype SAV F-10024) DNAsequences from the holotype ITS = KC595259 28S =KC595260 RPB2 = KX382880

Etymology from pratum (Latin) meadow growingin meadows and pastures

Ascocarps (17ndash)268ndash496(ndash62) mm high tongue-like or club-shaped stipitate Hymenium (10ndash)124ndash277(ndash40) times (15ndash)22ndash47(ndash9) mm mace-shapedcylindrical clavate truncate or lanceolate verticallygrooved usually twisted glabrous naked blue-greenor gray-green dark green and often with lateral crackswhen dry Hymenium usually occupying the upper 12of the ascocarp or more Stipe (7ndash)118ndash242(ndash32) times1ndash3 mm often flattened and flexuous blue-green orconcolorous with hymenium (54 dry ascocarps exam-ined) Asci (67ndash)781ndash915(ndash105) times (5ndash)71ndash85(ndash9) μm8-spored cylindrical to clavate apex rounded narrowlytapered towards the base biseriate above uniseriatebelow the pore light bluing in Melzerrsquos reagentAscospores (11ndash)135ndash165(ndash20) times (4ndash)45ndash5 μm Qvalue = 32ndash41 (av = 37) ellipsoidal to oblong usuallyslightly curved or sigmoid ends obtuse or taperinghyaline or with several (lt4) lipid bodies real septanot observed Paraphyses filiform straight branchedin basal part some of the paraphyses branched at apicalpart the apical cells filiform 1ndash2) μm or only slightlyswollen lt3 μm

Habitat Meadows or pastures in north and centralEurope on soil among grasses The type collection wasfound in a site with soil pH 52ndash56

Distribution Georgia Norway Russia SlovakiaSweden

Additional specimens examined SeeSUPPLEMENTARY TABLE 2

Notes Microglossum pratense is characterized byhaving green or gray-green hymenium lacking abrown tint Ascocarps are usually produced in biggerclusters of lt10 When dry the ascigerous part oftentransversely cracked The hymenium takes more than ahalf of the whole ascocarp and the stipe is often flat-tened Microglossum pratense could be confused withM tenebrosum or M clavatum but M pratense is palerin color and the ascospores are bigger (135ndash165 times 4ndash5

μm) than in M tenebrosum (12ndash15 times 4ndash45 μm) andsmaller than in M clavatum (15ndash20 times 4ndash5 μm)Collections in fungaria are mostly misidentified asM olivaceum or M rufescens In general M pratensewas the most common green Microglossum species withnaked stipe in the examined areas

Microglossum tenebrosum V Kučera TomšovskyacuteLizoň amp F Hampe sp nov FIG 3e fMycoBank MB817358

Typification GERMANY Hessen LangenthalEberschuumltz (51deg36prime269PrimeN 09deg23prime226PrimeE alt 225 m)calcareous neglected open grassland shell limestonewith Juniperus sp 12 Oct 2009 F Hampe (holotypeSAV F-11278) DNA sequences from the holotype ITS= KX382845 28S = KX382845 RPB2 = KX382891

Etymology From tenebrosus (Latin) dark ascocarpsdark-colored

Ascocarps (27ndash)306ndash482(ndash53) [40ndash60] mmhigh ton-gue-like or club-shaped stipitate Hymenium (14ndash)15ndash257(ndash30) times (21ndash)32ndash5 [20ndash30 times 3ndash6] mm mace-shapedcylindrical clavate truncate or lanceolate verticallygrooved glabrous naked at maturity dark green or darkviolet-green or bronze-green to brown-green Hymeniumusually occupying the upper 12 of the ascocarp or moreStipe (12ndash)132ndash258(ndash33) times (15ndash)25ndash3 [20ndash30 times 25ndash6]mm often flattened and flexuous blue-green and oftenconcolorous with hymenium at the base (11 dry ascocarpsexamined) Asci (70ndash)795ndash954(ndash120) times (6ndash)73ndash86(ndash9)[(100ndash)1154ndash1243(ndash1398) times (75ndash)86ndash92(ndash104)] μm8-spored cylindrical to clavate apex rounded narrowlytapered towards the base biseriate above uniseriatebelow the pore bluing in Melzerrsquos reagent Ascospores(10ndash)118ndash154(ndash20) times (3ndash)37ndash45(ndash55) [(116ndash)138ndash148(ndash17) times (31ndash)42ndash46(ndash57)] μm Q value = 25ndash47(av = 34) [24ndash41 (av = 329)] ellipsoidal to oblongusually slightly curved or sigmoid ends obtuse or taper-ing hyaline or with several (lt4) lipid bodies real septa notobserved Paraphyses filiform straight branched in basalpart some of the paraphyses branched at middle part theapical cells filiform 1ndash2(ndash25) μm or slightly clavate orcapitate (lt3 μm)

Habitat On soil among grasses or in forest clear-ings meadows and pastures with Juniperus sp underBuxus sp and calcareous bedrock in western and cen-tral Europe

Distribution France Germany SpainAdditional specimens examined See

SUPPLEMENTARY TABLE 2Notes Microglossum tenebrosum is characterized by

shades of dark green The color of the hymenium isgenerally repeated at the basal part of the stipeMicroglossum tenebrosum is distinguished by the Q

52 V KUČERA ET AL NEW MICROGLOSSUM TAXA

value of the ascospores (34) compared with M nudipes(39) M clavatum (38) M pratense (37) M truncatum(38) and M parvisporum (305) Most collections of Mtenebrosum are from open spaces and not forests Thecombination of relatively large asci (795ndash95 times 7ndash9 μm)and small ascospores (12ndash15 times 4ndash45 μm) is characteristicThe data for fresh collections were taken from RibesRipoll (2013)Microglossum clavatum V Kučera Lizoň ampTomšovskyacute sp novMycoBank MB817351

Typification SPAIN Huesca Puente de Reina deJaca (42deg34prime0418PrimeN 0deg45prime4464PrimeW 665 m) underBuxus sempervirens in calcareous soil 6 Dec 2006 JHernanz (holotype SAV F-11276) DNA sequencesfrom the holotype ITS = KX382864 28S = KX382864RPB2 = KX382884

Etymology From clava (Latin) club ascocarps typi-cally club-shaped

Ascocarps 15ndash31(ndash37) mm high tongue-like club-shaped or clavate usually compressed stipitateHymenium 10ndash199(ndash25) times 1ndash4 mm clavate truncateor lanceolate slightly vertically grooved glabrousnaked dark green sometimes with a brown tintHymenium occupying more than the upper 12 ofthe ascocarp Stipe (4ndash)52ndash125 times (1ndash)25ndash3 mmcylindrical or flattened concolorous with hymenium(14 dry ascocarps examined) Asci (715ndash)966ndash1178(130) times (65ndash)82ndash101(ndash11) μm 8-spored cylindricalto clavate apex rounded narrowly tapered towardsthe base biseriate above uniseriate below the porebluing in Melzerrsquos reagent Ascospores (14ndash)155ndash204(ndash23) times (4ndash)45ndash5 μm Q value = 3ndash57 (av = 38)ellipsoidal to oblong usually slightly curved or sig-moid ends obtuse or tapering hyaline or with several(lt4) lipid bodies true septa not observed Paraphysesfiliform straight branched in basal part some of theparaphyses branched at middle part the apical cellsfiliform 1ndash2(ndash25) μm or slightly clavate or capitate(lt35 μm)

Habitat On calcareous soil under Buxus sp treeDistribution SpainAdditional specimens examined SeeSUPPLEMENTARY TABLE 2Notes Microglossum clavatum is similar to M

nudipes based on the size of asci and shape of para-physes However the values of all examined charactersof M clavatum are slightly smaller the paraphyses arenot apically branched and the ascus pore does not reactas strongly in Melzerrsquos reagent as it does in M nudipes

Examined specimens of other Microglossum spp SeeSUPPLEMENTARY TABLE 2

DISCUSSION

During our research on theMicroglossumnudipes complexwe recognized sevenwell-delimited taxa Three of themMnudipes (Boudier 1917) M parvisporum (Kučera et al2014a) and M fuscorubens (Boudier 1907) were alreadydescribed Four new species are described in this paper butthere is a high likelihood that additional morphotypes andor taxa in this complex will be discovered

In all species we observed that the apical part of theasci and paraphyses are surrounded by a green-brownamorphous matrix that is lt20 μm thick Moreoverascospores occurring in the apical part of the asci aresmaller than others

Ascocarps of all taxa in theM nudipes complex lack thepink ochraceous or red colors that are typical for Mrufescens Five specimens studied (SAV F-11274 F-11051F-11285 F-11271 F-11053) are morphologically similar toeach other but differ from lectotype of M nudipes anddiffer from other taxa in the ITS region and LSU andRPB2 genes we temporarily maintain these entities underthe name M nudipes aff The ascocarps of M nudipes affmay have in addition to green different tints of brown onthe stipe and according to the phylogenetic data the groupis closely related to M fuscorubens

Occasionally several species of the M nudipes com-plex grow in one site We observed three species (Mtruncatum M pretense and M nudipes aff) at Gruacuteň inthe Biele Karpaty Mts Slovakia where the ascocarpswere growing in a range of less than 2 m

Taxa of green naked-stipe species preferentially growamong grass on soil with calcareous bedrock in openspaces such as mowed meadows or pastures It is alsopossible to find them in forest or bushes with Quercus spBuxus sp Chamaecyparis sp or Laurus sp especially inwarmer regions The main period of sporulation is fromOctober to January depending on geographic location andprecipitation

ACKNOWLEDGMENTS

The authors thank P Marstad T Kristiansen S and J MMoingeon Z Egertovaacute J Hernanz K Bergelin M A RibesRipoll M Della Maggiora V Kaounas P Tanchaud JDubus J P Priou E Popov E Smikovaacute P Smik APolhorskyacute M Krivuš V Kautman and I Kautmanovaacute forgraciously providing specimens for study and analyses andcurators of fungaria O PC LE BRA and SAV for the loan ofspecimens Alvalab is acknowledged for processing selectedDNA samples We acknowledge the following sources offunding Slovak Academy of Sciences (grant VEGA 2000815) The European Social Fund and the State Budget of theCzech Republic Project No CZ1072300200265

MYCOLOGIA 53

ORCID

Viktor Kučera httporcidorg0000-0002-9815-4782Pavel Lizoň httporcidorg0000-0003-0283-2831

LITERATURE CITED

Boudier JLEacute 1907 Histoire et classification des DiscomycegravetesdrsquoEurope Paris France Libraire des SciencesNaturelles 223 p

Boudier JLEacute 1917 Dernieacuteres eacutetincelles mycologiquesBulletin de la Socieacuteteacute mycologique de France 337ndash22

Brock PM Doring H Bidartondo MI 2009 How to knowunknown fungi the role of a herbarium New Phytologist181719ndash724

Darriba D Taboada GL Doallo R Posada D 2012jModelTest 2 more models new heuristics and parallelcomputing Nature Methods 9772ndash773

Durand EJ 1908 The Geoglossaceae of North AmericaAnnales Mycologici 6387ndash477

Gillet CC 1879 Champignons de France Les DiscomycegravetesVol 1 Alenccedilon France E De Broise 230 p

Hansen K LoBuglio KF Pfister DH 2005 Evolutionaryrelationships of the cup-fungus genus Peziza andPezizaceae inferred from multiple nuclear genes RPB2β-tubulin and LSU rDNA Molecular Phylogenetics andEvolution 361ndash23

Hustad VP Miller AN Dentinger BTM Cannon PF 2013Generic circumscriptions in Geoglossomycetes Persoonia31100ndash111

Hustad VP Miller AN Moingeon JM Priou JP 2011 Inclusionof Nothomitra in Geoglossomycetes Mycosphere 2646ndash654

Iglesias P Arauzo S 2013 Un Microglossum nuevo recolec-tado en Orintildeoacuten (Cantabria) Errotari 1013ndash26

Imai S 1942 Contributiones ad studia monographicaGeoglossacearum Botanical Magazine Tokyo 56523ndash527

Katoh K Toh H 2008 Recent developments in the MAFFTmultiple sequence alignment program Briefings inBioinformatics 9276ndash285

Kučera V Lizoň P Tomšovskyacute M 2014a A new greenearth-tongue Microglossum parvisporum sp nov Sydowia66335ndash343

Kučera V Lizoň P Tomšovskyacute M Kučera J Gaisler J 2014b Re-evaluation of the morphological variability of Microglossumviride and M griseoviride sp nov Mycologia 106282ndash290

Liu YL Whelen S Hall BD 1999 Phylogenetic relationshipsamong ascomycetes evidence from an RNA polymerase IIsubunit Molecular Biology and Evolution 161799ndash1808

Moingeon S Moingeon JM 2004 Contributions agrave lrsquoeacutetude desGeoglossaceae agrave spores hyalines Miscellanea Mycologica80ndash8125ndash35

Moncalvo JM Lutzoni FM Rehner SA Johson J Vilgalys R2000 Phylogenetic relationships of agaric fungi based onnuclear large subunit ribosomal DNA Systematic Biology49278ndash305

Ohenoja E Wang Z Townsend JP Mitchel D Voitk A 2010Northern species of earth tongue genus Thuemenidiumrevisited considering morphology ecology and molecularphylogeny Mycologia 1021089ndash1095

Persoon CH 1794 Neuer Versuch einer systematischenEintheilung der Schwaumlmme Neues Magazin fuumlr dieBotanik 163ndash128

Persoon CH 1796 Observationes mycologicae Vol 1Leipzig Apud Petrum Philippum Wolf 115 p

Rambaut A Suchard MA Xie D Drummond AJ 2014Tracer version 16 [cited 3 June 2016] Available fromhttpbeastbioedacukTracer

Ribes Ripoll MA 2013 Microglossum nudipes Boud (1917)[cited 15 June 2016] httpwwwmicobotanicajaencomRevistaArticulosMARibesRPirineoAragones001Microglossum20nudipes201110092081pdf

Ronquist F Teslenko M van der Mark P Ayres DL DarlingA Houmlhna S Larget B Liu L Suchard MA Huelsenbeck JP2012 MrBayes 32 efficient Bayesian phylogenetic infer-ence and model choice across a large model spaceSystematic Biology 61539ndash542

Saccardo PA 1884 Conspectus generum Discomycetumhucusque cognitorum Botanisches Centralblatt 18213ndash220

Sandnes ACS 2006 Phylogenetic relationships among spe-cies and genera of Geoglossaceae (Helotiales) based on ITSand LSU nrDNA sequences [Cand Scient thesis] OsloNorway University of Oslo 32 p

Schoch CL Wang Z Townsend JP Spatafora JW 2009Geoglossomycetes cl nov Geoglossales ord nov andtaxa above class rank in the Ascomycota tree of lifePersoonia 22129ndash138

Stamatakis A 2014 RAxML version 8 a tool for phylogeneticanalysis and post-analysis of large phylogeniesBioinformatics 301312ndash1313

Tamura K Stecher G Peterson D Filipski A Kumar S 2013MEGA6 Molecular Evolutionary Genetics Analysis ver-sion 60 Molecular Biology and Evolution 302725ndash2729

Thiers B [continuously updated] Index herbariorum a glo-bal directory of public herbaria and associated staff NewYork Botanical Gardenrsquos Virtual Herbarium [cited 27 June2016] Available from httpsweetgumnybgorgih

Van Brummelen J 1985 Introductory note to the re-editionof Boudierrsquos Icones Mycologicae In Van Brummelen JKorf RP Cleacutemencon H Julich W Demoulin V eds VIcones Mycologicae par Emile Boudier V Listepreacuteliminaire amp explication des planches [reprint edition]Lausanne Switzerland Editions Piantanida p xxivndashxxvi

Wang Z Binder M Hibbett DS 2005 Life history andsystematics of the aquatic discomycete Mitrula(Helotiales Ascomycota) based on cultural morphologicaland molecular studies American Journal of Botany921565ndash1574

Wang Z Binder M Schoch CL Johnston PR Spatafora JWHibbett DS 2006 Evolution of helotialean fungi(Leotiomycetes Pezizomycotina) a nuclear rDNA phylo-geny Molecular Phylogenetics and Evolution 41295ndash312

White TJ Bruns TD Lee SB Taylor JW 1990 Amplificationand direct sequencing of fungal ribosomal RNA genes forphylogenetics In Innis MA Gelfand DH Sninsky JJWhite TJ eds PCR protocols a guide to methods andapplications New York NY Academic Press p 315ndash322

54 V KUČERA ET AL NEW MICROGLOSSUM TAXA

Microglossum parvisporumV Kučera Lizoňamp Tomšovskyacutehas the smallest ascospores in this complex of green naked-stipe species

Phylogenetic analysesmdashThe aligned ITS data set wascomposed of 513 positions and included 325 conserved159 variable and 36 unique sites as determined by MEGA606 (Tamura et al 2013) The combined ITS-28S-RPB2data set had 2546 positions including 2139 conserved 398variable and 333 unique sites The sequence data sets aredeposited in TreeBASE (Submission ID 19742)

The phylogenetic trees (FIGS 1 2) revealed that theMnudipes complex was composed of M fuscorubens BoudM parvisporum and four new species M clavatum Mpratense M tenebrosum andM truncatum Microglossumnudipes sensu stricto still seems to be variable and could bean aggregate of more species so we use the indication Mnudipes aff in this paper The M nudipes complex is

proximal to M rufum M rufescens M olivaceum andM cyanobasis The previously reported sequenced speci-men M cf nudipes (SAV 10024) (Kučera et al 2014b) fellwithinM pratense Microglossum griseoviride andM virideform a basal clade

TAXONOMY

Microglossum Gillet Champignons de France Discom125 1879

= Helote Hazsl Magyar Tud Akad Eacutertes 11(19)81881

= Ochroglossum S Imai Sci Rep Yokohama NatlUniv Sect 2 46 1955

Type species Microglossum viride (Pers Fr) GilletChampignons de France Discom 125 1879

Ascocarps fleshy erect stipitate clavate ascigerousportion only in upper part with the general form and

Figure 1 Phylogenetic tree of the internal transcribed spacer (ITS) region conducted by Bayesian analysis (for legends to collectionnumbers see SUPPLEMENTARY TABLE 1) Numbers at branches indicate maximum likelihood bootstrap proportion (left) andBayesian posterior probability (right) values The type specimens are highlighted in bold The asterisk () indicates different topologyin the two analyses Bar = number of expected substitutions per position

48 V KUČERA ET AL NEW MICROGLOSSUM TAXA

habit resembling species of the genus Geoglossum cla-vate or tongue-shaped somewhat compressed alwayscolorful (green brown or reddish brown olivaceousyellow clay) never dark brown or black Asci clavate-cylindrical opening by an amyloid pore Ascospores 8biseriate hyaline smooth ellipsoidal fusiform orcylindrical to oblong aseptate or septate paraphysesstraight slightly curved or circinate at the apex

Two basic groups of green earth tongues can bedistinguished easily (i) a group characterized by scalystipes including M viride M rickii Imai and M gri-seoviride (Kučera et al 2014b) and (ii) a group withnaked smooth stipe including M nudipes and M oli-vaceum both in a broad sense

KEY TO GREEN MICROGLOSSUM SPECIES

1 Stipe covered with scales ascocarp green yellowish-green grayish-green apex of paraphyses with a ldquocaprdquoof pigment 2

1prime Stipe naked green pinkish-green olive-greenblue-green apex of paraphyses lacking a ldquocaprdquo ofpigment 4

2 Ascocarp lt15 cm tall asci lt80 μm (65ndash75 times 9ndash10μm) long ascospores 10ndash14 times 4ndash5 μm known onlyfrom Brasilia M rickiib

2prime Ascocarps lt8 cm tall asci gt80 μm long 3

3 Ascocarp yellow-green growing in wet places oftenwith liverworts asci 106ndash134 times 95ndash12 μm ascos-pores 18ndash22 times 5ndash7 μm M viridea

3prime Ascocarp gray-green growing on soil in the forestasci 105ndash139 times 8ndash10 μm ascospores 16ndash20 times 4ndash5μm M griseoviridea

4 Stipe constituting 12ndash34 of the mature ascocarpdominant color of the hymenium buff pink olivestipe is light brown usually mixed with other colorsascospores 13ndash15 times 4 μm M rufescensc

4prime Stipe equal to or shorter in length than hyme-nium ascocarps without pink and olivecolor 5

Figure 2 Phylogenetic tree for a combined analysis of the ITS region and 28S and RPB2 genes conducted by Bayesian analysis (forlegends to collection numbers see SUPPLEMENTARY TABLE 1) Numbers at branches indicate maximum likelihood bootstrapproportion (left) and Bayesian posterior probability (right) values Type specimens are highlighted in bold Bar = number ofexpected substitutions per position

MYCOLOGIA 49

5 Ascospores le16 um long growing in open areas oncalcareous bedrock 6

5prime Ascospores gt16 um long growing in mesophilousmeadows or forests 7

6 Ascocarps white-green when old with violac-eous color asci lt80 μm long M parvisporuma

6prime Ascocarps dark green when old dark greenwith dark violet tint asci 80ndash95 μm long M tenebrosum

7 Asci gt105 μm long growing in theforests 8

7prime Asci lt105 μm long growing on mesophilousmeadows pastures 9

8 Ascocarp green hymenium with brownish tintstipe blue-green paraphyses branched also inupper 13 and in the middle 108prime Ascocarp with brown color present also on thestipe paraphyses tips swollen lt5 μm branched inbasal 13 M nudipes aff

9 Hymenium concolorous with sterile ascospores135ndash165 times 4ndash5 μm M pratense

9prime Hymenium usually brownish-green or brownstipe blue-green ascospores 15ndash18 times 4ndash5μm M truncatum

10 Ascocarp flattened hymenium club-shapedusually longer than the stipe paraphyses branchedat the base and in the middle M clavatum10prime Hymeniummace-shaped as long as stipe para-physes branched both at basal and apical part M nudipesd

In the sense of aKučera et al 2014a 2014b bImai 1942cMoingeon 2004 dBoudier 1917

Microglossum nudipes Boud Bull Soc Mycol France3316 1917

Typification FRANCE Vienne Savigneacute Dec 1913A Greacutelet (lectotype designated here ex herb BoudierPC 0139818) MBT 373899

Description of the lectotype Ascocarps (10ndash)168ndash313(ndash38) mm high tongue-like or club-shaped stipitateHymenium (5ndash)78ndash152(ndash18) times 1ndash38(ndash6) mm mace-shaped cylindrical clavate truncate or lanceolate long-itudinally grooved sometimes twisted and almost lacu-nose or compressed on the side glabrous nakedsubolivaceous dark green or grayish-green Hymeniumconstituting approximately the upper 12 of the ascocarpStipe (5ndash)86ndash166(ndash20) times 05ndash4 mm cylindrical some-times flexuous naked blue-green or concolorous withhymenium not paler at the base flesh yellow-green (20dry ascocarps examined) Asci (80ndash)914ndash1103(ndash130) times(6ndash)71ndash95(ndash11) μm 8-spored clavate rounded at the

apex narrowly tapered towards the base biseriate aboveuniseriate below pore dark blue in Melzerrsquos reagentAscospores (12ndash)166ndash205(ndash23) times (4ndash)42ndash52 μm Qvalue = 24ndash55 (av = 39) usually slightly curved orsigmoid ends obtuse or tapering hyaline without clearlyvisible lipid bodies true septa not observed Paraphysesfiliform 1ndash2 μmwide straight branched at the base someof the paraphyses branched in apical part apical cellsfiliform (1ndash)11ndash25(ndash3) μm or rarely slightly swollenlt35 μm

Habitat Growing among the mosses underBuxus sp

Distribution FranceAdditional specimens examined See

SUPPLEMENTARY TABLE 2Notes Microglossum nudipes was described by Boudier

(1917) from Savigneacute (Vienne France) in December 1913where it was as noted by him first collected by D Arnouldand consequently by L Greacutelet The description presentedboth in Latin and French has important data but unfortu-nately lacks details to allow it to be connected unequivo-cally to a type specimen There are four specimens in PClabeled Microglossum nudipes all collected by Greacutelet twodated 1913 two collected later in 1938 Arnould is notmentioned as collector on any label The collections from1938 are not conspecific with those from 1913 and accord-ing to our observations represent M rufescens MoreoverBoudierrsquos description does not fully correspondwith any ofthe specimens As noted by Van Brummelen (1985)Boudierrsquos microscopic measurements were often exagger-ated in his descriptions as a result of an error in theconstruction of hismeasuring scale and his sporemeasure-ments after 1885 were usually about 10 too large Whenmeasurement of asci and spore size in his descriptions areadjusted by lowering them by 20 allowing for shrinkageby drying and Boudierrsquos inaccurate scale they roughlycorrespond with our observations of PC 0139818 Thuswe emended the description of Microglossum nudipes toinclude these details DNA studies using Boudierrsquos originalmaterial were not done because of the age of the specimenUnfortunately no specimen among recently collectedmaterial corresponds with the lectotype of M nudipesand the second collection gathered from the type localityin 1913 Our visit to the original locality in France in 2015to recollect the species was unsuccessful

Microglossum truncatum V Kučera Tomšovskyacute ampLizoň sp nov FIG 3a bMycoBank MB817350

Typification SLOVAKIA Biele Karpaty Mts NovaacuteBošaacuteca ca 17 km SE from the church Španie settle-ment Natural Monument Blažejovaacute (48deg52prime333PrimeN17deg49prime034PrimeE alt 415 m) in a meadow 7 Nov 2013

50 V KUČERA ET AL NEW MICROGLOSSUM TAXA

V Kučera V Kautman and I Kautmanovaacute (holotypeSAV F-11280) DNA sequences from the holotypeITS = KX382861 28S = KX382861 RPB2 = KX382875

Etymology From truncatus (Latin) ending abruptlyascocarps truncate when young

Ascocarps (19ndash)254ndash443(ndash55) [(20ndash)302ndash555(ndash60)]mm high tongue-like or club-shaped stipitateHymenium (8ndash)222ndash285(ndash30) times (1ndash)32ndash41(ndash7) [(10ndash)253ndash323(ndash44) times (1ndash)31ndash52(ndash8)] mm cylindrical club-shaped truncate or lanceolate only slightly grooved ver-tically glabrous naked brownish-green or light brown orgreen-brown dark green or brownish-green when dryHymenium usually constituting more than the upper 12of the ascocarp Stipe (10ndash)152ndash201(ndash27) times 1ndash22(ndash3)[(11ndash)165ndash236(ndash29) times 12ndash31(ndash4)] mm cylindricalblue-green not paler at the base (15 ascocarps examined)Asci (75ndash)851ndash987(ndash105) times (8ndash)84ndash96(ndash10) [(95ndash)1206ndash1353(ndash150) times (8ndash)101ndash115(12)] μm 8-sporedclavate rounded at the apex narrowly tapered towardsthe base biseriate above uniseriate below pore bluing in

Melzerrsquos reagent Ascospores (14ndash)154ndash183(ndash20) times 4ndash47(ndash5) [(17ndash)182ndash198(ndash21) times 4ndash5(55)] μm Q value = 33ndash48 (av = 38) hyaline ellipsoidal to oblong aseptateParaphyses filiform straight branched in basal partsome of the paraphyses branched in apical part apicalcells filiform 1ndash2 μm or only slightly swollen (lt3 μm)

Habitat On soil among grass in mesophilous mea-dows with soil pH 57ndash61

Distribution France Russia Slovakia SwedenAdditional specimens examined See

SUPPLEMENTARY TABLE 2Notes Ascocarps of Microglossum truncatum are

brown or brownish-green in the hymenium whereasthe stipe is blue-green and oval in cross-section Themost similar species is M nudipes aff which differs bya brown coloration on the stipe and is usually dumb-bell-shaped in cross-section Microglossum truncatummay be macroscopically confused with M parvisporumbut it clearly differs in the size of asci (85ndash98 times 8ndash9 μmvs 66ndash79 times 6 μm) and ascospores (15ndash18 times 4ndash5 μm vs

Figure 3 Microglossum species with green naked stipes A M truncatum SAV F-11261 (M Krivuš) B M truncatum SAV F-11280 (VKučera) C M pratense SAV-11020 (P Marstad) D M pratense SAV-10024 (V Kučera) E F M tenebrosum SAV F-11278 (F Hampe) GM nudipes aff SAV F-11051 (T Knutsson) H M nudipes aff SAV F-11285 (V Kaounas) I M nudipes aff SAV F-11271 (U Pera) Bars =25 cm (A) and 5 cm (BndashI)

MYCOLOGIA 51

11ndash14 times 3ndash4 μm) and acidity of the substrate (pH 57ndash61 vs pH 635)

Microglossum pratense V Kučera Tomšovskyacute ampLizoň sp nov FIG 3c dMycoBank MB808093

Typification SLOVAKIA Stolickeacute vrchy MtsMuraacutenska Huta village Prednaacute Hora recreation area for-mer ski slope ca 2 km SSW from the village (48deg45prime3124PrimeN 20deg06prime2698PrimeE alt 789 m) in grass 13 Oct 2010 VKautman and V Kučera (holotype SAV F-10024) DNAsequences from the holotype ITS = KC595259 28S =KC595260 RPB2 = KX382880

Etymology from pratum (Latin) meadow growingin meadows and pastures

Ascocarps (17ndash)268ndash496(ndash62) mm high tongue-like or club-shaped stipitate Hymenium (10ndash)124ndash277(ndash40) times (15ndash)22ndash47(ndash9) mm mace-shapedcylindrical clavate truncate or lanceolate verticallygrooved usually twisted glabrous naked blue-greenor gray-green dark green and often with lateral crackswhen dry Hymenium usually occupying the upper 12of the ascocarp or more Stipe (7ndash)118ndash242(ndash32) times1ndash3 mm often flattened and flexuous blue-green orconcolorous with hymenium (54 dry ascocarps exam-ined) Asci (67ndash)781ndash915(ndash105) times (5ndash)71ndash85(ndash9) μm8-spored cylindrical to clavate apex rounded narrowlytapered towards the base biseriate above uniseriatebelow the pore light bluing in Melzerrsquos reagentAscospores (11ndash)135ndash165(ndash20) times (4ndash)45ndash5 μm Qvalue = 32ndash41 (av = 37) ellipsoidal to oblong usuallyslightly curved or sigmoid ends obtuse or taperinghyaline or with several (lt4) lipid bodies real septanot observed Paraphyses filiform straight branchedin basal part some of the paraphyses branched at apicalpart the apical cells filiform 1ndash2) μm or only slightlyswollen lt3 μm

Habitat Meadows or pastures in north and centralEurope on soil among grasses The type collection wasfound in a site with soil pH 52ndash56

Distribution Georgia Norway Russia SlovakiaSweden

Additional specimens examined SeeSUPPLEMENTARY TABLE 2

Notes Microglossum pratense is characterized byhaving green or gray-green hymenium lacking abrown tint Ascocarps are usually produced in biggerclusters of lt10 When dry the ascigerous part oftentransversely cracked The hymenium takes more than ahalf of the whole ascocarp and the stipe is often flat-tened Microglossum pratense could be confused withM tenebrosum or M clavatum but M pratense is palerin color and the ascospores are bigger (135ndash165 times 4ndash5

μm) than in M tenebrosum (12ndash15 times 4ndash45 μm) andsmaller than in M clavatum (15ndash20 times 4ndash5 μm)Collections in fungaria are mostly misidentified asM olivaceum or M rufescens In general M pratensewas the most common green Microglossum species withnaked stipe in the examined areas

Microglossum tenebrosum V Kučera TomšovskyacuteLizoň amp F Hampe sp nov FIG 3e fMycoBank MB817358

Typification GERMANY Hessen LangenthalEberschuumltz (51deg36prime269PrimeN 09deg23prime226PrimeE alt 225 m)calcareous neglected open grassland shell limestonewith Juniperus sp 12 Oct 2009 F Hampe (holotypeSAV F-11278) DNA sequences from the holotype ITS= KX382845 28S = KX382845 RPB2 = KX382891

Etymology From tenebrosus (Latin) dark ascocarpsdark-colored

Ascocarps (27ndash)306ndash482(ndash53) [40ndash60] mmhigh ton-gue-like or club-shaped stipitate Hymenium (14ndash)15ndash257(ndash30) times (21ndash)32ndash5 [20ndash30 times 3ndash6] mm mace-shapedcylindrical clavate truncate or lanceolate verticallygrooved glabrous naked at maturity dark green or darkviolet-green or bronze-green to brown-green Hymeniumusually occupying the upper 12 of the ascocarp or moreStipe (12ndash)132ndash258(ndash33) times (15ndash)25ndash3 [20ndash30 times 25ndash6]mm often flattened and flexuous blue-green and oftenconcolorous with hymenium at the base (11 dry ascocarpsexamined) Asci (70ndash)795ndash954(ndash120) times (6ndash)73ndash86(ndash9)[(100ndash)1154ndash1243(ndash1398) times (75ndash)86ndash92(ndash104)] μm8-spored cylindrical to clavate apex rounded narrowlytapered towards the base biseriate above uniseriatebelow the pore bluing in Melzerrsquos reagent Ascospores(10ndash)118ndash154(ndash20) times (3ndash)37ndash45(ndash55) [(116ndash)138ndash148(ndash17) times (31ndash)42ndash46(ndash57)] μm Q value = 25ndash47(av = 34) [24ndash41 (av = 329)] ellipsoidal to oblongusually slightly curved or sigmoid ends obtuse or taper-ing hyaline or with several (lt4) lipid bodies real septa notobserved Paraphyses filiform straight branched in basalpart some of the paraphyses branched at middle part theapical cells filiform 1ndash2(ndash25) μm or slightly clavate orcapitate (lt3 μm)

Habitat On soil among grasses or in forest clear-ings meadows and pastures with Juniperus sp underBuxus sp and calcareous bedrock in western and cen-tral Europe

Distribution France Germany SpainAdditional specimens examined See

SUPPLEMENTARY TABLE 2Notes Microglossum tenebrosum is characterized by

shades of dark green The color of the hymenium isgenerally repeated at the basal part of the stipeMicroglossum tenebrosum is distinguished by the Q

52 V KUČERA ET AL NEW MICROGLOSSUM TAXA

value of the ascospores (34) compared with M nudipes(39) M clavatum (38) M pratense (37) M truncatum(38) and M parvisporum (305) Most collections of Mtenebrosum are from open spaces and not forests Thecombination of relatively large asci (795ndash95 times 7ndash9 μm)and small ascospores (12ndash15 times 4ndash45 μm) is characteristicThe data for fresh collections were taken from RibesRipoll (2013)Microglossum clavatum V Kučera Lizoň ampTomšovskyacute sp novMycoBank MB817351

Typification SPAIN Huesca Puente de Reina deJaca (42deg34prime0418PrimeN 0deg45prime4464PrimeW 665 m) underBuxus sempervirens in calcareous soil 6 Dec 2006 JHernanz (holotype SAV F-11276) DNA sequencesfrom the holotype ITS = KX382864 28S = KX382864RPB2 = KX382884

Etymology From clava (Latin) club ascocarps typi-cally club-shaped

Ascocarps 15ndash31(ndash37) mm high tongue-like club-shaped or clavate usually compressed stipitateHymenium 10ndash199(ndash25) times 1ndash4 mm clavate truncateor lanceolate slightly vertically grooved glabrousnaked dark green sometimes with a brown tintHymenium occupying more than the upper 12 ofthe ascocarp Stipe (4ndash)52ndash125 times (1ndash)25ndash3 mmcylindrical or flattened concolorous with hymenium(14 dry ascocarps examined) Asci (715ndash)966ndash1178(130) times (65ndash)82ndash101(ndash11) μm 8-spored cylindricalto clavate apex rounded narrowly tapered towardsthe base biseriate above uniseriate below the porebluing in Melzerrsquos reagent Ascospores (14ndash)155ndash204(ndash23) times (4ndash)45ndash5 μm Q value = 3ndash57 (av = 38)ellipsoidal to oblong usually slightly curved or sig-moid ends obtuse or tapering hyaline or with several(lt4) lipid bodies true septa not observed Paraphysesfiliform straight branched in basal part some of theparaphyses branched at middle part the apical cellsfiliform 1ndash2(ndash25) μm or slightly clavate or capitate(lt35 μm)

Habitat On calcareous soil under Buxus sp treeDistribution SpainAdditional specimens examined SeeSUPPLEMENTARY TABLE 2Notes Microglossum clavatum is similar to M

nudipes based on the size of asci and shape of para-physes However the values of all examined charactersof M clavatum are slightly smaller the paraphyses arenot apically branched and the ascus pore does not reactas strongly in Melzerrsquos reagent as it does in M nudipes

Examined specimens of other Microglossum spp SeeSUPPLEMENTARY TABLE 2

DISCUSSION

During our research on theMicroglossumnudipes complexwe recognized sevenwell-delimited taxa Three of themMnudipes (Boudier 1917) M parvisporum (Kučera et al2014a) and M fuscorubens (Boudier 1907) were alreadydescribed Four new species are described in this paper butthere is a high likelihood that additional morphotypes andor taxa in this complex will be discovered

In all species we observed that the apical part of theasci and paraphyses are surrounded by a green-brownamorphous matrix that is lt20 μm thick Moreoverascospores occurring in the apical part of the asci aresmaller than others

Ascocarps of all taxa in theM nudipes complex lack thepink ochraceous or red colors that are typical for Mrufescens Five specimens studied (SAV F-11274 F-11051F-11285 F-11271 F-11053) are morphologically similar toeach other but differ from lectotype of M nudipes anddiffer from other taxa in the ITS region and LSU andRPB2 genes we temporarily maintain these entities underthe name M nudipes aff The ascocarps of M nudipes affmay have in addition to green different tints of brown onthe stipe and according to the phylogenetic data the groupis closely related to M fuscorubens

Occasionally several species of the M nudipes com-plex grow in one site We observed three species (Mtruncatum M pretense and M nudipes aff) at Gruacuteň inthe Biele Karpaty Mts Slovakia where the ascocarpswere growing in a range of less than 2 m

Taxa of green naked-stipe species preferentially growamong grass on soil with calcareous bedrock in openspaces such as mowed meadows or pastures It is alsopossible to find them in forest or bushes with Quercus spBuxus sp Chamaecyparis sp or Laurus sp especially inwarmer regions The main period of sporulation is fromOctober to January depending on geographic location andprecipitation

ACKNOWLEDGMENTS

The authors thank P Marstad T Kristiansen S and J MMoingeon Z Egertovaacute J Hernanz K Bergelin M A RibesRipoll M Della Maggiora V Kaounas P Tanchaud JDubus J P Priou E Popov E Smikovaacute P Smik APolhorskyacute M Krivuš V Kautman and I Kautmanovaacute forgraciously providing specimens for study and analyses andcurators of fungaria O PC LE BRA and SAV for the loan ofspecimens Alvalab is acknowledged for processing selectedDNA samples We acknowledge the following sources offunding Slovak Academy of Sciences (grant VEGA 2000815) The European Social Fund and the State Budget of theCzech Republic Project No CZ1072300200265

MYCOLOGIA 53

ORCID

Viktor Kučera httporcidorg0000-0002-9815-4782Pavel Lizoň httporcidorg0000-0003-0283-2831

LITERATURE CITED

Boudier JLEacute 1907 Histoire et classification des DiscomycegravetesdrsquoEurope Paris France Libraire des SciencesNaturelles 223 p

Boudier JLEacute 1917 Dernieacuteres eacutetincelles mycologiquesBulletin de la Socieacuteteacute mycologique de France 337ndash22

Brock PM Doring H Bidartondo MI 2009 How to knowunknown fungi the role of a herbarium New Phytologist181719ndash724

Darriba D Taboada GL Doallo R Posada D 2012jModelTest 2 more models new heuristics and parallelcomputing Nature Methods 9772ndash773

Durand EJ 1908 The Geoglossaceae of North AmericaAnnales Mycologici 6387ndash477

Gillet CC 1879 Champignons de France Les DiscomycegravetesVol 1 Alenccedilon France E De Broise 230 p

Hansen K LoBuglio KF Pfister DH 2005 Evolutionaryrelationships of the cup-fungus genus Peziza andPezizaceae inferred from multiple nuclear genes RPB2β-tubulin and LSU rDNA Molecular Phylogenetics andEvolution 361ndash23

Hustad VP Miller AN Dentinger BTM Cannon PF 2013Generic circumscriptions in Geoglossomycetes Persoonia31100ndash111

Hustad VP Miller AN Moingeon JM Priou JP 2011 Inclusionof Nothomitra in Geoglossomycetes Mycosphere 2646ndash654

Iglesias P Arauzo S 2013 Un Microglossum nuevo recolec-tado en Orintildeoacuten (Cantabria) Errotari 1013ndash26

Imai S 1942 Contributiones ad studia monographicaGeoglossacearum Botanical Magazine Tokyo 56523ndash527

Katoh K Toh H 2008 Recent developments in the MAFFTmultiple sequence alignment program Briefings inBioinformatics 9276ndash285

Kučera V Lizoň P Tomšovskyacute M 2014a A new greenearth-tongue Microglossum parvisporum sp nov Sydowia66335ndash343

Kučera V Lizoň P Tomšovskyacute M Kučera J Gaisler J 2014b Re-evaluation of the morphological variability of Microglossumviride and M griseoviride sp nov Mycologia 106282ndash290

Liu YL Whelen S Hall BD 1999 Phylogenetic relationshipsamong ascomycetes evidence from an RNA polymerase IIsubunit Molecular Biology and Evolution 161799ndash1808

Moingeon S Moingeon JM 2004 Contributions agrave lrsquoeacutetude desGeoglossaceae agrave spores hyalines Miscellanea Mycologica80ndash8125ndash35

Moncalvo JM Lutzoni FM Rehner SA Johson J Vilgalys R2000 Phylogenetic relationships of agaric fungi based onnuclear large subunit ribosomal DNA Systematic Biology49278ndash305

Ohenoja E Wang Z Townsend JP Mitchel D Voitk A 2010Northern species of earth tongue genus Thuemenidiumrevisited considering morphology ecology and molecularphylogeny Mycologia 1021089ndash1095

Persoon CH 1794 Neuer Versuch einer systematischenEintheilung der Schwaumlmme Neues Magazin fuumlr dieBotanik 163ndash128

Persoon CH 1796 Observationes mycologicae Vol 1Leipzig Apud Petrum Philippum Wolf 115 p

Rambaut A Suchard MA Xie D Drummond AJ 2014Tracer version 16 [cited 3 June 2016] Available fromhttpbeastbioedacukTracer

Ribes Ripoll MA 2013 Microglossum nudipes Boud (1917)[cited 15 June 2016] httpwwwmicobotanicajaencomRevistaArticulosMARibesRPirineoAragones001Microglossum20nudipes201110092081pdf

Ronquist F Teslenko M van der Mark P Ayres DL DarlingA Houmlhna S Larget B Liu L Suchard MA Huelsenbeck JP2012 MrBayes 32 efficient Bayesian phylogenetic infer-ence and model choice across a large model spaceSystematic Biology 61539ndash542

Saccardo PA 1884 Conspectus generum Discomycetumhucusque cognitorum Botanisches Centralblatt 18213ndash220

Sandnes ACS 2006 Phylogenetic relationships among spe-cies and genera of Geoglossaceae (Helotiales) based on ITSand LSU nrDNA sequences [Cand Scient thesis] OsloNorway University of Oslo 32 p

Schoch CL Wang Z Townsend JP Spatafora JW 2009Geoglossomycetes cl nov Geoglossales ord nov andtaxa above class rank in the Ascomycota tree of lifePersoonia 22129ndash138

Stamatakis A 2014 RAxML version 8 a tool for phylogeneticanalysis and post-analysis of large phylogeniesBioinformatics 301312ndash1313

Tamura K Stecher G Peterson D Filipski A Kumar S 2013MEGA6 Molecular Evolutionary Genetics Analysis ver-sion 60 Molecular Biology and Evolution 302725ndash2729

Thiers B [continuously updated] Index herbariorum a glo-bal directory of public herbaria and associated staff NewYork Botanical Gardenrsquos Virtual Herbarium [cited 27 June2016] Available from httpsweetgumnybgorgih

Van Brummelen J 1985 Introductory note to the re-editionof Boudierrsquos Icones Mycologicae In Van Brummelen JKorf RP Cleacutemencon H Julich W Demoulin V eds VIcones Mycologicae par Emile Boudier V Listepreacuteliminaire amp explication des planches [reprint edition]Lausanne Switzerland Editions Piantanida p xxivndashxxvi

Wang Z Binder M Hibbett DS 2005 Life history andsystematics of the aquatic discomycete Mitrula(Helotiales Ascomycota) based on cultural morphologicaland molecular studies American Journal of Botany921565ndash1574

Wang Z Binder M Schoch CL Johnston PR Spatafora JWHibbett DS 2006 Evolution of helotialean fungi(Leotiomycetes Pezizomycotina) a nuclear rDNA phylo-geny Molecular Phylogenetics and Evolution 41295ndash312

White TJ Bruns TD Lee SB Taylor JW 1990 Amplificationand direct sequencing of fungal ribosomal RNA genes forphylogenetics In Innis MA Gelfand DH Sninsky JJWhite TJ eds PCR protocols a guide to methods andapplications New York NY Academic Press p 315ndash322

54 V KUČERA ET AL NEW MICROGLOSSUM TAXA

habit resembling species of the genus Geoglossum cla-vate or tongue-shaped somewhat compressed alwayscolorful (green brown or reddish brown olivaceousyellow clay) never dark brown or black Asci clavate-cylindrical opening by an amyloid pore Ascospores 8biseriate hyaline smooth ellipsoidal fusiform orcylindrical to oblong aseptate or septate paraphysesstraight slightly curved or circinate at the apex

Two basic groups of green earth tongues can bedistinguished easily (i) a group characterized by scalystipes including M viride M rickii Imai and M gri-seoviride (Kučera et al 2014b) and (ii) a group withnaked smooth stipe including M nudipes and M oli-vaceum both in a broad sense

KEY TO GREEN MICROGLOSSUM SPECIES

1 Stipe covered with scales ascocarp green yellowish-green grayish-green apex of paraphyses with a ldquocaprdquoof pigment 2

1prime Stipe naked green pinkish-green olive-greenblue-green apex of paraphyses lacking a ldquocaprdquo ofpigment 4

2 Ascocarp lt15 cm tall asci lt80 μm (65ndash75 times 9ndash10μm) long ascospores 10ndash14 times 4ndash5 μm known onlyfrom Brasilia M rickiib

2prime Ascocarps lt8 cm tall asci gt80 μm long 3

3 Ascocarp yellow-green growing in wet places oftenwith liverworts asci 106ndash134 times 95ndash12 μm ascos-pores 18ndash22 times 5ndash7 μm M viridea

3prime Ascocarp gray-green growing on soil in the forestasci 105ndash139 times 8ndash10 μm ascospores 16ndash20 times 4ndash5μm M griseoviridea

4 Stipe constituting 12ndash34 of the mature ascocarpdominant color of the hymenium buff pink olivestipe is light brown usually mixed with other colorsascospores 13ndash15 times 4 μm M rufescensc

4prime Stipe equal to or shorter in length than hyme-nium ascocarps without pink and olivecolor 5

Figure 2 Phylogenetic tree for a combined analysis of the ITS region and 28S and RPB2 genes conducted by Bayesian analysis (forlegends to collection numbers see SUPPLEMENTARY TABLE 1) Numbers at branches indicate maximum likelihood bootstrapproportion (left) and Bayesian posterior probability (right) values Type specimens are highlighted in bold Bar = number ofexpected substitutions per position

MYCOLOGIA 49

5 Ascospores le16 um long growing in open areas oncalcareous bedrock 6

5prime Ascospores gt16 um long growing in mesophilousmeadows or forests 7

6 Ascocarps white-green when old with violac-eous color asci lt80 μm long M parvisporuma

6prime Ascocarps dark green when old dark greenwith dark violet tint asci 80ndash95 μm long M tenebrosum

7 Asci gt105 μm long growing in theforests 8

7prime Asci lt105 μm long growing on mesophilousmeadows pastures 9

8 Ascocarp green hymenium with brownish tintstipe blue-green paraphyses branched also inupper 13 and in the middle 108prime Ascocarp with brown color present also on thestipe paraphyses tips swollen lt5 μm branched inbasal 13 M nudipes aff

9 Hymenium concolorous with sterile ascospores135ndash165 times 4ndash5 μm M pratense

9prime Hymenium usually brownish-green or brownstipe blue-green ascospores 15ndash18 times 4ndash5μm M truncatum

10 Ascocarp flattened hymenium club-shapedusually longer than the stipe paraphyses branchedat the base and in the middle M clavatum10prime Hymeniummace-shaped as long as stipe para-physes branched both at basal and apical part M nudipesd

In the sense of aKučera et al 2014a 2014b bImai 1942cMoingeon 2004 dBoudier 1917

Microglossum nudipes Boud Bull Soc Mycol France3316 1917

Typification FRANCE Vienne Savigneacute Dec 1913A Greacutelet (lectotype designated here ex herb BoudierPC 0139818) MBT 373899

Description of the lectotype Ascocarps (10ndash)168ndash313(ndash38) mm high tongue-like or club-shaped stipitateHymenium (5ndash)78ndash152(ndash18) times 1ndash38(ndash6) mm mace-shaped cylindrical clavate truncate or lanceolate long-itudinally grooved sometimes twisted and almost lacu-nose or compressed on the side glabrous nakedsubolivaceous dark green or grayish-green Hymeniumconstituting approximately the upper 12 of the ascocarpStipe (5ndash)86ndash166(ndash20) times 05ndash4 mm cylindrical some-times flexuous naked blue-green or concolorous withhymenium not paler at the base flesh yellow-green (20dry ascocarps examined) Asci (80ndash)914ndash1103(ndash130) times(6ndash)71ndash95(ndash11) μm 8-spored clavate rounded at the

apex narrowly tapered towards the base biseriate aboveuniseriate below pore dark blue in Melzerrsquos reagentAscospores (12ndash)166ndash205(ndash23) times (4ndash)42ndash52 μm Qvalue = 24ndash55 (av = 39) usually slightly curved orsigmoid ends obtuse or tapering hyaline without clearlyvisible lipid bodies true septa not observed Paraphysesfiliform 1ndash2 μmwide straight branched at the base someof the paraphyses branched in apical part apical cellsfiliform (1ndash)11ndash25(ndash3) μm or rarely slightly swollenlt35 μm

Habitat Growing among the mosses underBuxus sp

Distribution FranceAdditional specimens examined See

SUPPLEMENTARY TABLE 2Notes Microglossum nudipes was described by Boudier

(1917) from Savigneacute (Vienne France) in December 1913where it was as noted by him first collected by D Arnouldand consequently by L Greacutelet The description presentedboth in Latin and French has important data but unfortu-nately lacks details to allow it to be connected unequivo-cally to a type specimen There are four specimens in PClabeled Microglossum nudipes all collected by Greacutelet twodated 1913 two collected later in 1938 Arnould is notmentioned as collector on any label The collections from1938 are not conspecific with those from 1913 and accord-ing to our observations represent M rufescens MoreoverBoudierrsquos description does not fully correspondwith any ofthe specimens As noted by Van Brummelen (1985)Boudierrsquos microscopic measurements were often exagger-ated in his descriptions as a result of an error in theconstruction of hismeasuring scale and his sporemeasure-ments after 1885 were usually about 10 too large Whenmeasurement of asci and spore size in his descriptions areadjusted by lowering them by 20 allowing for shrinkageby drying and Boudierrsquos inaccurate scale they roughlycorrespond with our observations of PC 0139818 Thuswe emended the description of Microglossum nudipes toinclude these details DNA studies using Boudierrsquos originalmaterial were not done because of the age of the specimenUnfortunately no specimen among recently collectedmaterial corresponds with the lectotype of M nudipesand the second collection gathered from the type localityin 1913 Our visit to the original locality in France in 2015to recollect the species was unsuccessful

Microglossum truncatum V Kučera Tomšovskyacute ampLizoň sp nov FIG 3a bMycoBank MB817350

Typification SLOVAKIA Biele Karpaty Mts NovaacuteBošaacuteca ca 17 km SE from the church Španie settle-ment Natural Monument Blažejovaacute (48deg52prime333PrimeN17deg49prime034PrimeE alt 415 m) in a meadow 7 Nov 2013

50 V KUČERA ET AL NEW MICROGLOSSUM TAXA

V Kučera V Kautman and I Kautmanovaacute (holotypeSAV F-11280) DNA sequences from the holotypeITS = KX382861 28S = KX382861 RPB2 = KX382875

Etymology From truncatus (Latin) ending abruptlyascocarps truncate when young

Ascocarps (19ndash)254ndash443(ndash55) [(20ndash)302ndash555(ndash60)]mm high tongue-like or club-shaped stipitateHymenium (8ndash)222ndash285(ndash30) times (1ndash)32ndash41(ndash7) [(10ndash)253ndash323(ndash44) times (1ndash)31ndash52(ndash8)] mm cylindrical club-shaped truncate or lanceolate only slightly grooved ver-tically glabrous naked brownish-green or light brown orgreen-brown dark green or brownish-green when dryHymenium usually constituting more than the upper 12of the ascocarp Stipe (10ndash)152ndash201(ndash27) times 1ndash22(ndash3)[(11ndash)165ndash236(ndash29) times 12ndash31(ndash4)] mm cylindricalblue-green not paler at the base (15 ascocarps examined)Asci (75ndash)851ndash987(ndash105) times (8ndash)84ndash96(ndash10) [(95ndash)1206ndash1353(ndash150) times (8ndash)101ndash115(12)] μm 8-sporedclavate rounded at the apex narrowly tapered towardsthe base biseriate above uniseriate below pore bluing in

Melzerrsquos reagent Ascospores (14ndash)154ndash183(ndash20) times 4ndash47(ndash5) [(17ndash)182ndash198(ndash21) times 4ndash5(55)] μm Q value = 33ndash48 (av = 38) hyaline ellipsoidal to oblong aseptateParaphyses filiform straight branched in basal partsome of the paraphyses branched in apical part apicalcells filiform 1ndash2 μm or only slightly swollen (lt3 μm)

Habitat On soil among grass in mesophilous mea-dows with soil pH 57ndash61

Distribution France Russia Slovakia SwedenAdditional specimens examined See

SUPPLEMENTARY TABLE 2Notes Ascocarps of Microglossum truncatum are

brown or brownish-green in the hymenium whereasthe stipe is blue-green and oval in cross-section Themost similar species is M nudipes aff which differs bya brown coloration on the stipe and is usually dumb-bell-shaped in cross-section Microglossum truncatummay be macroscopically confused with M parvisporumbut it clearly differs in the size of asci (85ndash98 times 8ndash9 μmvs 66ndash79 times 6 μm) and ascospores (15ndash18 times 4ndash5 μm vs

Figure 3 Microglossum species with green naked stipes A M truncatum SAV F-11261 (M Krivuš) B M truncatum SAV F-11280 (VKučera) C M pratense SAV-11020 (P Marstad) D M pratense SAV-10024 (V Kučera) E F M tenebrosum SAV F-11278 (F Hampe) GM nudipes aff SAV F-11051 (T Knutsson) H M nudipes aff SAV F-11285 (V Kaounas) I M nudipes aff SAV F-11271 (U Pera) Bars =25 cm (A) and 5 cm (BndashI)

MYCOLOGIA 51

11ndash14 times 3ndash4 μm) and acidity of the substrate (pH 57ndash61 vs pH 635)

Microglossum pratense V Kučera Tomšovskyacute ampLizoň sp nov FIG 3c dMycoBank MB808093

Typification SLOVAKIA Stolickeacute vrchy MtsMuraacutenska Huta village Prednaacute Hora recreation area for-mer ski slope ca 2 km SSW from the village (48deg45prime3124PrimeN 20deg06prime2698PrimeE alt 789 m) in grass 13 Oct 2010 VKautman and V Kučera (holotype SAV F-10024) DNAsequences from the holotype ITS = KC595259 28S =KC595260 RPB2 = KX382880

Etymology from pratum (Latin) meadow growingin meadows and pastures

Ascocarps (17ndash)268ndash496(ndash62) mm high tongue-like or club-shaped stipitate Hymenium (10ndash)124ndash277(ndash40) times (15ndash)22ndash47(ndash9) mm mace-shapedcylindrical clavate truncate or lanceolate verticallygrooved usually twisted glabrous naked blue-greenor gray-green dark green and often with lateral crackswhen dry Hymenium usually occupying the upper 12of the ascocarp or more Stipe (7ndash)118ndash242(ndash32) times1ndash3 mm often flattened and flexuous blue-green orconcolorous with hymenium (54 dry ascocarps exam-ined) Asci (67ndash)781ndash915(ndash105) times (5ndash)71ndash85(ndash9) μm8-spored cylindrical to clavate apex rounded narrowlytapered towards the base biseriate above uniseriatebelow the pore light bluing in Melzerrsquos reagentAscospores (11ndash)135ndash165(ndash20) times (4ndash)45ndash5 μm Qvalue = 32ndash41 (av = 37) ellipsoidal to oblong usuallyslightly curved or sigmoid ends obtuse or taperinghyaline or with several (lt4) lipid bodies real septanot observed Paraphyses filiform straight branchedin basal part some of the paraphyses branched at apicalpart the apical cells filiform 1ndash2) μm or only slightlyswollen lt3 μm

Habitat Meadows or pastures in north and centralEurope on soil among grasses The type collection wasfound in a site with soil pH 52ndash56

Distribution Georgia Norway Russia SlovakiaSweden

Additional specimens examined SeeSUPPLEMENTARY TABLE 2

Notes Microglossum pratense is characterized byhaving green or gray-green hymenium lacking abrown tint Ascocarps are usually produced in biggerclusters of lt10 When dry the ascigerous part oftentransversely cracked The hymenium takes more than ahalf of the whole ascocarp and the stipe is often flat-tened Microglossum pratense could be confused withM tenebrosum or M clavatum but M pratense is palerin color and the ascospores are bigger (135ndash165 times 4ndash5

μm) than in M tenebrosum (12ndash15 times 4ndash45 μm) andsmaller than in M clavatum (15ndash20 times 4ndash5 μm)Collections in fungaria are mostly misidentified asM olivaceum or M rufescens In general M pratensewas the most common green Microglossum species withnaked stipe in the examined areas

Microglossum tenebrosum V Kučera TomšovskyacuteLizoň amp F Hampe sp nov FIG 3e fMycoBank MB817358

Typification GERMANY Hessen LangenthalEberschuumltz (51deg36prime269PrimeN 09deg23prime226PrimeE alt 225 m)calcareous neglected open grassland shell limestonewith Juniperus sp 12 Oct 2009 F Hampe (holotypeSAV F-11278) DNA sequences from the holotype ITS= KX382845 28S = KX382845 RPB2 = KX382891

Etymology From tenebrosus (Latin) dark ascocarpsdark-colored

Ascocarps (27ndash)306ndash482(ndash53) [40ndash60] mmhigh ton-gue-like or club-shaped stipitate Hymenium (14ndash)15ndash257(ndash30) times (21ndash)32ndash5 [20ndash30 times 3ndash6] mm mace-shapedcylindrical clavate truncate or lanceolate verticallygrooved glabrous naked at maturity dark green or darkviolet-green or bronze-green to brown-green Hymeniumusually occupying the upper 12 of the ascocarp or moreStipe (12ndash)132ndash258(ndash33) times (15ndash)25ndash3 [20ndash30 times 25ndash6]mm often flattened and flexuous blue-green and oftenconcolorous with hymenium at the base (11 dry ascocarpsexamined) Asci (70ndash)795ndash954(ndash120) times (6ndash)73ndash86(ndash9)[(100ndash)1154ndash1243(ndash1398) times (75ndash)86ndash92(ndash104)] μm8-spored cylindrical to clavate apex rounded narrowlytapered towards the base biseriate above uniseriatebelow the pore bluing in Melzerrsquos reagent Ascospores(10ndash)118ndash154(ndash20) times (3ndash)37ndash45(ndash55) [(116ndash)138ndash148(ndash17) times (31ndash)42ndash46(ndash57)] μm Q value = 25ndash47(av = 34) [24ndash41 (av = 329)] ellipsoidal to oblongusually slightly curved or sigmoid ends obtuse or taper-ing hyaline or with several (lt4) lipid bodies real septa notobserved Paraphyses filiform straight branched in basalpart some of the paraphyses branched at middle part theapical cells filiform 1ndash2(ndash25) μm or slightly clavate orcapitate (lt3 μm)

Habitat On soil among grasses or in forest clear-ings meadows and pastures with Juniperus sp underBuxus sp and calcareous bedrock in western and cen-tral Europe

Distribution France Germany SpainAdditional specimens examined See

SUPPLEMENTARY TABLE 2Notes Microglossum tenebrosum is characterized by

shades of dark green The color of the hymenium isgenerally repeated at the basal part of the stipeMicroglossum tenebrosum is distinguished by the Q

52 V KUČERA ET AL NEW MICROGLOSSUM TAXA

value of the ascospores (34) compared with M nudipes(39) M clavatum (38) M pratense (37) M truncatum(38) and M parvisporum (305) Most collections of Mtenebrosum are from open spaces and not forests Thecombination of relatively large asci (795ndash95 times 7ndash9 μm)and small ascospores (12ndash15 times 4ndash45 μm) is characteristicThe data for fresh collections were taken from RibesRipoll (2013)Microglossum clavatum V Kučera Lizoň ampTomšovskyacute sp novMycoBank MB817351

Typification SPAIN Huesca Puente de Reina deJaca (42deg34prime0418PrimeN 0deg45prime4464PrimeW 665 m) underBuxus sempervirens in calcareous soil 6 Dec 2006 JHernanz (holotype SAV F-11276) DNA sequencesfrom the holotype ITS = KX382864 28S = KX382864RPB2 = KX382884

Etymology From clava (Latin) club ascocarps typi-cally club-shaped

Ascocarps 15ndash31(ndash37) mm high tongue-like club-shaped or clavate usually compressed stipitateHymenium 10ndash199(ndash25) times 1ndash4 mm clavate truncateor lanceolate slightly vertically grooved glabrousnaked dark green sometimes with a brown tintHymenium occupying more than the upper 12 ofthe ascocarp Stipe (4ndash)52ndash125 times (1ndash)25ndash3 mmcylindrical or flattened concolorous with hymenium(14 dry ascocarps examined) Asci (715ndash)966ndash1178(130) times (65ndash)82ndash101(ndash11) μm 8-spored cylindricalto clavate apex rounded narrowly tapered towardsthe base biseriate above uniseriate below the porebluing in Melzerrsquos reagent Ascospores (14ndash)155ndash204(ndash23) times (4ndash)45ndash5 μm Q value = 3ndash57 (av = 38)ellipsoidal to oblong usually slightly curved or sig-moid ends obtuse or tapering hyaline or with several(lt4) lipid bodies true septa not observed Paraphysesfiliform straight branched in basal part some of theparaphyses branched at middle part the apical cellsfiliform 1ndash2(ndash25) μm or slightly clavate or capitate(lt35 μm)

Habitat On calcareous soil under Buxus sp treeDistribution SpainAdditional specimens examined SeeSUPPLEMENTARY TABLE 2Notes Microglossum clavatum is similar to M

nudipes based on the size of asci and shape of para-physes However the values of all examined charactersof M clavatum are slightly smaller the paraphyses arenot apically branched and the ascus pore does not reactas strongly in Melzerrsquos reagent as it does in M nudipes

Examined specimens of other Microglossum spp SeeSUPPLEMENTARY TABLE 2

DISCUSSION

During our research on theMicroglossumnudipes complexwe recognized sevenwell-delimited taxa Three of themMnudipes (Boudier 1917) M parvisporum (Kučera et al2014a) and M fuscorubens (Boudier 1907) were alreadydescribed Four new species are described in this paper butthere is a high likelihood that additional morphotypes andor taxa in this complex will be discovered

In all species we observed that the apical part of theasci and paraphyses are surrounded by a green-brownamorphous matrix that is lt20 μm thick Moreoverascospores occurring in the apical part of the asci aresmaller than others

Ascocarps of all taxa in theM nudipes complex lack thepink ochraceous or red colors that are typical for Mrufescens Five specimens studied (SAV F-11274 F-11051F-11285 F-11271 F-11053) are morphologically similar toeach other but differ from lectotype of M nudipes anddiffer from other taxa in the ITS region and LSU andRPB2 genes we temporarily maintain these entities underthe name M nudipes aff The ascocarps of M nudipes affmay have in addition to green different tints of brown onthe stipe and according to the phylogenetic data the groupis closely related to M fuscorubens

Occasionally several species of the M nudipes com-plex grow in one site We observed three species (Mtruncatum M pretense and M nudipes aff) at Gruacuteň inthe Biele Karpaty Mts Slovakia where the ascocarpswere growing in a range of less than 2 m

Taxa of green naked-stipe species preferentially growamong grass on soil with calcareous bedrock in openspaces such as mowed meadows or pastures It is alsopossible to find them in forest or bushes with Quercus spBuxus sp Chamaecyparis sp or Laurus sp especially inwarmer regions The main period of sporulation is fromOctober to January depending on geographic location andprecipitation

ACKNOWLEDGMENTS

The authors thank P Marstad T Kristiansen S and J MMoingeon Z Egertovaacute J Hernanz K Bergelin M A RibesRipoll M Della Maggiora V Kaounas P Tanchaud JDubus J P Priou E Popov E Smikovaacute P Smik APolhorskyacute M Krivuš V Kautman and I Kautmanovaacute forgraciously providing specimens for study and analyses andcurators of fungaria O PC LE BRA and SAV for the loan ofspecimens Alvalab is acknowledged for processing selectedDNA samples We acknowledge the following sources offunding Slovak Academy of Sciences (grant VEGA 2000815) The European Social Fund and the State Budget of theCzech Republic Project No CZ1072300200265

MYCOLOGIA 53

ORCID

Viktor Kučera httporcidorg0000-0002-9815-4782Pavel Lizoň httporcidorg0000-0003-0283-2831

LITERATURE CITED

Boudier JLEacute 1907 Histoire et classification des DiscomycegravetesdrsquoEurope Paris France Libraire des SciencesNaturelles 223 p

Boudier JLEacute 1917 Dernieacuteres eacutetincelles mycologiquesBulletin de la Socieacuteteacute mycologique de France 337ndash22

Brock PM Doring H Bidartondo MI 2009 How to knowunknown fungi the role of a herbarium New Phytologist181719ndash724

Darriba D Taboada GL Doallo R Posada D 2012jModelTest 2 more models new heuristics and parallelcomputing Nature Methods 9772ndash773

Durand EJ 1908 The Geoglossaceae of North AmericaAnnales Mycologici 6387ndash477

Gillet CC 1879 Champignons de France Les DiscomycegravetesVol 1 Alenccedilon France E De Broise 230 p

Hansen K LoBuglio KF Pfister DH 2005 Evolutionaryrelationships of the cup-fungus genus Peziza andPezizaceae inferred from multiple nuclear genes RPB2β-tubulin and LSU rDNA Molecular Phylogenetics andEvolution 361ndash23

Hustad VP Miller AN Dentinger BTM Cannon PF 2013Generic circumscriptions in Geoglossomycetes Persoonia31100ndash111

Hustad VP Miller AN Moingeon JM Priou JP 2011 Inclusionof Nothomitra in Geoglossomycetes Mycosphere 2646ndash654

Iglesias P Arauzo S 2013 Un Microglossum nuevo recolec-tado en Orintildeoacuten (Cantabria) Errotari 1013ndash26

Imai S 1942 Contributiones ad studia monographicaGeoglossacearum Botanical Magazine Tokyo 56523ndash527

Katoh K Toh H 2008 Recent developments in the MAFFTmultiple sequence alignment program Briefings inBioinformatics 9276ndash285

Kučera V Lizoň P Tomšovskyacute M 2014a A new greenearth-tongue Microglossum parvisporum sp nov Sydowia66335ndash343

Kučera V Lizoň P Tomšovskyacute M Kučera J Gaisler J 2014b Re-evaluation of the morphological variability of Microglossumviride and M griseoviride sp nov Mycologia 106282ndash290

Liu YL Whelen S Hall BD 1999 Phylogenetic relationshipsamong ascomycetes evidence from an RNA polymerase IIsubunit Molecular Biology and Evolution 161799ndash1808

Moingeon S Moingeon JM 2004 Contributions agrave lrsquoeacutetude desGeoglossaceae agrave spores hyalines Miscellanea Mycologica80ndash8125ndash35

Moncalvo JM Lutzoni FM Rehner SA Johson J Vilgalys R2000 Phylogenetic relationships of agaric fungi based onnuclear large subunit ribosomal DNA Systematic Biology49278ndash305

Ohenoja E Wang Z Townsend JP Mitchel D Voitk A 2010Northern species of earth tongue genus Thuemenidiumrevisited considering morphology ecology and molecularphylogeny Mycologia 1021089ndash1095

Persoon CH 1794 Neuer Versuch einer systematischenEintheilung der Schwaumlmme Neues Magazin fuumlr dieBotanik 163ndash128

Persoon CH 1796 Observationes mycologicae Vol 1Leipzig Apud Petrum Philippum Wolf 115 p

Rambaut A Suchard MA Xie D Drummond AJ 2014Tracer version 16 [cited 3 June 2016] Available fromhttpbeastbioedacukTracer

Ribes Ripoll MA 2013 Microglossum nudipes Boud (1917)[cited 15 June 2016] httpwwwmicobotanicajaencomRevistaArticulosMARibesRPirineoAragones001Microglossum20nudipes201110092081pdf

Ronquist F Teslenko M van der Mark P Ayres DL DarlingA Houmlhna S Larget B Liu L Suchard MA Huelsenbeck JP2012 MrBayes 32 efficient Bayesian phylogenetic infer-ence and model choice across a large model spaceSystematic Biology 61539ndash542

Saccardo PA 1884 Conspectus generum Discomycetumhucusque cognitorum Botanisches Centralblatt 18213ndash220

Sandnes ACS 2006 Phylogenetic relationships among spe-cies and genera of Geoglossaceae (Helotiales) based on ITSand LSU nrDNA sequences [Cand Scient thesis] OsloNorway University of Oslo 32 p

Schoch CL Wang Z Townsend JP Spatafora JW 2009Geoglossomycetes cl nov Geoglossales ord nov andtaxa above class rank in the Ascomycota tree of lifePersoonia 22129ndash138

Stamatakis A 2014 RAxML version 8 a tool for phylogeneticanalysis and post-analysis of large phylogeniesBioinformatics 301312ndash1313

Tamura K Stecher G Peterson D Filipski A Kumar S 2013MEGA6 Molecular Evolutionary Genetics Analysis ver-sion 60 Molecular Biology and Evolution 302725ndash2729

Thiers B [continuously updated] Index herbariorum a glo-bal directory of public herbaria and associated staff NewYork Botanical Gardenrsquos Virtual Herbarium [cited 27 June2016] Available from httpsweetgumnybgorgih

Van Brummelen J 1985 Introductory note to the re-editionof Boudierrsquos Icones Mycologicae In Van Brummelen JKorf RP Cleacutemencon H Julich W Demoulin V eds VIcones Mycologicae par Emile Boudier V Listepreacuteliminaire amp explication des planches [reprint edition]Lausanne Switzerland Editions Piantanida p xxivndashxxvi

Wang Z Binder M Hibbett DS 2005 Life history andsystematics of the aquatic discomycete Mitrula(Helotiales Ascomycota) based on cultural morphologicaland molecular studies American Journal of Botany921565ndash1574

Wang Z Binder M Schoch CL Johnston PR Spatafora JWHibbett DS 2006 Evolution of helotialean fungi(Leotiomycetes Pezizomycotina) a nuclear rDNA phylo-geny Molecular Phylogenetics and Evolution 41295ndash312

White TJ Bruns TD Lee SB Taylor JW 1990 Amplificationand direct sequencing of fungal ribosomal RNA genes forphylogenetics In Innis MA Gelfand DH Sninsky JJWhite TJ eds PCR protocols a guide to methods andapplications New York NY Academic Press p 315ndash322

54 V KUČERA ET AL NEW MICROGLOSSUM TAXA

5 Ascospores le16 um long growing in open areas oncalcareous bedrock 6

5prime Ascospores gt16 um long growing in mesophilousmeadows or forests 7

6 Ascocarps white-green when old with violac-eous color asci lt80 μm long M parvisporuma

6prime Ascocarps dark green when old dark greenwith dark violet tint asci 80ndash95 μm long M tenebrosum

7 Asci gt105 μm long growing in theforests 8

7prime Asci lt105 μm long growing on mesophilousmeadows pastures 9

8 Ascocarp green hymenium with brownish tintstipe blue-green paraphyses branched also inupper 13 and in the middle 108prime Ascocarp with brown color present also on thestipe paraphyses tips swollen lt5 μm branched inbasal 13 M nudipes aff

9 Hymenium concolorous with sterile ascospores135ndash165 times 4ndash5 μm M pratense

9prime Hymenium usually brownish-green or brownstipe blue-green ascospores 15ndash18 times 4ndash5μm M truncatum

10 Ascocarp flattened hymenium club-shapedusually longer than the stipe paraphyses branchedat the base and in the middle M clavatum10prime Hymeniummace-shaped as long as stipe para-physes branched both at basal and apical part M nudipesd

In the sense of aKučera et al 2014a 2014b bImai 1942cMoingeon 2004 dBoudier 1917

Microglossum nudipes Boud Bull Soc Mycol France3316 1917

Typification FRANCE Vienne Savigneacute Dec 1913A Greacutelet (lectotype designated here ex herb BoudierPC 0139818) MBT 373899

Description of the lectotype Ascocarps (10ndash)168ndash313(ndash38) mm high tongue-like or club-shaped stipitateHymenium (5ndash)78ndash152(ndash18) times 1ndash38(ndash6) mm mace-shaped cylindrical clavate truncate or lanceolate long-itudinally grooved sometimes twisted and almost lacu-nose or compressed on the side glabrous nakedsubolivaceous dark green or grayish-green Hymeniumconstituting approximately the upper 12 of the ascocarpStipe (5ndash)86ndash166(ndash20) times 05ndash4 mm cylindrical some-times flexuous naked blue-green or concolorous withhymenium not paler at the base flesh yellow-green (20dry ascocarps examined) Asci (80ndash)914ndash1103(ndash130) times(6ndash)71ndash95(ndash11) μm 8-spored clavate rounded at the

apex narrowly tapered towards the base biseriate aboveuniseriate below pore dark blue in Melzerrsquos reagentAscospores (12ndash)166ndash205(ndash23) times (4ndash)42ndash52 μm Qvalue = 24ndash55 (av = 39) usually slightly curved orsigmoid ends obtuse or tapering hyaline without clearlyvisible lipid bodies true septa not observed Paraphysesfiliform 1ndash2 μmwide straight branched at the base someof the paraphyses branched in apical part apical cellsfiliform (1ndash)11ndash25(ndash3) μm or rarely slightly swollenlt35 μm

Habitat Growing among the mosses underBuxus sp

Distribution FranceAdditional specimens examined See

SUPPLEMENTARY TABLE 2Notes Microglossum nudipes was described by Boudier

(1917) from Savigneacute (Vienne France) in December 1913where it was as noted by him first collected by D Arnouldand consequently by L Greacutelet The description presentedboth in Latin and French has important data but unfortu-nately lacks details to allow it to be connected unequivo-cally to a type specimen There are four specimens in PClabeled Microglossum nudipes all collected by Greacutelet twodated 1913 two collected later in 1938 Arnould is notmentioned as collector on any label The collections from1938 are not conspecific with those from 1913 and accord-ing to our observations represent M rufescens MoreoverBoudierrsquos description does not fully correspondwith any ofthe specimens As noted by Van Brummelen (1985)Boudierrsquos microscopic measurements were often exagger-ated in his descriptions as a result of an error in theconstruction of hismeasuring scale and his sporemeasure-ments after 1885 were usually about 10 too large Whenmeasurement of asci and spore size in his descriptions areadjusted by lowering them by 20 allowing for shrinkageby drying and Boudierrsquos inaccurate scale they roughlycorrespond with our observations of PC 0139818 Thuswe emended the description of Microglossum nudipes toinclude these details DNA studies using Boudierrsquos originalmaterial were not done because of the age of the specimenUnfortunately no specimen among recently collectedmaterial corresponds with the lectotype of M nudipesand the second collection gathered from the type localityin 1913 Our visit to the original locality in France in 2015to recollect the species was unsuccessful

Microglossum truncatum V Kučera Tomšovskyacute ampLizoň sp nov FIG 3a bMycoBank MB817350

Typification SLOVAKIA Biele Karpaty Mts NovaacuteBošaacuteca ca 17 km SE from the church Španie settle-ment Natural Monument Blažejovaacute (48deg52prime333PrimeN17deg49prime034PrimeE alt 415 m) in a meadow 7 Nov 2013

50 V KUČERA ET AL NEW MICROGLOSSUM TAXA

V Kučera V Kautman and I Kautmanovaacute (holotypeSAV F-11280) DNA sequences from the holotypeITS = KX382861 28S = KX382861 RPB2 = KX382875

Etymology From truncatus (Latin) ending abruptlyascocarps truncate when young

Ascocarps (19ndash)254ndash443(ndash55) [(20ndash)302ndash555(ndash60)]mm high tongue-like or club-shaped stipitateHymenium (8ndash)222ndash285(ndash30) times (1ndash)32ndash41(ndash7) [(10ndash)253ndash323(ndash44) times (1ndash)31ndash52(ndash8)] mm cylindrical club-shaped truncate or lanceolate only slightly grooved ver-tically glabrous naked brownish-green or light brown orgreen-brown dark green or brownish-green when dryHymenium usually constituting more than the upper 12of the ascocarp Stipe (10ndash)152ndash201(ndash27) times 1ndash22(ndash3)[(11ndash)165ndash236(ndash29) times 12ndash31(ndash4)] mm cylindricalblue-green not paler at the base (15 ascocarps examined)Asci (75ndash)851ndash987(ndash105) times (8ndash)84ndash96(ndash10) [(95ndash)1206ndash1353(ndash150) times (8ndash)101ndash115(12)] μm 8-sporedclavate rounded at the apex narrowly tapered towardsthe base biseriate above uniseriate below pore bluing in

Melzerrsquos reagent Ascospores (14ndash)154ndash183(ndash20) times 4ndash47(ndash5) [(17ndash)182ndash198(ndash21) times 4ndash5(55)] μm Q value = 33ndash48 (av = 38) hyaline ellipsoidal to oblong aseptateParaphyses filiform straight branched in basal partsome of the paraphyses branched in apical part apicalcells filiform 1ndash2 μm or only slightly swollen (lt3 μm)

Habitat On soil among grass in mesophilous mea-dows with soil pH 57ndash61

Distribution France Russia Slovakia SwedenAdditional specimens examined See

SUPPLEMENTARY TABLE 2Notes Ascocarps of Microglossum truncatum are

brown or brownish-green in the hymenium whereasthe stipe is blue-green and oval in cross-section Themost similar species is M nudipes aff which differs bya brown coloration on the stipe and is usually dumb-bell-shaped in cross-section Microglossum truncatummay be macroscopically confused with M parvisporumbut it clearly differs in the size of asci (85ndash98 times 8ndash9 μmvs 66ndash79 times 6 μm) and ascospores (15ndash18 times 4ndash5 μm vs

Figure 3 Microglossum species with green naked stipes A M truncatum SAV F-11261 (M Krivuš) B M truncatum SAV F-11280 (VKučera) C M pratense SAV-11020 (P Marstad) D M pratense SAV-10024 (V Kučera) E F M tenebrosum SAV F-11278 (F Hampe) GM nudipes aff SAV F-11051 (T Knutsson) H M nudipes aff SAV F-11285 (V Kaounas) I M nudipes aff SAV F-11271 (U Pera) Bars =25 cm (A) and 5 cm (BndashI)

MYCOLOGIA 51

11ndash14 times 3ndash4 μm) and acidity of the substrate (pH 57ndash61 vs pH 635)

Microglossum pratense V Kučera Tomšovskyacute ampLizoň sp nov FIG 3c dMycoBank MB808093

Typification SLOVAKIA Stolickeacute vrchy MtsMuraacutenska Huta village Prednaacute Hora recreation area for-mer ski slope ca 2 km SSW from the village (48deg45prime3124PrimeN 20deg06prime2698PrimeE alt 789 m) in grass 13 Oct 2010 VKautman and V Kučera (holotype SAV F-10024) DNAsequences from the holotype ITS = KC595259 28S =KC595260 RPB2 = KX382880

Etymology from pratum (Latin) meadow growingin meadows and pastures

Ascocarps (17ndash)268ndash496(ndash62) mm high tongue-like or club-shaped stipitate Hymenium (10ndash)124ndash277(ndash40) times (15ndash)22ndash47(ndash9) mm mace-shapedcylindrical clavate truncate or lanceolate verticallygrooved usually twisted glabrous naked blue-greenor gray-green dark green and often with lateral crackswhen dry Hymenium usually occupying the upper 12of the ascocarp or more Stipe (7ndash)118ndash242(ndash32) times1ndash3 mm often flattened and flexuous blue-green orconcolorous with hymenium (54 dry ascocarps exam-ined) Asci (67ndash)781ndash915(ndash105) times (5ndash)71ndash85(ndash9) μm8-spored cylindrical to clavate apex rounded narrowlytapered towards the base biseriate above uniseriatebelow the pore light bluing in Melzerrsquos reagentAscospores (11ndash)135ndash165(ndash20) times (4ndash)45ndash5 μm Qvalue = 32ndash41 (av = 37) ellipsoidal to oblong usuallyslightly curved or sigmoid ends obtuse or taperinghyaline or with several (lt4) lipid bodies real septanot observed Paraphyses filiform straight branchedin basal part some of the paraphyses branched at apicalpart the apical cells filiform 1ndash2) μm or only slightlyswollen lt3 μm

Habitat Meadows or pastures in north and centralEurope on soil among grasses The type collection wasfound in a site with soil pH 52ndash56

Distribution Georgia Norway Russia SlovakiaSweden

Additional specimens examined SeeSUPPLEMENTARY TABLE 2

Notes Microglossum pratense is characterized byhaving green or gray-green hymenium lacking abrown tint Ascocarps are usually produced in biggerclusters of lt10 When dry the ascigerous part oftentransversely cracked The hymenium takes more than ahalf of the whole ascocarp and the stipe is often flat-tened Microglossum pratense could be confused withM tenebrosum or M clavatum but M pratense is palerin color and the ascospores are bigger (135ndash165 times 4ndash5

μm) than in M tenebrosum (12ndash15 times 4ndash45 μm) andsmaller than in M clavatum (15ndash20 times 4ndash5 μm)Collections in fungaria are mostly misidentified asM olivaceum or M rufescens In general M pratensewas the most common green Microglossum species withnaked stipe in the examined areas

Microglossum tenebrosum V Kučera TomšovskyacuteLizoň amp F Hampe sp nov FIG 3e fMycoBank MB817358

Typification GERMANY Hessen LangenthalEberschuumltz (51deg36prime269PrimeN 09deg23prime226PrimeE alt 225 m)calcareous neglected open grassland shell limestonewith Juniperus sp 12 Oct 2009 F Hampe (holotypeSAV F-11278) DNA sequences from the holotype ITS= KX382845 28S = KX382845 RPB2 = KX382891

Etymology From tenebrosus (Latin) dark ascocarpsdark-colored

Ascocarps (27ndash)306ndash482(ndash53) [40ndash60] mmhigh ton-gue-like or club-shaped stipitate Hymenium (14ndash)15ndash257(ndash30) times (21ndash)32ndash5 [20ndash30 times 3ndash6] mm mace-shapedcylindrical clavate truncate or lanceolate verticallygrooved glabrous naked at maturity dark green or darkviolet-green or bronze-green to brown-green Hymeniumusually occupying the upper 12 of the ascocarp or moreStipe (12ndash)132ndash258(ndash33) times (15ndash)25ndash3 [20ndash30 times 25ndash6]mm often flattened and flexuous blue-green and oftenconcolorous with hymenium at the base (11 dry ascocarpsexamined) Asci (70ndash)795ndash954(ndash120) times (6ndash)73ndash86(ndash9)[(100ndash)1154ndash1243(ndash1398) times (75ndash)86ndash92(ndash104)] μm8-spored cylindrical to clavate apex rounded narrowlytapered towards the base biseriate above uniseriatebelow the pore bluing in Melzerrsquos reagent Ascospores(10ndash)118ndash154(ndash20) times (3ndash)37ndash45(ndash55) [(116ndash)138ndash148(ndash17) times (31ndash)42ndash46(ndash57)] μm Q value = 25ndash47(av = 34) [24ndash41 (av = 329)] ellipsoidal to oblongusually slightly curved or sigmoid ends obtuse or taper-ing hyaline or with several (lt4) lipid bodies real septa notobserved Paraphyses filiform straight branched in basalpart some of the paraphyses branched at middle part theapical cells filiform 1ndash2(ndash25) μm or slightly clavate orcapitate (lt3 μm)

Habitat On soil among grasses or in forest clear-ings meadows and pastures with Juniperus sp underBuxus sp and calcareous bedrock in western and cen-tral Europe

Distribution France Germany SpainAdditional specimens examined See

SUPPLEMENTARY TABLE 2Notes Microglossum tenebrosum is characterized by

shades of dark green The color of the hymenium isgenerally repeated at the basal part of the stipeMicroglossum tenebrosum is distinguished by the Q

52 V KUČERA ET AL NEW MICROGLOSSUM TAXA

value of the ascospores (34) compared with M nudipes(39) M clavatum (38) M pratense (37) M truncatum(38) and M parvisporum (305) Most collections of Mtenebrosum are from open spaces and not forests Thecombination of relatively large asci (795ndash95 times 7ndash9 μm)and small ascospores (12ndash15 times 4ndash45 μm) is characteristicThe data for fresh collections were taken from RibesRipoll (2013)Microglossum clavatum V Kučera Lizoň ampTomšovskyacute sp novMycoBank MB817351

Typification SPAIN Huesca Puente de Reina deJaca (42deg34prime0418PrimeN 0deg45prime4464PrimeW 665 m) underBuxus sempervirens in calcareous soil 6 Dec 2006 JHernanz (holotype SAV F-11276) DNA sequencesfrom the holotype ITS = KX382864 28S = KX382864RPB2 = KX382884

Etymology From clava (Latin) club ascocarps typi-cally club-shaped

Ascocarps 15ndash31(ndash37) mm high tongue-like club-shaped or clavate usually compressed stipitateHymenium 10ndash199(ndash25) times 1ndash4 mm clavate truncateor lanceolate slightly vertically grooved glabrousnaked dark green sometimes with a brown tintHymenium occupying more than the upper 12 ofthe ascocarp Stipe (4ndash)52ndash125 times (1ndash)25ndash3 mmcylindrical or flattened concolorous with hymenium(14 dry ascocarps examined) Asci (715ndash)966ndash1178(130) times (65ndash)82ndash101(ndash11) μm 8-spored cylindricalto clavate apex rounded narrowly tapered towardsthe base biseriate above uniseriate below the porebluing in Melzerrsquos reagent Ascospores (14ndash)155ndash204(ndash23) times (4ndash)45ndash5 μm Q value = 3ndash57 (av = 38)ellipsoidal to oblong usually slightly curved or sig-moid ends obtuse or tapering hyaline or with several(lt4) lipid bodies true septa not observed Paraphysesfiliform straight branched in basal part some of theparaphyses branched at middle part the apical cellsfiliform 1ndash2(ndash25) μm or slightly clavate or capitate(lt35 μm)

Habitat On calcareous soil under Buxus sp treeDistribution SpainAdditional specimens examined SeeSUPPLEMENTARY TABLE 2Notes Microglossum clavatum is similar to M

nudipes based on the size of asci and shape of para-physes However the values of all examined charactersof M clavatum are slightly smaller the paraphyses arenot apically branched and the ascus pore does not reactas strongly in Melzerrsquos reagent as it does in M nudipes

Examined specimens of other Microglossum spp SeeSUPPLEMENTARY TABLE 2

DISCUSSION

During our research on theMicroglossumnudipes complexwe recognized sevenwell-delimited taxa Three of themMnudipes (Boudier 1917) M parvisporum (Kučera et al2014a) and M fuscorubens (Boudier 1907) were alreadydescribed Four new species are described in this paper butthere is a high likelihood that additional morphotypes andor taxa in this complex will be discovered

In all species we observed that the apical part of theasci and paraphyses are surrounded by a green-brownamorphous matrix that is lt20 μm thick Moreoverascospores occurring in the apical part of the asci aresmaller than others

Ascocarps of all taxa in theM nudipes complex lack thepink ochraceous or red colors that are typical for Mrufescens Five specimens studied (SAV F-11274 F-11051F-11285 F-11271 F-11053) are morphologically similar toeach other but differ from lectotype of M nudipes anddiffer from other taxa in the ITS region and LSU andRPB2 genes we temporarily maintain these entities underthe name M nudipes aff The ascocarps of M nudipes affmay have in addition to green different tints of brown onthe stipe and according to the phylogenetic data the groupis closely related to M fuscorubens

Occasionally several species of the M nudipes com-plex grow in one site We observed three species (Mtruncatum M pretense and M nudipes aff) at Gruacuteň inthe Biele Karpaty Mts Slovakia where the ascocarpswere growing in a range of less than 2 m

Taxa of green naked-stipe species preferentially growamong grass on soil with calcareous bedrock in openspaces such as mowed meadows or pastures It is alsopossible to find them in forest or bushes with Quercus spBuxus sp Chamaecyparis sp or Laurus sp especially inwarmer regions The main period of sporulation is fromOctober to January depending on geographic location andprecipitation

ACKNOWLEDGMENTS

The authors thank P Marstad T Kristiansen S and J MMoingeon Z Egertovaacute J Hernanz K Bergelin M A RibesRipoll M Della Maggiora V Kaounas P Tanchaud JDubus J P Priou E Popov E Smikovaacute P Smik APolhorskyacute M Krivuš V Kautman and I Kautmanovaacute forgraciously providing specimens for study and analyses andcurators of fungaria O PC LE BRA and SAV for the loan ofspecimens Alvalab is acknowledged for processing selectedDNA samples We acknowledge the following sources offunding Slovak Academy of Sciences (grant VEGA 2000815) The European Social Fund and the State Budget of theCzech Republic Project No CZ1072300200265

MYCOLOGIA 53

ORCID

Viktor Kučera httporcidorg0000-0002-9815-4782Pavel Lizoň httporcidorg0000-0003-0283-2831

LITERATURE CITED

Boudier JLEacute 1907 Histoire et classification des DiscomycegravetesdrsquoEurope Paris France Libraire des SciencesNaturelles 223 p

Boudier JLEacute 1917 Dernieacuteres eacutetincelles mycologiquesBulletin de la Socieacuteteacute mycologique de France 337ndash22

Brock PM Doring H Bidartondo MI 2009 How to knowunknown fungi the role of a herbarium New Phytologist181719ndash724

Darriba D Taboada GL Doallo R Posada D 2012jModelTest 2 more models new heuristics and parallelcomputing Nature Methods 9772ndash773

Durand EJ 1908 The Geoglossaceae of North AmericaAnnales Mycologici 6387ndash477

Gillet CC 1879 Champignons de France Les DiscomycegravetesVol 1 Alenccedilon France E De Broise 230 p

Hansen K LoBuglio KF Pfister DH 2005 Evolutionaryrelationships of the cup-fungus genus Peziza andPezizaceae inferred from multiple nuclear genes RPB2β-tubulin and LSU rDNA Molecular Phylogenetics andEvolution 361ndash23

Hustad VP Miller AN Dentinger BTM Cannon PF 2013Generic circumscriptions in Geoglossomycetes Persoonia31100ndash111

Hustad VP Miller AN Moingeon JM Priou JP 2011 Inclusionof Nothomitra in Geoglossomycetes Mycosphere 2646ndash654

Iglesias P Arauzo S 2013 Un Microglossum nuevo recolec-tado en Orintildeoacuten (Cantabria) Errotari 1013ndash26

Imai S 1942 Contributiones ad studia monographicaGeoglossacearum Botanical Magazine Tokyo 56523ndash527

Katoh K Toh H 2008 Recent developments in the MAFFTmultiple sequence alignment program Briefings inBioinformatics 9276ndash285

Kučera V Lizoň P Tomšovskyacute M 2014a A new greenearth-tongue Microglossum parvisporum sp nov Sydowia66335ndash343

Kučera V Lizoň P Tomšovskyacute M Kučera J Gaisler J 2014b Re-evaluation of the morphological variability of Microglossumviride and M griseoviride sp nov Mycologia 106282ndash290

Liu YL Whelen S Hall BD 1999 Phylogenetic relationshipsamong ascomycetes evidence from an RNA polymerase IIsubunit Molecular Biology and Evolution 161799ndash1808

Moingeon S Moingeon JM 2004 Contributions agrave lrsquoeacutetude desGeoglossaceae agrave spores hyalines Miscellanea Mycologica80ndash8125ndash35

Moncalvo JM Lutzoni FM Rehner SA Johson J Vilgalys R2000 Phylogenetic relationships of agaric fungi based onnuclear large subunit ribosomal DNA Systematic Biology49278ndash305

Ohenoja E Wang Z Townsend JP Mitchel D Voitk A 2010Northern species of earth tongue genus Thuemenidiumrevisited considering morphology ecology and molecularphylogeny Mycologia 1021089ndash1095

Persoon CH 1794 Neuer Versuch einer systematischenEintheilung der Schwaumlmme Neues Magazin fuumlr dieBotanik 163ndash128

Persoon CH 1796 Observationes mycologicae Vol 1Leipzig Apud Petrum Philippum Wolf 115 p

Rambaut A Suchard MA Xie D Drummond AJ 2014Tracer version 16 [cited 3 June 2016] Available fromhttpbeastbioedacukTracer

Ribes Ripoll MA 2013 Microglossum nudipes Boud (1917)[cited 15 June 2016] httpwwwmicobotanicajaencomRevistaArticulosMARibesRPirineoAragones001Microglossum20nudipes201110092081pdf

Ronquist F Teslenko M van der Mark P Ayres DL DarlingA Houmlhna S Larget B Liu L Suchard MA Huelsenbeck JP2012 MrBayes 32 efficient Bayesian phylogenetic infer-ence and model choice across a large model spaceSystematic Biology 61539ndash542

Saccardo PA 1884 Conspectus generum Discomycetumhucusque cognitorum Botanisches Centralblatt 18213ndash220

Sandnes ACS 2006 Phylogenetic relationships among spe-cies and genera of Geoglossaceae (Helotiales) based on ITSand LSU nrDNA sequences [Cand Scient thesis] OsloNorway University of Oslo 32 p

Schoch CL Wang Z Townsend JP Spatafora JW 2009Geoglossomycetes cl nov Geoglossales ord nov andtaxa above class rank in the Ascomycota tree of lifePersoonia 22129ndash138

Stamatakis A 2014 RAxML version 8 a tool for phylogeneticanalysis and post-analysis of large phylogeniesBioinformatics 301312ndash1313

Tamura K Stecher G Peterson D Filipski A Kumar S 2013MEGA6 Molecular Evolutionary Genetics Analysis ver-sion 60 Molecular Biology and Evolution 302725ndash2729

Thiers B [continuously updated] Index herbariorum a glo-bal directory of public herbaria and associated staff NewYork Botanical Gardenrsquos Virtual Herbarium [cited 27 June2016] Available from httpsweetgumnybgorgih

Van Brummelen J 1985 Introductory note to the re-editionof Boudierrsquos Icones Mycologicae In Van Brummelen JKorf RP Cleacutemencon H Julich W Demoulin V eds VIcones Mycologicae par Emile Boudier V Listepreacuteliminaire amp explication des planches [reprint edition]Lausanne Switzerland Editions Piantanida p xxivndashxxvi

Wang Z Binder M Hibbett DS 2005 Life history andsystematics of the aquatic discomycete Mitrula(Helotiales Ascomycota) based on cultural morphologicaland molecular studies American Journal of Botany921565ndash1574

Wang Z Binder M Schoch CL Johnston PR Spatafora JWHibbett DS 2006 Evolution of helotialean fungi(Leotiomycetes Pezizomycotina) a nuclear rDNA phylo-geny Molecular Phylogenetics and Evolution 41295ndash312

White TJ Bruns TD Lee SB Taylor JW 1990 Amplificationand direct sequencing of fungal ribosomal RNA genes forphylogenetics In Innis MA Gelfand DH Sninsky JJWhite TJ eds PCR protocols a guide to methods andapplications New York NY Academic Press p 315ndash322

54 V KUČERA ET AL NEW MICROGLOSSUM TAXA

V Kučera V Kautman and I Kautmanovaacute (holotypeSAV F-11280) DNA sequences from the holotypeITS = KX382861 28S = KX382861 RPB2 = KX382875

Etymology From truncatus (Latin) ending abruptlyascocarps truncate when young

Ascocarps (19ndash)254ndash443(ndash55) [(20ndash)302ndash555(ndash60)]mm high tongue-like or club-shaped stipitateHymenium (8ndash)222ndash285(ndash30) times (1ndash)32ndash41(ndash7) [(10ndash)253ndash323(ndash44) times (1ndash)31ndash52(ndash8)] mm cylindrical club-shaped truncate or lanceolate only slightly grooved ver-tically glabrous naked brownish-green or light brown orgreen-brown dark green or brownish-green when dryHymenium usually constituting more than the upper 12of the ascocarp Stipe (10ndash)152ndash201(ndash27) times 1ndash22(ndash3)[(11ndash)165ndash236(ndash29) times 12ndash31(ndash4)] mm cylindricalblue-green not paler at the base (15 ascocarps examined)Asci (75ndash)851ndash987(ndash105) times (8ndash)84ndash96(ndash10) [(95ndash)1206ndash1353(ndash150) times (8ndash)101ndash115(12)] μm 8-sporedclavate rounded at the apex narrowly tapered towardsthe base biseriate above uniseriate below pore bluing in

Melzerrsquos reagent Ascospores (14ndash)154ndash183(ndash20) times 4ndash47(ndash5) [(17ndash)182ndash198(ndash21) times 4ndash5(55)] μm Q value = 33ndash48 (av = 38) hyaline ellipsoidal to oblong aseptateParaphyses filiform straight branched in basal partsome of the paraphyses branched in apical part apicalcells filiform 1ndash2 μm or only slightly swollen (lt3 μm)

Habitat On soil among grass in mesophilous mea-dows with soil pH 57ndash61

Distribution France Russia Slovakia SwedenAdditional specimens examined See

SUPPLEMENTARY TABLE 2Notes Ascocarps of Microglossum truncatum are

brown or brownish-green in the hymenium whereasthe stipe is blue-green and oval in cross-section Themost similar species is M nudipes aff which differs bya brown coloration on the stipe and is usually dumb-bell-shaped in cross-section Microglossum truncatummay be macroscopically confused with M parvisporumbut it clearly differs in the size of asci (85ndash98 times 8ndash9 μmvs 66ndash79 times 6 μm) and ascospores (15ndash18 times 4ndash5 μm vs

Figure 3 Microglossum species with green naked stipes A M truncatum SAV F-11261 (M Krivuš) B M truncatum SAV F-11280 (VKučera) C M pratense SAV-11020 (P Marstad) D M pratense SAV-10024 (V Kučera) E F M tenebrosum SAV F-11278 (F Hampe) GM nudipes aff SAV F-11051 (T Knutsson) H M nudipes aff SAV F-11285 (V Kaounas) I M nudipes aff SAV F-11271 (U Pera) Bars =25 cm (A) and 5 cm (BndashI)

MYCOLOGIA 51

11ndash14 times 3ndash4 μm) and acidity of the substrate (pH 57ndash61 vs pH 635)

Microglossum pratense V Kučera Tomšovskyacute ampLizoň sp nov FIG 3c dMycoBank MB808093

Typification SLOVAKIA Stolickeacute vrchy MtsMuraacutenska Huta village Prednaacute Hora recreation area for-mer ski slope ca 2 km SSW from the village (48deg45prime3124PrimeN 20deg06prime2698PrimeE alt 789 m) in grass 13 Oct 2010 VKautman and V Kučera (holotype SAV F-10024) DNAsequences from the holotype ITS = KC595259 28S =KC595260 RPB2 = KX382880

Etymology from pratum (Latin) meadow growingin meadows and pastures

Ascocarps (17ndash)268ndash496(ndash62) mm high tongue-like or club-shaped stipitate Hymenium (10ndash)124ndash277(ndash40) times (15ndash)22ndash47(ndash9) mm mace-shapedcylindrical clavate truncate or lanceolate verticallygrooved usually twisted glabrous naked blue-greenor gray-green dark green and often with lateral crackswhen dry Hymenium usually occupying the upper 12of the ascocarp or more Stipe (7ndash)118ndash242(ndash32) times1ndash3 mm often flattened and flexuous blue-green orconcolorous with hymenium (54 dry ascocarps exam-ined) Asci (67ndash)781ndash915(ndash105) times (5ndash)71ndash85(ndash9) μm8-spored cylindrical to clavate apex rounded narrowlytapered towards the base biseriate above uniseriatebelow the pore light bluing in Melzerrsquos reagentAscospores (11ndash)135ndash165(ndash20) times (4ndash)45ndash5 μm Qvalue = 32ndash41 (av = 37) ellipsoidal to oblong usuallyslightly curved or sigmoid ends obtuse or taperinghyaline or with several (lt4) lipid bodies real septanot observed Paraphyses filiform straight branchedin basal part some of the paraphyses branched at apicalpart the apical cells filiform 1ndash2) μm or only slightlyswollen lt3 μm

Habitat Meadows or pastures in north and centralEurope on soil among grasses The type collection wasfound in a site with soil pH 52ndash56

Distribution Georgia Norway Russia SlovakiaSweden

Additional specimens examined SeeSUPPLEMENTARY TABLE 2

Notes Microglossum pratense is characterized byhaving green or gray-green hymenium lacking abrown tint Ascocarps are usually produced in biggerclusters of lt10 When dry the ascigerous part oftentransversely cracked The hymenium takes more than ahalf of the whole ascocarp and the stipe is often flat-tened Microglossum pratense could be confused withM tenebrosum or M clavatum but M pratense is palerin color and the ascospores are bigger (135ndash165 times 4ndash5

μm) than in M tenebrosum (12ndash15 times 4ndash45 μm) andsmaller than in M clavatum (15ndash20 times 4ndash5 μm)Collections in fungaria are mostly misidentified asM olivaceum or M rufescens In general M pratensewas the most common green Microglossum species withnaked stipe in the examined areas

Microglossum tenebrosum V Kučera TomšovskyacuteLizoň amp F Hampe sp nov FIG 3e fMycoBank MB817358

Typification GERMANY Hessen LangenthalEberschuumltz (51deg36prime269PrimeN 09deg23prime226PrimeE alt 225 m)calcareous neglected open grassland shell limestonewith Juniperus sp 12 Oct 2009 F Hampe (holotypeSAV F-11278) DNA sequences from the holotype ITS= KX382845 28S = KX382845 RPB2 = KX382891

Etymology From tenebrosus (Latin) dark ascocarpsdark-colored

Ascocarps (27ndash)306ndash482(ndash53) [40ndash60] mmhigh ton-gue-like or club-shaped stipitate Hymenium (14ndash)15ndash257(ndash30) times (21ndash)32ndash5 [20ndash30 times 3ndash6] mm mace-shapedcylindrical clavate truncate or lanceolate verticallygrooved glabrous naked at maturity dark green or darkviolet-green or bronze-green to brown-green Hymeniumusually occupying the upper 12 of the ascocarp or moreStipe (12ndash)132ndash258(ndash33) times (15ndash)25ndash3 [20ndash30 times 25ndash6]mm often flattened and flexuous blue-green and oftenconcolorous with hymenium at the base (11 dry ascocarpsexamined) Asci (70ndash)795ndash954(ndash120) times (6ndash)73ndash86(ndash9)[(100ndash)1154ndash1243(ndash1398) times (75ndash)86ndash92(ndash104)] μm8-spored cylindrical to clavate apex rounded narrowlytapered towards the base biseriate above uniseriatebelow the pore bluing in Melzerrsquos reagent Ascospores(10ndash)118ndash154(ndash20) times (3ndash)37ndash45(ndash55) [(116ndash)138ndash148(ndash17) times (31ndash)42ndash46(ndash57)] μm Q value = 25ndash47(av = 34) [24ndash41 (av = 329)] ellipsoidal to oblongusually slightly curved or sigmoid ends obtuse or taper-ing hyaline or with several (lt4) lipid bodies real septa notobserved Paraphyses filiform straight branched in basalpart some of the paraphyses branched at middle part theapical cells filiform 1ndash2(ndash25) μm or slightly clavate orcapitate (lt3 μm)

Habitat On soil among grasses or in forest clear-ings meadows and pastures with Juniperus sp underBuxus sp and calcareous bedrock in western and cen-tral Europe

Distribution France Germany SpainAdditional specimens examined See

SUPPLEMENTARY TABLE 2Notes Microglossum tenebrosum is characterized by

shades of dark green The color of the hymenium isgenerally repeated at the basal part of the stipeMicroglossum tenebrosum is distinguished by the Q

52 V KUČERA ET AL NEW MICROGLOSSUM TAXA

value of the ascospores (34) compared with M nudipes(39) M clavatum (38) M pratense (37) M truncatum(38) and M parvisporum (305) Most collections of Mtenebrosum are from open spaces and not forests Thecombination of relatively large asci (795ndash95 times 7ndash9 μm)and small ascospores (12ndash15 times 4ndash45 μm) is characteristicThe data for fresh collections were taken from RibesRipoll (2013)Microglossum clavatum V Kučera Lizoň ampTomšovskyacute sp novMycoBank MB817351

Typification SPAIN Huesca Puente de Reina deJaca (42deg34prime0418PrimeN 0deg45prime4464PrimeW 665 m) underBuxus sempervirens in calcareous soil 6 Dec 2006 JHernanz (holotype SAV F-11276) DNA sequencesfrom the holotype ITS = KX382864 28S = KX382864RPB2 = KX382884

Etymology From clava (Latin) club ascocarps typi-cally club-shaped

Ascocarps 15ndash31(ndash37) mm high tongue-like club-shaped or clavate usually compressed stipitateHymenium 10ndash199(ndash25) times 1ndash4 mm clavate truncateor lanceolate slightly vertically grooved glabrousnaked dark green sometimes with a brown tintHymenium occupying more than the upper 12 ofthe ascocarp Stipe (4ndash)52ndash125 times (1ndash)25ndash3 mmcylindrical or flattened concolorous with hymenium(14 dry ascocarps examined) Asci (715ndash)966ndash1178(130) times (65ndash)82ndash101(ndash11) μm 8-spored cylindricalto clavate apex rounded narrowly tapered towardsthe base biseriate above uniseriate below the porebluing in Melzerrsquos reagent Ascospores (14ndash)155ndash204(ndash23) times (4ndash)45ndash5 μm Q value = 3ndash57 (av = 38)ellipsoidal to oblong usually slightly curved or sig-moid ends obtuse or tapering hyaline or with several(lt4) lipid bodies true septa not observed Paraphysesfiliform straight branched in basal part some of theparaphyses branched at middle part the apical cellsfiliform 1ndash2(ndash25) μm or slightly clavate or capitate(lt35 μm)

Habitat On calcareous soil under Buxus sp treeDistribution SpainAdditional specimens examined SeeSUPPLEMENTARY TABLE 2Notes Microglossum clavatum is similar to M

nudipes based on the size of asci and shape of para-physes However the values of all examined charactersof M clavatum are slightly smaller the paraphyses arenot apically branched and the ascus pore does not reactas strongly in Melzerrsquos reagent as it does in M nudipes

Examined specimens of other Microglossum spp SeeSUPPLEMENTARY TABLE 2

DISCUSSION

During our research on theMicroglossumnudipes complexwe recognized sevenwell-delimited taxa Three of themMnudipes (Boudier 1917) M parvisporum (Kučera et al2014a) and M fuscorubens (Boudier 1907) were alreadydescribed Four new species are described in this paper butthere is a high likelihood that additional morphotypes andor taxa in this complex will be discovered

In all species we observed that the apical part of theasci and paraphyses are surrounded by a green-brownamorphous matrix that is lt20 μm thick Moreoverascospores occurring in the apical part of the asci aresmaller than others

Ascocarps of all taxa in theM nudipes complex lack thepink ochraceous or red colors that are typical for Mrufescens Five specimens studied (SAV F-11274 F-11051F-11285 F-11271 F-11053) are morphologically similar toeach other but differ from lectotype of M nudipes anddiffer from other taxa in the ITS region and LSU andRPB2 genes we temporarily maintain these entities underthe name M nudipes aff The ascocarps of M nudipes affmay have in addition to green different tints of brown onthe stipe and according to the phylogenetic data the groupis closely related to M fuscorubens

Occasionally several species of the M nudipes com-plex grow in one site We observed three species (Mtruncatum M pretense and M nudipes aff) at Gruacuteň inthe Biele Karpaty Mts Slovakia where the ascocarpswere growing in a range of less than 2 m

Taxa of green naked-stipe species preferentially growamong grass on soil with calcareous bedrock in openspaces such as mowed meadows or pastures It is alsopossible to find them in forest or bushes with Quercus spBuxus sp Chamaecyparis sp or Laurus sp especially inwarmer regions The main period of sporulation is fromOctober to January depending on geographic location andprecipitation

ACKNOWLEDGMENTS

The authors thank P Marstad T Kristiansen S and J MMoingeon Z Egertovaacute J Hernanz K Bergelin M A RibesRipoll M Della Maggiora V Kaounas P Tanchaud JDubus J P Priou E Popov E Smikovaacute P Smik APolhorskyacute M Krivuš V Kautman and I Kautmanovaacute forgraciously providing specimens for study and analyses andcurators of fungaria O PC LE BRA and SAV for the loan ofspecimens Alvalab is acknowledged for processing selectedDNA samples We acknowledge the following sources offunding Slovak Academy of Sciences (grant VEGA 2000815) The European Social Fund and the State Budget of theCzech Republic Project No CZ1072300200265

MYCOLOGIA 53

ORCID

Viktor Kučera httporcidorg0000-0002-9815-4782Pavel Lizoň httporcidorg0000-0003-0283-2831

LITERATURE CITED

Boudier JLEacute 1907 Histoire et classification des DiscomycegravetesdrsquoEurope Paris France Libraire des SciencesNaturelles 223 p

Boudier JLEacute 1917 Dernieacuteres eacutetincelles mycologiquesBulletin de la Socieacuteteacute mycologique de France 337ndash22

Brock PM Doring H Bidartondo MI 2009 How to knowunknown fungi the role of a herbarium New Phytologist181719ndash724

Darriba D Taboada GL Doallo R Posada D 2012jModelTest 2 more models new heuristics and parallelcomputing Nature Methods 9772ndash773

Durand EJ 1908 The Geoglossaceae of North AmericaAnnales Mycologici 6387ndash477

Gillet CC 1879 Champignons de France Les DiscomycegravetesVol 1 Alenccedilon France E De Broise 230 p

Hansen K LoBuglio KF Pfister DH 2005 Evolutionaryrelationships of the cup-fungus genus Peziza andPezizaceae inferred from multiple nuclear genes RPB2β-tubulin and LSU rDNA Molecular Phylogenetics andEvolution 361ndash23

Hustad VP Miller AN Dentinger BTM Cannon PF 2013Generic circumscriptions in Geoglossomycetes Persoonia31100ndash111

Hustad VP Miller AN Moingeon JM Priou JP 2011 Inclusionof Nothomitra in Geoglossomycetes Mycosphere 2646ndash654

Iglesias P Arauzo S 2013 Un Microglossum nuevo recolec-tado en Orintildeoacuten (Cantabria) Errotari 1013ndash26

Imai S 1942 Contributiones ad studia monographicaGeoglossacearum Botanical Magazine Tokyo 56523ndash527

Katoh K Toh H 2008 Recent developments in the MAFFTmultiple sequence alignment program Briefings inBioinformatics 9276ndash285

Kučera V Lizoň P Tomšovskyacute M 2014a A new greenearth-tongue Microglossum parvisporum sp nov Sydowia66335ndash343

Kučera V Lizoň P Tomšovskyacute M Kučera J Gaisler J 2014b Re-evaluation of the morphological variability of Microglossumviride and M griseoviride sp nov Mycologia 106282ndash290

Liu YL Whelen S Hall BD 1999 Phylogenetic relationshipsamong ascomycetes evidence from an RNA polymerase IIsubunit Molecular Biology and Evolution 161799ndash1808

Moingeon S Moingeon JM 2004 Contributions agrave lrsquoeacutetude desGeoglossaceae agrave spores hyalines Miscellanea Mycologica80ndash8125ndash35

Moncalvo JM Lutzoni FM Rehner SA Johson J Vilgalys R2000 Phylogenetic relationships of agaric fungi based onnuclear large subunit ribosomal DNA Systematic Biology49278ndash305

Ohenoja E Wang Z Townsend JP Mitchel D Voitk A 2010Northern species of earth tongue genus Thuemenidiumrevisited considering morphology ecology and molecularphylogeny Mycologia 1021089ndash1095

Persoon CH 1794 Neuer Versuch einer systematischenEintheilung der Schwaumlmme Neues Magazin fuumlr dieBotanik 163ndash128

Persoon CH 1796 Observationes mycologicae Vol 1Leipzig Apud Petrum Philippum Wolf 115 p

Rambaut A Suchard MA Xie D Drummond AJ 2014Tracer version 16 [cited 3 June 2016] Available fromhttpbeastbioedacukTracer

Ribes Ripoll MA 2013 Microglossum nudipes Boud (1917)[cited 15 June 2016] httpwwwmicobotanicajaencomRevistaArticulosMARibesRPirineoAragones001Microglossum20nudipes201110092081pdf

Ronquist F Teslenko M van der Mark P Ayres DL DarlingA Houmlhna S Larget B Liu L Suchard MA Huelsenbeck JP2012 MrBayes 32 efficient Bayesian phylogenetic infer-ence and model choice across a large model spaceSystematic Biology 61539ndash542

Saccardo PA 1884 Conspectus generum Discomycetumhucusque cognitorum Botanisches Centralblatt 18213ndash220

Sandnes ACS 2006 Phylogenetic relationships among spe-cies and genera of Geoglossaceae (Helotiales) based on ITSand LSU nrDNA sequences [Cand Scient thesis] OsloNorway University of Oslo 32 p

Schoch CL Wang Z Townsend JP Spatafora JW 2009Geoglossomycetes cl nov Geoglossales ord nov andtaxa above class rank in the Ascomycota tree of lifePersoonia 22129ndash138

Stamatakis A 2014 RAxML version 8 a tool for phylogeneticanalysis and post-analysis of large phylogeniesBioinformatics 301312ndash1313

Tamura K Stecher G Peterson D Filipski A Kumar S 2013MEGA6 Molecular Evolutionary Genetics Analysis ver-sion 60 Molecular Biology and Evolution 302725ndash2729

Thiers B [continuously updated] Index herbariorum a glo-bal directory of public herbaria and associated staff NewYork Botanical Gardenrsquos Virtual Herbarium [cited 27 June2016] Available from httpsweetgumnybgorgih

Van Brummelen J 1985 Introductory note to the re-editionof Boudierrsquos Icones Mycologicae In Van Brummelen JKorf RP Cleacutemencon H Julich W Demoulin V eds VIcones Mycologicae par Emile Boudier V Listepreacuteliminaire amp explication des planches [reprint edition]Lausanne Switzerland Editions Piantanida p xxivndashxxvi

Wang Z Binder M Hibbett DS 2005 Life history andsystematics of the aquatic discomycete Mitrula(Helotiales Ascomycota) based on cultural morphologicaland molecular studies American Journal of Botany921565ndash1574

Wang Z Binder M Schoch CL Johnston PR Spatafora JWHibbett DS 2006 Evolution of helotialean fungi(Leotiomycetes Pezizomycotina) a nuclear rDNA phylo-geny Molecular Phylogenetics and Evolution 41295ndash312

White TJ Bruns TD Lee SB Taylor JW 1990 Amplificationand direct sequencing of fungal ribosomal RNA genes forphylogenetics In Innis MA Gelfand DH Sninsky JJWhite TJ eds PCR protocols a guide to methods andapplications New York NY Academic Press p 315ndash322

54 V KUČERA ET AL NEW MICROGLOSSUM TAXA

11ndash14 times 3ndash4 μm) and acidity of the substrate (pH 57ndash61 vs pH 635)

Microglossum pratense V Kučera Tomšovskyacute ampLizoň sp nov FIG 3c dMycoBank MB808093

Typification SLOVAKIA Stolickeacute vrchy MtsMuraacutenska Huta village Prednaacute Hora recreation area for-mer ski slope ca 2 km SSW from the village (48deg45prime3124PrimeN 20deg06prime2698PrimeE alt 789 m) in grass 13 Oct 2010 VKautman and V Kučera (holotype SAV F-10024) DNAsequences from the holotype ITS = KC595259 28S =KC595260 RPB2 = KX382880

Etymology from pratum (Latin) meadow growingin meadows and pastures

Ascocarps (17ndash)268ndash496(ndash62) mm high tongue-like or club-shaped stipitate Hymenium (10ndash)124ndash277(ndash40) times (15ndash)22ndash47(ndash9) mm mace-shapedcylindrical clavate truncate or lanceolate verticallygrooved usually twisted glabrous naked blue-greenor gray-green dark green and often with lateral crackswhen dry Hymenium usually occupying the upper 12of the ascocarp or more Stipe (7ndash)118ndash242(ndash32) times1ndash3 mm often flattened and flexuous blue-green orconcolorous with hymenium (54 dry ascocarps exam-ined) Asci (67ndash)781ndash915(ndash105) times (5ndash)71ndash85(ndash9) μm8-spored cylindrical to clavate apex rounded narrowlytapered towards the base biseriate above uniseriatebelow the pore light bluing in Melzerrsquos reagentAscospores (11ndash)135ndash165(ndash20) times (4ndash)45ndash5 μm Qvalue = 32ndash41 (av = 37) ellipsoidal to oblong usuallyslightly curved or sigmoid ends obtuse or taperinghyaline or with several (lt4) lipid bodies real septanot observed Paraphyses filiform straight branchedin basal part some of the paraphyses branched at apicalpart the apical cells filiform 1ndash2) μm or only slightlyswollen lt3 μm

Habitat Meadows or pastures in north and centralEurope on soil among grasses The type collection wasfound in a site with soil pH 52ndash56

Distribution Georgia Norway Russia SlovakiaSweden

Additional specimens examined SeeSUPPLEMENTARY TABLE 2

Notes Microglossum pratense is characterized byhaving green or gray-green hymenium lacking abrown tint Ascocarps are usually produced in biggerclusters of lt10 When dry the ascigerous part oftentransversely cracked The hymenium takes more than ahalf of the whole ascocarp and the stipe is often flat-tened Microglossum pratense could be confused withM tenebrosum or M clavatum but M pratense is palerin color and the ascospores are bigger (135ndash165 times 4ndash5

μm) than in M tenebrosum (12ndash15 times 4ndash45 μm) andsmaller than in M clavatum (15ndash20 times 4ndash5 μm)Collections in fungaria are mostly misidentified asM olivaceum or M rufescens In general M pratensewas the most common green Microglossum species withnaked stipe in the examined areas

Microglossum tenebrosum V Kučera TomšovskyacuteLizoň amp F Hampe sp nov FIG 3e fMycoBank MB817358

Typification GERMANY Hessen LangenthalEberschuumltz (51deg36prime269PrimeN 09deg23prime226PrimeE alt 225 m)calcareous neglected open grassland shell limestonewith Juniperus sp 12 Oct 2009 F Hampe (holotypeSAV F-11278) DNA sequences from the holotype ITS= KX382845 28S = KX382845 RPB2 = KX382891

Etymology From tenebrosus (Latin) dark ascocarpsdark-colored

Ascocarps (27ndash)306ndash482(ndash53) [40ndash60] mmhigh ton-gue-like or club-shaped stipitate Hymenium (14ndash)15ndash257(ndash30) times (21ndash)32ndash5 [20ndash30 times 3ndash6] mm mace-shapedcylindrical clavate truncate or lanceolate verticallygrooved glabrous naked at maturity dark green or darkviolet-green or bronze-green to brown-green Hymeniumusually occupying the upper 12 of the ascocarp or moreStipe (12ndash)132ndash258(ndash33) times (15ndash)25ndash3 [20ndash30 times 25ndash6]mm often flattened and flexuous blue-green and oftenconcolorous with hymenium at the base (11 dry ascocarpsexamined) Asci (70ndash)795ndash954(ndash120) times (6ndash)73ndash86(ndash9)[(100ndash)1154ndash1243(ndash1398) times (75ndash)86ndash92(ndash104)] μm8-spored cylindrical to clavate apex rounded narrowlytapered towards the base biseriate above uniseriatebelow the pore bluing in Melzerrsquos reagent Ascospores(10ndash)118ndash154(ndash20) times (3ndash)37ndash45(ndash55) [(116ndash)138ndash148(ndash17) times (31ndash)42ndash46(ndash57)] μm Q value = 25ndash47(av = 34) [24ndash41 (av = 329)] ellipsoidal to oblongusually slightly curved or sigmoid ends obtuse or taper-ing hyaline or with several (lt4) lipid bodies real septa notobserved Paraphyses filiform straight branched in basalpart some of the paraphyses branched at middle part theapical cells filiform 1ndash2(ndash25) μm or slightly clavate orcapitate (lt3 μm)

Habitat On soil among grasses or in forest clear-ings meadows and pastures with Juniperus sp underBuxus sp and calcareous bedrock in western and cen-tral Europe

Distribution France Germany SpainAdditional specimens examined See

SUPPLEMENTARY TABLE 2Notes Microglossum tenebrosum is characterized by

shades of dark green The color of the hymenium isgenerally repeated at the basal part of the stipeMicroglossum tenebrosum is distinguished by the Q

52 V KUČERA ET AL NEW MICROGLOSSUM TAXA

value of the ascospores (34) compared with M nudipes(39) M clavatum (38) M pratense (37) M truncatum(38) and M parvisporum (305) Most collections of Mtenebrosum are from open spaces and not forests Thecombination of relatively large asci (795ndash95 times 7ndash9 μm)and small ascospores (12ndash15 times 4ndash45 μm) is characteristicThe data for fresh collections were taken from RibesRipoll (2013)Microglossum clavatum V Kučera Lizoň ampTomšovskyacute sp novMycoBank MB817351

Typification SPAIN Huesca Puente de Reina deJaca (42deg34prime0418PrimeN 0deg45prime4464PrimeW 665 m) underBuxus sempervirens in calcareous soil 6 Dec 2006 JHernanz (holotype SAV F-11276) DNA sequencesfrom the holotype ITS = KX382864 28S = KX382864RPB2 = KX382884

Etymology From clava (Latin) club ascocarps typi-cally club-shaped

Ascocarps 15ndash31(ndash37) mm high tongue-like club-shaped or clavate usually compressed stipitateHymenium 10ndash199(ndash25) times 1ndash4 mm clavate truncateor lanceolate slightly vertically grooved glabrousnaked dark green sometimes with a brown tintHymenium occupying more than the upper 12 ofthe ascocarp Stipe (4ndash)52ndash125 times (1ndash)25ndash3 mmcylindrical or flattened concolorous with hymenium(14 dry ascocarps examined) Asci (715ndash)966ndash1178(130) times (65ndash)82ndash101(ndash11) μm 8-spored cylindricalto clavate apex rounded narrowly tapered towardsthe base biseriate above uniseriate below the porebluing in Melzerrsquos reagent Ascospores (14ndash)155ndash204(ndash23) times (4ndash)45ndash5 μm Q value = 3ndash57 (av = 38)ellipsoidal to oblong usually slightly curved or sig-moid ends obtuse or tapering hyaline or with several(lt4) lipid bodies true septa not observed Paraphysesfiliform straight branched in basal part some of theparaphyses branched at middle part the apical cellsfiliform 1ndash2(ndash25) μm or slightly clavate or capitate(lt35 μm)

Habitat On calcareous soil under Buxus sp treeDistribution SpainAdditional specimens examined SeeSUPPLEMENTARY TABLE 2Notes Microglossum clavatum is similar to M

nudipes based on the size of asci and shape of para-physes However the values of all examined charactersof M clavatum are slightly smaller the paraphyses arenot apically branched and the ascus pore does not reactas strongly in Melzerrsquos reagent as it does in M nudipes

Examined specimens of other Microglossum spp SeeSUPPLEMENTARY TABLE 2

DISCUSSION

During our research on theMicroglossumnudipes complexwe recognized sevenwell-delimited taxa Three of themMnudipes (Boudier 1917) M parvisporum (Kučera et al2014a) and M fuscorubens (Boudier 1907) were alreadydescribed Four new species are described in this paper butthere is a high likelihood that additional morphotypes andor taxa in this complex will be discovered

In all species we observed that the apical part of theasci and paraphyses are surrounded by a green-brownamorphous matrix that is lt20 μm thick Moreoverascospores occurring in the apical part of the asci aresmaller than others

Ascocarps of all taxa in theM nudipes complex lack thepink ochraceous or red colors that are typical for Mrufescens Five specimens studied (SAV F-11274 F-11051F-11285 F-11271 F-11053) are morphologically similar toeach other but differ from lectotype of M nudipes anddiffer from other taxa in the ITS region and LSU andRPB2 genes we temporarily maintain these entities underthe name M nudipes aff The ascocarps of M nudipes affmay have in addition to green different tints of brown onthe stipe and according to the phylogenetic data the groupis closely related to M fuscorubens

Occasionally several species of the M nudipes com-plex grow in one site We observed three species (Mtruncatum M pretense and M nudipes aff) at Gruacuteň inthe Biele Karpaty Mts Slovakia where the ascocarpswere growing in a range of less than 2 m

Taxa of green naked-stipe species preferentially growamong grass on soil with calcareous bedrock in openspaces such as mowed meadows or pastures It is alsopossible to find them in forest or bushes with Quercus spBuxus sp Chamaecyparis sp or Laurus sp especially inwarmer regions The main period of sporulation is fromOctober to January depending on geographic location andprecipitation

ACKNOWLEDGMENTS

The authors thank P Marstad T Kristiansen S and J MMoingeon Z Egertovaacute J Hernanz K Bergelin M A RibesRipoll M Della Maggiora V Kaounas P Tanchaud JDubus J P Priou E Popov E Smikovaacute P Smik APolhorskyacute M Krivuš V Kautman and I Kautmanovaacute forgraciously providing specimens for study and analyses andcurators of fungaria O PC LE BRA and SAV for the loan ofspecimens Alvalab is acknowledged for processing selectedDNA samples We acknowledge the following sources offunding Slovak Academy of Sciences (grant VEGA 2000815) The European Social Fund and the State Budget of theCzech Republic Project No CZ1072300200265

MYCOLOGIA 53

ORCID

Viktor Kučera httporcidorg0000-0002-9815-4782Pavel Lizoň httporcidorg0000-0003-0283-2831

LITERATURE CITED

Boudier JLEacute 1907 Histoire et classification des DiscomycegravetesdrsquoEurope Paris France Libraire des SciencesNaturelles 223 p

Boudier JLEacute 1917 Dernieacuteres eacutetincelles mycologiquesBulletin de la Socieacuteteacute mycologique de France 337ndash22

Brock PM Doring H Bidartondo MI 2009 How to knowunknown fungi the role of a herbarium New Phytologist181719ndash724

Darriba D Taboada GL Doallo R Posada D 2012jModelTest 2 more models new heuristics and parallelcomputing Nature Methods 9772ndash773

Durand EJ 1908 The Geoglossaceae of North AmericaAnnales Mycologici 6387ndash477

Gillet CC 1879 Champignons de France Les DiscomycegravetesVol 1 Alenccedilon France E De Broise 230 p

Hansen K LoBuglio KF Pfister DH 2005 Evolutionaryrelationships of the cup-fungus genus Peziza andPezizaceae inferred from multiple nuclear genes RPB2β-tubulin and LSU rDNA Molecular Phylogenetics andEvolution 361ndash23

Hustad VP Miller AN Dentinger BTM Cannon PF 2013Generic circumscriptions in Geoglossomycetes Persoonia31100ndash111

Hustad VP Miller AN Moingeon JM Priou JP 2011 Inclusionof Nothomitra in Geoglossomycetes Mycosphere 2646ndash654

Iglesias P Arauzo S 2013 Un Microglossum nuevo recolec-tado en Orintildeoacuten (Cantabria) Errotari 1013ndash26

Imai S 1942 Contributiones ad studia monographicaGeoglossacearum Botanical Magazine Tokyo 56523ndash527

Katoh K Toh H 2008 Recent developments in the MAFFTmultiple sequence alignment program Briefings inBioinformatics 9276ndash285

Kučera V Lizoň P Tomšovskyacute M 2014a A new greenearth-tongue Microglossum parvisporum sp nov Sydowia66335ndash343

Kučera V Lizoň P Tomšovskyacute M Kučera J Gaisler J 2014b Re-evaluation of the morphological variability of Microglossumviride and M griseoviride sp nov Mycologia 106282ndash290

Liu YL Whelen S Hall BD 1999 Phylogenetic relationshipsamong ascomycetes evidence from an RNA polymerase IIsubunit Molecular Biology and Evolution 161799ndash1808

Moingeon S Moingeon JM 2004 Contributions agrave lrsquoeacutetude desGeoglossaceae agrave spores hyalines Miscellanea Mycologica80ndash8125ndash35

Moncalvo JM Lutzoni FM Rehner SA Johson J Vilgalys R2000 Phylogenetic relationships of agaric fungi based onnuclear large subunit ribosomal DNA Systematic Biology49278ndash305

Ohenoja E Wang Z Townsend JP Mitchel D Voitk A 2010Northern species of earth tongue genus Thuemenidiumrevisited considering morphology ecology and molecularphylogeny Mycologia 1021089ndash1095

Persoon CH 1794 Neuer Versuch einer systematischenEintheilung der Schwaumlmme Neues Magazin fuumlr dieBotanik 163ndash128

Persoon CH 1796 Observationes mycologicae Vol 1Leipzig Apud Petrum Philippum Wolf 115 p

Rambaut A Suchard MA Xie D Drummond AJ 2014Tracer version 16 [cited 3 June 2016] Available fromhttpbeastbioedacukTracer

Ribes Ripoll MA 2013 Microglossum nudipes Boud (1917)[cited 15 June 2016] httpwwwmicobotanicajaencomRevistaArticulosMARibesRPirineoAragones001Microglossum20nudipes201110092081pdf

Ronquist F Teslenko M van der Mark P Ayres DL DarlingA Houmlhna S Larget B Liu L Suchard MA Huelsenbeck JP2012 MrBayes 32 efficient Bayesian phylogenetic infer-ence and model choice across a large model spaceSystematic Biology 61539ndash542

Saccardo PA 1884 Conspectus generum Discomycetumhucusque cognitorum Botanisches Centralblatt 18213ndash220

Sandnes ACS 2006 Phylogenetic relationships among spe-cies and genera of Geoglossaceae (Helotiales) based on ITSand LSU nrDNA sequences [Cand Scient thesis] OsloNorway University of Oslo 32 p

Schoch CL Wang Z Townsend JP Spatafora JW 2009Geoglossomycetes cl nov Geoglossales ord nov andtaxa above class rank in the Ascomycota tree of lifePersoonia 22129ndash138

Stamatakis A 2014 RAxML version 8 a tool for phylogeneticanalysis and post-analysis of large phylogeniesBioinformatics 301312ndash1313

Tamura K Stecher G Peterson D Filipski A Kumar S 2013MEGA6 Molecular Evolutionary Genetics Analysis ver-sion 60 Molecular Biology and Evolution 302725ndash2729

Thiers B [continuously updated] Index herbariorum a glo-bal directory of public herbaria and associated staff NewYork Botanical Gardenrsquos Virtual Herbarium [cited 27 June2016] Available from httpsweetgumnybgorgih

Van Brummelen J 1985 Introductory note to the re-editionof Boudierrsquos Icones Mycologicae In Van Brummelen JKorf RP Cleacutemencon H Julich W Demoulin V eds VIcones Mycologicae par Emile Boudier V Listepreacuteliminaire amp explication des planches [reprint edition]Lausanne Switzerland Editions Piantanida p xxivndashxxvi

Wang Z Binder M Hibbett DS 2005 Life history andsystematics of the aquatic discomycete Mitrula(Helotiales Ascomycota) based on cultural morphologicaland molecular studies American Journal of Botany921565ndash1574

Wang Z Binder M Schoch CL Johnston PR Spatafora JWHibbett DS 2006 Evolution of helotialean fungi(Leotiomycetes Pezizomycotina) a nuclear rDNA phylo-geny Molecular Phylogenetics and Evolution 41295ndash312

White TJ Bruns TD Lee SB Taylor JW 1990 Amplificationand direct sequencing of fungal ribosomal RNA genes forphylogenetics In Innis MA Gelfand DH Sninsky JJWhite TJ eds PCR protocols a guide to methods andapplications New York NY Academic Press p 315ndash322

54 V KUČERA ET AL NEW MICROGLOSSUM TAXA

value of the ascospores (34) compared with M nudipes(39) M clavatum (38) M pratense (37) M truncatum(38) and M parvisporum (305) Most collections of Mtenebrosum are from open spaces and not forests Thecombination of relatively large asci (795ndash95 times 7ndash9 μm)and small ascospores (12ndash15 times 4ndash45 μm) is characteristicThe data for fresh collections were taken from RibesRipoll (2013)Microglossum clavatum V Kučera Lizoň ampTomšovskyacute sp novMycoBank MB817351

Typification SPAIN Huesca Puente de Reina deJaca (42deg34prime0418PrimeN 0deg45prime4464PrimeW 665 m) underBuxus sempervirens in calcareous soil 6 Dec 2006 JHernanz (holotype SAV F-11276) DNA sequencesfrom the holotype ITS = KX382864 28S = KX382864RPB2 = KX382884

Etymology From clava (Latin) club ascocarps typi-cally club-shaped

Ascocarps 15ndash31(ndash37) mm high tongue-like club-shaped or clavate usually compressed stipitateHymenium 10ndash199(ndash25) times 1ndash4 mm clavate truncateor lanceolate slightly vertically grooved glabrousnaked dark green sometimes with a brown tintHymenium occupying more than the upper 12 ofthe ascocarp Stipe (4ndash)52ndash125 times (1ndash)25ndash3 mmcylindrical or flattened concolorous with hymenium(14 dry ascocarps examined) Asci (715ndash)966ndash1178(130) times (65ndash)82ndash101(ndash11) μm 8-spored cylindricalto clavate apex rounded narrowly tapered towardsthe base biseriate above uniseriate below the porebluing in Melzerrsquos reagent Ascospores (14ndash)155ndash204(ndash23) times (4ndash)45ndash5 μm Q value = 3ndash57 (av = 38)ellipsoidal to oblong usually slightly curved or sig-moid ends obtuse or tapering hyaline or with several(lt4) lipid bodies true septa not observed Paraphysesfiliform straight branched in basal part some of theparaphyses branched at middle part the apical cellsfiliform 1ndash2(ndash25) μm or slightly clavate or capitate(lt35 μm)

Habitat On calcareous soil under Buxus sp treeDistribution SpainAdditional specimens examined SeeSUPPLEMENTARY TABLE 2Notes Microglossum clavatum is similar to M

nudipes based on the size of asci and shape of para-physes However the values of all examined charactersof M clavatum are slightly smaller the paraphyses arenot apically branched and the ascus pore does not reactas strongly in Melzerrsquos reagent as it does in M nudipes

Examined specimens of other Microglossum spp SeeSUPPLEMENTARY TABLE 2

DISCUSSION

During our research on theMicroglossumnudipes complexwe recognized sevenwell-delimited taxa Three of themMnudipes (Boudier 1917) M parvisporum (Kučera et al2014a) and M fuscorubens (Boudier 1907) were alreadydescribed Four new species are described in this paper butthere is a high likelihood that additional morphotypes andor taxa in this complex will be discovered

In all species we observed that the apical part of theasci and paraphyses are surrounded by a green-brownamorphous matrix that is lt20 μm thick Moreoverascospores occurring in the apical part of the asci aresmaller than others

Ascocarps of all taxa in theM nudipes complex lack thepink ochraceous or red colors that are typical for Mrufescens Five specimens studied (SAV F-11274 F-11051F-11285 F-11271 F-11053) are morphologically similar toeach other but differ from lectotype of M nudipes anddiffer from other taxa in the ITS region and LSU andRPB2 genes we temporarily maintain these entities underthe name M nudipes aff The ascocarps of M nudipes affmay have in addition to green different tints of brown onthe stipe and according to the phylogenetic data the groupis closely related to M fuscorubens

Occasionally several species of the M nudipes com-plex grow in one site We observed three species (Mtruncatum M pretense and M nudipes aff) at Gruacuteň inthe Biele Karpaty Mts Slovakia where the ascocarpswere growing in a range of less than 2 m

Taxa of green naked-stipe species preferentially growamong grass on soil with calcareous bedrock in openspaces such as mowed meadows or pastures It is alsopossible to find them in forest or bushes with Quercus spBuxus sp Chamaecyparis sp or Laurus sp especially inwarmer regions The main period of sporulation is fromOctober to January depending on geographic location andprecipitation

ACKNOWLEDGMENTS

The authors thank P Marstad T Kristiansen S and J MMoingeon Z Egertovaacute J Hernanz K Bergelin M A RibesRipoll M Della Maggiora V Kaounas P Tanchaud JDubus J P Priou E Popov E Smikovaacute P Smik APolhorskyacute M Krivuš V Kautman and I Kautmanovaacute forgraciously providing specimens for study and analyses andcurators of fungaria O PC LE BRA and SAV for the loan ofspecimens Alvalab is acknowledged for processing selectedDNA samples We acknowledge the following sources offunding Slovak Academy of Sciences (grant VEGA 2000815) The European Social Fund and the State Budget of theCzech Republic Project No CZ1072300200265

MYCOLOGIA 53

ORCID

Viktor Kučera httporcidorg0000-0002-9815-4782Pavel Lizoň httporcidorg0000-0003-0283-2831

LITERATURE CITED

Boudier JLEacute 1907 Histoire et classification des DiscomycegravetesdrsquoEurope Paris France Libraire des SciencesNaturelles 223 p

Boudier JLEacute 1917 Dernieacuteres eacutetincelles mycologiquesBulletin de la Socieacuteteacute mycologique de France 337ndash22

Brock PM Doring H Bidartondo MI 2009 How to knowunknown fungi the role of a herbarium New Phytologist181719ndash724

Darriba D Taboada GL Doallo R Posada D 2012jModelTest 2 more models new heuristics and parallelcomputing Nature Methods 9772ndash773

Durand EJ 1908 The Geoglossaceae of North AmericaAnnales Mycologici 6387ndash477

Gillet CC 1879 Champignons de France Les DiscomycegravetesVol 1 Alenccedilon France E De Broise 230 p

Hansen K LoBuglio KF Pfister DH 2005 Evolutionaryrelationships of the cup-fungus genus Peziza andPezizaceae inferred from multiple nuclear genes RPB2β-tubulin and LSU rDNA Molecular Phylogenetics andEvolution 361ndash23

Hustad VP Miller AN Dentinger BTM Cannon PF 2013Generic circumscriptions in Geoglossomycetes Persoonia31100ndash111

Hustad VP Miller AN Moingeon JM Priou JP 2011 Inclusionof Nothomitra in Geoglossomycetes Mycosphere 2646ndash654

Iglesias P Arauzo S 2013 Un Microglossum nuevo recolec-tado en Orintildeoacuten (Cantabria) Errotari 1013ndash26

Imai S 1942 Contributiones ad studia monographicaGeoglossacearum Botanical Magazine Tokyo 56523ndash527

Katoh K Toh H 2008 Recent developments in the MAFFTmultiple sequence alignment program Briefings inBioinformatics 9276ndash285

Kučera V Lizoň P Tomšovskyacute M 2014a A new greenearth-tongue Microglossum parvisporum sp nov Sydowia66335ndash343

Kučera V Lizoň P Tomšovskyacute M Kučera J Gaisler J 2014b Re-evaluation of the morphological variability of Microglossumviride and M griseoviride sp nov Mycologia 106282ndash290

Liu YL Whelen S Hall BD 1999 Phylogenetic relationshipsamong ascomycetes evidence from an RNA polymerase IIsubunit Molecular Biology and Evolution 161799ndash1808

Moingeon S Moingeon JM 2004 Contributions agrave lrsquoeacutetude desGeoglossaceae agrave spores hyalines Miscellanea Mycologica80ndash8125ndash35

Moncalvo JM Lutzoni FM Rehner SA Johson J Vilgalys R2000 Phylogenetic relationships of agaric fungi based onnuclear large subunit ribosomal DNA Systematic Biology49278ndash305

Ohenoja E Wang Z Townsend JP Mitchel D Voitk A 2010Northern species of earth tongue genus Thuemenidiumrevisited considering morphology ecology and molecularphylogeny Mycologia 1021089ndash1095

Persoon CH 1794 Neuer Versuch einer systematischenEintheilung der Schwaumlmme Neues Magazin fuumlr dieBotanik 163ndash128

Persoon CH 1796 Observationes mycologicae Vol 1Leipzig Apud Petrum Philippum Wolf 115 p

Rambaut A Suchard MA Xie D Drummond AJ 2014Tracer version 16 [cited 3 June 2016] Available fromhttpbeastbioedacukTracer

Ribes Ripoll MA 2013 Microglossum nudipes Boud (1917)[cited 15 June 2016] httpwwwmicobotanicajaencomRevistaArticulosMARibesRPirineoAragones001Microglossum20nudipes201110092081pdf

Ronquist F Teslenko M van der Mark P Ayres DL DarlingA Houmlhna S Larget B Liu L Suchard MA Huelsenbeck JP2012 MrBayes 32 efficient Bayesian phylogenetic infer-ence and model choice across a large model spaceSystematic Biology 61539ndash542

Saccardo PA 1884 Conspectus generum Discomycetumhucusque cognitorum Botanisches Centralblatt 18213ndash220

Sandnes ACS 2006 Phylogenetic relationships among spe-cies and genera of Geoglossaceae (Helotiales) based on ITSand LSU nrDNA sequences [Cand Scient thesis] OsloNorway University of Oslo 32 p

Schoch CL Wang Z Townsend JP Spatafora JW 2009Geoglossomycetes cl nov Geoglossales ord nov andtaxa above class rank in the Ascomycota tree of lifePersoonia 22129ndash138

Stamatakis A 2014 RAxML version 8 a tool for phylogeneticanalysis and post-analysis of large phylogeniesBioinformatics 301312ndash1313

Tamura K Stecher G Peterson D Filipski A Kumar S 2013MEGA6 Molecular Evolutionary Genetics Analysis ver-sion 60 Molecular Biology and Evolution 302725ndash2729

Thiers B [continuously updated] Index herbariorum a glo-bal directory of public herbaria and associated staff NewYork Botanical Gardenrsquos Virtual Herbarium [cited 27 June2016] Available from httpsweetgumnybgorgih

Van Brummelen J 1985 Introductory note to the re-editionof Boudierrsquos Icones Mycologicae In Van Brummelen JKorf RP Cleacutemencon H Julich W Demoulin V eds VIcones Mycologicae par Emile Boudier V Listepreacuteliminaire amp explication des planches [reprint edition]Lausanne Switzerland Editions Piantanida p xxivndashxxvi

Wang Z Binder M Hibbett DS 2005 Life history andsystematics of the aquatic discomycete Mitrula(Helotiales Ascomycota) based on cultural morphologicaland molecular studies American Journal of Botany921565ndash1574

Wang Z Binder M Schoch CL Johnston PR Spatafora JWHibbett DS 2006 Evolution of helotialean fungi(Leotiomycetes Pezizomycotina) a nuclear rDNA phylo-geny Molecular Phylogenetics and Evolution 41295ndash312

White TJ Bruns TD Lee SB Taylor JW 1990 Amplificationand direct sequencing of fungal ribosomal RNA genes forphylogenetics In Innis MA Gelfand DH Sninsky JJWhite TJ eds PCR protocols a guide to methods andapplications New York NY Academic Press p 315ndash322

54 V KUČERA ET AL NEW MICROGLOSSUM TAXA

ORCID

Viktor Kučera httporcidorg0000-0002-9815-4782Pavel Lizoň httporcidorg0000-0003-0283-2831

LITERATURE CITED

Boudier JLEacute 1907 Histoire et classification des DiscomycegravetesdrsquoEurope Paris France Libraire des SciencesNaturelles 223 p

Boudier JLEacute 1917 Dernieacuteres eacutetincelles mycologiquesBulletin de la Socieacuteteacute mycologique de France 337ndash22

Brock PM Doring H Bidartondo MI 2009 How to knowunknown fungi the role of a herbarium New Phytologist181719ndash724

Darriba D Taboada GL Doallo R Posada D 2012jModelTest 2 more models new heuristics and parallelcomputing Nature Methods 9772ndash773

Durand EJ 1908 The Geoglossaceae of North AmericaAnnales Mycologici 6387ndash477

Gillet CC 1879 Champignons de France Les DiscomycegravetesVol 1 Alenccedilon France E De Broise 230 p

Hansen K LoBuglio KF Pfister DH 2005 Evolutionaryrelationships of the cup-fungus genus Peziza andPezizaceae inferred from multiple nuclear genes RPB2β-tubulin and LSU rDNA Molecular Phylogenetics andEvolution 361ndash23

Hustad VP Miller AN Dentinger BTM Cannon PF 2013Generic circumscriptions in Geoglossomycetes Persoonia31100ndash111

Hustad VP Miller AN Moingeon JM Priou JP 2011 Inclusionof Nothomitra in Geoglossomycetes Mycosphere 2646ndash654

Iglesias P Arauzo S 2013 Un Microglossum nuevo recolec-tado en Orintildeoacuten (Cantabria) Errotari 1013ndash26

Imai S 1942 Contributiones ad studia monographicaGeoglossacearum Botanical Magazine Tokyo 56523ndash527

Katoh K Toh H 2008 Recent developments in the MAFFTmultiple sequence alignment program Briefings inBioinformatics 9276ndash285

Kučera V Lizoň P Tomšovskyacute M 2014a A new greenearth-tongue Microglossum parvisporum sp nov Sydowia66335ndash343

Kučera V Lizoň P Tomšovskyacute M Kučera J Gaisler J 2014b Re-evaluation of the morphological variability of Microglossumviride and M griseoviride sp nov Mycologia 106282ndash290

Liu YL Whelen S Hall BD 1999 Phylogenetic relationshipsamong ascomycetes evidence from an RNA polymerase IIsubunit Molecular Biology and Evolution 161799ndash1808

Moingeon S Moingeon JM 2004 Contributions agrave lrsquoeacutetude desGeoglossaceae agrave spores hyalines Miscellanea Mycologica80ndash8125ndash35

Moncalvo JM Lutzoni FM Rehner SA Johson J Vilgalys R2000 Phylogenetic relationships of agaric fungi based onnuclear large subunit ribosomal DNA Systematic Biology49278ndash305

Ohenoja E Wang Z Townsend JP Mitchel D Voitk A 2010Northern species of earth tongue genus Thuemenidiumrevisited considering morphology ecology and molecularphylogeny Mycologia 1021089ndash1095

Persoon CH 1794 Neuer Versuch einer systematischenEintheilung der Schwaumlmme Neues Magazin fuumlr dieBotanik 163ndash128

Persoon CH 1796 Observationes mycologicae Vol 1Leipzig Apud Petrum Philippum Wolf 115 p

Rambaut A Suchard MA Xie D Drummond AJ 2014Tracer version 16 [cited 3 June 2016] Available fromhttpbeastbioedacukTracer

Ribes Ripoll MA 2013 Microglossum nudipes Boud (1917)[cited 15 June 2016] httpwwwmicobotanicajaencomRevistaArticulosMARibesRPirineoAragones001Microglossum20nudipes201110092081pdf

Ronquist F Teslenko M van der Mark P Ayres DL DarlingA Houmlhna S Larget B Liu L Suchard MA Huelsenbeck JP2012 MrBayes 32 efficient Bayesian phylogenetic infer-ence and model choice across a large model spaceSystematic Biology 61539ndash542

Saccardo PA 1884 Conspectus generum Discomycetumhucusque cognitorum Botanisches Centralblatt 18213ndash220

Sandnes ACS 2006 Phylogenetic relationships among spe-cies and genera of Geoglossaceae (Helotiales) based on ITSand LSU nrDNA sequences [Cand Scient thesis] OsloNorway University of Oslo 32 p

Schoch CL Wang Z Townsend JP Spatafora JW 2009Geoglossomycetes cl nov Geoglossales ord nov andtaxa above class rank in the Ascomycota tree of lifePersoonia 22129ndash138

Stamatakis A 2014 RAxML version 8 a tool for phylogeneticanalysis and post-analysis of large phylogeniesBioinformatics 301312ndash1313

Tamura K Stecher G Peterson D Filipski A Kumar S 2013MEGA6 Molecular Evolutionary Genetics Analysis ver-sion 60 Molecular Biology and Evolution 302725ndash2729

Thiers B [continuously updated] Index herbariorum a glo-bal directory of public herbaria and associated staff NewYork Botanical Gardenrsquos Virtual Herbarium [cited 27 June2016] Available from httpsweetgumnybgorgih

Van Brummelen J 1985 Introductory note to the re-editionof Boudierrsquos Icones Mycologicae In Van Brummelen JKorf RP Cleacutemencon H Julich W Demoulin V eds VIcones Mycologicae par Emile Boudier V Listepreacuteliminaire amp explication des planches [reprint edition]Lausanne Switzerland Editions Piantanida p xxivndashxxvi

Wang Z Binder M Hibbett DS 2005 Life history andsystematics of the aquatic discomycete Mitrula(Helotiales Ascomycota) based on cultural morphologicaland molecular studies American Journal of Botany921565ndash1574

Wang Z Binder M Schoch CL Johnston PR Spatafora JWHibbett DS 2006 Evolution of helotialean fungi(Leotiomycetes Pezizomycotina) a nuclear rDNA phylo-geny Molecular Phylogenetics and Evolution 41295ndash312

White TJ Bruns TD Lee SB Taylor JW 1990 Amplificationand direct sequencing of fungal ribosomal RNA genes forphylogenetics In Innis MA Gelfand DH Sninsky JJWhite TJ eds PCR protocols a guide to methods andapplications New York NY Academic Press p 315ndash322

54 V KUČERA ET AL NEW MICROGLOSSUM TAXA