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mycoscience 56 (2015) 572e579

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Short communication Maasoglossum, a basal in Geoglossomycetes

* Vincent P. Hustad a,b, , Andrew N. Miller b a Department of Plant Biology, University of Illinois at Urbana-Champaign, 505 S. Goodwin Ave., Urbana, IL 61801, USA b Illinois Natural History Survey, University of Illinois, 1816 S. Oak St., Champaign, IL 61820, USA article info abstract

Article history: The genus Maasoglossum is examined using morphology, ecology, and molecular system- Received 19 January 2015 atics of the internal transcribed spacer region and large subunit of the nuclear ribosomal Received in revised form RNA gene, all of which support the placement of Maasoglossum among the basal members 22 May 2015 of Geoglossomycetes. The morphology of the genus extends the range of ascocarp and Accepted 24 May 2015 development in Geoglossomycetes. The ecology and conservational significance Available online 24 June 2015 of the genus is discussed, a nomenclatural transfer of aseptatum to Maaso- glossum is made, and an emended description of Maasoglossum is provided. Keywords: © 2015 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. Earth tongues Geoglossum Phylogenetics Systematics

The class Geoglossomycetes is a basal ascocarp-producing character not found in any other member of the genus Geo- member of the ‘’ (Schoch et al. 2009). Kirk et al. glossum. Preliminary molecular analysis (data not shown) (2008) suggest 48 in four genera belong in the class, suggested that this belonged in a separate lineage from while lists over 200 names for these same Geoglossum and a review of the literature suggested a possible four genera. Recent research indicates eight genera belong in link between G. aseptatum and the genus Maasoglossum Thind the class: Geoglossum Pers., Glutinoglossum Hustad et al., Hem- & R. Sharma. ileucoglossum Arauzo, Leucoglossum S. Imai, Nothomitra Maas The genus Maasoglossum was described by Thind and Geest., Sabuloglossum Hustad et al., S. Ito & S. Imai, Sharma (1984) to accommodate a single species, M. verrucis- and Boud. (Hustad et al. 2013; Arauzo and porum, described from a collection made in 1981 from the Iglesias 2014). The prevalence of rarely collected species has temperate Eastern Himalayan forest of Bhutan. The authors made an accurate estimation of the number of species in the noted the macroscopic similarity of the ascocarp to that of class difficult. Geoglossum, but that it differed in the of M. verru- The rare species, Geoglossum aseptatum Hakelier ex Nitare, cisporum contained small ridges and grooves that became is characterized by light brown, aseptate mature , a more prominent upon drying, a character rarely encountered

* Corresponding author. Department of Plant Biology, University of Illinois at Urbana-Champaign, 505 S. Goodwin Ave., Urbana IL 61801, USA. Tel.: þ01 618 843 0784; fax: þ01 217 265 4678. E-mail address: [email protected] (V.P. Hustad). http://dx.doi.org/10.1016/j.myc.2015.05.003 1340-3540/© 2015 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. mycoscience 56 (2015) 572e579 573

in Geoglossum. Additionally, the one-celled, lightly-colored, minimum length of a block ¼ 2, and allowed gap positions in warted ascospores precluded the placement of this fungus in 50% of sequences; for LSU: same as ITS except minimum Geoglossum. Since no other members of the number of sequences for both conserved and flanking sensu lato contain ornamented ascospores, they were unsure regions ¼ 14. of the placement of this genus within Geoglossaceae sensu lato The GTR þ I þ G model was determined to be the best-fit or (). The genus is currently model of evolution for both ribosomal regions using the regarded as incertae sedis within Leotiomycetes (Lumbsch and Akaike Information Criterion (AIC) (Posada and Buckley 2004) Huhndorf 2010). In this paper we provide an in-depth in jModelTest 2.0 v. 0.1.1 (Guindon and Gascuel 2003; Darriba assessment of the genus Maasoglossum based on et al. 2012) on the XSEDE platform of the CIPRES Science morphology, ecology, and molecular systematics, and test the Gateway Teragrid (Miller et al. 2010) and was used in phylo- hypotheses that Maasoglossum is a basal member of Geo- genetic analyses of each individual dataset. Maximum likeli- glossomycetes and that G. aseptatum belongs in the genus hood (ML) analyses were performed using PhyML 3.0 (Guindon Maasoglossum. et al. 2010) on the ATGC server (http://www.atgc-montpellier. For this study, the specimens of both Geoglossum fr/phyml/). The best of subtree pruning and regrafting (SPR) aseptatum and Maasoglossum verrucisporum were obtained and and nearest neighbor interchange (NNI) was implemented examined. Dried ascomata were hand-sectioned and squash during the heuristic search. Nonparametric bootstrap support mounted in 5% KOH and micromorphological characters were (Felsenstein 1985) (BS) was determined with 1000 replicates. observed. Images of pertinent micromorphological characters Clades with BS values of 70% were considered significant were captured using a QImaging QColor 3 digital camera (Hillis and Bull 1993). mounted on an Olympus BX51 compound microscope using Bayesian inference employing a Markov Chain Monte Carlo differential interference microscopy. Images were processed (MCMC) algorithm was performed using MrBayes v. 3.2.2 with Adobe Photoshop v. 7.0 (Adobe Systems Inc., Mountain (Ronquist et al. 2012) on the XSEDE platform. Four indepen- View, California). A minimum of 30 measurements was made dent chains of MCMC were run for 10 million generations. for each micromorphological character using NIH Image v. Clades with Bayesian posterior probability (BPP) values of 1.63 (National Institutes of Health, Bethesda, Maryland). 95% were considered significant (Alfaro et al. 2003). Tracer v. Taxonomic novelties and associated data were deposited in 1.5 (Rambaut and Drummond 2009) was used to estimate MycoBank (Crous et al. 2004). effective sample size (ESS) using the standard deviation of Total genomic DNA was extracted from approximately split frequencies produced by Bayesian analysis. 0.5 cm2 of hymenium tissue from a single dried ascoma with Individual ITS and LSU datasets were examined for po- the DNeasy Plant Mini Kit (QIAGEN Inc., Valencia, California) tential conflict before concatenation into a single dataset for for recently-collected specimens (collected within 20 years). total evidence analysis (Kluge 1989; Eernisse and Kluge 1993). For older specimens, DNA was extracted from approximately Individual gene phylogenies were considered to be incon- 0.2 cm2 of hymenium tissue using the EZNA Forensic DNA kit gruent if clades with significant ML BS and BPP support were (Omega Bio-Tek, Norcross, Georgia). Gene fragments were PCR conflicting in individual tree topologies (Wiens 1998; Alfaro amplified following the methods outlined in Promputtha and et al. 2003; Lutzoni et al. 2004). Since no incongruencies were Miller (2010) and purified using a Wizard SV Gel and PCR found among individual phylogenies, the ITS and LSU data- Clean-Up System (Promega Corp., Fitchburg, Wisconsin). Se- sets were concatenated and final ML and Bayesian analyses quences were generated on an ABI Applied Biosystems 3730XL were performed on the combined dataset. Alignments and high-throughput DNA capillary sequencer at the UIUC Keck trees are deposited in TreeBASE (http://treebase.org) under Center for Comparative and Functional Genomics. Two re- submission ID 16591. gions of the nuclear rRNA gene were used for molecular Ten new sequences were generated in this study, five ITS phylogenetic analysis: the ca. 570 bp internal transcribed and five LSU sequences (Table 1). These were analyzed spacer (ITS) region, consisting of the ITS1, 5.8S, and ITS2 re- together with 23 ITS and 21 LSU sequences from our previous gions, was amplified and sequenced using a combination of studies (Hustad and Miller 2011; Hustad et al. 2011, 2013, 2014) the primers ITS5, ITS1, ITS2, ITS3, and ITS4 (White et al. 1990); along with sequences of Geoglossomycetes from previously and a ca. 630 bp fragment of the 28S large subunit (LSU) region published research (Pfister 1997; Zhao et al. 2001; Wang et al. was amplified and sequenced with the primers JS1 (Landvik 2002, 2005, 2006, 2011; Peterson 2008; Brock et al. 2009; 1996) and LR3 (Vilgalys and Hester 1990). Due to the age of Ohenoja et al. 2010). Twenty-eight collections, representing a the material, attempts to PCR amplify single-copy protein total of 15 Geoglossomycetes species (including sequences coding genes (i.e. RPB1, RPB2, MCM7) were unsuccessful. Out- from type species of each genus in Geoglossomycetes except group species were chosen from closely related taxa based on Sarcoleotia) and eleven outgroup species, were included in the previous studies (Wang et al. 2006; Hustad et al. 2013). analyses. Of the 28 collections included in the final dataset, Sequence alignments were created by eye in Sequencher none lack ITS and only two lack LSU, with sequences for both 5.0.1 (Gene Codes Corp., Ann Arbor, Michigan) and optimized markers available for 93% of the collections (Table 1). if necessary using Muscle v. 3.8.31 (Edgar 2004) in SeaView v. The final combined data matrix had an aligned length of 4.4.2 (Gouy et al. 2010). Ambiguous regions were removed 2712 bp, which was reduced to 1110 bp after the removal of from each dataset using Gblocks 0.91b (Castresana 2000) 1602 bp of ambiguously aligned regions by Gblocks. Of the under the following parameters: for ITS: minimum number of 1110 characters used in the final phylogenetic analyses, 554 sequences for both conserved and flanking regions ¼ 15, were constant and 556 were variable. A burn-in of 10% was maximum number of contiguous, nonconserved regions ¼ 2, estimated using Tracer v. 1.5 to be sufficient to remove the 574 mycoscience 56 (2015) 572e579

Table 1 e List of taxa, locality, collection/strain numbers of voucher specimens, fungarium accession numbers, and GenBank accession numbers for specimens used in this study. Species Locality Voucher Fungarium no. GenBank Accession no. ITS 28S Aspergillus fumigatus USA, Connecticut C. Thom 118 ATCC 1022 EF669931 U28460 A. nidulans USA, Texas 97-726 GenBank AF078899 AF109338 Geoglossum difforme USA, North Carolina ASM 10498 ILLS 67349 KC222124 KC222137 G. glabrum Czech Republic J. Gaisler s.n. ILLS 72358 KP657559 KP657564 G. simile USA, New York TJB 9613 ILLS 71160 KF944381 KF944383 Glutinoglossum glutinosum Czech Republic J. Gaisler s.n. ILLS 67353 KC222128 KC222141 G. heptaseptatum Czech Republic J. Gaisler s.n. ILLS 63754 KC222130 KC222143 Graddonia coracina USA, Tennessee ANM 2018 ILLS 60491 JQ256423 JN012009 Hemileucoglossum alveolatum USA, Michigan Imshaug 3640 MICH s.n. KP657560 KP657565 H. littorale Denmark Lœssøe 32VNJ2704 C: 35673 KP657561 KP657566 Leucoglossum durandii China s.n. HMAS 70090 HQ222875 N/A Maasoglossum aseptatum Sweden J. Nitare s.n. UPS: F-118883 KP657562 KP657567 M. verrucisporum Bhutan Sharma 17725 CUP-IN-000606 KP657563 KP657568 Unknown FH-DSH97-103 GenBank AY789398 AY789397 M. rufum Unknown Ingo-Clark-Geo 163 GenBank DQ257360 DQ470981 Nothomitra cinnamomea France Moingeon s.n. ILLS 61042 JQ256424 JQ256439 Orbilia auricolor United Kingdom AFTOL-ID 906 CBS 547.63 DQ491512 DQ470953 O. delicatula USA, Maine DHP 108 GenBank U72595 N/A phyllogena USA, Massachusetts WZ-Geo44-Clark GenBank AY789329 AY789328 P. varia Unknown WZ-Geo94-Clark GenBank AY789392 AY789391 Sabuloglossum arenarium The Netherlands CFR 181007 ILLS 61043 JQ256426 JQ256440 S. arenarium Finland Ohenoja s.n. OULU-F077201 GU324765 GU324764 Sarcoleotia globosa USA, Washington Trappe 26123 OSC 63633 AY789410 AY789409 S. globosa Unknown s.n. MBH 52476 AY789429 AY789428 flavida Unknown wz95 GenBank AF433155 AF433144 Thuemenidium atropurpureum United Kingdom S. Kelly s.n. K(M): 135612 EU784253 AY789307 USA, Tennessee ANM 2233 ILLS 67355 KC222132 KC222145 T. octopartitum USA, Tennessee ANM 2227 ILLS 67356 KC222134 KC222147

Collector acronyms: ANM ¼ Andrew N. Miller, ASM ¼ Andrew Methven, CFR ¼ Kees Roobeek, DHP ¼ Donald Pfister, DSH ¼ David Hibbett, TJB ¼ Timothy Baroni, WZ ¼ Zheng Wang. Fungarium acronyms: ATCC ¼ American Type Culture Collection, C ¼ University of Copenhagen, CBS ¼ Centraalbureau voor Schimmelcultures, CUP ¼ Cornell University, HMAS ¼ Kunming University, ILLS ¼ Illinois Natural History Survey, K(M) ¼ Kew (), MBH ¼ Marlborough College, MICH ¼ University of Michigan, OSC ¼ Oregon State University, OULU ¼ University of Oulu, UPS ¼ Uppsala University. Newly generated sequences are in bold. All other sequences are from GenBank. s.n. ¼ sine numero (no voucher or fungarium number is available).

pre-stationary posterior probability distribution, resulting in porrecta, with dark-colored narrow hyphae intertwining an ESS value of 2894.54. The standard deviation of split fre- inflated longitudinal cells. Paraphyses longer than the asci, quencies was 0.000284 at the end of the Bayesian analysis. hyaline to light brown, straight to curved or circinate, enlarged Fig. 1 represents the most likely tree produced by PhyML of the at the apex (up to 15 mm broad), agglutinated into an epi- final combined dataset. thecum by a light brown amorphous matter above. Asci Geoglossomycetes was supported as a clade with 90% ML clavate to clavate-cylindric, up to 165 mm in length and 16 mm BS, although with less than 0.95 BPP. Maasoglossum was wide, mostly 8-spored, tip Jþ in Melzer's. Ascospores light recovered as a distinct genus within Geoglossomycetes, with brown in maturity, cymbiform to ellipsoid, straight to slightly the clade containing the genera Maasoglossum, Nothomitra, and curved, aseptate, occasionally with 1e2 oil drops. Growing in Sarcoleotia well-supported (99% ML BS, 1.0 BPP) and distinct soil, often in disturbed habitats. from the remaining members of Geoglossomycetes. Maaso- The light brown, aseptate ascospores separate this genus glossum appears most closely related to Sarcoleotia, although from other members of Geoglossomycetes. Sabuloglossum also branch support is lacking. Geoglossum aseptatum and M. ver- has aseptate ascospores, although mature, septate ascospores rucisporum appear as separate species in the Maasoglossum are rarely encountered and the ascospores in that genus are clade, with 12% ITS and 9% LSU sequence divergence between typically hyaline except for occasional yellowish to light these two species (data not shown). brown pigments in mature ascospores. Maasoglossum can be distinguished from Hemileucoglossum in that the paraphyses, Maasoglossum K.S. Thind & R. Sharma emend. Hustad & A.N. while strongly agglutinated into an epithecum of brown Mill., Kavaka 12: 37 (1984) amorphous matter, are often curved to circinate at the apex in Mycobank no.: MB 25055. Maasoglossum, whereas the paraphyses are straight in Hemi- Ascomata scattered to gregarious, black, glabrous, leucoglossum. Furthermore, the agglutinating material in becoming slightly longitudinally rugulose upon drying, Maasoglossum is a paler brown than in Hemileucoglossum. 2e8 cm tall, up to 2 mm wide. terete, glabrous, textura Hemileucoglossum also has ascospores that long remain mycoscience 56 (2015) 572e579 575

79 Geoglossum glabrum ILLS 72358 97 Geoglossum difforme ILLS 67349 92 Geoglossum simile ILLS 71160 0.01 Leucoglossum durandii HMAS 70090 Sabuloglossum arenarium ILLS 61043 Sabuloglossum arenarium OULU-F077201 100 Trichoglossum hirsutum ILLS 67355 87 Trichoglossum octopartitum ILLS 67356 97 Glutinoglossum glutinosum ILLS 67353 Geoglossomycetes Glutinoglossum heptaseptatum ILLS 63754 100 Hemileucoglossum alveolatum Imshaug 3640 90 Hemileucoglossum littorale C: 35673 100 Maasoglossum aseptatum UPS: F-118883 Maasoglossum verrucisporum CUP-IN-000606 99 91 Sarcoleotia globosa OSC 63633 Sarcoleotia globosa MBH 52476 73 Nothomitra cinnamomea ILLS 61042 90 Microglossum rufum Ingo-Clark-Geo 163 97 Thuemenidium atropurpureum K(M): 135612 97 Microglossum olivaceum FH-DSH97-103 92 wz95 90 Graddonia coracina ILLS 60491 100 Aspergillus fumigatus NRRL 163 Aspergillus nidulans 97-726 100 91 Orbilia delicatula DHP 108 Orbilia auricolor AFTOL-ID 906 Peziza phyllogena WZ-Geo44-Clark Peziza varia ZW-Geo94-Clark

Fig. 1 e PhyML maximum likelihood phylogeny showing the position of Maasoglossum within Geoglossomycetes based on a combined dataset (1110 bp) of ITS and LSU nrDNA sequences ((-ln)L score ¼ 9595.503). Numbers at nodes indicate significant BS values (≥70%) based on 100 replicates; thickened branches indicate significant BPP (≥95%). Numbers associated with taxon names are fungarium accession numbers or strain numbers obtained from GenBank. Type species for genera in the Geoglossomycetes are shown in bold.

hyaline, but the ascospores rapidly develop septation and Distribution: Finland (Bonsdorff von et al. 2012), the some species (e.g. Hemileucoglossum littorale (Rostr.) Arauzo) Netherlands (Nitare 2012; www.gbif.org), Sweden (Nitare 1982, have pigmented mature ascospores. Sarcoleotia produces a 1984, 2012, www.gbif.org). pileate ascocarp and ascospores that remain hyaline and Specimens examined: Holotype (UPS:F-118883); SWEDEN, develop up to five septa at maturity. Vastra€ Gotaland,€ Bohuslan,€ Goteborg;€ Goteborgs€ Botaniska Tradga€ ˚rd (Gothenburg Botanic Garden), in soil among moss, 1 Maasoglossum aseptatum (Hakelier ex Nitare) Hustad & A.N. Sep 1987, J. Nitare s.n. Mill. comb. nov. Macroscopically, Maasoglossum aseptatum is hardly distin- Basionym ¼ Geoglossum aseptatum Hakelier ex Nitare, Wind- guishable from other species in Geoglossum. The ascocarp is ahlia 14: 40 (1984) [as “asaeptatum”]. within normal size ranges of other species of Geoglossum and Mycobank no.: MB 811291 Fig. 2. is black throughout. Upon drying, the hymenium becomes € Type: SWEDEN, Orebro, Narke,€ Axberg, Vitmossen, on soil slightly longitudinally rugulose. The small, light brown, in moss in the drip line of a small building, 1 Sep 1977, leg. N. aseptate ascospores are the most noticeable difference be- Hakelier s.n. (holotype, UPS:F-116948). tween this species and other Geoglossum. Additionally, the Etymology: Refers to the aseptate mature ascospores. paraphyses are slightly agglutinated with an amorphous Ascomata black throughout, lanceolate-linear, becoming brown material. This combination of characters led Nitare slightly longitudinally rugulose upon drying, 3.1e8.2 cm tall, (1982) to suggest M. aseptatum as a potential link between 0.1e0.5 cm wide. Stipe smooth, black, terete, not distinctly the genus Geoglossum and Thuemenidium atropurpureum different from fertile hymenium, up to 13 mm wide. Paraphy- (Batsch) Kuntze, a fungus formerly considered to belong in ses hyaline to subhyaline, straight to curved or circinate at tips, Geoglossaceae but which is now considered to belong in often enlarged at the apex (Fig. 2C), longer than asci, the upper Leotiomycetes (Ohenoja et al. 2010; Hustad et al. 2013). portions strongly agglutinated by a light brown amorphous Maasoglossum aseptatum was described from collections matter (Fig. 2E). Asci clavate, (80e)104e131(e141) (9.6e) made by Nils Hakelier from a single location in central Sweden 10.5e13.1(e14.2) mm (average 117.12 ± 13.21 11.8 ± 1.23 mm, in 1977 (Nitare 1984). To date, the fungus has only been found n ¼ 30), 8-spored, having zero or at most two uniseriate asco- in four sites: two locations in Sweden (Bohuslan,€ in the below, biseriate or triseriate above (Fig. 2A), tip Gothenburg Botanic Garden and the type locality in Narke),€ lightly Jþ in Melzer's(Fig. 2D). Ascospores cymbiform to one location in Finland (Tavastia, Hameenlinna,€ Levonkallio), obovoid or clavate, often enlarged at one end, (16.2e) and one location in the Netherlands (Gelderland, Voorst, 23.1e29.9(e35.0) (4.9e)5.4e6.3(e6.9) mm (average Gietelse Bos). Each location where ascocarps of M. aseptatum 25.47 ± 2.54 5.96 ± 0.48 mm, n ¼ 30), aseptate, hyaline to light were found fruiting in disturbed soil has a history of cultiva- golden brown, sometimes with one or two oil drops (Fig. 2B). tion, gardening, forestry, or other soil disturbance. The species 576 mycoscience 56 (2015) 572e579

Fig. 2 e Maasoglossum aseptatum from dried holotype material (UPS:BOT:F-116948). A: Ascus; B: Ascospores; C: Paraphyses; D: Ascus tip lightly Jþ; E: Hymenium. Bars:10mm.

has been reported from gardens and forests and possible as- sociations may exist between the fungus and plants including Rumex acetosa L., Luzula pilosa (L.) Willd., mosses including Atrichum undulatum (Hedw.) P. Beauv. and Fissidens adianthoides Hedw., and trees including Prunus padus L., Ulmus glabra Huds., and Picea abies (L.) H. Karst. (Nitare 1984, 2012; Bonsdorff von et al. 2012). Nitare (2012) suggested that the fungus acts as a decomposer in these ecosystems, although he also hypothe- sized the possibility that the fungus may form ericoid mycorrhizae with shrubs and trees. Although Maasoglossum aseptatum is regarded as Data Deficient in regional red lists of Swedish Fungi (Gardenfors€ 2010), this fungus should almost certainly be regarded as Critically Endangered due to the rarity of occurrence and the fragility of its habitat. Of the two Swedish localities for M. aseptatum, the site at the Gothenburg Botanic Garden was destroyed during renovations and the fungus has not been collected there since 2005 (Gardenfors€ 2010; Nitare 2012). The site in the Netherlands was also destroyed. Of the four known locations of M. aseptatum in the world, only the type location in Sweden (Narke)€ and the location in Finland remain.

Maasoglossum verrucisporum K.S. Thind & R. Sharma, Kavaka 12: 37 (1984) [as “verrucosporum”]. Mycobank no.: MB 544188 Fig. 3. Type: BHUTAN, Thimphu, Dochula, on soil among mosses Fig. 3 e Maasoglossum verrucisporum from dried isotype in mixed forest, 10,000 feet elev., 3 Aug 1984, R. Sharma 17725, material (CUP-IN 000606). A: Ascospores; B: Paraphyses; C: (holotype PAN 17725, isotype CUP-IN-000606). Ascus; D: Transverse section of axial stipe hyphae. Bars: Etymology: Refers to the warted ascospores. 10 mm. mycoscience 56 (2015) 572e579 577

Ascomata scattered to gregarious, black throughout, members of Geoglossomycetes. Lastly, the name Phaeoglossum cylindrical-clavate, distinctly rugose with longitudinal ridges Petch is illegitimate pursuant to Article 53.1 of the ICN especially prominent in dried specimens, 2e6 cm tall, (McNeill 2012) as it is a later homonym of Phaeoglossum 0.1e0.5 cm wide. Stipe smooth, black, terete, not distinctly Skottsb., a brown alga described by Skottsberg (1907). different from fertile hymenium, up to 1.5 mm wide. Pa- Maasoglossum has previously been misidentified as Thue- raphyses hyaline to subhyaline, straight to curved at tips, menidium (Geoglossum) atropurpureum (Nitare 2012) and more somewhat enlarged at the apex (Fig. 3B), longer than asci, the collections of it may exist under that misnomer in fungaria in upper portions agglutinated by a light brown amorphous Northern Europe. Both species of Maasoglossum and T. atro- matter. Asci clavate-cylindric, (112e)130e151(e165) (10.2e) purpureum possess hyaline paraphyses that are agglutinated 12.5e14.4(e16.0) mm (average 141.2 ± 10.1 13.3 ± 0.9 mm, into an epithecum, although in T. atropurpureum the epi- n ¼ 30), 8-spored (occasionally 4-spored), ascospores biseriate thecum is a vinous-brown compared to a light brown in both (Fig. 3C), ascus tip Jþ in Melzer's. Ascospores cymbiform to species of Maasoglossum. The ascomata of T. atropurpureum are obovoid or clavate, (23.9e)26.1e30.0(e32.0) (5.8e) distinctly purplish-brown when fresh, and the fertile portion 6.2e7.3(e7.9) mm (average 28.05 ± 1.95 6.78 ± 0.52 mm, n ¼ 30), is often irregular to spathulate and distinct from the sterile aseptate, hyaline to light brown, distinctly covered with stipe. Maasoglossum and T. atropurpureum are also easily minute, profuse warts (Fig. 3A). separated based on ascospore morphology: ascospores in T. Distribution: Bhutan (Thind and Sharma 1984). atropurpureum are initially continuous but become multi- Specimen examined: Isotype, CUP-IN-000606. guttulate and finally septate when overmature and remain Maasoglossum verrucisporum is known only from the type hyaline throughout development, whereas in both species of collection found in Bhutan. The continuous, light brown, Maasoglossum the ascospores are continuous and light brown warted ascospores distinguish this species from all other with distinct warts present in M. verrucisporum. members of Geoglossomycetes. The fungus is not named in The hyphae at the apex of the stipe in Maasoglossum the 2009 Biodiversity Action Plan for Bhutan (NBSAP 2009), contain inflated, hyaline, longitudinal elements and an although it should certainly be regarded as Critically Endan- anastomosing network of brown, narrow hyphae intertwining gered due to the extreme rarity of occurrence. them (Fig. 3D). The more derived members of Geo- Our results suggest that Maasoglossum belongs in Geo- glossomycetes also contain anastomosing hyphae in the stipe, glossomycetes and is likely most closely related to Nothomitra but the longitudinal cells are not as strongly inflated. The and Sarcoleotia, although the long-branch connecting Maaso- inflated longitudinal cells and narrower binding hyphae in the glossum to these genera suggests that Maasoglossum has long axial stipe of Maasoglossum are similar to those found in been evolving independently of Nothomitra and Sarcoleotia. Thuemenidium atropurpureum, but different from Microglossum These three genera have ecological and morphological simi- (Maas Geesteranus 1964). In Microglossum, the axial stipe hy- larities. Each of these genera is characterized by mature as- phae are comprised primarily of agglutinated, narrower lon- cospores that are hyaline or lightly colored and are aseptate, gitudinal elements, with binding hyphae very rarely observed. although they can become 1- to 3-septate when overmature in Nothomitra and Sarcoleotia also contain a densely interwoven Nothomitra and 3- to 5-septate when overmature in Sarcoleotia. internal stipe hyphal system, although the longitudinal ele- The derived members of Geoglossomycetes are characterized ments are not inflated as they are in Maasoglossum. by ascospores that are more consistently darkly pigmented In summary, our study supported the inclusion of Maa- and generally multiseptate when mature. These species may soglossum as a basal member of Geoglossomycetes. Our also be ecologically linked by fruiting in response to soil findings suggest that genera with hyaline- to lightly-pig- disturbance. mented and aseptate ascospores form the base of the Geo- Production of fruiting bodies following soil disturbance is glossomycetes clade, with the more derived members of the common in several fungi (Sagara 1992). Many discomycetous clade exhibiting darkly-pigmented and multiseptate asco- fungi are known to fruit following soil disturbance (e.g. Geo- spores (e.g. Geoglossum and Trichoglossum). The ascocarp of pyxis (Pers.) Sacc., Octospora Hedw., Peziza Dill. ex Fries, and Maasoglossum is morphologically similar to the more derived Trichophaea Cooke & W. Phillips) (Petersen 1970; Dix and members of Geoglossomycetes, with the fertile hymenium Webster 1995). Production of fruiting bodies in response to intergrading entirely with the sterile stipe. The morphology disturbance appears to be a basal character in Geo- of the ascospores and internal stipe hyphae of Maasoglossum glossomycetes, with Sarcoleotia globosa (Sommerf.: Fr) Korf hints at the common ancestry between Geoglossomycetes being most commonly encountered on recently disturbed soil and stalked earth tongues in Leotiomycetes, such as (Schumacher and Sivertsen 1987; Jumpponen et al. 1997). Thuemenidium. Petch (1922) described the genus Phaeoglossum Petch in Geoglossaceae to accommodate the species Phaeoglossum zeylanicum Petch from Sri Lanka. While we have not seen material of this species, we concur with Nitare (1984) that this Acknowledgments genus name does not apply to Maasoglossum based on differ- ences in morphology and habitat between the two genera. We wish to thank the following for graciously providing Petch's Phaeoglossum was described as a black club-shaped specimens for study: T Baroni, T Læssøe, P Lizon, AS Methven, fungus with white internal hyphae not known to be present J-M Moingeon, CF Roobeek, and the fungaria at CUP, MICH, in Geoglossomycetes. It is also described as growing on SAV, and WTU. The authors wish to acknowledge the decaying Loranthus L. fruits, a habitat not found in other following sources of funding to VPH: American Society of 578 mycoscience 56 (2015) 572e579

Plant Taxonomists, Discover Life in America, the Graduate Hustad VP, Kucera V, Rybarikov a N, Lizon P, Gaisler J, Baroni TJ, College at the University of Illinois at Urbana-Champaign, Miller AN, 2014. Geoglossum simile of North America and Mycological Society of America, and the North American Europe: distribution of a widespread earth tongue species and designation of an epitype. Mycological Progress 13: 857e866. Mycological Association. http://dx.doi.org/10.1007/s11557-014-0969-z. Hustad VP, Miller AN, 2011. Phylogenetic placement of four e references genera within the Leotiomycetes. North American Fungi 6: 1 13. Hustad VP, Miller AN, Dentinger BTM, Cannon PF, 2013. Generic circumscriptions in Geoglossomycetes. Persoonia 31: 101e111. http://dx.doi.org/10.3767/003158513X671235. Alfaro ME, Zoller S, Lutzoni F, 2003. Bayes or bootstrap? A Hustad VP, Miller AN, Moingeon J-M, Priou J-P, 2011. Inclusion of simulation study comparing the performance of Bayesian Nothomitra in Geoglossomycetes. Mycosphere 2: 646e654. http:// Markov Chain Monte Carlo sampling and bootstrapping in dx.doi.org/10.5943/mycosphere/2/6/5. assessing phylogenetic confidence. Molecular Biology and Jumpponen A, Weber NS, Trappe JM, Cazares E, 1997. Distribution Evolution 20: 255e266. http://dx.doi.org/10.1093/molbev/ and ecology of the ascomycete Sarcoleotia globosa in the United msg028. States. Canadian Journal of Botany 75: 2228e2231. Arauzo S, Iglesias P, 2014. La familia Geoglossaceae ss. str. en la Kirk PM, Cannon PF, Minter DW, Stalpers JA, 2008. Dictionary of the Peninsula Iberica y la Macaronesia (in Spanish and Basque). fungi, 10th edn. CAB International, Wallingford. Errotari 11: 166e259. Kluge AG, 1989. A concern for evidence and phylogenetic Bonsdorff von T, Niskanen T, Kytovuori€ I, Liimatainen K, Vauras J, hypothesis of relationships among Epicrates (Boidae, Kokkonen K, Huhtinen S, Puolasmaa A, Kosonen L, 2012. New Serpentes). Systematic Biology 38: 7e25. http://dx.doi.org/10. national and regional biological records for Finland 1. 1093/sysbio/38.1.7. Contributions to agaricoid and ascomycetoid taxa of fungi. Landvik S, 1996. , a fruit-body producing genus of the Memoranda Societatis pro Fauna et Flora Fennica 88: 53e60. basal ascomycetes, as shown by SSU and LSU rDNA Brock PM, Doring€ H, Bidartondo MI, 2009. How to know the sequences. Mycological Research 100: 199e202. http://dx.doi. unknown fungi: the role of a herbarium. New Phytologist 181: org/10.1016/S0953-7562(96)80122-5. 719e724. http://dx.doi.org/10.1111/j.1469-8137.2008.02703.x. Lumbsch HT, Huhndorf SM, 2010. Myconet Volume 14. Part 1. Castresana J, 2000. Selection of conserved blocks from multiple Outline of Ascomycotad2009. Part two. Notes on ascomycete alignments and their use in phylogenetic analysis. Molecular systematics Nos. 4751e5113. Fieldiana Life and Earth Sciences 1: Biology and Evolution 17: 540e552. http://dx.doi.org/10.1093/ 1e64; http://dx.doi.org/10.3158/1557.1. oxfordjournals.molbev.a026334. Lutzoni F, Kauff F, Cox CJ, McLaughlin D, Celio G, Dentinger B, Crous PW, Gams W, Stalpers JA, Robert V, Stegehuis G, 2004. Padamsee M, Hibbett D, James TY, Baloch E, Grube M, Reeb V, MycoBank: an online initiative to launch mycology into the Hofstetter V, Schoch C, Arnold AE, Miadlikowska J, Spatafora J, 21st century. Studies in Mycology 50: 19e22. Johnson D, Hambleton S, Crockett M, Shoemaker R, Sung G-H, Darriba D, Taboada GL, Doallo R, Posada D, 2012. jModelTest 2: Lucking R, Lumbsch T, O'Donnell K, Binder M, Diederich P, more models, new heuristics and parallel computing. Nature Ertz D, Gueidan C, Hansen K, Harris RC, Hosaka K, Lim Y-W, Methods 9: 772. http://dx.doi.org/10.1038/nmeth.2109. Matheny B, Nishida H, Pfister D, Rogers J, Rossman A, Schmitt I, Dix NJ, Webster J, 1995. Fungal ecology. Chapman and Hall, Sipman H, Stone J, Sugiyama J, Yahr R, Vilgalys R, 2004. London. Assembling the fungal tree of life: progress, classification, and Edgar R, 2004. MUSCLE: multiple sequence alignment with high evolution of subcellular traits. American Journal of Botany 91: accuracy and high throughput. Nucleic Acids Research 32: 1446e1480. http://dx.doi.org/10.3732/ajb.91.10.1446. 1792e1797. http://dx.doi.org/10.1093/nar/gkh340. Maas Geesteranus RA, 1964. On some white-spored Eernise DJ, Kluge AG, 1993. Taxonomic congruence versus total Geoglossaceae. Persoonia 3: 81e96. evidence, and amniote phylogeny inferred from fossils, McNeill J, 2012. International code of nomenclature for algae, fungi, and molecules, and morphology. Molecular Biology and Evolution 10: plants (Melbourne Code): adopted by the Eighteenth International 1170e1195. Botanical Congress Melbourne Australia, July 2011. Koeltz Felsenstein J, 1985. Confidence limits on phylogenies: an Scientific Books, Konigstein€ . approach using the bootstrap. Evolution 39: 783e791. http://dx. Miller MA, Pfeiffer W, Schwartz T, 2010. Creating the CIPRES doi.org/10.2307/2408678. Science Gateway for inference of large phylogenetic trees. In: Gardenfors€ U, 2010. Rodlistade€ arter i Sverige 2010 (in Swedish). Proceedings of the Gateway Computing Environments Workshop Artdatabanken SLU, Uppsala. (GCE), New Orleans, November 14,pp1e8. Gouy M, Guindon S, Gascuel O, 2010. SeaView version 4: a NBSAP Bhutan, 2009. National biodiversity action plan for Bhutan. multiplatform graphical user interface for sequence alignment National Biodiversity Center, Ministry of Agriculture, and phylogenetic tree building. Molecular Biology and Evolution Thimphu. 27: 221e224. http://dx.doi.org/10.1093/molbev/msp259. Nitare J, 1982. Geoglossum arenarium, en problematisk jordtunga Guindon S, Dufayard JF, Lefort V, Anisomova M, Hordijk W, (in Swedish). Goteborgs€ Svampklubbs A˚ rsskrift 1981: 61e68. Gascuel O, 2010. New algorithms and methods to estimate Nitare J, 1984. Geoglossum asaeptatum n. sp., a new earth tongue maximum-likelihood phylogenies: assessing the performance with brown continuous spores. Windahlia 14: 37e42. of PhyML 3.0. Systematic Biology 59: 307e321. http://dx.doi.org/ Nitare J, 2012. Geoglossum aseptatum, slat jordtunga (in Swedish). 10.1093/sysbio/syq010. Artdatabanken, Uppsala. http://www.artfakta.se/artfaktablad/ Guindon S, Gascuel O, 2003. A simple, fast and accurate method Geoglossum_Aseptatum_700.pdf. Accessed 15 Jan 2015. to estimate large phylogenies by maximum-likelihood. Ohenoja E, Wang Z, Townsend JP, Mitchell D, Voitk A, 2010. Systematic Biology 52: 696e704. http://dx.doi.org/10.1080/ Northern species of the earth tongue genus Thuemenidium 10635150390235520. revisited, considering morphology, ecology and molecular Hillis DM, Bull JJ, 1993. An empirical test of bootstrapping as a phylogeny. Mycologia 102: 1089e1095. http://dx.doi.org/10. method for assessing confidence in phylogenetic analysis. 3852/09-317. Systematic Biology 42: 182e192. http://dx.doi.org/10.2307/ Petch T, 1922. Additions to Ceylon fungi (II). Annals of the Royal 2992540. Botanic Gardens of Peradeniya 7: 279e322. mycoscience 56 (2015) 572e579 579

Petersen PM, 1970. Danish fireplace fungi. An ecological Thind KS, Sharma R, 1984. Maasoglossum, a new genus of investigation on fungi on burns. Dansk Botanisk Arkiv 27: 1e97. Geoglossaceae from the Eastern Himalayas. Kavaka 12: Peterson SW, 2008. Phylogenetic analysis of Aspergillus species 37e40. using DNA sequences from four loci. Mycologia 100: 205e226. Vilgalys R, Hester M, 1990. Rapid identification and mapping of http://dx.doi.org/10.3852/mycologia.100.2.205. enzymatically amplified ribosomal DNA from several Pfister DH, 1997. Castor, pollux and life histories of fungi. Cryptococcus species. Journal of Bacteriology 172: 4238e4246. Mycologia 89: 1e23. http://dx.doi.org/10.2307/3761168. Wang Z, Binder M, Hibbett DS, 2002. A new species of Cudonia Posada D, Buckley TR, 2004. Model selection and model averaging based on morphological and molecular data. Mycologia 94: in phylogenetics: advantages of Akaike information Criterion 641e650. http://dx.doi.org/10.2307/3761715. and Bayesian approaches over likelihood ratio tests. Systematic Wang Z, Binder M, Hibbett DS, 2005. Life history and systematics Biology 53: 793e808. http://dx.doi.org/10.1080/ of the aquatic discomycete Mitrula (, Ascomycota) 10635150490522304. based on cultural, morphological, and molecular studies. Promputtha I, Miller AN, 2010. Three new species of American Journal of Botany 92: 1565e1574. http://dx.doi.org/10. Acanthostigma (Tubeufiaceae, Dothidiomycetes) from the Great 3732/ajb.92.9.1565. Smoky Mountains National Park. Mycologia 102: 574e587. Wang Z, Binder M, Schoch CL, Johnston PR, Spatafora JW, http://dx.doi.org/10.3852/09-051. Hibbett DS, 2006. Evolution of Helotialean fungi Rambaut A, Drummond AJ, 2009. Tracer v.1.5. http://tree.bio.ed.ac. (Leotiomycetes, ): a nuclear rDNA phylogeny. uk/software/tracer. Accessed 15 Jan 2015. Molecular Phylogenetics and Evolution 41: 295e312. http://dx.doi. Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, org/10.1016/j.ympev.2006.05.031. Hohna€ S, Larget B, Liu L, Suchard MA, Huelsenbeck JP, 2012. Wang Z, Nilsson RH, Lopez-Giraldez F, Zhuang W, Dai Y, MrBayes 3.2: efficient Bayesian phylogenetic inference and Johnston PR, Townsend JP, 2011. Tasting soil fungal diversity model choice across a large model space. Systematic Biology 61: with earth tongues: phylogenetic test of SATe alignments for 539e542. http://dx.doi.org/10.1093/sysbio/sys029. environmental ITS data. PLoS One 6: e19039. Sagara N, 1992. Experimental disturbances and epigeous fungi. In: White TJ, Bruns T, Lee S, Taylor JW, 1990. Amplification and direct Carroll GC, Wicklow DT (eds), The fungal community, 2nd edn. sequencing of fungal ribosomal RNA genes for phylogenetics. Marcel Dekker, New York, pp 427e454. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds), PCR Schoch CL, Wang Z, Townsend JP, Spatafora JW, 2009. Protocol: a guide to methods and applications. Academic Press, San Geoglossomycetes cl. nov., Geoglossales ord. nov. and taxa Diego, pp 315e322. above class rank in the Ascomycota tree of life. Persoonia 22: Wiens JJ, 1998. Combining data sets with different phylogenetic 129e138. http://dx.doi.org/10.3767/003158509X461486. histories. Systematic Biology 47: 568e581. http://dx.doi.org/10. Schumacher T, Sivertsen S, 1987. Sarcoleotia globosa (Sommerf.: 1080/106351598260581. Fr.) Korf, , ecology, and distribution. In: Laursen GA, Zhao J, Kong F, Li R, Wang X, Wan Z, Wang D, 2001. Identification Ammirati JF, Redhead SA (eds), Arctic and Alpine mycology, vol. of Aspergillus fumigatus and related species by nested PCR 2. Plenum Press, New York and London, pp 163e176. targeting ribosomal DNA internal transcribed spacer regions. Skottsberg C, 1907. Zur Kenntnis der subantarktischen und Journal of Clinical Microbiology 39: 2261e2266. http://dx.doi.org/ antarktischen Meeresalgen. I. Phaeophyceen (in German). 10.1128/JCM.39.6.2261-2266.2001. Lithographisches Institut des Generalstabs, Stockholm.