Mycosphere Doi 10.5943/mycosphere/2/6/5/

Inclusion of Nothomitra in Geoglossomycetes

Hustad VP1,2*, Miller AN2, Moingeon J-M3 and Priou J-P4

1Department of Plant Biology, University of Illinois at Urbana-Champaign, 505 S. Goodwin Ave., Urbana, IL 61801 2Illinois Natural History Survey, University of Illinois, 1816 S. Oak St., Champaign, IL 61820 328 Grande Rue, 25520 Goux-les-Usiers, France 47 Rue De Picardie, F- 56200 La Gacilly, France

Hustad VP, Miller AN, Moingeon J-M, Priou J-P 2011 – Inclusion of Nothomitra in Geoglossomycetes. Mycosphere 2(6), 646-654, Doi 10.5943/mycosphere/2/6/5/

Nothomitra is a small of earth tongues consisting of three species. Historically placed within the sensu lato, the genus is currently considered incertae sedis within the . We reviewed the morphology and analyzed the phylogenetic relationships of Nothomitra using a combined dataset of ITS, LSU and Mcm7 DNA sequences representing 22 species. The placement of Nothomitra was strongly supported within the Geoglossomycetes clade, forming part of the ancestral base of the class with Sarcoleotia globosa and Thuemenidium arenarium. The inclusion of Nothomitra within the Geoglossomycetes is confirmed.

Key words – – earth tongues – Geoglossaceae – – phylogeny

Article Information Received 1 December 2011 Accepted 5 December 2011 Published online 29 December 2011 *Corresponding author: Vincent Hustad – e-mail – [email protected]

Introduction Trichoglossum (19 species) (Kirk et al. 2008). Earth tongues are among the most However, several genera formerly included widely distributed groups of fungi on earth and within the Geoglossaceae sensu lato are have been a subject of mycological inquiry currently considered incertae sedis and the since Persoon first described Geoglossum in placement of these taxa within the Pezizomy- the late 18th century. Genera typically referred cotina is unknown. to as earth tongues include Geoglossum, The monotypic genus Nothomitra was Trichoglossum, Microglossum, , and introduced by Maas Geesteranus (1964) to Spathularia. During the last 200 years, numer- accommodate N. cinnamomea Maas Geest., ous genera and species have been included and which was described from specimens collected removed from this group based primarily on in Upper Austria during the autumn of 1962. morphological data. Recent molecular studies Three species are accepted in the current (Pfister and Kimbrough 2001, Wang et al. concept of the genus following the additions of 2006a and b, Schoch et al. 2009, Ohenoja et al. Nothomitra kovalii Raitviir (1971) from 2010) have suggested earth tongues are not a Kunashir in the Kuril Islands and Nothomitra monophyletic group and this resulted in the sinensis Zhuang and Wang (1997) from China. introduction of the class Geoglossomycetes At present, Nothomitra is only known to occur (Schoch et al. 2009), which contains four in Europe and Asia, though extensive distri- genera and approximately 50 species. Currently bution data is lacking. All species in included within the Geoglossomycetes are Nothomitra are terrestrial with N. cinnamomea Geoglossum (22 species), Sarcoleotia (4 reported growing amongst Sphagnum, N. species), Thuemenidium (5 species), and kovalii reported from rocky soil, and N.

646 Mycosphere Doi 10.5943/mycosphere/2/6/5/ sinensis reported from mossy soil in coniferous the following sets of primers: ITS1 and ITS4 forests. Nothomitra is found across a wide (White et al. 1990) for the internal transcribed range of altitudes. N. cinnamomea is recorded spacer (ITS) region of nrDNA; JS1 (Landvik from the European Alps from 670 to 1100 m 1996) and LR6 (Vilgal-ys and Hester 1990) for elevation, Nothomitra kovalii is found between the partial 28S nuclear ribosomal large subunit 400-800 m elevation on Mt. Mendeleyeva in (LSU) of nrDNA; 709F and 1348R (Schmitt et the Kuril Islands, whereas N. sinensis is descri- al. 2009) for the DNA replication licensing bed from the Qilian Mountains in Northern factor MS456 (Mcm7). China at 2850 m elevation. These genes were chosen because: a) Nothomitra is characterized by the they provide appropriate resolution at various distinct free edge of the hymenium at the taxonomic levels (i.e. species to class), b) junction of the stipe, unlike Microglossum in fungal and ascomycete–specific primers have which the hymenium intergrades with the stipe been developed for these genes, c) a large on the flattened sides (see Fig 1C). Nothomitra number of available sequences are available is also differentiated from Microglossum in that from GenBank because previous researchers the fertile head of the ascocarp is not flattened (e.g. Wang et al. 2006a and b, Schoch et al. as in Microglossum, and the internal stipe 2009, Ohenoja et al. 2010, Hustad and Miller hyphae of Nothomitra are parallel and easily 2011) have used the nuclear ribosomal genes to separable versus the interwoven and effectively reconstruct phylogenies within Geo- agglutinated hyphae found in Microglossum. glossomycetes and neighboring groups, d) These morphological differences were cited by based on our preliminary data (Raja et al. Maas Geesteranus (1964) as evidence that 2011), Mcm7 shows promise for reconstruction Nothomitra is not congeneric with Microglo- of accurate species-level to class-level phylog- ssum. However, Moingeon and Moingeon enies, and, e) incorporating both ribosomal and (2004) argued that these characters were not protein–coding genes allows for higher sufficient to support Nothomitra as a separate certainty in assessing phylogenetic relation- genus and advocated the placement of N. ships. cinnamomea into Microglossum, thereby rend- ering the genus Nothomitra a synonym. Sequence Alignment and Phylogenetic Since the importance of the morpholo- Analyses gical differences between Nothomitra and Each generated ITS and LSU sequence Microglossum are disputed as is the taxonomic fragment was subjected to an individual blast placement of Nothomitra, it is necessary to search to verify its identity. Mcm7 sequences evaluate molecular characters in order to deter- were only used from specimens which provid- mine the phylogenetic relationships of this ed reliable ITS and/or LSU sequences. Sequen- genus. As such, the purpose of this study is to ces were assembled using Sequencher 4.9 include Nothomitra in a modern phylogenetic (Gene Codes Corp., Ann Arbor, Michigan), analysis for the first time to determine its optimized by eye and manually corrected when placement within the and to necessary. Alignments of individual genes provide detailed insight into the systematics of were created manually by eye in Sequencher the Geoglossomycetes using a multi-gene phy- 4.9 or using Muscle 3.7 (Edgar 2004) in logeny. Seaview 4.2 (Galtier et al. 1996). Individual gene datasets were then analyzed using Gbloc- Methods ks 0.91b (Castresana 2000) to identify and Generation of Molecular Data remove ambiguous regions from the alignment. Total genomic DNA was extracted from The Akaike Information Criterion dried ascomata using a QIAGEN DNeasy Plant (AIC) (Posada and Buckley 2004) as imple- Mini Kit (QIAGEN Inc., Valencia, California) mented in jModelTest 0.1.1 (Posada 2008) and gene fragments were PCR amplified and determined GTR+I+G as the best fit model of sequenced following the meth-ods outlined in evolution for both maximum likelihood and Promputtha and Miller (2010) and Raja et al. Bayesian inference. Maximum likelihood (2011). Gene fragments were amplified using analyses were performed using PhyML

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(Guindon and Gascuel 2003) under the GTR Ascomata scattered to gregarious occurring in substitution model with six rate classes and soil, 1–3.3 cm high, hymenium borne on vari- invariable sites optimized. A BioNJ starting ously-shaped fertile heads, head glabrous, spa- tree was constructed and the best of nearest thulate to obovoid or subglobose with concolo- neighbor interchange (NNI) and subtree rous wavy lobes, pale cinnamon to olivaceous, pruning and regrafting (SPR) tree improvement darkening with age, 3–9 mm broad (Fig 1A, was implemented. Bootstrap support B), hymenium distinctly separated from stipe (Felsenstein 1985) (BS) was determined with (Fig 1C), stipe straight or flexuous, terete, 100 bootstrap replicates. Clades with >70% BS tapering towards base, ochraceous above were considered significant and highly becoming paler toward base, squamulose supported (Hillis and Bull 1993). above, becoming glabrous at base, 0.7–2.4 cm Bayesian inference employing a high. Hyphae at center of stipe easily separated, Markov Chain Monte Carlo (MCMC) often swollen at the septa, thin–walled and algorithm was performed using MrBayes 3.1.2 often branched. Hyphae near the periphery of (Huelsenbeck and Ronquist 2001) as an addi- the stipe thin-walled and tightly bundled. tional means of assessing branch support. The Paraphyses filiform, upper cells hyaline, with GTR+I+G model with six rate classes was brownish guttules in lower cells, septate, employed. Four independent chains of MCMC sometimes branched at apex or base, curved at were run for 10 million generations to insure the apex, slightly longer than asci, 1–1.5 m that trees were not trapped in local optima. wide, expanding to 2–3 m wide at apex. Asci Clades with Bayesian posterior probability cylindrical–clavate, with crosiers, inoperculate, (BPP) >95% were considered significant and apical ring euamyloid, deep blue in IKI, small, highly supported (Alfaro et al. 2003). not occupying entire apex, 150–180 × 9.5– The individual ITS, LSU, and Mcm7 12.5m (Fig 1B), 8–spored, biseriate. Asco- datasets were examined for potential conflict spores fusiform to narrowly obclavate, rounded before concatenation into a single dataset for at apex, acute at base, hyaline, smooth, multi- total evidence analysis (Kluge 1989, Eernisse guttulate, single–celled in ascus, becoming up and Kluge 1993). The individual gene phylog- to 5–septate when mature or old, 35–47 (–55) × enies were considered incongruent if clades 3.5–5.5 (–6) m (Fig 1D). with significant ML bootstrap and Bayesian Habitat: Growing among Sphagnum and posterior probability (>70% BS or >95% BPP) Aulocomnium palustre (Hedw.) Schwägr., were conflicting in the individual tree often accompanying Geoglossum sphagno- topologies (Wiens 1998, Alfaro et al. 2003, philum Ehrenb. September–October. Lutzoni et al. 2004). As no incongruencies Distribution: Known from Austria and France. were found among the three individual data Anamorph: Unknown. sets, they were concatenated using Seaview 4.2 Material examined – France, Jura, Belle- and subjected to phylogenetic analyses as fontaine, September 2001, 1100 m, leg. J.M. above. Moingeon s.n., ILLS Acc. ANM463; ILLS Acc. ANM538; ILLS Acc. ANM540; October Results 2001, leg. J.M. Moingeon s.n., ILLS Acc. Morphology ANM549. Nothomitra cinnamomea Maas Geest., Persoonia 3(1): 92, 1964. Phylogenetic analyses = Microglossum cinnamomeum S. Twenty–two taxa were included in the Moingeon & J.M. Moingeon, final analyses (Table 1). Mcm7 data for Micro- Miscellannea Mycologica 80–81: glossum olivaceum and Sarcoleotia globosa 31, 2004. were not available. The final data matrix had an aligned length of 2720 base pairs, which was Type: Austria, Attergau, Fehra Moos, SW of reduced to 2091 after the removal of 629 St. Georgen, 29 September 1969, J.T. Palmer ambiguous characters by Gblocks. Of the 2091 11391. L 962.271-144. characters used in the final analyses, 76 were

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Figs 1 (A-D) – Nothomitra cinnamomea. A In situ photograph of ascomata. B Ascus, total magnification = 400X. C Close up of fertile tip, arrow denotes separation of head and stipe. D Ascospores illustrating variable septation, total magnification = 800X. constant, 819 were parsimony–uninformative, of Geoglossomycetes, closely aligned with and 1196 were parsimony informative. The Sarcoleotia globosa as the most basal members maximum likelihood tree produced from the of the class. Morphologically, S. globosa is combined ITS, LSU, and Mcm7 dataset is pres- rather similar to N. cinnamomea (Fig 3). Both ented in Fig 2. The topology of Geoglossomy- species possess a distinct capitate hymenium cetes is congruent with those produced from that is clearly separated from the stipe when similar analyses including Geoglossomycetes mature, but the margin of the hymenium is taxa (Schoch et al. 2009, Ohenoja et al. 2010, completely free in N. cinnamomea and comple- Wang et al. 2011). Two major clades are pre- tely inrolled in S. globosa. Both species also sent and strongly supported in our analyses: the possess hyaline ascospores that develop 3-5 Leotiomycetes clade (BP=100%, PP=1.0) and septa upon maturation. Lastly, both species are the Geoglossomycetes clade (BP=100%, terrestrial and collection data suggests that an PP=1.0). Nothomitra cinnamomea was placed association with mosses exists in both species within Geoglossomycetes as a sister taxon to (Maas Geesteranus 1964, Schumacher and Sarcoleotia globosa with moderate support Silvertsen 1987). These morphological and (BS=78%). Geoglossum occurred as a strongly ecological similarities support the close phylo- supported monophyletic group (BP=100%, genetic relationship of N. cinnamomea and S. PP=1.0), whereas Trichoglossum was paraphy- globosa revealed by the molecular phylogeny letic. (Fig 2).

Discussion Another morphological feature that links N. Our analyses confirm Nothomitra cinnamomea within Geoglossomycetes is that cinnamonmea as a strongly supported member the hyphae at the axis of the stipe are not 649 Mycosphere Doi 10.5943/mycosphere/2/6/5/

Table 1 List of taxa, GenBank and herbarium accession numbers, collections numbers, and locality for specimens used in this study. Collection Herbarium Locality Name Number # ITS LSU Mcm7 ILLS61033 Champaign Bisporella citrine VPH s.n. JQ256414 JQ256432 JN672971 County, Illinois ILLS60488 GSMNP, Cudoniella clavus ANM2087 JQ256415 JN012006 JN672988 Tennessee Geoglossum barlae Moingeon s.n. ILLS61034 JQ256416 JQ256433 JQ256444 France ILLS61035 GSMNP, North Geoglossum cookeanum ANM2257 JQ256417 JQ256434 JQ256445 Carolina ILLS61036 Cades Cove, GSMNP, Geoglossum difforme ANM2169 JQ256418 JN673044 JN672990 Tennessee ILLS61037 Hamrstejn, Geoglossum fallax J. Gaisler s.n. JQ256419 JQ256435 JQ256446 Czech Republic ILLS61038 GSMNP, Geoglossum glabrum ANM2267 JQ256420 JQ256436 JQ256447 Tennessee ILLS61039 GSMNP, Geoglossum simile ANM2171 JQ256421 JQ256437 JQ256448 Tennessee ILLS61040 Kennemerland, Geoglossum umbratile CFR251108 JQ256422 JQ256438 JQ256449 Netherlands ILLS60491 GSMNP, Graddonia coracina ANM2018 JQ256423 JN012009 JN672993 Tennessee Hymenoscyphus fructigenus ASM10619 ILLS61041 JQ268558 JN673046 JN672997 Samara, Russia GenBank N/A AY789398 AY789397 N/A N/A Neurospora crassa GenBank N/A JN198494 AF286411 XM958785 N/A ILLS61042 Bellefontaine, Nothomitra cinnamomea Moingeon s.n. JQ256424 JQ256439 JQ256450 Jura, France ILLS60497 GSMNP, North Propolis versicolor ANM2050 JQ256425 JN012015 JQ256451 Carolina Sarcoleotia globosa GenBank N/A AY789300 AY789299 N/A N/A ILLS61043 Kennemerland, Thuemenidium arenarium CFR181007 JQ256426 JQ256440 JQ256452 Netherlands ILLS61044 Cortland Thuemenidium County, New atropurpureum ASM4931 JQ256427 JQ256441 JQ256453 York ILLS61045 Hamrstejn, Trichoglossum hirsutum J. Gaisler s.n. JQ256428 JQ256442 JQ256454 Czech Republic ILLS61046 Senavelle, Trichoglossum octopartitum JPP10191 JQ256429 JQ256443 JQ256455 France ILLS61047 GSMNP, North Trichoglossum walteri ANM2203 JQ256430 JN673053 JN673022 Carolina Vibrissia filisporia f. ILLS60499 GSMNP, North filisporia ANM2064 JQ256431 JN012017 JN673023 Carolina agglutinated and easily separable, a character Both Microglossum and Thuemenidium possess commonly seen in Geoglossomycetes. Maas hyaline ascospores but this character is not Geesteranus (1964) cited this character in his sufficient to exclude these genera from original proposal to separate Nothomitra from Geoglossomycetes since several Geoglossum Microglossum, and this character appears to be species possess hyaline ascospores. one of the few conserved characters throughout Microglossum can be delineated from the class. As in previous molecular based Geoglossomycetes based on its ascomata that phylogenies (Wang et al. 2006a and b, Schoch range from brightly colored to brown. et al. 2009, Ohenoja et al. 2010), Microglossum Thuemenidium is a polyphyletic genus olivaceum and Thuemenidium atropurpureum composed of at least two disparate species, T. were shown to occur in the Leotiomycetes. arenarium, which belongs in Geoglosso

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Fig 2 – Maximum likelihood phylogeny of Geoglossomycetes based on a combined dataset (2091 bp) of ITS, LSU, and Mcm7 DNA sequences representing 22 taxa using PhyML ((-ln)L score = 13700). Thickened branches indicate significant Bayesian posterior probabilities (>95%); numbers refer to PhyML bootstrap support values >70% based on 1000 replicates. Neurospora crassa and the Leotiomycetes were used as outgroup taxa.

Fig 3 – Sarcoleotia globosa. Arrow indicates distinct separation of fertile head and stipe.

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mycetes, and T. atropurpureum, shown by Carlo Sampling and Bootstrapping in this study and Ohenoja et al. (2010) to assessing phylogenetic confidence. belong in Leotiomycetes. Thuemenidium Molecular Biology and Evolution 20, atropurpureum produces ascomata ranging 255–266. from brown to purplish black, whereas T. Castresana J. 2000 – Selection of conserved arenarium does not possess any purplish blocks from multiple alignments and coloration. their use in phylogenetic analysis. The Geoglossomycetes are an early- Molecular Biology and Evolution 17, diverging lineage appearing on a long 540–552. branch within the Ascomycota and further Edgar R. 2004 – MUSCLE: multiple molecular research is needed in the group to sequence alignment with high construct a comprehensive phylogeny of the accuracy and high throughput. class. Several genera have historically been Nucleic Acids Research 32, 1792– associated within this group which are now 1797. considered incertae sedis (e.g. Hemiglossum Eernisse DJ, Kluge AG. 1993 – Taxonomic Pat., Leucoglossum Imai, and Maasoglos- congruence versus total evidence, sum Thind and Sharma), and representatives and amniote phylogeny inferred from from these genera need to be examined fossils, molecules, and morphology. using molecular phylogenies to fully Molecular Biology and Evolution 10, understand their place within the 1170–1195. Pezizomycotina. Moreover, several species Felsenstein J. 1985 – Confidence limits on complexes are likely present within the phylogenies: An approach using the group and Australasian lineages appear to bootstrap. Evolution 39, 783–791. have origins entirely separate from Northern Galtier N, Gouy M, Goutier C. 1996 – Hemisphere counterparts (Wang et al. SEAVIEW and PHYLO_WIN: two 2011). Further molecular data are also graphic tools for sequence alignment needed to provide accurate reference and molecular phylogeny. Computer sequences for environmental sampling as Applications in the Biosciences 12, ongoing efforts in this field may shed some 543–48. light on the enigmatic host associations Guindon S, Gascuel O. 2003 – A simple, within Geoglossomycetes. fast, and accurate algorithm to estimate large phylogenies by Acknowledgements maximum likelihood. Systematic This project was funded in part by an Biology 52, 696–704. American Society of Plant Taxonomists Hillis DM, Bull JJ. 1993 – An empirical test Graduate Student Research Grant to VPH. of bootstrapping as a method for The authors wish to thank Bohumil assessing confidence in phylogenetic Dolensky, Jan Gaisler, Andrew Methven, analysis. Systematic Biology 42, and Kees Roobeek for kindly providing 182–192. specimens used in this study. Huelsenbeck JP, Ronquist F. 2001 – MrBa- yes: Bayesian inference of phyloge- References netic trees. Bioinformatics 17, 754– Alfaro ME, Zoller S, Lutzoni F. 2003 – 755. Bayes or Bootstrap? A simulation Hustad VP, Miller AN. 2011 – Phylogenetic study comparing the performance of placement of four genera within the Bayesian Markov Chain Monte Leotiomycetes (Ascomycota). North

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American Fungi 6, 1–13. Pfister DH, Kimbrough JW. 2000 – Kirk PM, Cannon, PF, Minter DW, Staples Discomycetes. In: The Mycota VII JA. 2008 – Dictionary of the fungi, Part A. Systematics and Evolution 10th edition. CAB International, (eds DJ McLaughlin, EG Wallingford, UK. McLaughlin, PA Lemke). Springer, Kluge AG. 1989 – A concern for evidence Berlin Heidelberg 257–281. and a phylogenetic hypothesis of Posada D, Buckley TR. 2004 – Model relationships among Epicrates (Boi- selection and model averaging in dae, Serpentes). Systematic Biology phylogenetics: advantages of Akaike 38, 7–25. information criterion and Bayesian Landvik S. 1996 – , a fruit-body- approaches over likelihood ratio producing genus of the basal tests. Systematic Biology 53, 793– ascomycetes, as shown by SSU and 808. LSU rDNA sequences. Mycological Posada D. 2008 – jModelTest: Phylogenetic Research 100, 199–202. model averaging. Molecular Biology Lutzoni F, Kauff F, Cox C, McLaughlin D, and Evolution 25, 1253–1256. Celio G, Dentinger B, Padamsee M, Promputtha I, Miller AN. 2010 – Three new Hibbett D, James T, Baloch E, Grube species of Acanthostigma (Tubeu- M, Reeb V, Hofstetter V, Schoch C, fiaceae, Dothidiomycetes) from the Arnold AE, Miadlikowskia J, Spata- Great Smoky Mountains National fora J, Johnson D, Hambleton S, Park. Mycologia 102, 574–587. Crockett M, Shoemaker R, Sung G, Raitviir A. 1971 – The Geoglossaceae of the Lucking R, Lumbsch T, O’Donnell Far East. In: Plants and animals of K, Binder M, Diederich P, Ertz D, the Far East (ed E Parmasto). Val- Gueidan C, Hansen K, Harris R, Ho- gus, Tallin 52–83. saka K, Lim Y, Matheny B, Nishida Raja HA, Schoch CL, Hustad VP, Shearer H, Pfister D, Rogers J, Rossman A, CA, Miller AN. 2011 – Testing the Schmitt I, Sipman H, Stone J, Sugi- phylogenetic utility of MCM7 in the yama J, Yahr R, Vilgalys R. 2004 – Ascomycota. MycoKeys 1, 46–56. Assembling the fungal tree of life: Schoch CL, Wang Z, Townsend JP, progress classifcation and evolution Spatafora JW. 2009 – Geoglossomy- of subcellular traits. American Journ- cetes cl. nov., Geoglossales ord. nov. al of Botany 91, 1446–1480. and taxa above class rank in the Maas Geesteranus RA. 1964 – On some Ascomycota Tree of Life. Persoonia white-spored Geoglossaceae. Persoo- 22, 129–136. nia 3, 81–96. Schmitt I, Crespo A, Divakar PK, Moingeon S, Moingeon J–M. 2004 – Fankhauser JD, Herman–Sackett E, Contributions à l’étude des Geoglos- Kalb K, Nelson MP, Nelson NA, saceae à spores hyalines. Miscell- Rivas–Plata E, Shimp AD, Widhelm anea Mycologica 80–81, 25–35. T, Lumbsch HT. 2009 – New pri- Ohenoja E, Wang Z, Townsend JP, Mitchell mers for promising single-copy D, Voitk A. 2010 – Northern species genes in fungal phylogenetics and of the earth tongue genus Thumeni- systematics. Persoonia 23, 35–40. dium revisited, considering morpho- Schumacher T, Silvertsen S. 1987 – Sarco- logy, ecology and molecular phylo- loetia globosa (Sommerf.: Fr.) Korf, geny. Mycologia 102, 1089–1095. , ecology, and distribution.

653 Mycosphere Doi 10.5943/mycosphere/2/6/5/

In: Arctic and Alpine Mycology 2 White TJ, Bruns T, Lee S, Taylor JW. 1990 (eds GA Larsen, JF Amirati, SA – Amplification and direct sequen- Redhead). Plenum Press, New York cing of fungal ribosomal RNA genes and London 163–176. for phylogenetics. In: PCR Protocol: Vilgalys R, Hester M. 1990 – Rapid identi- a guide to methods and applications fication and mapping of enzymatic- (eds MA Innis, DH Gelfand, JJ cally amplified ribosomal DNA from Sninsky, TJ White). Academic Press, several Cryptococcus species. Journ- San Diego. 315–322. al of Bacteriology 172, 4238–4246. Wiens JJ. 1998 – Combining data sets with Wang Z, Binder M, Schoch CL, Johnston different phylogenetic histories. PR, Spatafora JW, Hibbett DS. Systematic Biology 47, 568–581. 2006a – Evolution of helotialean Zhuang W, Wang Z. 1997 – Some new fungi (Leotiomycetes, Pezizomy- species and new records of discomy- cotina): A nuclear rDNA phylogeny. cetes in China, 7. Mycotaxon 63, Molecular Phylogenetics and Evolu- 307–321. tion 41, 295–312. Wang Z, Johnston PR, Takamatsu S, Spatafora JW, Hibbett DS. 2006b – Toward a phylogenetic classification of the Leotiomycetes based on rDNA data. Mycologia 98, 1065–1075. Wang Z, Nilsson RH, Lopez-Giraldez F, Zhuang W, Dai Y, Johnston PR, Townsend JP. 2011 –Tasting soil fungal diversity with earth tongues: Phylogenetic test of SATé align- ments for environmental ITS data. PLos ONE 6, e19039.

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