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Short communication

Maasoglossum, a basal genus 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, USAb Illinois Natural History Survey, University of Illinois, 1816 S. Oak St., Champaign, IL 61820, USA

a r t i c l e i n f o

Article history:

Received 19 January 2015

Received in revised form

22 May 2015

Accepted 24 May 2015

Available online 24 June 2015

Keywords:

Ascomycota

Earth tongues

Geoglossum

Phylogenetics

Systematics

* Corresponding author. Department of PlantUSA. Tel.: þ01 618 843 0784; fax: þ01 217 265

E-mail address: vhustad@illinois.edu (V.Phttp://dx.doi.org/10.1016/j.myc.2015.05.0031340-3540/© 2015 The Mycological Society of

a b s t r a c t

The genus Maasoglossum is examined using morphology, ecology, and molecular system-

atics of the internal transcribed spacer region and large subunit of the nuclear ribosomal

RNA gene, all of which support the placement of Maasoglossum among the basal members

of Geoglossomycetes. The morphology of the genus extends the range of ascocarp and

ascospore development in Geoglossomycetes. The ecology and conservational significance

of the genus is discussed, a nomenclatural transfer of Geoglossum aseptatum to Maaso-

glossum is made, and an emended description of Maasoglossum is provided.

© 2015 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.

The class Geoglossomycetes is a basal ascocarp-producing

member of the ‘Leotiomyceta’ (Schoch et al. 2009). Kirk et al.

(2008) suggest 48 species in four genera belong in the class,

while Index Fungorum lists over 200 names for these same

four genera. Recent research indicates eight genera belong in

the class: Geoglossum Pers., Glutinoglossum Hustad et al., Hem-

ileucoglossum Arauzo, Leucoglossum S. Imai, Nothomitra Maas

Geest., Sabuloglossum Hustad et al., Sarcoleotia S. Ito & S. Imai,

and Trichoglossum Boud. (Hustad et al. 2013; Arauzo and

Iglesias 2014). The prevalence of rarely collected species has

made an accurate estimation of the number of species in the

class difficult.

The rare species, Geoglossum aseptatum Hakelier ex Nitare,

is characterized by light brown, aseptatemature ascospores, a

Biology, University of Illi4678.

. Hustad).

Japan. Published by Else

character not found in any other member of the genus Geo-

glossum. Preliminary molecular analysis (data not shown)

suggested that this fungus belonged in a separate lineage from

Geoglossum and a review of the literature suggested a possible

link between G. aseptatum and the genus Maasoglossum Thind

& R. Sharma.

The genus Maasoglossum was described by Thind and

Sharma (1984) to accommodate a single species, M. verrucis-

porum, described from a collection made in 1981 from the

temperate Eastern Himalayan forest of Bhutan. The authors

noted the macroscopic similarity of the ascocarp to that of

Geoglossum, but that it differed in the hymenium of M. verru-

cisporum contained small ridges and grooves that became

more prominent upon drying, a character rarely encountered

nois at Urbana-Champaign, 505 S. Goodwin Ave., Urbana IL 61801,

vier B.V. All rights reserved.

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myc o s c i e n c e 5 6 ( 2 0 1 5 ) 5 7 2e5 7 9 573

in Geoglossum. Additionally, the one-celled, lightly-colored,

warted ascospores precluded the placement of this fungus in

Geoglossum. Since no other members of the Geoglossaceae

sensu lato contain ornamented ascospores, they were unsure

of the placement of this genus within Geoglossaceae sensu lato

or Leotiomycetes (Microglossum). The genus is currently

regarded as incertae sedis within Leotiomycetes (Lumbsch and

Huhndorf 2010). In this paper we provide an in-depth

assessment of the genus Maasoglossum based on

morphology, ecology, and molecular systematics, and test the

hypotheses that Maasoglossum is a basal member of Geo-

glossomycetes and that G. aseptatum belongs in the genus

Maasoglossum.

For this study, the type specimens of both Geoglossum

aseptatum and Maasoglossum verrucisporum were obtained and

examined. Dried ascomata were hand-sectioned and squash

mounted in 5% KOH andmicromorphological characters were

observed. Images of pertinent micromorphological characters

were captured using a QImaging QColor 3 digital camera

mounted on an Olympus BX51 compound microscope using

differential interference microscopy. Images were processed

with Adobe Photoshop v. 7.0 (Adobe Systems Inc., Mountain

View, California). A minimum of 30 measurements was made

for each micromorphological character using NIH Image v.

1.63 (National Institutes of Health, Bethesda, Maryland).

Taxonomic novelties and associated data were deposited in

MycoBank (Crous et al. 2004).

Total genomic DNA was extracted from approximately

0.5 cm2 of hymenium tissue from a single dried ascoma with

the DNeasy Plant Mini Kit (QIAGEN Inc., Valencia, California)

for recently-collected specimens (collected within 20 years).

For older specimens, DNA was extracted from approximately

0.2 cm2 of hymenium tissue using the EZNA Forensic DNA kit

(Omega Bio-Tek, Norcross, Georgia). Gene fragments were PCR

amplified following the methods outlined in Promputtha and

Miller (2010) and purified using a Wizard SV Gel and PCR

Clean-Up System (Promega Corp., Fitchburg, Wisconsin). Se-

quenceswere generated on an ABI Applied Biosystems 3730XL

high-throughput DNA capillary sequencer at the UIUC Keck

Center for Comparative and Functional Genomics. Two re-

gions of the nuclear rRNA gene were used for molecular

phylogenetic analysis: the ca. 570 bp internal transcribed

spacer (ITS) region, consisting of the ITS1, 5.8S, and ITS2 re-

gions, was amplified and sequenced using a combination of

the primers ITS5, ITS1, ITS2, ITS3, and ITS4 (White et al. 1990);

and a ca. 630 bp fragment of the 28S large subunit (LSU) region

was amplified and sequenced with the primers JS1 (Landvik

1996) and LR3 (Vilgalys and Hester 1990). Due to the age of

the material, attempts to PCR amplify single-copy protein

coding genes (i.e. RPB1, RPB2, MCM7) were unsuccessful. Out-

group species were chosen from closely related taxa based on

previous studies (Wang et al. 2006; Hustad et al. 2013).

Sequence alignments were created by eye in Sequencher

5.0.1 (Gene Codes Corp., Ann Arbor, Michigan) and optimized

if necessary using Muscle v. 3.8.31 (Edgar 2004) in SeaView v.

4.4.2 (Gouy et al. 2010). Ambiguous regions were removed

from each dataset using Gblocks 0.91b (Castresana 2000)

under the following parameters: for ITS: minimum number of

sequences for both conserved and flanking regions ¼ 15,maximum number of contiguous, nonconserved regions ¼ 2,

minimum length of a block ¼ 2, and allowed gap positions in50% of sequences; for LSU: same as ITS except minimum

number of sequences for both conserved and flanking

regions ¼ 14.The GTR þ I þ G model was determined to be the best-fit

model of evolution for both ribosomal regions using the

Akaike Information Criterion (AIC) (Posada and Buckley 2004)

in jModelTest 2.0 v. 0.1.1 (Guindon and Gascuel 2003; Darriba

et al. 2012) on the XSEDE platform of the CIPRES Science

Gateway Teragrid (Miller et al. 2010) and was used in phylo-

genetic analyses of each individual dataset. Maximum likeli-

hood (ML) analyses were performed using PhyML 3.0 (Guindon

et al. 2010) on the ATGC server (http://www.atgc-montpellier.

fr/phyml/). The best of subtree pruning and regrafting (SPR)

and nearest neighbor interchange (NNI) was implemented

during the heuristic search. Nonparametric bootstrap support

(Felsenstein 1985) (BS) was determined with 1000 replicates.

Clades with BS values of �70% were considered significant(Hillis and Bull 1993).

Bayesian inference employing aMarkov ChainMonte Carlo

(MCMC) algorithm was performed using MrBayes v. 3.2.2

(Ronquist et al. 2012) on the XSEDE platform. Four indepen-

dent chains of MCMC were run for 10 million generations.

Clades with Bayesian posterior probability (BPP) values of

�95% were considered significant (Alfaro et al. 2003). Tracer v.1.5 (Rambaut and Drummond 2009) was used to estimate

effective sample size (ESS) using the standard deviation of

split frequencies produced by Bayesian analysis.

Individual ITS and LSU datasets were examined for po-

tential conflict before concatenation into a single dataset for

total evidence analysis (Kluge 1989; Eernisse and Kluge 1993).

Individual gene phylogenies were considered to be incon-

gruent if clades with significant ML BS and BPP support were

conflicting in individual tree topologies (Wiens 1998; Alfaro

et al. 2003; Lutzoni et al. 2004). Since no incongruencies were

found among individual phylogenies, the ITS and LSU data-

sets were concatenated and final ML and Bayesian analyses

were performed on the combined dataset. Alignments and

trees are deposited in TreeBASE (http://treebase.org) under

submission ID 16591.

Ten new sequences were generated in this study, five ITS

and five LSU sequences (Table 1). These were analyzed

together with 23 ITS and 21 LSU sequences from our previous

studies (Hustad and Miller 2011; Hustad et al. 2011, 2013, 2014)

along with sequences of Geoglossomycetes from previously

published research (Pfister 1997; Zhao et al. 2001; Wang et al.

2002, 2005, 2006, 2011; Peterson 2008; Brock et al. 2009;

Ohenoja et al. 2010). Twenty-eight collections, representing a

total of 15 Geoglossomycetes species (including sequences

from type species of each genus in Geoglossomycetes except

Sarcoleotia) and eleven outgroup species, were included in the

analyses. Of the 28 collections included in the final dataset,

none lack ITS and only two lack LSU, with sequences for both

markers available for 93% of the collections (Table 1).

The final combined data matrix had an aligned length of

2712 bp, which was reduced to 1110 bp after the removal of

1602 bp of ambiguously aligned regions by Gblocks. Of the

1110 characters used in the final phylogenetic analyses, 554

were constant and 556 were variable. A burn-in of 10% was

estimated using Tracer v. 1.5 to be sufficient to remove the

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Table 1 e List of taxa, locality, collection/strain numbers of voucher specimens, fungarium accession numbers, andGenBank 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 KP657566Leucoglossum 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

Microglossum olivaceum 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

Peziza 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

Spathularia flavida Unknown wz95 GenBank AF433155 AF433144

Thuemenidium atropurpureum United Kingdom S. Kelly s.n. K(M): 135612 EU784253 AY789307

Trichoglossum hirsutum 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,TJB¼ Timothy Baroni, WZ¼ ZhengWang. Fungarium acronyms: ATCC¼ American Type Culture Collection, C¼University of Copenhagen, CBS¼ Centraalbureau voor Schimmelcultures, CUP ¼ Cornell University, HMAS ¼ Kunming University, ILLS ¼ Illinois Natural History Survey,K(M)¼Kew (Mycology), MBH¼Marlborough College, MICH¼University ofMichigan, OSC¼Oregon State University, OULU¼University 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 orfungarium number is available).

my c o s c i e n c e 5 6 ( 2 0 1 5 ) 5 7 2e5 7 9574

pre-stationary posterior probability distribution, resulting in

an ESS value of 2894.54. The standard deviation of split fre-

quencies was 0.000284 at the end of the Bayesian analysis.

Fig. 1 represents themost likely tree produced by PhyML of the

final combined dataset.

Geoglossomycetes was supported as a clade with 90% ML

BS, although with less than 0.95 BPP. Maasoglossum was

recovered as a distinct genus within Geoglossomycetes, with

the clade containing the generaMaasoglossum,Nothomitra, and

Sarcoleotia well-supported (99% ML BS, 1.0 BPP) and distinct

from the remaining members of Geoglossomycetes. Maaso-

glossum appears most closely related to Sarcoleotia, although

branch support is lacking. Geoglossum aseptatum and M. ver-

rucisporum appear as separate species in the Maasoglossum

clade, with 12% ITS and 9% LSU sequence divergence between

these two species (data not shown).

Maasoglossum K.S. Thind & R. Sharma emend. Hustad & A.N.

Mill., Kavaka 12: 37 (1984)

Mycobank no.: MB 25055.

Ascomata scattered to gregarious, black, glabrous,

becoming slightly longitudinally rugulose upon drying,

2e8 cm tall, up to 2 mm wide. Stipe terete, glabrous, textura

porrecta, with dark-colored narrow hyphae intertwining

inflated longitudinal cells. Paraphyses longer than the asci,

hyaline to light brown, straight to curved or circinate, enlarged

at the apex (up to 15 mm broad), agglutinated into an epi-

thecum by a light brown amorphous matter above. Asci

clavate to clavate-cylindric, up to 165 mm in length and 16 mm

wide, mostly 8-spored, tip Jþ in Melzer's. Ascospores lightbrown in maturity, cymbiform to ellipsoid, straight to slightly

curved, aseptate, occasionally with 1e2 oil drops. Growing in

soil, often in disturbed habitats.

The light brown, aseptate ascospores separate this genus

from other members of Geoglossomycetes. Sabuloglossum also

has aseptate ascospores, althoughmature, septate ascospores

are rarely encountered and the ascospores in that genus are

typically hyaline except for occasional yellowish to light

brown pigments in mature ascospores. Maasoglossum can be

distinguished from Hemileucoglossum in that the paraphyses,

while strongly agglutinated into an epithecum of brown

amorphousmatter, are often curved to circinate at the apex in

Maasoglossum, whereas the paraphyses are straight in Hemi-

leucoglossum. Furthermore, the agglutinating material in

Maasoglossum is a paler brown than in Hemileucoglossum.

Hemileucoglossum also has ascospores that long remain

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Geoglossum glabrum ILLS 72358Geoglossum difforme ILLS 67349

79

Geoglossum simile ILLS 7116097

Leucoglossum durandii HMAS 7009092

Sabuloglossum arenarium ILLS 61043Sabuloglossum arenarium OULU-F077201

Trichoglossum hirsutum ILLS 67355Trichoglossum octopartitum ILLS 67356

100

Glutinoglossum glutinosum ILLS 67353Glutinoglossum heptaseptatum ILLS 63754

97

Hemileucoglossum alveolatum Imshaug 3640Hemileucoglossum littorale C: 35673

100

87

Maasoglossum aseptatum UPS: F-118883Maasoglossum verrucisporum CUP-IN-000606

100

Sarcoleotia globosa OSC 63633Sarcoleotia globosa MBH 52476

91

Nothomitra cinnamomea ILLS 61042

99

90

Microglossum rufum Ingo-Clark-Geo 163Thuemenidium atropurpureum K(M): 135612

90

Microglossum olivaceum FH-DSH97-10397

Spathularia flavida wz95Graddonia coracina ILLS 60491

9297

Aspergillus fumigatus NRRL 163Aspergillus nidulans 97-726

100

90

73

Orbilia delicatula DHP 108Orbilia auricolor AFTOL-ID 906

91

Peziza phyllogena WZ-Geo44-ClarkPeziza varia ZW-Geo94-Clark

100

0.01

Geoglossomycetes

Fig. 1 e PhyMLmaximum likelihood phylogeny showing the position ofMaasoglossumwithin Geoglossomycetes based on a

combined dataset (1110 bp) of ITS and LSU nrDNA sequences ((-ln)L score¼ 9595.503). Numbers at nodes indicate significantBS values (≥70%) based on 100 replicates; thickened branches indicate significant BPP (≥95%). Numbers associated withtaxon names are fungarium accession numbers or strain numbers obtained from GenBank. Type species for genera in the

Geoglossomycetes are shown in bold.

myc o s c i e n c e 5 6 ( 2 0 1 5 ) 5 7 2e5 7 9 575

hyaline, but the ascospores rapidly develop septation and

some species (e.g. Hemileucoglossum littorale (Rostr.) Arauzo)

have pigmented mature ascospores. Sarcoleotia produces a

pileate ascocarp and ascospores that remain hyaline and

develop up to five septa at maturity.

Maasoglossum aseptatum (Hakelier ex Nitare) Hustad & A.N.

Mill. comb. nov.

Basionym ¼ Geoglossum aseptatum Hakelier ex Nitare, Wind-ahlia 14: 40 (1984) [as “asaeptatum”].

Mycobank no.: MB 811291 Fig. 2.

Type: SWEDEN, €Orebro, N€arke, Axberg, Vitmossen, on soil

in moss in the drip line of a small building, 1 Sep 1977, leg. N.

Hakelier s.n. (holotype, UPS:F-116948).

Etymology: Refers to the aseptate mature ascospores.

Ascomata black throughout, lanceolate-linear, becoming

slightly longitudinally rugulose upon drying, 3.1e8.2 cm tall,

0.1e0.5 cm wide. Stipe smooth, black, terete, not distinctly

different from fertile hymenium, up to 13 mm wide. Paraphy-

seshyaline to subhyaline, straight to curvedor circinate at tips,

often enlarged at the apex (Fig. 2C), longer than asci, the upper

portions strongly agglutinated by a light brown amorphous

matter (Fig. 2E). Asci clavate, (80e)104e131(e141) � (9.6e)10.5e13.1(e14.2) mm (average 117.12 ± 13.21 � 11.8 ± 1.23 mm,n ¼ 30), 8-spored, having zero or at most two uniseriate asco-spores below, biseriate or triseriate above (Fig. 2A), ascus tip

lightly Jþ in Melzer's (Fig. 2D). Ascospores cymbiform toobovoid or clavate, often enlarged at one end, (16.2e)

23.1e29.9(e35.0) � (4.9e)5.4e6.3(e6.9) mm (average25.47 ± 2.54 � 5.96 ± 0.48 mm, n ¼ 30), aseptate, hyaline to lightgolden brown, sometimes with one or two oil drops (Fig. 2B).

Distribution: Finland (Bonsdorff von et al. 2012), the

Netherlands (Nitare 2012; www.gbif.org), Sweden (Nitare 1982,

1984, 2012, www.gbif.org).

Specimens examined: Holotype (UPS:F-118883); SWEDEN,

V€astra G€otaland, Bohusl€an, G€oteborg; G€oteborgs Botaniska

Tr€adgård (Gothenburg Botanic Garden), in soil among moss, 1

Sep 1987, J. Nitare s.n.

Macroscopically, Maasoglossum aseptatum is hardly distin-

guishable from other species in Geoglossum. The ascocarp is

within normal size ranges of other species of Geoglossum and

is black throughout. Upon drying, the hymenium becomes

slightly longitudinally rugulose. The small, light brown,

aseptate ascospores are the most noticeable difference be-

tween this species and other Geoglossum. Additionally, the

paraphyses are slightly agglutinated with an amorphous

brown material. This combination of characters led Nitare

(1982) to suggest M. aseptatum as a potential link between

the genus Geoglossum and Thuemenidium atropurpureum

(Batsch) Kuntze, a fungus formerly considered to belong in

Geoglossaceae but which is now considered to belong in

Leotiomycetes (Ohenoja et al. 2010; Hustad et al. 2013).

Maasoglossum aseptatum was described from collections

made byNils Hakelier froma single location in central Sweden

in 1977 (Nitare 1984). To date, the fungus has only been found

in four sites: two locations in Sweden (Bohusl€an, in the

Gothenburg Botanic Garden and the type locality in N€arke),

one location in Finland (Tavastia, H€ameenlinna, Levonkallio),

and one location in the Netherlands (Gelderland, Voorst,

Gietelse Bos). Each location where ascocarps of M. aseptatum

were found fruiting in disturbed soil has a history of cultiva-

tion, gardening, forestry, or other soil disturbance. The species

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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: 10 mm.

Fig. 3 e Maasoglossum verrucisporum from dried isotype

material (CUP-IN 000606). A: Ascospores; B: Paraphyses; C:

Ascus; D: Transverse section of axial stipe hyphae. Bars:

10 mm.

my c o s c i e n c e 5 6 ( 2 0 1 5 ) 5 7 2e5 7 9576

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

Atrichumundulatum (Hedw.) P. Beauv. and Fissidens adianthoides

Hedw., and trees including Prunus padus L.,Ulmus glabraHuds.,

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 (G€ardenfors

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 (G€ardenfors 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 (N€arke) 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

in mixed forest, 10,000 feet elev., 3 Aug 1984, R. Sharma 17725,

(holotype PAN 17725, isotype CUP-IN-000606).

Etymology: Refers to the warted ascospores.

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myc o s c i e n c e 5 6 ( 2 0 1 5 ) 5 7 2e5 7 9 577

Ascomata scattered to gregarious, black throughout,

cylindrical-clavate, distinctly rugose with longitudinal ridges

especially prominent in dried specimens, 2e6 cm tall,

0.1e0.5 cm wide. Stipe smooth, black, terete, not distinctly

different from fertile hymenium, up to 1.5 mm wide. Pa-

raphyses hyaline to subhyaline, straight to curved at tips,

somewhat enlarged at the apex (Fig. 3B), longer than asci, the

upper portions agglutinated by a light brown amorphous

matter. Asci clavate-cylindric, (112e)130e151(e165) � (10.2e)12.5e14.4(e16.0) mm (average 141.2 ± 10.1 � 13.3 ± 0.9 mm,n ¼ 30), 8-spored (occasionally 4-spored), ascospores biseriate(Fig. 3C), ascus tip Jþ in Melzer's. Ascospores cymbiform toobovoid or clavate, (23.9e)26.1e30.0(e32.0) � (5.8e)6.2e7.3(e7.9) mm (average 28.05 ± 1.95� 6.78 ± 0.52 mm, n¼ 30),aseptate, hyaline to light brown, distinctly covered with

minute, profuse warts (Fig. 3A).

Distribution: Bhutan (Thind and Sharma 1984).

Specimen examined: Isotype, CUP-IN-000606.

Maasoglossum verrucisporum is known only from the type

collection found in Bhutan. The continuous, light brown,

warted ascospores distinguish this species from all other

members of Geoglossomycetes. The fungus is not named in

the 2009 Biodiversity Action Plan for Bhutan (NBSAP 2009),

although it should certainly be regarded as Critically Endan-

gered due to the extreme rarity of occurrence.

Our results suggest that Maasoglossum belongs in Geo-

glossomycetes and is likely most closely related to Nothomitra

and Sarcoleotia, although the long-branch connecting Maaso-

glossum to these genera suggests that Maasoglossum has long

been evolving independently of Nothomitra and Sarcoleotia.

These three genera have ecological and morphological simi-

larities. Each of these genera is characterized by mature as-

cospores that are hyaline or lightly colored and are aseptate,

although they can become 1- to 3-septate when overmature in

Nothomitra and 3- to 5-septate when overmature in Sarcoleotia.

The derived members of Geoglossomycetes are characterized

by ascospores that are more consistently darkly pigmented

and generally multiseptate when mature. These species may

also be ecologically linked by fruiting in response to soil

disturbance.

Production of fruiting bodies following soil disturbance is

common in several fungi (Sagara 1992). Many discomycetous

fungi are known to fruit following soil disturbance (e.g. Geo-

pyxis (Pers.) Sacc., Octospora Hedw., Peziza Dill. ex Fries, and

Trichophaea Cooke & W. Phillips) (Petersen 1970; Dix and

Webster 1995). Production of fruiting bodies in response to

disturbance appears to be a basal character in Geo-

glossomycetes, with Sarcoleotia globosa (Sommerf.: Fr) Korf

being most commonly encountered on recently disturbed soil

(Schumacher and Sivertsen 1987; Jumpponen et al. 1997).

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

genus name does not apply to Maasoglossum based on differ-

ences in morphology and habitat between the two genera.

Petch's Phaeoglossum was described as a black club-shapedfungus with white internal hyphae not known to be present

in Geoglossomycetes. It is also described as growing on

decaying Loranthus L. fruits, a habitat not found in other

members of Geoglossomycetes. Lastly, the name Phaeoglossum

Petch is illegitimate pursuant to Article 53.1 of the ICN

(McNeill 2012) as it is a later homonym of Phaeoglossum

Skottsb., a brown alga described by Skottsberg (1907).

Maasoglossum has previously been misidentified as Thue-

menidium (Geoglossum) atropurpureum (Nitare 2012) and more

collections of it may exist under that misnomer in fungaria in

Northern Europe. Both species of Maasoglossum and T. atro-

purpureum possess hyaline paraphyses that are agglutinated

into an epithecum, although in T. atropurpureum the epi-

thecum is a vinous-brown compared to a light brown in both

species ofMaasoglossum. The ascomata of T. atropurpureum are

distinctly purplish-brown when fresh, and the fertile portion

is often irregular to spathulate and distinct from the sterile

stipe. Maasoglossum and T. atropurpureum are also easily

separated based on ascospore morphology: ascospores in T.

atropurpureum are initially continuous but become multi-

guttulate and finally septate when overmature and remain

hyaline throughout development, whereas in both species of

Maasoglossum the ascospores are continuous and light brown

with distinct warts present in M. verrucisporum.

The hyphae at the apex of the stipe in Maasoglossum

contain inflated, hyaline, longitudinal elements and an

anastomosing network of brown, narrow hyphae intertwining

them (Fig. 3D). The more derived members of Geo-

glossomycetes also contain anastomosing hyphae in the stipe,

but the longitudinal cells are not as strongly inflated. The

inflated longitudinal cells and narrower binding hyphae in the

axial stipe of Maasoglossum are similar to those found in

Thuemenidium atropurpureum, but different from Microglossum

(Maas Geesteranus 1964). In Microglossum, the axial stipe hy-

phae are comprised primarily of agglutinated, narrower lon-

gitudinal elements, with binding hyphae very rarely observed.

Nothomitra and Sarcoleotia also contain a densely interwoven

internal stipe hyphal system, although the longitudinal ele-

ments are not inflated as they are in Maasoglossum.

In summary, our study supported the inclusion of Maa-

soglossum as a basal member of Geoglossomycetes. Our

findings suggest that genera with hyaline- to lightly-pig-

mented and aseptate ascospores form the base of the Geo-

glossomycetes clade, with the more derived members of the

clade exhibiting darkly-pigmented and multiseptate asco-

spores (e.g. Geoglossum and Trichoglossum). The ascocarp of

Maasoglossum is morphologically similar to the more derived

members of Geoglossomycetes, with the fertile hymenium

intergrading entirely with the sterile stipe. The morphology

of the ascospores and internal stipe hyphae of Maasoglossum

hints at the common ancestry between Geoglossomycetes

and stalked earth tongues in Leotiomycetes, such as

Thuemenidium.

Acknowledgments

We wish to thank the following for graciously providing

specimens for study: T Baroni, T Læssøe, P Lizon, AS Methven,

J-M Moingeon, CF Roobeek, and the fungaria at CUP, MICH,

SAV, and WTU. The authors wish to acknowledge the

following sources of funding to VPH: American Society of

http://dx.doi.org/10.1016/j.myc.2015.05.003http://dx.doi.org/10.1016/j.myc.2015.05.003

my c o s c i e n c e 5 6 ( 2 0 1 5 ) 5 7 2e5 7 9578

Plant Taxonomists, Discover Life in America, the Graduate

College at the University of Illinois at Urbana-Champaign,

Mycological Society of America, and the North American

Mycological Association.

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Maasoglossum, a basal genus in GeoglossomycetesAcknowledgmentsReferences