Persoonia 31, 2013: 101–111 www.ingentaconnect.com/content/nhn/pimj RESEARCH ARTICLE http://dx.doi.org/10.3767/003158513X671235

Generic circumscriptions in Geoglossomycetes

V.P. Hustad1,2, A.N. Miller 2, B.T.M. Dentinger 3, P.F. Cannon 3

Key words Abstract The class Geoglossomycetes is a recently created class of Ascomycota, currently comprised of one family (GeoglossaceaeF#)"+#$%&#'&"&()#*Geoglossum, Nothomitra, Sarcoleotia, Thuemenidium and Trichoglos­ Ascomycota sum). These fungi, commonly known as earth tongues, have long been a subject of mycological research. However, earth tongues the taxonomy within the group has historically been hindered by the lack of reliable morphological characters, Geoglossum uncertain ecological associations, and the inability to grow these fungi in culture. The phylogenetic relationships of Microglossum Geoglossomycetes were investigated by conducting maximum likelihood and Bayesian analyses using a 4­gene phylogenetics dataset (ITS, LSU, MCM7, RPB1). Five well­supported monophyletic clades were found that did not correspond systematics exactly with the currently recognised genera, necessitating a taxonomic revision of the group. Two new genera are proposed: Glutinoglossum to accommodate G. glutinosum and the newly described species G. heptaseptatum, and Sabuloglossum to accommodate S. arenarium. The type species of Thuemenidium, traditionally included within the Geoglossaceae;#!0#2,"$(<&+#)0#4&:,"'!"'#-,#)#0&1)()-&#:!"&)'&#-.)-#!0#,":8#+!0-)"-:8#(&:)-&+#-,#Geoglossomycetes.

Article info Received: 19 October 2012; Accepted: 24 February 2013; Published: 24 July 2013.

INTRODUCTION 2011), clear ecological connections between these fungi and plant hosts are lacking. Furthermore, the ascospores of these Schoch et al. (2009b) created the class Geoglossomycetes taxa do not germinate in culture, and their anamorphic states and the order Geoglossales to contain three genera (Geoglos­ (if they exist) are unknown (Wang et al. 2006). sum, Sarcoleotia and Trichoglossum) in Geoglossaceae, which Despite being the focus of a number of morphological stud­ was previously placed in Leotiomycetes. Fifty­three species ies, modern molecular phylogenetic analyses of Geoglosso­ !"#$%&#'&"&()#*Geoglossum (22 species), Nothomitra (3 spe­ mycetes are sparse. GenBank currently houses sequences cies), Sarcoleotia (4 species), Thuemenidium (5 species), and from only 17 of 53 Geoglossomycetes. Preliminary molecular Trichoglossum (19 species)) are currently accepted in Geo­ 0-9+!&0#*@$0-&(#A#B!<4(,9'.#677C;#D)"'#&-#):>#677EF#!"+!2)-&# glosso mycetes (Kirk et al. 2008, Hustad et al. 2011); though that Geoglossaceae (as circumscribed at the time) does not many synonyms, dubious names and invalid names have been form a monophyletic clade within Leotiomycetes leading these published in the group to date. Furthermore, several varieties authors to propose removal of several taxa from Geoglossa­ )"+#,-.&(#!"/()01&2!$2#-)3)#.)%&&"#(&2,'"!0&+#5!-.!"#-.&# ceae. These studies suggest that the inoperculate method of genera Geoglossum and Trichoglossum. )0290#+&.!02&"2&#!0#",-#)#09/$2!&"-:8#0!'"!$2)"-#2.)()2-&(#-,# Geoglossomycetes are typically characterised by large, dark, continue to group all earth tongues within Leotiomycetes and club­shaped, terrestrial ascocarps with a fertile hymenium ori­ that several taxa form a separate monophyletic clade basal to ginating at the apex of the ascocarp, eventually intergrading Leotiomycetes deserving of a higher taxonomic rank. Sandnes with (Geoglossum and Trichoglossum) or abruptly terminating at (2006) examined nrDNA and found Geoglossum, Sarcoleotia (Nothomitra and Sarcoleotia) a sterile stipe. Geoglossomycetes and Trichoglossum to form a monophyletic clade. Using a ascospores range from dark brown to black, fusiform and multi­ GH'&"
.8:,'&"8#5!-.#$%&#!"'(,91#01&2!&0;#I2.,2.#&-#):># septate (Geoglossum and Trichoglossum), to light­coloured to (2009b) found these genera to form a monophyletic group hyaline, ellipsoid­fusiform and sparsely septate (Nothomitra and basal to Leotiomycetes and proposed the class Geoglosso­ Sarcoleotia). Many morphological characters used to separate mycetes and order Geoglossales to contain Geoglossum, taxa are ambiguous within the group, as evidenced by more Sarcoleotia and Trichoglossum within a single family, the -.)"#677#8&)(0#,/#2,"/90!"'#2:)00!$2)-!,"#)-#",-#,":8#-.
&­ Geoglossaceae. Ohenoja et al. (2010) recognised the wide 2!&0#:&%&:;#49-#):0,#)-#.!'.&(#-)3,",#?+&"-!$2)-!,"#,/# separation between Geoglossum and the type species of species is frequently compromised by a lack of appreciation Thuemenidium, although they did not complete the taxonomic that spore pigmentation and septation may not develop until a work necessary to revise the latter genus. Recently, Hustad very late stage. Geoglossomycetes have been reported from et al. (2011) included Notho mitra in Geoglossomycetes based every continent except Antarctica and are common components on a 3­gene phylogeny. of many temperate and tropical mycobiota. Although previously !+&"-!$&+#!"#<,:&29:)(#&"%!(,"<&"-):#0)<1:&0#,/#0,!:#.81.)&#)"+# Taxa assigned to Geoglossomycetes are considered to be of root endophytes (Bergemann & Garbelotto 2006, Wang et al. 2,"0&(%)-!,"#0!'"!$2)"2&#!"#0&%&():#J9(,1&)"#2,9"-(!&0>#K.&(&;# many species are typical members of ‘unimproved grassland’ 1 Department of Plant Biology, University of Illinois, 505 S. Goodwin Ave., (non­intensively managed semi­natural grassland habitats that Urbana, IL 61801, USA; have not been treated with nitrogen fertiliser). The number of corresponding author e­mail: [email protected]. species present, along with those from three other fungal groups 2 Illinois Natural History Survey, University of Illinois, 1816 S. Oak St., Champaign, IL 61820, USA. (the Clavariaceae, Entolomataceae and Hygrophoraceae) 3 Jodrell Laboratory, Royal Botanic Gardens, Kew, Surrey TW9 3AB, UK. has been used as a proxy for grassland health, impacting on

© 2013 Naturalis Biodiversity Center & Centraalbureau voor Schimmelcultures You are free to share ­ to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non­commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by­nc­nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. 102 Persoonia – Volume 31, 2013 conservation value assessments (Newton et al. 2003, Gen­ in this analysis was the RNA polymerase II subunit 1 (RPB1) ney et al. 2009). Thuemenidium atropurpureum, until recently gene. RPB1 codes the largest subunit of RNA polymerase II, assumed to be part of the Geoglossomycetes clade, is listed the polymerase responsible for synthesising messenger RNA under UK legislation (under its synonym Geoglossum atro­ in eukaryotes. RPB1 was shown by Schoch et al. (2009a) to purpureum) as a UK Priority species (http://jncc.defra.gov. have the highest per­site informativeness (Townsend 2007) uk/_speciespages/2290.pdf) within the national Biodiversity across six genes in the Ascomycota. The primers RPB1af and Action Plan. Species of Geoglossomycetes are included in the T@\C2(#*Q)-.&"8#&-#):>#6776F#5&(Z&+#/,(#)<1:!$2)-!,"#)"+# Norwegian (Kålås et al. 2010), Swedish (Gärdenfors 2010) and sequencing of the RPB1 gene. Swiss (Senn­Irlet et al. 2007) Red Data Lists. Phylogenetic analyses The goal of this study was to examine the phylogenetic rela­ tionships within Geoglossomycetes and its component genera Alignments of individual genes were created manually by eye in using a robust 4­gene phylogeny with the largest sampling of I&U9&"2.&(#[>Y#,(#48#90!"'#Q902:&#%>#Z>]#*J+')(#677[F#!"#I&)H species to date. view v. 4.2 (Galtier et al. 1996). Ambiguous regions were removed from the individual gene datasets using Gblocks v. 0.91b (Cas­ tresana 2000) under the following parameters: minimum number MATERIALS AND METHODS of sequences for both conserved and flanking regions = 22, Morphological analysis maximum number of contiguous nonconserved positions = 8, minimum length of a block = 10, and allowed gap positions in 30 I1&2!<&"0#5&(&#!+&"-!$&+#4)0&+#,"#-.&#<,(1.,:,'8#,/#)02,­ sequences. The Akaike Information Criterion (AIC) (Posada & mata and microscopic characters using the pertinent literature Buckley 2004), implemented using jModelTest v. 0.1.1 (Posada (e.g., Massee 1897, Durand 1908, Imai 1941, Nannfeldt 1942, 677VF;#+&-&(#S:)+&0#5&(,"0!+&(&+#0!'"!$2)"-#)"+#.!'.:8# was taken for all micromorphological structures when pos­ 0911,(-&+#5.&"#\I#_#]7#`#*a!::!0#A#\9::#CYYZF># sible using NIH Image v. 1.63 (National Institutes of Health, Bayesian inference employing a Markov Chain Monte Carlo Bethesda, Maryland). Taxonomic novelties and nomenclatural (MCMC) algorithm was performed using MrBayes v. 3.1.2 (Huel­ data were deposited in MycoBank (Crous et al. 2004). 0&"4&2=#A#T,"U9!0-#677CF#,"#-.&#S?@TJI#I2!&"2&#N)-&5)8# Teragrid (Miller et al. 2010) as an additional means of branch Molecular procedures support. The GTR+I+G model with six rate classes was em­ Total genomic DNA was extracted from dried ascomata using a ployed. Four independent chains of MCMC were run for 10 L?MNJO#PO&)08#@:)"-#Q!"!#B!-#*L?MNJO#?"2>;#R):&"2!);#S):!H million generations to ensure that trees were not trapped in /,("!)F#)"+#'&"&#/()'<&"-0#5&(&#@ST#)<1:!$&+#)"+#0&U9&"2&+# local optima. Clades with Bayesian posterior probability (BPP) following the methods outlined in Promputtha & Miller (2010) _#YE#`#5&(,"0!+&(&+#0!'"!$2)"-#)"+#.!'.:8#0911,(-&+#*M:­ and Raja et al. (2011). The internal transcribed spacer (ITS) /)(,#&-#):>#677ZF>#J//&2-!%�)<1:�!b&#*JIIF#5)0#&0-!<)-&+# region of nuclear ribosomal DNA (nrDNA), consisting of the using Tracer v. 1.5 (Rambaut & Drummond 2009). Individual ?KIC;#E>VI#)"+#?KI6#(&'!,"0;#5)0#)<1:!$&+#)"+#0&U9&"2&+# datasets of ITS, LSU, MCM7 and RPB1 were examined for using a combination of the primers ITS1F (Gardes & Bruns potential conflict before concatenated into a single dataset for 1993), ITS5, ITS1, ITS4 (White et al. 1990) and ITS4A (Larena -,-):#&%!+&"2&#)"):80!0#*B:9'&#CYVY;#J&("!00&#A#B:9'&#CYYZF># et al. 1999). A variety of primer combinations were used dur­ Individual gene phylogenies were considered to be incongruent !"'#)<1:!$2)-!,"#+9&#-,#-.&#/(&U9&"-#1(&0&"2&#,/#!"-(,"0#!"#-.&# !/#2:)+&0#5!-.#0!'"!$2)"-#QX#\I#)"+#\@@#*_#]7#`#\I#)"+c,(# 3’ end of the adjacent 18S ribosomal small subunit in Geo­ _#YE#`#\@@F#5&(,"/:!2-!"'#!"#-.&#!"+!%!+9):#-(&&#-,1,:,'!&0# glossomycetes. The 28S large subunit (LSU) nrDNA region *D!&"0#CYYV;#M:/)(,#&-#):>#677Z;#X9-b,"!#&-#):>#677[F>#I!"2&#-.&(&# 5)0#)<1:!$&+#90!"'#WIC#*X)"+%!=#CYYGF#)"+#XTG#*R!:'):80#A# were no incongruencies found among the individual datasets, Hester 1990) and sequenced with these primers in addition to all genes were concatenated using Seaview v. 4.2 with the the internal primers LR3 (Vilgalys & Hester 1990) and LR3R following gene order: ITS, LSU, MCM7, RPB1. Phylogenetic (Rehner & Samuels 1995). analyses were then performed on the concatenated dataset as In addition to the ribosomal genes, two protein coding genes )4,%&>#M:!'"<&"-0#)"+#)"):80&0#5&(&#+&1,0!-&+#!"#K(&&\MIJ# were also used to infer taxonomic relationships at both lower (http://treebase.org) under submission ID 13597. and higher taxonomic levels. The minichromosome mainte­ Shimodaira­Hasegawa (S­H) tests (Shimodaira & Hasegawa nance complex component 7 (MCM7) gene is a single­copy 1999) were performed in PAUP v. 4.0b10 (Swofford 2003) to test gene that codes for a DNA replication licensing factor required generic hypotheses. Separate maximum likelihood analyses for DNA replication initiation and cell proliferation (Moir et were conducted with: 1) all taxa in Geoglossum constrained to al. 1982, Kearsey & Labib 1998). MCM7 has been found to be monophyletic; and 2) Thuemenidium species constrained produce highly accurate phylogenies in fungi (Aguileta et !"#)#<,",1.8:&-!2#'&"90>#IHa#-&0-0#90!"'#TJXX#)11(,3!<)-!,"# al. 2008, Schmitt et al. 2009), and it has been shown to be and 1 000 bootstrap replicates were then conducted to compare reliable across a wide range of ascomycete taxa, including these constrained trees to the most­likely tree. Geoglossomycetes (Raja et al. 2011). The primers 709F and CZ[VT#*I2.