MYCOTAXON THE INTERNATIONAL JOURNAL OF FUNGAL TAXONOMY & NOMENCLATURE VOLUME 135 (1) JANUARY-MARCH 2020 Pseudocercospora seropedicensis sp. nov. on leaves of Solanum asperum (Andrade & al.— PLATE 1, p. 123) ISSN (PRINT) 0093-4666 https://doi.org/10.5248/135-1 ISSN (ONLINE) 2154-8889 MYXNAE 135(1): 1-234 (2020) EDITORIAL ADVISORY BOARD KAREN HANSEN (2014-2021), Chair Stockholm, Sweden BRANDON MATHENY (2013-2020), Past Chair Knoxville, Tennessee, U.S.A. ELSE VELLINGA (2019-2022) Oakland, California, U.S.A. XINLI WEI (2019-2023) Beijing, China ToDD OSMUNDSON (2019-2024) La Crosse, Wisconsin, U.S.A. ELAINE MALosso (2019-2025) Recife, Brazil ISSN 0093-4666 (PRINT) ISSN 2154-8889 (ONLINE) MYCOTAXON THE INTERNATIONAL JOURNAL OF FUNGAL TAXONOMY & NOMENCLATURE JANUARY-MARCH 2020 VOLUME 135 (1) http://dx.doi.org/10.5248/135-1 EDITOR-IN-CHIEF LORELEI L. NORVELL editor@mycotaxon.com Pacific Northwest Mycology Service 6720 NW Skyline Boulevard Portland, Oregon 97229-1309 USA NOMENCLATURE EDITOR SHAUN R. PENNYCOOK PennycookS@LandcareResearch.co.nz Manaaki Whenua Landcare Research Auckland, New Zealand MyYcoTAXxONn, LTD. © 2020 www.mycotaxon.com & www.ingentaconnect.com/content/mtax/mt P.O. BOX 264, ITHACA, NY 14581-0264, USA IV ... MYCOTAXON 135(1) MYCOTAXON VOLUME ONE HUNDRED THIRTY-FIVE (1) — TABLE OF CONTENTS Nomenclatural novelties & typifications ....... 0... eee eee eens vii DRONA OWES, 6 ote an 8 oy wie nt aterg YS cig asl ata gene ert Meth ot ae iy rh le ogee Ray ix Coriivenda.... Samy. <6. bx MP ee Mc on 6 Rpt CRS AO SU ein aR wR % EP OE TTI ABOU 9 cect ante Bigs Rigas POL Rl Baca te lies ete et IE age cae ky xi DOLOSADUIISSION PROCEG HTC. § «AW Dons Sant Perret hea cab ine he apenas ahd Hote hhen sey xiii TAXONOMY & NOMENCLATURE Two new genera of gymnopoid/marasmioid euagarics RONALD H. PETERSEN & KAREN W. HuGHeEs_ 1 Caliciopsis sambaibae sp. nov. from the Brazilian Cerrado JosE Luiz BEZERRA, MARUZANETE PEREIRA MELO, JosE EVANDO AGUIAR BESERRA JR, ELLIOT WATANABE KITAJIMA, SAMARA RAQUEL SOUSA, CRISTIANE DUARTE SANTOS 97 Morphological and molecular identification of Phlebia wuliangshanensis sp. nov. in China Ruo-X1A HUANG, KAI-YUE Luo, RuI-XIN Ma, CHANG-LIN ZHAO 103 Pseudocercospora seropedicensis & P. solani-cernui spp. nov. on Solanum K.M. ANDRADE, P.S. MEDEIROS, JESSICA REMBINSKI, JUCIMAR M. OLIVEIRA, C.A. INAcIo 119 The nomenclatural history of Umbilicaria spodochroa and nomenclatural corrections in Umbilicariaceae Evceny A. Davypov, TEUvo AHTI, ALEXANDER N. SENNIKOV 131 Ochroconis terricola sp. nov. from China XIN ZHANG, KUN-YING WANG, PENG-PENG REN, YU-LAN JIANG 143 Neomyrmecridium asymmetricum sp. nov. from Ecuador LIZETTE SERRANO, DAYNET SOSA, FREDDY MAGDAMA, FERNANDO ESPINOZA, ADELA QUEVEDO, MARCOS VERA, MIRIAM VILLAVICENCIO, GABRIELA MARIDUENA, SIMON PEREZ-MARTINEZ, ELAINE MALOosso, BEATRIZ RaAMosS-GARCiA, RAFAEL F, CASTANEDA-Ru1z 151 A comparison of anamorphs of some Pachyphlodes species and the type of Chromelosporium: are they congeneric? GREGOIRE L. HENNEBERT & Cony DECOCK 167 Pseudosperma flavorimosum sp. nov. from Pakistan SANA JABEEN & ABDUL NASIR KHALID 183 Three Rinodina species new to China X1A0-JiA ZHENG & QIANG REN 195 JANUARY-MARCH 2020... V Gymnopus barbipes and G. dysodes, new records for Pakistan MALKA SABA, JUNAID KHAN, SAMINA SARWAR, HASSAN SHER, ABDUL NASIR KHALID 203 Agaricus, Steccherinum, and Typhula species new for Turkey Hakan Istk 213 Tubakia koreana sp. nov. causing Quercus leaf blight Hyg YOUNG YUN & YouNG Ho Kim 223 MycosBioTa (FUNGA) NEW TO THE MYCOTAXON WEBSITE Macrofungi from the Hebron and Jerusalem Hills of Palestine (SUMMARY) Maximus THALER, AYSHA AL-WAHSH, ALEA Meusrr, ALyssA Rooks, MAzIN QumsrveEH 231 Checklist of Bolivian Agaricales. 2: Species with white or pale spore prints (SUMMARY) ELIZABETH MELGAREJO-ESTRADA, DIANA ROCABADO, Marfa EUGENIA SUAREZ, OSWALDO MAILLARD, BERNARDO ERNESTO LECHNER 233 vI ... MYCOTAXON 135(1) PUBLICATION DATE FOR VOLUME ONE HUNDRED THIRTY-FOUR (4) MYCOTAXON for OCTOBER—DECEMBER 2019 (I-xI + 491-740) was issued on January 13, 2020 JANUARY-MARCH 2020... NOMENCLATURAL NOVELTIES AND TYPIFICATIONS PROPOSED IN MYCOTAXON 135(1) Caliciopsis sambaibae J.L. Bezerra, M.P. Melo & Beserra [MB 819221], p. 99 Neomyrmecridium asymmetricum R.F. Castaneda, Serrano & D. Sosa [MB 831330], p. 157 Ochroconis terricola Xin Zhang & Y.L. Jiang [MB 831137], p. 146 Paramycetinis R.H. Petersen [IF 555792], p. 9 Paramycetinis austrobrevipes R.H. Petersen [IF 555793], p. 11 Paramycetinis caulocystidiatus R.H. Petersen [IF 555794], p. 21 Phlebia wuliangshanensis C.L. Zhao [MB 830801], p. 111 Pseudocercospora seropedicensis Andrade, Medeiros & Inacio [MB 819383], p. 121 Pseudocercospora solani-cernui Rembinski, Oliveira & Inacio [MB 824359], p. 124 Pseudomarasmius R.H. Petersen & K.W. Hughes [IF 317324], p. 28 Pseudomarasmius efibulatus R.H. Petersen [IF 555730], p. 31 Pseudomarasmius glabrocystidiatus (Antonin, Ryoo & Ka) R.H. Petersen [IF 555747], p. 37 Pseudomarasmius nidus-avis (César, Bandala & Montoya) R.H. Petersen [IF 555746], p. 38 Pseudomarasmius obscurus R.H. Petersen [IF 555731], p. 50 Pseudomarasmius pallidocephalus (Gilliam) R.H. Petersen [IF 555745], p.55 Pseudomarasmius patagonianus R.H. Petersen [IF 555733], p. 68 Pseudomarasmius quercophiloides R.H. Petersen [IF 555732], p. 73 Pseudomarasmius straminipes (Peck) R.H. Petersen [IF 555734], p. 83 Pseudosperma flavorimosum Jabeen & Khalid [MB 823494], p. 187 VII Vil ... MYCOTAXON 135(1) Tubakia koreana H.Y. Yun [MB 814540], p. 225 Umbilicaria subg. Papillophora Davydov, Ahti & Sennikov [MB 830067], p. 139 = “Umbilicaria subg. Gyrophora” sensu Davydov & al., Taxon 66: 1297. 2017. JANUARY-MARCH 2020... REVIEWERS — VOLUME ONE HUNDRED THIRTY-FIVE (1) The Editors express their appreciation to the following individuals who have, prior to acceptance for publication, reviewed one or more of the papers prepared for this issue. Ahmed M. Abdel-Azeem Vladimir Antonin Timothy J. Baroni Jadson D.P. Bezerra Uwe Braun Hui Deng Muhammad Fiaz Shouyu Guo Laura Guzman- Davalos Khalid M. Hameed Mei-Ling Han Rosanne Healy Yuuri Hirooka Jason M. Karakehian Abdullah Kaya Ali Keles Paul M. Kirk K.P. Deepna Latha De-Wei Li Helmut Mayrhofer George A. Meindl Roger Fagner Ribeiro Melo Josiane Santana Monteiro Arooj Naseer Lorelei L. Norvell Yoshitaka Ono Clark L. Ovrebo Shaun R. Pennycook Christian Printzen Gerhard Rambold Gonzalo Romano Keith A. Seifert Wen-Xiu Sun Ix xX ... MYCOTAXON 135(1) CORRIGENDA FOR MYCOTAXON 135(1) Cited below are mistakes present in files submitted for PDF conversion in the current issue but not detected by the authors until after the paper had gone to press. p. 30, Fic. 15, label on “green” clade in Pseudomarasmius FOR: Ps. nitus-avis READ: Ps. nidus-avis p. 129, line 8 FOR: 2:1-388 READ: 2: 1-388 p. 129, line 21 FOR: V. 13, p. 365-371. READ: 13: 365-371. p. 224, line 29 FOR: (Maddison & Maddison 2015). READ: (Maddison & Maddison 2015). JANUARY-MARCH 2020... XI FROM THE EDITOR-IN-CHIEF MyYCOTAXON, RANGE EXTENSIONS, AND TYPE DESCRIPTIONS — ‘There are many places to publish brief “field guide” descriptions accompanying a photo of a species collected outside its reported range, but MycoTAxon is not one of them. For each range extension, MYCOTAXON requires authors to provide a correct scientific name, a technical description based entirely on the specimens collected (without adding data the species is “supposed” to have but which the specimen lacks), a section listing the specimens examined and vouchered, illustrations, and a comparison of the similarities and differences between the new collections and previously published descriptions. What we have perhaps NOT stressed enough is that researchers must first closely compare their find with the TyPE description, even though it may not be possible to examine the actual type itself. Other critical literature to be consulted includes type studies and monographic treatments of the putative species. While field guides (such as the oft-cited Breitenbach & Kranzlin) are helpful in reaching a species identification, they cannot replace primary references for reliability. Remember that species are named based on the type collection and not on an ephemeral ‘general’ concept. How BIG IS THAT SPORE AGAIN? — Scale “bars” (lines indicating the relative size of a specimen or structure ina scientific illustration) should be unobtrusive (i.e. relatively narrow) and accurate. We urge all authors to “keep it simple” by selecting only a few dimensions divisible by 5 or 10. Measure macroscopic features in millimeters (using cm only when a feature is larger than 10 mm) and limit microscopic measurements to 5 um (small spores), 10 um (larger spores or cells), or 50 or 100 um (complex hyphal tissues). Scales (and their accompanying structures) should lengthen or shorten as needed to match precisely the dimensions cited in the legend. Authors should group dimensions at the end of each legend for easy reference. An acceptable legend ends with: “Scale bars: A-C = 500 um; D, F = 10 um; E,G,H.=5 pm? An unacceptable legend ends with: “Scale bars: A = 488 um; B = 503.5 um; C = 425 um; D = 6.8 um; E = 3.2; F= 12.5 um; G = 6.3 um; B = 4.8 um.” When your editors encounter manuscripts with scale information presented in the second format, they become very surly indeed. MyYCOTAXON 135(1) contains 13 papers by 60 authors (representing 13 countries) as revised by 33 expert reviewers and the editors. The 2020 January-March MycoTAxon proposes Two new genera (Paramycetinis & Pseudomarasmius), ONE new subgenus (Umbilicaria subg. Gyrophora), and 12 species new to science representing Caliciopsis & Pseudocercospora from BRAZIL; Neomyrmecridium from Ecuapor; Ochroconis & Phlebia from CHINA; xl ... MYCOTAXON 135(1) Paramycetinis from AUSTRALIA & NEW ZEALAND; Pseudomarasmius from CHILE, CHINA, and Costa Rica; Pseudosperma from PAKISTAN; and Tubakia from SOUTH Korea. We also offer four new combinations in Pseudomarasmius. New species range extensions are reported for [basidiomycetes] Agaricus, Steccherinum, and Typhula for TuRKEY, Gymnopus for PAKISTAN, and [lichens] Rinodina for Cutna. Also included are an excellent historical exploration of the nomenclature (and spelling!) of Umbilicaria spodochroa and a thoughtful comparison between Pachyphlodes and Chromelosporium anamorphs. MycotTaxon 135(1) closes with the announcements of two mycobiota [now posted on www.mycotaxon.com] covering [1] macrofungi in Palestine and [2] white and pink-spored agarics in Bolivia. Covib-19—This issue goes to press (late as usual, unfortunately) in the middle of a global endemic caused by a new corona-virus. The fallout from this plague will unfortunately alter our lives on many fronts beyond the current deaths and isolation we now face. Take heart, persevere, and strive ever upward. I close with these lines by Alicia Jo Rabins: “Through windows and screens We sing and raise a glass Simultaneous but not together. Oh my friends my beloved strangers I never knew our closeness Until it was gone” For now— work safely apart; stay healthy together. Lorelei Norvell (Editor-in-Chief) 14 April 2020 JANUARY-MARCH 2020... XIII 2020 MyCOTAXON SUBMISSION PROCEDURE Prospective MycoTaxon authors should download the MycotTaxon 2020 guide, review & submission forms, and MycoTaxon sample manuscript by clicking the ‘file download page’ link on our INSTRUCTIONS TO AUTHORS page before preparing their manuscript. This page briefly summarizes our ‘4-step’ submission process. 1—PEER REVIEW: Authors first contact peer reviewers (two for journal papers; three for mycobiota/fungae) before sending them formatted text & illustration files and the appropriate 2020 MycoTaxon journal or mycota reviewer comment form. Experts return revisions & comments to BoTH the Editor-in-Chief and authors. ALL co-authors MuST correct and proof- read their files before submitting them to the Nomenclature Editor. 2—NOMENCLATURAL REVIEW: Authors email all ERROR-FREE text & illustration files to the Nomenclature Editor . Place first author surname + genus + ‘Mycotaxon’ on the subject line, and (required) attach a completed sUBMISSION FORM. The Nomenclature Editor will (i) immediately assign the accession number and (ii) after a few weeks return his notes and suggested revisions to the author(s) and Editor-in-Chief. 3—FINAL SUBMISSION: All coauthors thoroughly revise and proof-read files to prepare error-free text and images ready for immediate publication. Poorly formatted copy willbe rejected or returned for revision. E-mail the final manuscript to the Editor-in-Chief , adding the accession number to the message and all files, which include a (i) revised 2020 submission form, all (ii) text files and (iii) jpg images, and (iv) FN, IF, or MB identifier verifications for each new name or typification. The Editor-in-Chief acknowledges submissions within two weeks of final submission but requests authors to wait at least 14 days before sending a follow-up query (without attachments). 4—FINAL EDITORIAL REVIEW & PUBLICATION: The Editor-in-Chief conducts a final grammatical and scientific review and returns her editorial revisions to all expert reviewers and coauthors for final author approval. Author-approved files are placed in the publication queue. The PDF proof and bibliographic & nomenclatural index entries are sent to all coauthors for final inspection. After PDF processing, the Editor-in-Chief corrects ONLY PDF editorial/conversion and index entry errors; corrections of all other errors are listed in the Errata of a subsequent issue for no charge. Authors will pay fees for mycobiota uploads, optional open access, and correction of major author errors to the Business Manager at this time. MyYcoTAxoON LTD— www.mycotaxon.com The Mycotaxon Webmaster posts announcements, subscription & publication information, and author forms & templates on the official MycotTaxon site. Our server also hosts the mycobiota web-page for free download of Fungae (regional annotated species lists). MYCOTAXON ONLINE— www.ingentaconnect.com/content/mtax/mt The Mycotaxon journal publishes four quarterly issues per year. Both open access and subscription articles are offered. MY COTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 January-March 2020—Volume 135, pp. 1-95 https://doi.org/10.5248/135.1 Two new genera of gymnopoid/marasmioid euagarics RONALD H. PETERSEN & KAREN W. HUGHES Ecology and Evolutionary Biology, University of Tennessee, Knoxville, TN 137996-1100, TN “CORRESPONDENCE TO: repete@utk.edu ABSTRACT— Twonewgenera, Paramycetinisand Pseudomarasmius, are placed phylogenetically within the /omphalotaceae among genera allied with Gymnopus. Paramycetinis comprises two Antipodal taxa related to Mycetinis. Both Paramycetinis species are characterized by luxuriant rhizomorphs, with basidiomata arising occasionally as side branches but also separately from rhizomorphs. Pseudomarasmius accommodates four new species plus four others previously placed in Marasmius. Pseudomarasmius encompasses significant basidiomatal variation, but nrLSU- and ITS-based phylogenies support its taxa in a monophyletic clade that occupies a position related to some Rhodocollybia. All Pseudomarasmius and Paramycetinis species are described (or redescribed) and illustrated. Both genera exhibit characters also found in other superficially similar lineages, making non-molecular based diagnoses of genera in the /gymnopus/marasmiellus clades difficult. KEY worps— biogeography, clamp connections, morphological taxonomy Introduction For the last few decades, numerous, small, “white-spored” saprotrophic mushrooms have undergone a comprehensive reclassification. Especially edifying has been the introduction of molecule-based phylogenetic analyses at about the turn of the 21* century. New taxa have been identified at several nomenclatural ranks, from infraspecific (Aldrovandi & al. 2015; Petersen & Hughes 2016, 2017a,b) to class (Moncalvo & al. 2002, Matheny & al. 2006, Dentinger & al. 2016). In many cases, detailed morphological descriptions have accompanied molecular analyses (or the reverse), but “morphological taxa’ are still being proposed without phylogenetic placement (Petersen & 2 ... Petersen & Hughes Hughes 2016a, 2017; Retnowati 2018) when materials are too old or degraded to produce molecular data or the appropriate instrumentation is unavailable. Desjardin (1989) summarized the classification and publication history of Marasmius and its segregation from Gymnopus (sensu Persoon 1801) or Collybia (sensu Fries 1821). The taxonomic history of Gymnopus (as Collybia) was outlined by Halling (1983). Subsequently, Antonin & al. (1997) discussed infrageneric taxonomy within Marasmius and Gymnopus (as Collybia), and Hughes & al. (2001) segregated Gymnopus (G. fusipes typus generis) from Collybia s.s. (C. tuberosa typus generis). Owings & Desjardin (1997) apparently conducted the first research into the collybioid/marasmioid complexes using DNA sequences based on a more expanded thesis (Owings 1997; nrITS-based). Their work revealed that marasmioid fungi were not monophyletic but segregated into three lineages: present-day Physalacriaceae, Omphalotaceae, and Marasmiaceae. Moncalvo & al. (2000, 2002; LSU-based) essentially inferred the same. Mata & al. (2004; ITS- & LSU-based) showed that Marasmiellus juniperinus (typus generis) belonged in the omphalotoid complex, Wilson & Desjardin (2005) tied the morphological fragmentation of Marasmius to an nrLSU-based phylogeny and refined phylogenetic relationships of a wider array of organisms, and Mata & al. (2007; ITS-based) offered a more comprehensive phylogeny of /omphalotaceae, with emphasis on Gymnopus. The nomenclatural consequence of these works was circumscription of two major clades/ families, Marasmiaceae and Omphalotaceae. A further consequence was more detailed fragmentation within these groups (Petersen & Hughes 2016a, 2017), in which Gymnopus as summarized by Mata & al. (2007) is clearly para- to polyphyletic with several related genera (Rhodocollybia, Lentinula, Mycetinis, Connopus) interspersed and Gymnopanella (Sandoval-Leiva & al. 2016) also associated. Three genera (Lentinula, Rhodocollybia, Mycetinis) are well- populated while two (Connopus, Gymnopanella) currently appear monotypic. If the concepts of Wilson & Desjardin (2005) are advanced to the present day, two large and complex clades contain traditional gymnopoid/marasmioid taxa: /Gymnopus incorporates Gymnopus sects. Levipedes, Striipedes, and Impudici, the traditional Micromphale (including sect. Perforantia), Caripia, and Marasmius sect. Androsacei; /Marasmiellus (including M. juniperinus, typus generis) includes Gymnopus sect. Vestipedes as well as Marasmius (Marasmiellus) ramealis, the type of Collybiopsis (J. Schrot.) Earle (Hughes & Petersen, ined.). /Marasmiellus) would appear to be a candidate for fractionation. Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 3 Oliveira & al. (2019) recognized their clade I (called “Gymnopanella”) based on the taxon described by Sandoval-Leiva & al. (2016) and which included two provisional names that had appeared in phylogenies by Petersen & Hughes (2016). We regard those two provisional names as belonging in a separate generic unit, here proposed as Paramycetinis, based on its placement as sister to Mycetinis. The intention of this paper is to propose two new genera, one comprising two new Antipodal species and the other accommodating several species. FiGureE | shows the relative placement of the new genera based on nrLSU- sequence resolution. Materials & methods Abbreviations: Pa = Paramycetinis; Ps = Pseudomarasmius; M = Marasmius; Ma = Marasmiellus; Mi = Micromphale; My = Mycetinis. Colors cited alphanumerically are from Kornerup & Wanscher (1967) or Munsell (1961), in quotation marks from Ridgway (1912), and special reference to Maerz & Paul (1950); BF = bright field microscopy; PhC = phase contrast microscopy (see also Petersen & Hughes 2017); TFB = Tennessee field-book number, assigned to fresh collections. In order to track information for collections from TENN, numbers are the last five digits of the fungal herbarium accession number (in MyCoPortal as TENN-F-0xxxxx); GSM (also GSMNP) = Great Smoky Mountains National Park, Tennessee/North Carolina. Percent similarity values within and between clades are given on the right. Culture media reported to encourage rhizomorph production followed directions by Farnet & al. (1999). Two different whole wheat flours (“whole wheat flour” and “stone-ground whole wheat flour,’ both obtained locally) produced no difference in rhizomorph occurrence, number, or vigor. The term “textura” to describe the intricate crust formed by many marasmioid, mycetinoid, marasmielloid, and gymnopoid isolates in culture has been borrowed from Korf (1973), who used it to describe the excipular layer of apothecia. Metrics of microstructures were calculated using a phase-contrast Olympus BX60 microscope fitted with phase contrast imagery and microphotographs were produced using an Olympus Qc color camera. Microscope mounts were usually in 3% KOH solution or Melzer’s reagent. Molecular methods for DNA extractions, PCR and Sanger sequencing of the nuclear ribosomal ITS and LSU regions follow Aldrovandi & al. (2015). PhyML phylogenetic analyses were performed in Geneious 11.0.3 (2017). Sequences have been deposited in GenBank (TABLE 1). Specimen data are available in MyCoPortal (2018). Nexis files containing nrLSU sequences and alignments were deposited in the Dryad Data Repository https://doi.org/10.5061/dryad.4081h). ITS sequence %-similarity is the average of all paired sequences computed by Geneious 11.0.3 (2017) as “percent identity”. 4 ... Petersen & Hughes Anthracophyllum DQ457670 Gymnopus contrarius 89 Neonothopanus <—— | Omphalotus 200 <— | Gymnopus pinophilus MGIII-Gordon 9 85. <— | Gymnopus foliiphilus ‘ : oy Gymnopus section Perforantia q Gymnopus perforans (Micromphale sect. Perforantia) <—_| Gymnopus foetidus (Micromphale sect. Micromphale) AY639432 Gymnopus vitellinipes Gymnopus ocior, G. dryophilus, G. bicolor, G. sepiiconicus, G. aurantiipes, G. earleae AF223172 Gymnopus acervatus sensu Moncalvo et al. <=] Gymnopus erythropus, G. spongiosus /C Mata et al. (2007) <——_] G. hariolorum, G. polyphyllus —Ice dysodes, G. barbipes, G. iocephalus <—] G. montagnei and AF261326 G. sp. /B Mata et al. (2007) Gymnopus neobrevipes, G. portoricensis (Micromphale sect. Rhizomorphigena) Petersen and Hughes, 2018 85 ——_] /novae-angliae, /frigidomarginatus, /adventitius <—] Gymnopus androsaceus (Marasmius sect. Androsacei) Wilson and Desjardin (2005) /gymnopus <=] /inflatotrama —] Gymnopus fusipes /A Mata et al. (2007) <——_]} /novomundi MGIIl Gordon 0.06 changes Paramycetinis (austrobrevipes, caulocystidiosus) 79 ——] Mycetinis (Marasmius) scorodonius My cetinis <——_| Mycetinis (Marasmiellus) opacus, My. copelandii Wilson and Desjar din (2005) AY639437 Mycetinis (Marasmius) applanatipes Petersen and Hughes (2017) — J mycetinis (Marasmius) alliaceus 22 ——_} Connopus acervatus 1 mm diam), forming loose nets; basidiomata arising from or separate from rhizomorphs; cheilocystidia clavate with clusters of apical SetwlaeGlasiiaarints: ete ee Ms eet fa et as 2) ee ce Pa Pa. austrobrevipes 1. Rhizomorphs slender (<1 mm diam), extensive; basidiomata arising separate from rhizomorphs; cheilocystidia clavate, without diverticula; fruiting on dead leaves and twigs of Nothofagus (New Zealand)......... Pa. caulocystidiatus Paramycetinis austrobrevipes R.H. Petersen, sp. nov. Figs 3-8 IF 555793 Differs from Paramycetinis caulocystidiatus by its broader rhizomorphs that form loose nets, by its basidiomata that arise as rhizomorph branches and/or from woody substrate, and by its broom cell-like cheilocystidia. Type: Australia, Tasmania, Gordon-Pedder National Heritage Area, Rainforest Nature Walk vicinity, 8.V1.1991, coll. RHP & KWH, TFB 4033 (Holotype TENN-F-050135). EryMoLoGey: referring to micromorphological similarities to Gymnopus neobrevipes. BASIDIOMATA (Fic. 3) marasmioid, diminutive, arising as branches from rhizomorphs and/or directly from woody substrate. PILEUs 2-12 mm broad, convex to shallowly conical, sometimes abruptly or broadly umbonate and even then abruptly umbilicate, deeply sulcate-striate to non-striate, matte, at high magnification (50x) telltale wispy white hairs can be seen in the sulcate depressions; disc (including umbo) 5F6 (“Prout’s brown”), 6E3 (“hair brown’), 9D3 (“benzo brown’), 7C4 (“wood brown’), 6E4 (“fuscous”), 7C6 (“Mikado brown’) to 9C3 (“cinnamon drab”); limb 6C5 (“sayal brown”), 6B5 (“cinnamon”), 17B2 (“light drab”), 7B2 (“tilleul buff”), to 9B2 (“vinaceous buff”), 8D5 (“army brown”), 9C3 (“cinnamon drab”), 9B3 (“light cinnamon drab”) to 7C5 (“fawn color”); margin 6A3 (“pinkish buff”) to 4A3 (“cartridge buff”). LAMELLAE adnexed, subdistant (total lamellae 20-24), about 1 mm broad, thickish, without anastomoses, 7B2 (“tilleul buff”), 9B2 (“vinaceous buff”), 6B4 (“cinnamon buff”) to 6A3 (“pinkish buff”), not marginate; dried 12 ... Petersen & Hughes lamellar trama dark brown and glassy, as though gelatinized. Stripe 18-85 x 0.8-1.2 mm, terete, stuffed (not hollow), appearing glabrous-shining but minutely pruinose at least upward and downward (35x), black to abruptly 6C5 (“sayal brown”), 6B3 (“cinnamon buff”) or mahogany at very apex (not concolorous with lamellae), downward totally black; medullary portion cloud gray, cortical layer thin, nearly black (40x); insertion non-insititious; vesture sparse, pallid, near 7B2 (“tilleul buff”). Ro1IzoMoRPHs >1 mm broad, gradually tapering to 0.2-0.5 mm distally, terete or somewhat compressed, extensive to almost absent, often forming a rudimentary net. ODor negligible; TASTE negligible or weak of garlic; consistency tough then mealy. HABITAT & PHENOLOGY: Nothofagus wood (and then densely gregarious) and twigs, at or near forest floor, Arthrotaxis dead branchlets; to this time, May-June. PILEIPELLIS constructed of the following: 1) slender hyphae 2-3.5 um diam, thick-walled (wall <0.5 um thick), sparsely encrusted (Fic. 4a) with ornamentation appearing as flakes adhered to outer hyphae wall; 2) slender hyphae 1.5-3 um diam, firm-walled with slender, sometimes awl-shaped diverticula (FIG. 4B, 54-D), obscurely clamped, forming a indiscrete layer in a mucoid matrix (i.e. significant debris including bacteria and collapsed spores, PhC); 3) slender, coralloid hyphal termini (Fic. 5£,F); 4) thicker hyphae 4-12 um diam, refringent (PhC), infrequently branched, meandering to appear a bit like lattice, thick or gelatinized—walled (wall <2.5 um thick), with outer profile smooth and clear but inner wall contour irregular, obscurely clamped; termini occasionally more or less erect as “pileal hairs.” PLEUROCYSTIDIA (FIGS 6B, 7A—C) common, 26-33 x 5-9 um, fusiform to mucronate, conspicuously clamped, usually with partitioned contents. BAsipIA 23-28 x 6-9 um, clavate, 4-sterigmate (sterigmata <6 um long, somewhat bowed, stout), obscurely clamped; contents homogeneous. BASIDIOSPORES (FIG. 6A) 6-9 x 3.5-4.5 (-5) um (Q = 1.502.43; Q™ = 1.92; L™ = 7.47 um), more or less pip-shaped but often tapered slightly proximally (that is, perhaps marasmioid), smooth, thin-walled, inamyloid; contents homogeneous. CHEILOCYSTIDIA (FIGS 6D, 7E-H) locally common to scattered among basidia, 17-32(-40) x 5-12 um, clavate to ventricose-rostrate, obscurely clamped, thin-walled below, firm- walled above, surmounted by a corona of extremely complex, vermiform to slender-digitate, refringent (PhC) diverticula; diverticula <7 x 0.71 um, gnarled, often branched. STIPE MEDULLARY HYPHAE hyaline, 2-5.5 um diam, firm- to thick-walled, seldom but conspicuously clamped; CORTICAL Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 13 Fic. 3. Paramycetinis austrobrevipes. Habit. A. (TENN 53181). B. (TENN-F- 050212). Scale bars: 20 mm. HYPHAE adherent, 3-7 um diam, yellow singly, dark ochraceous brown and refringent in mass (PhC), involved in a thin film of mucus, with side branches ranging from simple lobes to caulocystidia. CAULOCYSTIDIA (FIG. 8) 14 ... Petersen & Hughes Fic. 4. Paramycetinis austrobrevipes. Structures of pileipellis. A. Encrusted hyphae (TENN 50119). B. Diverticulate hyphae (TENN-F-050135). C. Thick-walled lattice hyphae (TENN-F-050135). D. Pileal hairs (TENN-F-050119. Scale bars: 20 um. Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 15 Fic. 5. Paramycetinis austrobrevipes (TENN-F-050135). Pileipellis elements. A. Layer of diverticulate hyphae as embedded within pileipellis. B. Layer of diverticulate hyphae. C,D. Individual diverticulate hyphae. E, F Broom cell-like hyphal termini. G,H. Hyphae with gelatinized walls. Scale bars: 10 um. scattered, <105 x 46 um (at widest point, narrowing to 3-3.5 um diam at base, 2-3 um diam at apex), gregarious but more or less evenly distributed, usually single, rarely in pairs, as side branches of stipe surface hyphae, apparently near the terminus of such hyphae (and therefore appearing as though from an asymmetrical origin, yellow and refringent (PhC), so refringent that wall thickness is hardly discernible (when wall seems to be discernible, 1-1.5 um thick), narrowly rounded apically and apparently without adherent debris, dextrinoid (Melzer’s reagent + PhC) or brown (Melzer’s reagent + BF), not internally septate. 16 ... Petersen & Hughes Fic. 6. Paramycetinis austrobrevipes (TENN-F-050135). A. Basidiospores. B. Pleurocystidium. C. Basidia. D. Cheilocystidia. E. Caulocystidia. Scale bars: A = 5 um; B-D = 20 um. ComMENTARY: Pileus shape in Pa. austrobrevipes varies from convex with an abrupt, distinct umbo to a gradual and shallow umbo to no evidence of an Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 17 Fic. 7. Paramycetinis austrobrevipes (TENN-F-050135). A-C. Pleurocystidia. D. Basidium. E-H. Cheilocystidia. Scale bars: 10 um. umbo. In all cases, the disc (with or without umbo) is significantly darker than the limb. The pileus micromorphology of Pa. austrobrevipes approaches that of the North American Marasmius brevipes [= Gymnopus neobrevipes R.H. Petersen & 18 ... Petersen & Hughes Fic. 8. Paramycetinis austrobrevipes (TENN-F-050135). Caulocystidia. Scale bars: 10 um. Hughes 2019)]. Basidiomata of both species arise on woody substrate (usually twigs with thin bark intact or occasionally as branches from an extensive rhizomorph net), but G. neobrevipes basidiomata are small and thumb tack- shaped while those of Pa. austrobrevipes are tall and mycenoid. Pileus colors are similar but also characteristic of several taxa in Gymnopus sect. Androsacei. Rhizomorphs and stipes are black, although stipe in G. neobrevipes is glabrous throughout its length. There is no record of a weak or latent taste of garlic for G. neobrevipes. Comparison of notes and drawings of G. neobrevipes specimens indicate that it also has a gelatinizing pileipellis, but with a more demonstrable “ramealis-structure.’” Gymnopus neobrevipes basidiomata also occur independently from rhizomorphs as well as branches from rhizomorphs, and its rhizomorphs are also thick and form an aerial network as well as on the substrate surface. Despite these similarities, molecular evidence shows Pa. austrobrevipes and G. neobrevipes to be only distantly related (see Fic. 1). Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 19 The surviving culture of TENN-F-053181 (TFB 3585) formed considerable aerial mycelium and what appears to be resupinate rhizomorphs against the side of the MEA-slanted storage test tube. This is different from other cultures of the same putative species but identical to a culture of TFB 3966, of which voucher basidiomata are no longer extant. Collections of Pa. austrobrevipes may be mistaken in the field for M. crinis-equi. The latter can be distinguished in the field by collariate lamellae, significantly smaller basidiomata with short, curved stipes and typical hymeniform pileipellis and cheilocystidia. Grgurinovic (1997) cited collections of M. crinis-equi from New South Wales and South Australia, and Pegler (1977) furnished a much wider range for the species. Close inspection of rhizomorph surfaces shows that when rhizomorphs branch, whether to produce a basidiome or a sterile branch, there is often a rupture of the parent rhizomorph cortex—splitting the cortex and revealing the medulla. This rupture is sparsely covered by a brown mycelium, which also covers the basal portion (<50 um) of the new branch. Whether sterile or fertile, the new branch is also vestured with caulocystidia, which peter out in less than a millimeter distally, leaving the greater portion of the rhizomorph branch (or stipe) subglabrous. On whole wheat flour agar (Farnett & al. 1999), cultures of Pa. austro- brevipes produced chiefly submerged mycelium, no textura intricata, and no rhizomorphs. TFB 3591 (TENN-F-053146) produced a minutely granular white mycelial mat with ill-defined margins. Text and illustrations of Paramycetinis species were generously pre-reviewed by Australian and New Zealand mycologists in order to avoid nomenclatural duplication of any taxon already described. While no present-day match was suggested (but see below), five historical names under Marasmius were pointed out, as follows: Marasmius eucalypti Berk., in Hooker, Fl. Tasman. 2: 249, 1859 [“1860”]. Pegler (1965: 331) examined the type specimen. While a basidioma is illustrated as arising as a side branch of a rhizomorph, pileipellis was described as “hymeniform, composed of diverticulate elements similar to the cheilocystidia.” Thick-walled caulocystidia are described and illustrated. All of this indicates identification as a Marasmius, probably M. sect. Sicci. Marasmius meloniformis Berk., in Hooker, Fl. Tasman. 2: 249, 1859 [“1860”]. Pegler’s (1965: 339) study of the type specimen described surviving material in poor condition. The “long, setaceous stipes, which arise as branches from 20 ... Petersen & Hughes creeping rhizomorphs” must resemble the habit of Paramycetinis austrobrevipes. Conversely, the following characters do not match: 1) pileus 1-2 mm broad, strongly plicate; 2) lamellae few; 3) epicutis hymeniform; 4) cheilocystidia broom cell-like. Pegler (1965) concluded: “The species clearly belongs in the genus Marasmius, sect. Marasmius.’ Marasmius subsupinus Berk., in Hooker, Fl. Tasman. 2: 249, 1859 [“1860”]. From Berkeley's description the following is inferred: Basidiomata apparently conchate with a very short, curved stipe. Lamellae “so thick and rigid that this pretty species might almost be placed in Lentinus” “It varies in color from nearly white to rufous.’ Marasmius emergens Cooke, Handb. Austral. Fungi: 88, 1892. Protologue: “Very minute, white, suberumpent; pileus convex (1 mm broad); stem abbreviated, or sometimes elongated, curved, ascending; gills distant, few, white. On wood. Tasmania.” Marasmius subroseus Cooke & Massee, Grevillea 21: 37, 1892. The protologue implies the following: basidiomata caespitose; no mention of rhizomorphs; “stem becoming a little reddish downwards and clad at the base with white pubescence;” gills distant. Unless proven otherwise, the organism can be accepted as a Marasmius. In addition to these names, an undescribed Australian species is well-known under the informal name of Marasmius “angina.” Online, numerous photos and one description (http://www.elfram.com/fungi/fungi_l/marspang_a.html; https://www.bushheritage.org.au/blog/fungi-and-citizen-science-in-the-liffey- valley) show an unmistakable resemblance to Paramycetinis austrobrevipes, except that the basidiomata are distinctly larger and no rhizomorphs appear in any photo. There is no reason (yet) to consider Marasmius “angina” as anything but a true Marasmius. ADDITIONAL SPECIMENS EXAMINED: AUSTRALIA, TasMANIA, Cradle Mountain National Park, Waldheim Nature Track, 31.V.1991, coll. RHP & KWH, TFB 3977 (TENN-F-053232); Geeveston, Tahune Forest Preserve, 5.VI.1991, coll. RHP & KWH, TFB 4008 (TENN-F-050195);Gordon-Pedder National Heritage Area, Rainforest Nature walk vicinity, 8.VI.1991, coll. RHP & KWH, TFB 4038 (TENN-F-050212); Hobart, Mt. Wellington, gully at 400 m elev., 25.V.1991, coll. KWH, TFB 3591 (TENN-F-053146); coll. RHP & KWH, TFB 3585 (TENN-F-053181); Lake Pedder National Heritage Area, 26.V.1991, coll. RHP & KWH, TFB 3917 (TENN-F-050119); Lake St. Clair National Park, Mt. Rufous track, 29.V.1991, coll. RHP & KWH, TFB 3945 (TENN-F-050073); 8-10 kms north of Rosebery, slopes of Mt. Murchison, 30.V.1991, coll. RHP & KWH, TFB 3949 (TENN-F-051333); coll. RHP, TFB 3966 (TENN-F-053231). Paramycetinis & Pseudomarasmius gen. & spp. nov.... 21 Fic. 9. Paramycetinis caulocystidiatus (TENN-F-053721). Basidiomata and rhizomorphs on natural substrate. Paramycetinis caulocystidiatus R.H. Petersen, sp. nov. Fics 9-14 IF 555794 Differs from Paramycetinis austrobrevipes by its 1) pileipellis with evidence of slime matrix; 2) high stipe length to pileus breadth ratio; 3) fruiting habit on black beech; and 4) gregarious (but separate) setoid caulocystidia. Type: New Zealand, North Island, Urewera National Park, track to Waipai Swamp, 35°45'13”S 177°09'17”E, 27.V.1994, coll. RHP, TFB 7148 (Holotype TENN-F-054050). EryMo toey: referring to caulocystidia of the vestured stipe. BASIDIOMATA (FIG. 9) slender, of medium stature, marasmioid. PILEUS 2-8 (-20) mm broad, convex at first, later campanulate to applanate with depressed disc and margin remaining downturned, minutely tuberculate, translucent- striate to sulcate at margin, surface moist, hygrophanous, glabrous; central dot 8E6 (“Natal brown”), 6D6-6D5 (“snuff brown” near “olive brown”), 5D6-5D5 (“buckthorn brown”), “hazel brown, 6D8-6D4 (“buffy brown”), fading to 5C5-5C4 (“tawny olive”); limb and margin 7C4 (“wood brown”) to 6C5 (“sayal 22 ... Petersen & Hughes brown”). LAMELLAE adnexed, suppressed and ridge-like to subventricose, subdistant, <2 mm broad, thickish, total lamellae = 35-50(-75), through lamellae = 10-17 per pileus with 1-2 ranks of lamellulae between each through- lamella, not forked or interveined, 4A2 (“pale ochraceous buff”), 5A2 (“pale cinnamon pink”), 9B2 (“vinaceous buff”), to 5B8 pastel burnt orange to near 6B3 (“tilleul buff” not white); edge entire, rounded (not sharp), not fimbriate, not marginate. StrpE 30-40(-75) x 1.3-2 mm, terete, equal (stipe base often slightly swollen), not insititious; surface moist to dry, pruinose, uniformly vestured as a turf of perpendicular setoid caulocystidia, upward 7C4 (“wood brown’) to 7D5 (“Rood’s brown”), downward 7E6 (“auburn”), 7E6 (“VanDyke brown”), 7F5 (“blackish brown (1)”) to black; “sterile stipes” often present, 20-32 x 1-2.5 mm, straight, stiff, vestured, without pileus, occasionally decapitated (Fic. 9) and then producing rhizomorph extension (black, slender, glabrous). RHIZOMORPHS <30 x 0.2-0.7 mm (at base), glabrous, gradually narrowing to flagelliform, mostly black, only slightly paler distally, when decapitated producing a rhizomorph or stipe (glabrous versus vestured) extension, but not producing pileate basidiomata from rhizomorphs. ODoR negligible; TAsTE negligible. HABITAT AND PHENOLOGY: On dead twigs, leaves and small branches of Nothofagus solandri; North Island, New Zealand; May-June. PILEIPELLIS a repent layer of generally radially oriented hyphae embedded in a thin slime matrix, tightly packed, of the following types: 1) pileal hairs (Frc. 10) <100 x 3-5 um, perhaps erect, produced as termini of repent hyphae (Fre. 11) finely encrusted, usually subtly capitulate; 2) repent hyphae 2-8(-13) um diam, at surface usually of the wider type, firm-walled, coarsely encrusted in thick scabs; profile calluses <2 um thick, sub-refringent (PhC); crust material often distributed with suggestion of striped or annular thickenings; 3) repent hyphae 5-10 um diam with gelatinizing walls (wall <2.5 um thick), variously encrusted; 4) subpellis hyphae usually thicker (6-14 um diam), smooth or with very fine encrustation appearing gritty on the hyphal surface; clamp connections ubiquitous. PLEUROCyYsTIDIA (Fic. 12) 24-32 x 6-9 um, common, broadly fusiform, conspicuously clamped; contents homogeneous to distinctly partitioned. Basip1a 25-30 x 7-11 um, clavate, often sub- urniform, 4-sterigmate, clamped; effete basidia (and pleurocystidia) do not disappear but leave residual lateral walls and shriveled sterigmata (“husking”). BASIDIOSPORES (5-)6.5-8(-9) x 3.5-4(-4.5) um (Q = 1.43-2.43; Q™ = 1.92; L™ = 7.41 um), ellipsoid, somewhat flattened adaxially, smooth, thin-walled, inamyloid. CHEILOocystTipIA (Fic. 13) hardly distinguishable from basidia, Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 23 ra ~ / Fic. 10. Paramycetinis caulocystidiatus (TENN-F-054050). Pileal hairs. A. Origin of two pileal hairs. B,C. Individual pileal hairs. Scale bars: 10 um. 29-35(-42) x 6-13 um, broadly truncate-clavate, hardly lobed apically, obscurely clamped. STIPE MEDULLARY HYPHAE 3-9 um diam, strictly parallel, adherent with minimal slime matrix, thin-walled inward, with gelatinizing walls (<2.5 um thick) near cortex, obscurely clamped, hyaline, inamyloid inward, increasingly dextrinoid toward stipe surface (to moderately so in Melzer’s reagent + BF). STIPE CORTICAL HYPHAE Strictly parallel, probably coherent and perhaps with thin slime layer, coarsely encrusted, thick-walled, yellow-brown 24 ... Petersen & Hughes Fic. 11. Paramycetinis caulocystidiatus (TENN-F-054050). Pileipellis elements. A. Heavily encrusted hypha with gelatinized wall. B,C. Encrusted hyphae with suggestion of striped or annular ornamentation. D. Hyphae with gelatinized walls. Note anastomosis in “H”-connection. E. Swollen subpellis hypha. Scale bars: 10 um. Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 25 Fic. 12. Paramycetinis caulocystidiatus (TENN-F-054050). Hymenial elements. A-D. Pleurocystidia. B. Note partitioned contents. E-H. Basidia. Scale bars: 10 um. (PhC), dark red-brown in Melzer’s reagent + BE. CauLocystip1a (Fic. 14) arising as side branches from thick-walled, smooth, superficial cortical hyphae, stiff, setoid, arising as short lobes (and then hyaline), elongating >70 x 6-9 um at base, 3-4.5 um diam at apex, narrowly rounded, thick-walled (wall <3 um thick when visible), sub-refringent and hyaline distally (150x + PhC), strongly dextrinoid (red-brown in Melzer’s reagent + BF; dark red-brown in Melzer’s reagent + PhC). 26 ... Petersen & Hughes A a Fic. 13. Paramycetinis caulocystidiatus (TENN-F-054050). Cheilocystidia. A. Grouped at lamellar edge. B-E. Individual cheilocystidia. Scale bars: 10 um. COMMENTARY: Pileipellis structure is typical of Gymnopus sect. Perforantia. Hyphal types match quite well, especially gelatinizing hyphal walls and presence of pileal hairs, and there is evidence of a thin slime matrix. Stipe to pileus ratio is high, with the stipe universally vestured and minutely barbed. Dried pileus now somewhat campanulate, with umbo slightly darker than limb or margin; pileus now appearing somewhat moist-laccate. Basidiomata are accompanied by thin, ascendant rhizomorphs. The “omnipotent” stipes and rhizomorphs are somewhat unique, and are quite obvious as abrupt, odd, asymmetrical joints of such structures (see Fie. 9). Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 27 Fic. 14. Paramycetinis caulocystidiatus (TENN-F-054050). Caulocystidia. A. Overview of stipe surface; note gregarious but separate individual caulocystidia. B-E. Individual caulocystidia. Scale bars: A = 20 um; B-E = 10 um. Paramycetinis caulocystidiatus joins Pa. austrobrevipes in a small clade sister to that of Mycetinis (Fic. 1). The latter is characterized by a roughly hymeniform pileipellis of inflated, naked, thin-walled cells and usually a basidiomatal alliaceous odor and taste. Wilson & Desjardin (2005) showed that 28 ... Petersen & Hughes Mycetinis taxa were found embedded among taxa of Gymnopus s.l. and assorted other genera. Petersen & Hughes (2017b) surveyed Mycetinis, with a detailed ITS-based phylogeny of the genus. On whole wheat flour agar (Farnet & al. 1999), cultures of Pa. caulocystidiatus produced no textura intricata, white to off-white felty mycelium, and no rhizomorphs. ADDITIONAL SPECIMENS EXAMINED: NEW ZEALAND, NortH ISLAND, Urewera National Park, Lake Waikaremoana, Black Beech Track, 28.V.1994, coll. A.S. Methven, TFB 7588 (TENN-F-053721); Lake Waikareiti Track, 27.V.1994, coll. A.S. Methven, TFB 7572 (TENN-F-053683). Pseudomarasmius Oliveira & al. (2019) resolved a clade they named /pallidocephalus (not a genus name). It is this clade that we propose as Pseudomarasmius. They encouraged generic status for the clade which, they admitted, needed more research. Oliveira & al. (2019) proposed Pusillomyces, closely related to Gymnopus asetosus and G. funalis (their clade B). Pusillomyces manuripioides (typus generis) was described as lacking clamp connections, a trait virtually diagnostic for Pseudomarasmius, proposed here, but relatively unrelated to their clade B. A PhyML analysis of nrITS sequences for species within Pseudomarasmius is given in Fic. 15 and broader placement based on the nrLSU region is given in Fic. 1. Pseudomarasmius comprises several species and an unidentified clade of fungal soil isolates from Guyana. Pseudomarasmius R.H. Petersen & K.W. Hughes, gen. nov. IF 317324 Differs from Marasmius by 1) diverticulate hyphae present in the pileipellis and 2) clamp connections absent. TYPE SPECIES: Pseudomarasmius pallidocephalus (Gilliam) R.H. Petersen Erymo .ocy: Referring to its similarity to Marasmius. Basidiomata of three types: 1) similar in size and stature to those of Gymnopus sect. Androsacei, with discrete, bristle-like stipe and convex to applanate cap; rhizomorphs slender and inconspicuous limited to rotting leaf or needle litter; 2) similar in size and shape to those of Marasmiellus, with small, curved stipe and shell-like pileus and usually dominant rhizomorphs which occasionally produce basidiomata as side branches, similar to this phenomenon in Marasmius crinis-equi; and 3) small, Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 29 slender, pallid yellowish mycenoid basidiomata lacking rhizomorphs, on rotting needles of Abies or Picea. Microstructures resembling those of Gymnopus sect. Androsacei and Marasmius sect. Sicci, including: 1) diverticulate hyphae in pileipellis (arranged in a “rameales-structure”); 2) thick-walled, gelatinizing hyphae and/or gelatinizing diverticulate hyphae in pileipellis and subpellis; 3) without clamp connections or clamps rare and restricted to pileus trama; 4) basidia (2—)-4-spored; 5) spores similar to those of other gymnopoid genera, ellipsoid, thin- walled, inamyloid; 6) molecular (LSU, ITS) profiles unique, grouping into a monophyletic clade related to Rhodocollybia but more distant from groups exhibiting similar basidiomatal types (Fic. 15). Key to species of Pseudomarasmius 1. Ratio of pileus breadth to stipe length <2 (“thumb-tack shaped”); basidiomata small, often arising as side branches of rhizomorphs............ 2 1. Ratio of pileus breadth to stipe length >4; basidiomata marasmioid or mycenoid, arising as rhizomorph branch or independently on leaf litter or woody ULES TAT sacks se cchcrcbe-lehoecls AT iel hc rencechcn ces ehcea are a Aries kar 3 2. Southern South America (Isla Chiloé, Chile); rhizomorphs inconspicuous, not producing basidiomata; fruiting on hardwood substrates; clamp connections absent; superficially inseparable from OS PTET SSAV ESN cee itt cnc Ride sala ade oa: Faults se desoy Pagans sme Retded «eRe oh Ps. patagonianus 2. North American Gulf Coastal Plain and subtropical eastern Mexico; rhizomorphs dominant, long, occasionally branched, occasionally producing basidiomata as side branches; fruiting on small dead branches and twigs of deciduous trees usually at some distance from the ground; clamp connections occasional in rhizomorphs and tramae but otherwise absent ................. Ps. nidus-avis 3. Antipodal (New Zealand); basidiomata minute (pileus 1-3.5 mm broad; stipe capillary, 6-8 mm long); fruiting on hanging dead terminal branches of DDGCT VATE Sere! PS ok AMT ae OE ae PTR, AAR soo BTA! ASU MRO Ps. efibulatus 3. Not Antipodal; fruiting substrate leaf or needle litter ......................00.. + 4. Fruiting on conifer needle litter; rhizomorphs usually inconspicuous............ 5 4, Fruiting on hardwood leaf litter (including Castanopsis); rhizomorphs conspicuous, PESUPINIALE ON Cal SUT LACES od Fe ntl (Oe ahd cae Phone APG eaeead Cai ceee eaten Pte eT shed 7 5. Temperate to northern North America; stipe black; basidiomata like those of Gymnopus sect. Androsacei (pileus <35 mm broad; stipe 12-43 x 0.2-0.8 mm); pileus white to off-white; rhizomorphs inconspicuous, usually found deep in ITUETIRLAS. >. «Sta h'p. «sha Nip tea Mi rata Wie ada bis fost.g be asta iret abe posi anit Ps. pallidocephalus 5. South Korea/eastern North America; basidiomata slender, mycenoid; stipe Ochtaceous LOrsthawaCOloreds hF 8 Fi ut Atal tna ali sa ha tants a ello arwsalt rk wall tak 6 30 ... Petersen & Hughes KC966049 Alaska AY313287 Greenland afn. Rhodocollybia FJ475743 Soil Sweden MK268235 China Ps. guercophiloides KF251072 G. glabrocystidiatus (not type) JN890249 Soil samples JN89021 9 Guyana JN890248 KY352649 Ps. patagonianus Chile MK268234 NZ MK268236 NZ KR673444 Marasmius sp. Korea | Ps. efibulatus FJ596763 USA, TN KY026691 USA, TN KY026636 USA, WA KY026635 USA, ID FJ596762 USA, TN 5015 culture, Canada, NS KY026684 USA, NY — Korea G. glabrocystidiatus type KY026732 USA, AL MK268237 USA, FL KY026723 USA, MS KY026646 USA, LA KY026733 USA, TX MH016872 USA, FL KY026753 USA, MS MK020094 USA, FL MK268238 USA, FL MH560575 Mexico MH560576 Mexico MH560577 Mexico MHS60579 Mexico MIH560578 Mexico Ps. pallidocephalus Pseudomarasmius 0.05 changes 8 3 Ps. nitus-avis Fic. 15. Maximum likelihood analysis of ribosomal RNA ITS sequences. Bootstrap values are given to the left of the supported node. Numbers are GenBank accession numbers (See TABLE 1). For collections from the United States and Canada, the state or province postal code is given. NZ = New Zealand. Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 31 6. South Korea (and probably Japan); basidiomata minute, fragile (pileus 48 mm broad; stipe 15-40 x 0.5 mm); pileus dark grey-green; stipe dark brown to black-brown; rhizomorphs inconspicuous; fruiting onideadmeedles OPA MICS. feiss. 8 ie eth, cok ented Ps. glabrocystidiatus 6. Eastern North America; basidiomata similar to those of Ps. glabrocystidiatus; pileus light brown or pale greyish brown; stipe brownish orange to yellow or straw-colored; on rotting needles of Picea and Abies ......... Ps, straminipes 7. Costa Rica; high-altitude Quercus forests ......... 0... cece eee eee Ps. obscurus 7. South Korea, China (Yunnan); on dead leaves of Castanopsis ... Ps. quercophiloides Pseudomarasmius efibulatus R.H. Petersen, sp. nov. FIGs 16-20 IF 555730 Differs from Pseudomarasmius taxa by its: 1) minute basidiomata; 2) fruiting habit on dead Dacrydium branchlets; 3) scattered pyramidal to digitate diverticula on pileipellis elements; 4) known distribution limited to the New Zealand type location. Type: New Zealand, South Island, Westland, Fox Glacier, Lake Matheson loop trail, 43°26'22”S 169°57'54’E 13.V.1994, coll. RHP, TFB 7070 (TENN-F-056187). EryMo_oey: from fibula (Latin) for clamp, buckle + e- (Latin), without. Referring to the clampless condition. BASIDIOMATA (FIG. 16A) diminutive. PrLEus 1-3.5 mm broad, convex when young, becoming everted by maturity, matte, pebbled; disc 7C5 (“fawn color’), outward 9B2 (“vinaceous buff”) to 6A3 (“pinkish buff”). LAMELLAE adnate, arcuate, not collariate, thickish, c. 1 mm broad, not marginate, off-white when fresh, becoming 5A4 (“light ochraceous buff”) over time; lamellulae occasional, short; total lamellae (10)1218; through lamellae 7-8. Stipe 6-8 x 0.2-0.4 mm, apically 8E6 (“Natal brown”) (and this color extending through lamellar attachment to pileus flesh), lower 7/8 black, glabrous-shining, insititious. RHIZOMORPHS (Fic. 164,17) common, <40 x 0.1-0.3 mm, more or less straight (not curly), rarely branched, arising separately from basidiomata. HABITAT AND PHENOLOGY: On dead, pendant Dacrydium foliage; Autumn. PILEIPELLIS with minimal slime matrix and composed of the following: 1) diverticulate hyphae (Fic. 18A-p) 4.5-10 um diam, thin-walled, with scattered diverticula <10 x 1.52 um (often c. 4 um diam at base and therefore more or less pyramidal), often dichotomous or saddle-shaped, not refringent [in fact appearing darker than the parent hyphae in PhC]; 2) broom cell- like termini (Fic. 18E-H) 15-25 x 9-16 um, stalked, usually dichotomously branched, surmounted with rows of diverticula; diverticula 3-8 x 1-1.5 um, hardly refringent (PhC). Pileus tramal hyphae 3-6 um diam, firm-walled, long-celled, apparently without clamp connections; wall somewhat obscure, 32 ... Petersen & Hughes Fic. 16. Pseudomarasmius efibulatus (TENN-F-056187, holotype). A. Basidiomata and rhizomorphs. B. Basidiospores. Scale bars: A= 5 mm; B = 5 um. as though semi-gelatinized. Lamellar tramal hyphae similar. Hymenium apparently involved in minimal gelatinized matrix (excessive hymenial remnants and other debris). PLEUROCYSTIDIA (FIG. 19A—D) 24-30 x 6-7 um, fusiform, often irregularly so, without content partitioning or clamp connection. Basidioles clavate, usually subcapitulate; Bastp1A (FIG. 19E-H) 22-30 x 6-9 um, clavate, 4-sterigmate, without clamp connections. BASIDIOSPORES (FIG. 16B) (7—)7.5-11 x 3.5-4.5 um (Q = 2.053.14; Q™ = 2.26; L™ = 8.67 um), elongate-ellipsoid to subcylindrical, more or less marasmioid (somewhat tapered proximally), flattened somewhat adaxially, thin-walled, smooth, inamyloid. Lamellar edge apparently fertile. CHEILOCYSTIDIA (FIG. 20) widely scattered, 22-35 x 10-12 um, stalked-clavate to saccate, entire or with a few slender, digitate apical processes, without clamp connections. STIPE MEDULLARY HYPHAE 1.5-12 um diam, strictly parallel, involved in a slime matrix, thin- to thick-walled (wall c. 0.7 um thick, hyaline), distinctly without clamp connections, relatively short-celled; subcortical hyphae 4-8 um diam, thick-walled (wall c. 1.5 um thick), strongly refringent, yellow (PhC). STIPE CORTICAL HYPHAE 4-7 um diam, firm-walled, short-celled, without clamp connections, pigmented yellow-olive (PhC); surface minutely roughened with dried thin slime. CAuLocysT1p1A absent. Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 33 Fic. 17. Pseudomarasmius efibulatus (TENN-F-056187, holotype). Dacrydium branchlets with rhizomorphs. Scale bar = 10 mm. COMMENTARY: Based on ribosomal nrRNA ITS sequences, Ps. efibulatus is related to but distinct from Ps. pallidocephalus, Ps. glabrocystidiatus, and Ps. nidus-avis. It is closely related to sequence JAC10695 received from J. Cooper, Landcare Research, New Zealand (Fic. 15) A blast of ITS sequence from Ps. efibulatus (TFB 7070, TENN-F-056187) showed an affinity with ITS sequences of Ps. glabrocystidiatus, a clampless taxon from South Korea, placed by its authors in Gymnopus sect. Androsacei. Sequences of Ps. glabrocystidiatus are virtually congruent to those of Ps. pallidocephalus (see above) found frequently across northern North America fruiting on dead conifer needles, also the habit of the Asian G. glabrocystidiatus. The nrITS-based phylogeny produced by Antonin & al. (2014) placed G. glabrocystidiatus within Gymnopus but on a long, independent branch described as part of G. sect. Androsacei. The nrLSU- based phylogeny in Fic. 1 (above) indicates even more significant isolation of Pseudomarasmius. Pseudomarasmius efibulatus belongs to a small complex of similar, slender, gracile basidiomata along with Ps. straminipes and Ps. glabrocystidiatus, but the basidiomata of all three species are so small and inconspicuous as to be 34 ... Petersen & Hughes Fic. 18. Pseudomarasmius efibulatus (TENN-F-056187, holotype). Pileipellis elements. A-D. Diverticulate hyphae. E-H. Broom cell-like hyphal termini. Scale bars: 10 um. easily overlooked. Pseudomarasmius efibulatus, however, is quite isolated in substrate and distribution. The few similarities between Ps. pallidocephalus and Ps. efibulatus include: 1) clampless condition; 2) temperate distribution (North- and South- Temperate climates); 3) stipe without caulocystidial ornamentation; 4) a complex “ramealis-structure” of pileipellis; 5) cheilocystidia that are usually smooth, broadly clavate or ampulliform; and 6) well-developed rhizomorphs. Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 35 Fic. 19. Pseudomarasmius efibulatus (TENN-F-056187, holotype). Hymenial elements. A-D. Pleurocystidia. E-H. Basidia. Scale bars: 10 um. Basidiospores are smooth, ellipsoid to elongate pip-shaped, and white in spore deposits throughout the /gymnopus/marasmiellus clade, with spore measurements distinguishing the taxa reported as follows: Ps. efibulatus = (7-)7.5-11 x 3.5-4.5 um (Q = 2.26); G. glabrocystidiatus = (7.7—)8.5-9.5 36 ... Petersen & Hughes Fic. 20. Pseudomarasmius efibulatus (TENN-F-056187, holotype). Cheilocystidia. Scale bars: 10 um. (-10) x 4-5 um (Q = 2); Ps. pallidocephalus = (5-)5.5-7(-9.5) x 3-3.5 um (QO. = 1,87). Macroscopically, basidiomata of Ps. nidus-avis and Ps. patagonianus, superficially resemble numerous Marasmiellus taxa fruiting on dead twigs and small branches of deciduous trees and exhibiting extensive, robust rhizomorphs. Although morphologically counterintuitive for inclusion in Pseudomarasmius, ITS- and LSU-based phylogenies place Ps. nidus-avis and Ps. patagonianus with the other taxa in the genus. Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 37 Perhaps most surprising about the sole collection of Ps. efibulatus was the absence of clamp connections. Collections of similar basidiomata found fruiting on the same somewhat unique substrate demonstrated ubiquitous clamp connections. Moreover, pileipellis elements of clamped and clampless organisms compare well: a complex pileipellis composed of both repent diverticulate hyphae (with diverticula often bifurcate) and broom cell-like hyphal termini beset with diverticula also often dichotomous. This may be analogous to the situation in Ps. straminipes (clampless) and its form fibulatus (clamp connections ubiquitous). DNA sequences not currently extant may help to clarify this situation. Pseudomarasmius glabrocystidiatus (Antonin, Ryoo & Ka) R.H. Petersen, comb. nov. FIGS 16-20 IF 555747 = Gymnopus glabrocystidiatus Antonin, Ryoo & Ka, Mycol. Prog. 13(3): 710. 2014. Republic of Korea, Inje, Baekdam-sa, 27. VI. 2008 leg. V. Antonin (08.36), R. Ryoo & J.G. Han (Holotype, BRNM 718676). ADDITIONAL PUTATIVE CONTAXIC COLLECTION (but see below): Culture. Korea, Jeju, Seonheul-gotjawal, 06.[X.2012, R. Ryoo, K.-H. Ka & H.D. Sou (KFRI 1935). For detailed description and illustrations see Antonin & al. (2014) Major distinguishing characters include: 1) basidiomata similar to those of Ps. straminipes; 2) clamp connections absent; 3) pileus dark (grey-)brown with paler margin when young, then brownish at center and paler, beige to pale brown at margin; 4) basidia 2- spored; 5) stipe smooth and glabrous, dark brown to black-brown; 6) cheilocystidia smooth, clavate or pyriform; 7) pileipellis composed of more or less smooth or diverticulate hyphae and single broom cells; 8) habitat on dead conifer (Abies) needles; 9) geographic distribution, South Korea (probably Japan). Hapsitat: needles of Abies holophylla. COMMENTARY: In the phylogeny presented by Antonin & al. (2014), two ITS sequences of putative Gymnopus glabrocystidiatus (GB NR152899, holotype; KF251072, ancillary sequence from culture) appeared in a single small clade but on individual short branches. This clade appears sister to Rhodocollybia, a position similar to that shown on our LSU-based phylogeny (Fic. 1) for Pseudomarasmius. An ITS sequence from holotype material of G. glabrocystidiatus differs from those of P. pallidocephalus by 31/694bp (4.46%) and while apparently in the same clade based on nrITS sequences alone (Fic. 15), nrITS sequence divergences warrant separate species designations. Further, G. glabrocystidiatus is an Asia taxon while 38 ... Petersen & Hughes Ps. pallidocephalus is North American in distribution. The description and illustrations of Ps. glabrocystidiatus furnished by Antonin & al. (2014) are adequate and require no enhancement here. Although two GenBank designations for ITS sequences of G. glabro- cystidiatus were reported in the phylogeny by Antonin & al. (2014), KP251073 (“NR152898” from holotype material) and KP251072, the latter can be traced to a culture from an ancillary specimen apparently misidentified. The two sequences appear only distantly related in our ITS-based phylogeny (Fie. 15). The non-type GenBank sequence (KF251072) virtually matches that of TFB 3162, here proposed as Ps. quercophiloides (q.v.). The latter was collected in China. GenBank sequence LC0148839, including the ITS region, was derived from an environmental sample from leaf litter of Picea jezoensis subsp. hondoensis (spruce) in Funabashi, Japan (Hagiwara & al. 2015). The sampling site was in central Honshu Island (35 36’N, 13728’E) and dealt with bleaching of substrate leaf debris, including conifer (Hintikka 1970; Miyamoto & al. 2000; Osono 2015). The spruce substrate generally agrees with that of Ps. glabrocystidiatus and, with the latter distribution in South Korea (Antonin & al. 2014), the Japanese report seems within the ecological pattern for the complex. Pseudomarasmius nidus-avis (César, Bandala & Montoya) R.H. Petersen, comb. nov. FIGS 21-31 IF 555746 = Gymnopus nidus-avis César, Bandala & Montoya, Mycokeys 43: 25. 2018 Type: Mexico. Veracruz: Municipality of Xalapa, Santuario del Bosque de Niebla, Instituto de Ecologia A.C., 1343 m a.s.l., gregarious, on fallen twigs of Quercus, 20 April 2016, Cesar 36 (Holotype, XAL). Diagnostic characters include 1) basidiomata that are small and thumb tack-shaped, often arising as side branches from rhizomorphs; 2) a short, curved stipe; 3) distribution in North American Gulf Coast and subtropical Mexico; 4) clamp connections lacking except rare in stipe medulla. BASIDIOMATA (Fics 21, 22) arising from woody substrate or (more often) as side branches of rhizomorphs. PILEus 2-6(2-10) mm broad, convex to plano-convex, usually somewhat depressed over the disc, occasionally shallowly umbonate, pulvinate, variously sulcate-striate, matt, uniformly more or less “wood brown” (7C4), pale brown (7.5 YR 7/4-6; 10YR 7/4; Munsell) to brown (7.5 YR 5/6; 10 YR 4/4; Munsell); context thin (<1 mm Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 39 Fic. 21. Pseudomarasmius nidus-avis (TENN-F-054912). Basidiomata. A. Habit photo. B. Basidiomata, macromorphological aspect. Scale bars: 10 mm. thick), soft, whitish. LAMELLAE (<1 mm broad; 13-26 total lamellae, 4-9 through lamellae, anastomoses lacking), adnate, adnexed to shallowly subdecurrent, thick with rounded edge, paler than pileus surface, ivory to off-white when fresh, very pale brown (2.5 Y 8/2; Munsell)], after drying suffused ruddy brownish outward from stipe (now “wood brown” 7C4, or “avellaneous” 7B3); lamellar edge entire. STIPE arising either from woody substratum or as side branches of rhizomorphs; 2—6(-12) x (0.2-)0.7(-1.6) mm, central to slightly eccentric, terete, equal or tapered downward, black, glabrous-shining, straight to strongly curved, insititious, reddish-brown at the apex (2.5 YR 4/6; Munsell), dark brown to black below (1OYR 2/1, 7.5YR 2.5/2; Munsell), brown; medulla light brown (2.5Y 6/4; Munsell; AO ... Petersen & Hughes Fic. 22. Pseudomarasmius nidus-avis (TENN-F-047666). Basidiomata. Scale bars: 10 mm. Photos courtesy David P. Lewis. basal tuft very sparse). RHIZOMORPHS (FIG. 21, 224,B) <500 x 0.1-0.3 mm, simple, black, wiry, abundant, black, glabrous-shining. TasTE and opor not distinctive. Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 41 Fic. 23. Pseudomarasmius nidus-avis (TENN-F-054912). Micromorphology. A. Pileipellis diverticulate hyphae. B. Pleurocystidium and basidia. C. Basidiospores. D. Cheilocystidia. Bars: A-C = 10 um; D = 5 um. HABITAT & PHENOLOGY: Subtropical forest, with dominant rhizomorphs scattered or gregarious on fallen or aerial twigs of trees, notably Quercus, with rhizomorphs used by birds as nesting material in Mexico; late spring and summer; North American Gulf of Mexico Coastal Plain (Alabama, Florida, Louisiana, Mississippi, Texas). PILEIPELLIS composed of: 1) pileal hairs (Fic. 244-c) <80 x 2-4 um, possibly erect, becoming apically subcapitulate, minutely ornamented; 2) repent diverticulate-ornamented hyphae (Fics 234,25), 2.5-5.5 um diam, 42 ... Petersen & Hughes Fic. 24. Pseudomarasmius nidus-avis (TENN-F-054912). Micromorphology. A-C. Pileal hairs. D. Broom cell-like pileipellis hyphal terminus. Bars = 10 um. thick-walled and diverticulate hyphae termini (Fics 24pD,25); diverticula 1-3 x 1-1.5 um, conical, slightly dextrinoid (fide César & al. 2018); clamp connections absent; 3) tightly interwoven, repent hyphae 2.5-6 um diam, thick-walled (wall <1 um thick), ornamented with annular deposits c. 0.5 um thick (Fic. 264,B); 4) repent hyphae 2-4.5 um diam, thick-walled (wall <1 um thick) with individual slime sheath. PILEUs TRAMA composed of interwoven hyphae 2.5-7.5 um diam, weakly dextrinoid (fide César & al. 2018), individually strongly gelatinized (wall <2.5 um thick, hyaline), implicitly ornamented with flake-like deposits (seen as carried by individual slime sheath; Fic. 26c,p); clamp connections absent. Lamellar trama loosely Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 43 Fic. 25. Pseudomarasmius nidus-avis [FLAS-F-60891]. Pileipellis diverticulate hyphae. Compare with Fics 38-40 (Ps. pallidocephalus). Bars = 10 um. interwoven; hyphae 2-3.5 um diam, firm-walled, hardly gelatinizing, not easily disarticulated, inamyloid to weakly dextrinoid (fide César & al. 2018); clamp connections absent. PLEUROCYSTIDIA (FIG. 23B, 27) common, 24-29 44 ... Petersen & Hughes Fic. 26. Pseudomarasmius nidus-avis [FLAS-F-60891]. Pileipellis hyphae. A. Hyphal encrustation in scabs. B. Annular ornamentation. C,D. Individual slime sheaths. Bars = 10 um. x (5-)6-7 um, fusiform to plump-fusiform, without clamp connections; contents multiguttulate; guttules refringent (PhC). Basidioles (Fic. 28a) clavate becoming subcapitulate, without clamp connections; BASIDIA (Fic. 28B-pD) (20-)27-32(-41) x (5-)9-11 um, clavate to subcapitulate, Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 45 Fic. 27. Pseudomarasmius nidus-avis [MES 1477 (FLAS)]. Pleurocystidia. Bars = 10 um. (2-)4-sterigmate, without clamp connections; contents multiguttulate; guttules refringent (PhC). BastDIOsPORES (FIGS 23C, 28E-H) (7—)8-10.5 x (3-)4-5.5 um (Q = 1.802.22; Q™ = 1.91; L™ = 9.2 um), ellipsoid to plump pip-shaped, flattened adaxially, usually somewhat tapered proximally, 46 ... Petersen & Hughes Fic. 28. Pseudomarasmius nidus-avis [MES 1477 (FLAS)]. A-D. Basidiole and individual basidia. E-H. Basidiospores. Bars = 10 um. hyaline, thin-walled, inamyloid; contents multiguttulate when mature; guttules refringent (PhC). CHEILOCysTIDIA (FIGs 23D, 29) 20-39 x 3-8 um, occasional on lamellar edge, hyphal (not inflated), stalked (stalk 2-2.5 um diam, thin-walled), irregularly lobed and/or branched, without clamp connections; contents homogeneous. STIPE MEDULLARY HYPHAE of two distinct forms: 1) stout hyphae (Fic. 30), 4-15(-30) um diam, highly refringent (PhC), apparently aseptate or with occasional cloissons d’retret; hyphal walls gelatinizing, occasionally strongly so (wall c. 4.5(-12) um thick), Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 47 Fic. 29. Pseudomarasmius nidus-avis [MES 1477 (FLAS)]. Cheilocystidia. Bars = 10 um. often ornamented with flake-like deposits in vague stripes and carried on the outside of the gelatinized sheath; and 2) slender hyphae (Fic. 31), 2-4.5 um diam, thick-walled (wall c 0.7 um thick), minutely ornamented, with occasional but conspicuous clamp connections. STIPE CORTICAL HYPHAE 2.5-7.5 um diam, repent, strictly parallel, strongly pigmented, thick-walled, minutely encrusted on stipe surface, dextrinoid (red-brown, Melzer’s reagent + PhC; reddish brown, Melzer’s reagent + BF); pigmented, diver- ticulate terminal elements 4—19(-—21) x 3-4(-5) um, thin-walled (fide César & al. 2018). COMMENTARY: A recent paper (César & al. 2018) described Gymnopus nidus-avis as clampless (except for scattered clamps in stipe medulla) and with basidiomata arising as side branches from copious, black rhizomorphs. 48 ... Petersen & Hughes Fic. 30. Pseudomarasmius nidus-avis [MES 1477 (FLAS)]. Stout stipe medullary hyphae. A Non-gelatinized hypha. B. Flake-like ornamentation on thin slime sheath. C. Maximum gelatinization. Bars = 10 um. ITS sequences from G. nidus-avis were intermixed with sequences deposited (by our lab) in GenBank as Marasmius brevipes. Independent discovery that voucher collections of these collections of putative M. brevipes were clampless confirmed the paraphyletic placement of putative M. brevipes. Typical Marasmius brevipes (Desjardin & Petersen 1989b) has been transferred to Gymnopus (as G. neobrevipes; see Petersen & Hughes 2019). Proposal of G. nidus-avis including several ITS sequences provides a name for the Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 49 Fic. 31. Pseudomarasmius nidus-avis [MES 1477 (FLAS)]. Slender stipe medullary hyphae. A. Non-gelatinized hypha. B. Flake-like ornamentation on thin slime sheath. C. Maximum gelatinization plus ornamentation. Bars = 10 um. non-typical collections of putative M. brevipes. Inherent is a significantly widened distribution for G. nidus-avis from subtropical Mexico to the North American Gulf Coast. A few specimens at FLAS (variously labelled as unidentified or as “aff. M. crinis-equi”) lack basidiomata but exhibit rhizomorphs of the G. nidus- avis morphology (black, smooth, very slender). Murrill (1915) proposed Polymarasmius for basidiomata which arise as branches of rhizomorphs but noted: “...lamellae adnate to a collar...” Polymarasmius was typified by Marasmius multiceps Berk. & M.A. Curtis from Cuba, with the other two species from Belize and Jamaica. Such a 50 ... Petersen & Hughes diagnosis seems to fit M. crinis-equi of Marasmius subsect. Sicciformes, which is known from the subtropics of the New World. SPECIMENS CONSULTED: MEXICO. VERACRUZ, Municipality of Xalapa, Santuario del Bosque de Niebla, Instituto de Ecologia A.C., 1343 m a.s.l., 18 May 2006, Bandala 4052; 7 July 2016, César 41; 10 Aug 2016, Ramos 682 (all at XAL). SPECIMENS EXAMINED: UNITED STATES. Atasama, Mobile Co., Univ. South Alabama, main campus, VII.2014, coll. unknown, det. J.L. Mata, TFB 14607 (TENN-F-69310). FLoripa, Alachua Co., Gainesville, Possum Creek Park, 20.IV.2016, coll. M.E. Smith [“On suppressed oak understory, fruiting from elevated branches.” ] MES1477 (FLAS-F-62667: as Marasmius ‘crinis-equi”); Gainesville, Sweetwater Reserve, 28.VII.18, coll. M.E. Smith, MES3139 (FLAS-F-62668 as Marasmius ‘crinis- equi’); Gainesville, Rock Creek neighborhood, 1.V.2015, coll. M.E. Smith, MES 884 (FLAS-F-62666; as Marasmius “crinis-equi”); Putnam Co., Ordway-Swisher Biological Station, near boat ramp of Lake Suggs, 14.VI.2017, coll. R. Healy & al., FLAS-F-60891 as Marasmius cf. brevipes. LOUISIANA, East Feliciana Parish, St. Francisville, James John Audubon Historic Site, 30°47.84’N 91°18.43’W, 22.V.1997, coll. RHP, TFB 9087 (TENN-F-054912). Mississippi, Harrison Co., Red Creek Wildlife Management Area, 30°40'35”N 88°54’02”W, 11.VII.2014, coll. KWH, TFB 14498 (TENN-F-069189). TEXAS, Orange Co., Vidor, Virginia Lane, 30°09.24’N 94°00.66W 26.VI.1976, coll. D.P. Lewis, det. D.E. Desjardin (as M. brevipes), Lewis 276 (TENN-F-047666). Pseudomarasmius obscurus R.H. Petersen, sp. nov. Figs: 42=35 IF 555731 Differs from Gymnopus pyracanthoides by its 1) marasmioid basidiomata (stipe slender, black; pileus small, gray, dry); 2) habit on dead, sclerophyllous Quercus leaves; 3) copious and obvious resupinate rhizomorphs; and 4) lack of clamp connections. TYPE: Costa Rica, Prov. San José, Jardin de Dota, 3.5 km W of Interamerican Highway at El Empalme, 9°4’52”N 83°58’28”W, 15.V1.1995, coll. RHP, TFB 7812 (Holotype, TENN-F-053787). EryMo_ocy: Referring to the difficulty in observing microscopic structures. BASIDIOMATA (FIG. 324) diminutive, very slender. PILEus 25 mm broad, convex at all ages, becoming essentially applanate with downturned margin by maturity, more or less unicolorous when dry, matte; disc mouse gray; outward to margin “tilleul buff” (7B2), not at all striate, scalloped. LAMELLAE adnexed to free, thickish, <1 mm broad, seceding in drying to appear pseudocollariate when dried, total lamellae 23-28, through lamellae 13-14, off-white; lamellulae in one rank, often ridge-like. Strrpz 11-22 x 0.3-0.6 mm, insititious, terete when fresh becoming channeled on drying, appearing superficially glabrous- shining but under magnification (40x) minutely pruinose upward, apically dark brown, extending into pseudocollarium, very dark brown downward to black-brown lower portion. RHIZOMORPHS common, often resupinate on leaf surface and there anchored by lateral brownish ciliatiform mycelium, Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 91 Fic. 32. Pseudomarasmius obscurus (TENN-F-053787). A. Basidioma and rhizomorphs with inset showing lamellar attachment. B. Basidioles. C. Pleurocystidium and basidia; D. Basidiospores. E. Cheilocystidia. F. Caulocystidia. Scale bars: A = 5 mm; B,C,E,F = 20 um; D = 5 um. <25 mm long where erect, occasionally producing a stellate growth form, very slender (0.1-0.3 mm diam), glabrous-shining, black, curly, frequently branched (similar to those of G. pyracanthoides; see Petersen & Hughes 2016). DISTRIBUTION & PHENOLOGY: At present known from a single collection; Costa Rica. Fruiting on sclerophyllous Quercus litter with resupinate rhizomorphs; summer. 52 ... Petersen & Hughes Fic. 33. Pseudomarasmius obscurus (TENN-F-053787). Encrusted hyphae of pileipellis. Scale bar = 10 um. PILEIPELLIS composed of two elements: 1) a tangle of free-form, broadly diverticulate hyphae (Fie. 34), 3.5-8 um diam, thin-walled, variously lobate to subsetulose, often septate, without clamp connections; and 2) strongly encrusted hyphae (Fic. 33) 3-7 um diam, firm- to thick-walled (wall c. 0.7 um thick, hyaline); crust material in bands or rings with profile calluses c. 1 um Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 53 Fic. 34. Pseudomarasmius obscurus (TENN-F-037987). Swollen diverticulate hyphae of pileipellis. Scale bars: 10 um. thick. PLEUROCYSTIDIAL STRUCTURES (FIG. 32c) obscure, 20-28 x 6-8 um, cylindrical to fusiform, hyaline, thin-walled, without clamp connections. Basidioles (Fic. 32B) clavate to subampulliform; BAsIDIA (Fic. 32c) 20-30 x 6-8 um, clavate to subcapitulate, 4-sterigmate, without clamp connections. BASIDIOSPORES (FIG. 32D) 5.5-7 x 3-3.5 um (Q = 1.71-2.33; Q™ = 1.90; L™ = 6.25 um), ellipsoid to slightly reniform, variable between tapered proximally and not so, thin-walled, smooth, inamyloid. CHEILOCYSTIDIA (F1G.32E) very obscure, 16-25 x 3-8 um, cylindrical to clavate, often furcate or lobate, thin-walled, hyaline, without clamp connections. STIPE MEDULLARY HYPHAE 4-7 um diam, thick-walled (wall <1 um thick, hyaline to weakly 54 ... Petersen & Hughes Fic. 35. Pseudomarasmius obscurus (TENN-F-053787). Caulocystidia. Scale bars: 10 um. pigmented), strictly parallel, with occasional slender, thin-walled, strangulate- digitate to branched hyphae, without clamp connections. STIPE CORTICAL HYPHAE 3.5-7.5 um diam, thick-walled (wall often occluding cell lumen), heavily pigmented, distal on stipe producing densely scattered caulocystidia. CAULOCYSTIDIA (Fics 328, 35) limited to distal third of stipe, 22-41 x 8-11 um, variously shaped from pyramidal to furcate to subampulliform, thick-walled (wall <1.0 um, hyaline), usually with broader base than apex and usually with narrow hyphal origin. Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 9D COMMENTARY: Without DNA sequences, accurate placement of TFB 7812 is problematic, but several morphological characters would suggest Pseudomarasmius. Production of resupinate rhizomorphs is similar to G. pyracanthoides (Petersen & Hughes 2016), also from Costa Rica, also fruiting on Quercus leaves and also without clamp connections. The two differ in pileipellis structure (G. pyracanthoides pileipellis an intricate thatch of narrow setulose hyphae) and stipe vesture (G. pyracanthoides vesture over entire stipe and caulocystidia longer). Pseudomarasmius pallidocephalus (Gilliam) R.H. Petersen, comb. nov. FIGs 36-42 TF 555745 = Marasmius pallidocephalus Gilliam, 1975 Mycologia 67: 818. Type: United States, Michigan, Chippewa Co., Gilliam 1165 (MICH, see below). Diagnostic characters include: Fruiting on dead spruce and fir needles in North America; 2) basidiomata diminutive, exhibiting a white to off-white pileus and off-black, bristle- like stipe; 3) pileipellis with diverticulate hyphae, free-form hyphal segments and broom cell-like hyphal termini; 4) caulocystidia occasional, ampulliform. BASIDIOMATA (FIG. 36A, 37) diminutive, marasmioid; ratio of stipe length to pileus breadth high (6-8:1). PILEUs 5-24(-35) mm broad, pulvinate to convex at first, then plano-convex or broadly conic-convex, finally plane to shallowly concave and often subumbonate or umbilicate, dry or subviscid in wet weather, dull opaque, pliant or membranous, reviving, smooth, subtly sulcate-striate, subtuberculate, even or faintly rugulose-striate to disc, minutely velutinous or matted-fibrillose, dark brown in primordia; disc and inner limb brown (7D4) when young, fading to light brownish grey (6C3), soon light yellowish brown (7.3YR/7.0/2.8 Munsell), brown (7E4) to light brown (7D4) to light greyish brown (7D3) overall, by maturity light grayish yellowish brown, light pinkish yellowish brown, light yellowish pink 7A2 (“light pinkish cinnamon’), pale orange yellow, 6A2 (“pale pinkish buff”), light yellowish brown 6B5, 7,3YR/7,0/2.8 (Munsell) (“cinnamon”), dark brown (Maerz & Paul 16A12) moderate 4.7Y/5.2/4.1 (Munsell) yellowish brown, fading to greyish orange (6B2); drying about 5A5 (“ochraceous buff”); outer limb and margin yellowish, at first entire, soon eroded or crenate, white (Maerz & Paul 9B2), pale orange yellow 6A2 (“pale pinkish buff”), 4D2 pale yellowish white to off-white to 7B2 (“tilleul buff”), smooth, brownish grey when young, becoming minutely rugulose-striate, buff in age — overall pallid coloration in age. Pileus trama thin, yellowish white (5.5Y/9.3/1.8 Munsell) to light yellowish brown (7.3YR/7.0/2.8 Munsell). 56 ... Petersen & Hughes Fic. 36. Pseudomarasmius pallidocephalus (TENN-F-063098). A. Basidioma and rhizomorphs. B-D. Pileipellis elements. B. Encrusted hyphal segment and hypha with slime sheath. C. Diverticulate hyphae. D. Diverticulate hyphal termini. E. Basidiospores. Scale bars: A = 20 mm. B-D. = 10 um. E=5 um. LAMELLAE narrow (c. 1 mm broad), thin, subdistant, total lamellae = 32-37, through lamellae = 15-17, unequal, adnate at first, becoming adnexed or free to sinuate in age, or sometimes attached to a partial adnate collar, seceding upon drying, pliant, entire or minutely fimbriate, thickish, narrow, straight at first, broader near the stipe in age, not intervenose or obscurely so in age, Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 97 = : rs ; ' | os . eA Fic. 37. Pseudomarasmius pallidocephalus. Basidiomata. A. Photo from nature (TENN-F-066344). Courtesy Steve Trudell. B. Field-dried basidiomata (TENN-F-052401). Scale bars: 10 mm. not forked, off-white at first, yellowish white (5.5Y/9.3/1.8 Munsell), pale orange yellow 3A2 (“light buff”), or light yellowish brown (7.3YR/7.0/2.8 58 ... Petersen & Hughes Fic. 38. Pseudomarasmius pallidocephalus (TENN-F-020486). Pileipellis elements. A-D. Repent, diverticulate hyphae. E-H. Broom cell-like, diverticulate hyphal termini. Scale bars: 10 um. Munsell), soon buff to pale greyish (5A2), buff to pale orange grey (<5B2), soon buff to pale greyish (<5A2), 6A2 (“pale pinkish cinnamon’), ca. 5A4 (“light ochraceous buff”), in age mellowing to 9B2 (“vinaceous buff”), to 6A2 (“pale pinkish cinnamon”), when dried buff to pale orange grey (<5B2); lamellulae in 12 tiers. StrpE 1243 x 0.20.8 mm thick, insititious, central or somewhat eccentric, terete, equal or tapering downward, straight when moist, soon curling and twisting on drying, shining, opaque, hollow, bristle-like but not tough (thin, stiff and easily cut), equal or tapering slightly downward, glabrous; apical 12 mm sometimes whitish-pruinose, more or less concolorous with lamellae (“ochraceous buft”), downward light yellowish brown (7.3YR/7.0/2.8 Munsell) to moderate yellow brown (0.7Y/5.2/4.1 Munsell) or dark reddish brown (10.0R/1.5/1.5 Munsell) to dark brown on the upper half, downward black-brown, brownish grey Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 59 Fic. 39. Pseudomarasmius pallidocephalus (MICH 51323). Pileipellis elements. A,B. Repent diverticulate hyphae. C-H. Broom cell-like, diverticulate hyphal termini. Note frequency of di- or trichotomously branched diverticula. Scale bars: 10 um. (6C3) to greyish brown (7D3), soon dark brown (7F4-8), 5F8 (“bister”), 6F4 (“fuscous black”), 7F8 (“bone brown’), olive-brown [6E4 (“fuscous”), 2F3 60 ... Petersen & Hughes Fic. 40. Pseudomarasmius pallidocephalus (WTU-F-8911). Free-form pileipellis elements. A-C. Non-diverticulate individuals. D-I. Diverticulate individuals. Scale bars: 10 um. (“chaetura black”)], never black downward; when dried glabrous-shining, delicately ridged, compressed; basal mycelium absent; sterile stipes absent. RHIZOMORPHS (FIG. 36A) <17 x 0.1-0.5 mm, slender, hair-like, scarce to Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 61 Fic. 41. Pseudomarasmius pallidocephalus. Pleurocystidia (A-D. MICH 51323. E-H. TENN-F-026266). Note frequent distal, vague partition of contents. Scale bars: 10 um. abundant, arising at intervals along the substrate, much branched with spur-branches often long and flagelliform, twisted and curled, sometimes forming a loose tangle. Opor negligible; TASTE negligible. HABITAT & PHENOLOGY: Occasional to common in troops on needles and debris of conifers, chiefly Picea and Abies in spruce-fir forest, rarely on Thuja debris: highest elevation of southern Appalachian Mountains, north through 62 ... Petersen & Hughes New England into and across the continent to the Pacific Coast temperate rain forest and there accompanied by Pseudotsuga menziesii, Arbutus menziesii, Quercus garryana, Acer macrophyllum, and occasionally Thuja plicata, with understory of Alnus, Salix, Shepherdia; apparently a mid-summer fungus. PILEIPELLIS composed of the following elements embedded in a thin slime matrix: 1) repent hyphae 3-7.5 um diam, firm- to thick-walled (wall c. 0.5 um thick), hyaline individually, yellowish in mass, coarsely ornamented variously from stripes with plate-like profile calluses to flake-like with the flakes very slightly separated from the hyphal outer wall as though with a very thin slime layer between; 2) scattered free-form hyphal segments (Fic. 40) as in a “dryophila-structure, but not articulated into a cutis; 3) repent hyphae (Fics 36B-D, 38A-D, 39) 4-40 x 4-10 um, inflated somewhat, with diverticulate, papillate to digitate (often forked) processes, 2-6 x 0.7-3 um at base, not thick-walled, sub-refringent, sometimes in pairs or dichotomously branched (ie. “saddle-shaped”), distributed more or less randomly (not unilateral); and 4) broom cell-like termini (Fics 36c, D, 38E-H, 39C-H, 40D-1) usually arising from lightly encrusted hyphae, stalked (stalk 15-25 x 3.5-5.5 um), thin-walled, often branched dichotomously and then often divaricate, beset by numerous diverticula; diverticula lobate to conical and sometimes with a combination of forms; clamp connections absent. PILEUS TRAMA loosely interwoven; hyphae 4.5-13 um diam, firm-walled, without clamp connections, involved in minimal individual slime sheath but not coherent in tissues, occasionally coarsely ornamented with annular incrustation <2 um thick, often ornamented with vague, poorly defined stripes. LAMELLAR TRAMA loosely interwoven; hyphae 2.5-8(-13) um diam, firm- to thick-walled (wall c. 0.5 um thick, hyaline), involved in minimal individual slime sheath but not coherent in tissues, occasionally coarsely ornamented with annular incrustation <2 um thick, often ornamented with vague, poorly defined stripes, easily disarticulated; clamp connections absent. PLEUROCYSTIDIA (Fic. 41) 23-33 x 5-8 um, narrowly fusiform to fusiform with narrowing rounded apex, usually without content partition, without clamp connections; contents homogeneous but with vague central vacuolated area (PhC). Basidioles clavate-subcapitate, clampless; contents vaguely multigranular; BAsIDIA (FIG. 42A-D) (20-)25-34 x (4—)5-8(-10) um, clavate, (2)4-sterigmate, without clamp connections; contents multigranular. BASIDIOSPORES (FIG. 36E) (5—)5.5-7(-9.5) x 3-3.5(-6) um (Q = 1.30-2.33; Q”™ = 1.87; L™ = 6.7 um), ellipsoid to plump ellipsoid to elongate pip-shaped, flattened adaxially, not tapered proximally, thin-walled, smooth, inamyloid; Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 63 Fic. 42. Pseudomarasmius pallidocephalus. Hymenial structures. A-D. Basidia (MICH 51323). E-H. Cheilocystidia (TENN-F-026266). Scale bars: 10 um. contents homogeneous. CHEILOCYSTIDIA (FIG. 42E-H) scattered, similar to basidioles, 21-30 x 5-8.5 um, clavate at first, becoming ampulliform by maturity, without clamp connections. STIPE MEDULLARY HYPHAE (Fic. 43) 64 ... Petersen & Hughes Fic. 43. Pseudomarasmius pallidocephalus (TENN-F-056761). Stipe medullary hyphae. A. Outer hyphae, with lines marking hyphal crust depositions. Stipe surface upward in photo. B, C. Individual inner medullary hyphae. B. Notes septum without clamp connection. C. Note wall thickness. Scale bars: 10 um. (inner) (2—)3.5-8.5 um diam, strictly parallel, firm- to thick-walled (wall <1.2 um thick, hyaline), coherent in tissues (ie. minimal slime matrix), lacking clamp connections; outer medullary hyphae 1-3.5(-7.5) um, firm- to thick-walled (wall <1 um thick, hyaline, ornamented with lens-shaped encrustation often appearing pitted). STIPE CORTICAL HYPHAE moderately to strongly dextrinoid, 3-5.5 um diam, thick-walled (wall often occluding cell lumen), smooth or covered with heterogeneous slime with inclusions. Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 65 COMMENTARY: Pseudomarasmius pallidocephalus combines characters seen in other related complexes. Basidiome stature, size, rhizomorphs, and habitat all mimic (or are mimicked by) Gymnopus sect. Androsacei and/or G. sect. Perforantia. The minimal slime matrix of the pileipellis and stipe medullary hyphae resemble those of Gymnopus sect. Perforantia, while dextrinoid stipe cortical tissue is like that of some members of /marasmiellus (ss Wilson & Desjardin (2005). The diverticulate hyphae of pileipellis resemble those of Mycetinis (including the inflated cells typical of that genus), not those of Marasmius (siccus-type) and the free-form hyphal segments of pileipellis are similar to such structures in Gymnopus sect. Levipedes (“dryophilus- structure”). Lack of clamp connections, however, seems quite unusual in related complexes. Several taxa in Gymnopus sect. Androsacei have been described as clampless, mostly by Singer, and they generally have been found in New- and Old-World tropical forests and none with accompanying molecular phylogenies. Desjardin (1989) reported Ps. (as Marasmius) pallidocephalus as “one of the more commonly collected, early-fruiting litter-decomposing agarics in the spruce-fir zone of the southern Appalachians.” In these spruce-fir forests, it has been and is easily misidentified as G. (as Micromphale) perforans, which usually exhibits a pigmented pileus toward cinnamon or avellaneous and vestured stipe. Gymnopus androsaceus is encountered only occasionally. Pleurocystidia in P pallidocephalus are typical of such structures found widely through gymnopoid/marasmioid fungi. Such structures are fusiform in shape, with narrowly rounded apex, as opposed to clavate to subcapitulate shapes of basidioles. Apparently without taxonomic relevance, pleurocystidial shapes are sometimes accompanied by vaguely partitioned contents. In Ps. pallidocephalus, however, such structures are often more broadly rounded apically. Basidioles and basidia, conversely seem to be consistently clavate from an early stage, and with heterogeneous contents (Fic. 42a-p), unlike pleurocystidial structures which remain homogeneous in content. In numerous descriptions (including our publications up to a couple years ago) the structures termed here as pleurocystidia were included under the perceived variability of basidioles. Gilliam (1975) mentioned clamp connections only once, under pileus tramal hyphae. This observation was erroneous: clamp connections are consistently absent on all basidiomatal hyphae in Ps. pallidocephalus. Desjardin (1990) examined cultures of Ps. pallidocephalus and reported the absence of clamp connections. 66 ... Petersen & Hughes Past experience (Petersen 1978, Petersen & Hughes 2010) has produced some links between absence of clamp connections, 2-spored_ basidia, and oversize basidiospores indicative of haploid state, together with unexplained sexual compatibility systems. These links seem irrelevant in Ps. pallidocephalus, where basidiomata lacking clamp connections normally produce 4-spored basidia. The mating system of this species remains unknown. Gilliam (1975) seemed intent on separating Ps. (as Marasmius) pallidocephalus from M. androsaceus, the often-reported taxon which fruits on similar substrates in Europe and North America. Her concept of M. androsaceus was revealed in the concluding lines of her discussion, as fruiting on a “variety of habitats.” Not only is this observation understandable given the state of prior literature and the date of her study, but it may have influenced Desjardin’s (1989) treatment of M. androsaceus in his dissertation, which also drew no attention to clamp connections. Currently, phylogenies indicate that M. androsaceus may be split into at least three species: M. androsaceus fruiting on conifer debris, plus two which fruit on dead deciduous leaves (Petersen & Hughes, ined.). Other mimics fruit on pine needles [Gymnopus (Micromphale) pinophilus complex; Petersen & Hughes 2016a], cedar twigs and redwood litter [Gymnopus (Micromphale) sequoiae complex]. In addition to presence/absence of clamp connections, Ps. pallidocephalus lacks conspicuous cheilocystidia [only a few ampulliform shapes reminiscent of those of G. trabzonensis (Vizzini & al. 2015)] while G. androsaceus exhibits common cheilocystidial structures. Finally, Gilliam (1975) reported the lack of “hymenial cystidia” (inclusive of pleuro- and cheilocystidia) in M. pallidocephalus, perhaps because pleurocystidia are not conspicuous. Our experience with other taxa indicates that pleurocystidial structures are present. Rhizomorph presence and activity are arcane in Ps. pallidocephalus. Basidiomata are usually formed on dead needles from the previous 1-2 years, but the needle bed on which they are found is usually deeper, with more decayed needles (differing in color and substance) and these decayed needles are held together by tangles of slender, branched, black rhizomorphs. Rhizomorphs at the surface of the needle bed, and therefore accompanying basidiomata, are usually individual, extremely slender, short, and therefore easily overlooked. Examination of numerous marasmioid specimens from Europe has not revealed a single specimen of Ps. pallidocephalus. Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 67 SPECIMENS EXAMINED: CANADA, BRITISH COLUMBIA, Cariboo Regional District, Ft. St. James, 54°26’34”N 124°15’04”’W, 13.VII.1940, coll. T.T. McCabe, det. J.W. Groves (as M. androsaceus), TTM 253 (UC 1319403); Stikine Region, Liard River Hotsprings Prov. Park, 59°25’06”N 126°05’27’W, 18.VIII.2000, coll. R. Winder, O. Ceska, det. O. Ceska (as M. androsaceus) (UBC F21036); Metro Vancouver Regional District, Mt. Seymour Prov. Park, base of chair lift, 49°22’51”N 122°56’24’”W, 27.VIII.1973, coll. S.A. Redhead (as M. androsaceus) (UBC F8178); Capital Regional District,, Saanich Peninsula, Observatory Hill, 48°31’44”N 123°25’19”W, 17.X.2008, coll. O. Ceska (as M. androsaceus) (UBC F29843); Observatory Hill, 48°31’44’N 123°25'19”W, 17.X.2008, coll. O. Ceska (as M. androsaceus) (UBC F29843); Observatory Hill, 48°51.67’N 123°42.20’W, 28.11.2010, coll. & det. O. Ceska (as M. androsaceus), UBC-F24293; Fraser Valley Regional District, Manning Prov. Park, Rhododendron Flats, 49°13’N 121°04’W, 11.VI.2006, coll. Paul Kroeger (as M. androsaceus), PK 4072 (UBC F17578). UNITED STATES, ALAsKaA, Fairbanks, Fairbanks North Star, Ballane Lake Marge, 64°54.82’N 147°42.31’W, 15.VII.2006, coll. G.A. Laursen [as Marasmius androsaceus], GAL 19442a (WTU-F-31851). IDAHO, Bonner Co., Priest Lake, Luby Bay, 48°32’50”N 116°55’24”W, 25.1X.1992, coll. RHP (as M. androsaceus), TFB 5632 (TENN 52407); Shoshone Co., vic. Wallace, Coeur d’Alene Nat. Forest., 47°30’N 115°53’W, 24.1X.1992, coll. RHP ( as M. androsaceus), TFB 5610 (TENN 52401); Maing, Hancock Co., rte 182, vic. Mud Pond and Salmon Pond, 44°3758’N 68°05'14”W, 21.VII.1992, coll. S.A. Gordon, TFB 4987 (TENN 56761). MICHIGAN, Cheboygan Co., vic. Univ. Michigan Biological Station, Hermit Swamp, 45°33’31”N 84°40’43” W, 26. V1.1957, coll. R.L. Shaffer (as M. cfr. androsaceus), RLS 1291 (MICH 51323); Chippewa Co., Tahquamenon Falls State Park, Lower Falls, 46°36'16”"N 85°12’11”’W, 22.VII.1971, coll. & det. M. Gilliam (HOLOTYPE), MSG 1165 (MICH 11402, portion only); Marquette Co., Pine Plains, W of Michigamme State Forest, “Triple A” road, 46°31’14”N 88°03’14”W, 15.VII.1970, coll. J.-. Ammirati & S.J. Mazzer, det. M. Gilliam (as M. pallidocephalus), SJM 4479 (MICH 51322). MINNESOTA, Clearwater Co., Itasca State Park, 9.VIII.1968, coll. M.G. Weaver (as Marasmius sp.), det. M. Gilliam (as M. pallidocephalus), MGW 1621 (MICH 175913). NEw York, Franklin Co., Paul Smith’s, NEMF pre-foray trip no. 1, Hazel Pond Rd., 44°28.89'N 74°16.50’W, 11.VIII.2011, coll. A.S. Methven, TFB 13933 (TENN 65829); NorTH CAROLINA, Swain Co., vic. Bryson City, GSMNP, Spruce-Fir Nature Trail, 35°34'02”N 83°28'54”W, 28.VIII.2009, coll. RHP (as M. androsaceus), TFB 13664 (TENN 63098); 8.VI.1991, coll. S.A. Gordon, TFB 3623 (TENN 50742); 28. VIII.2009, coll. RHP, TFB 13664 (TENN 63098); vic. Bryson City, GSMNP, Clingman’s Dome, 35°33’40”N 83°29'45”W, 10.1X.1985, coll. D.E. Desjardin, DED 3412 (TENN 54660); 35°33’47’"'N 83°29'55”W, 27.1V.1954, coll. A.H. Smith & L.R. Hesler (TENN 021343). TENNESSEE, Blount Co., GSMNP, Appalachian Trail vic Clingman’ss Dome, near old Indian Gap road, 28.V.2011, coll. Steve Trudell, SAT-11-11-179-05 (TENN 66344); Sevier Co., GSMNP, Indian Gap, Appalachian Trail, 35°36’35”N 83°26'19”’W, 29.V.2004, coll. RHP (as M. androsaceus), TFB 11778 (TENN 59896); 36°36’35”N 83°26'19’W, 13.V1I.2004, coll. S.C. McCleneghan & E.B. Lickey, TFB 11970 (TENN 59975); Old Indian Gap Road, 28.VI.1986, coll. D.E. Desjardin, DED 3691 (TENN 54667); 19. VII.1988, coll. D.E. Desjardin, DED 4615 (TENN 54650); 11.V1.1986, coll. D.E. Desjardin, DED 3581 (TENN 54646); WASHINGTON, Jefferson Co., Olympic 68 ... Petersen & Hughes National Park, Enchanted Valley, 47.6675°N 123.3939°W, coll. J.W. Lennox (as M. androsaceus), JWL 511 (WTU-F-8911); Lewis Co., Skate Creek Rd., between Horse Creek and milepost 17, 2.X.1993, coll. RHP (as M. androsaceus), TFB 5698 (TENN 53475). Pseudomarasmius patagonianus R.H. Petersen, sp. nov. Fics 44-48 IF 555733 Differs from Pseudomarasmius nidus-avis by its 1) slender rhizomorphs that are present but not dominant; 2) basidiomata arising as branches of rhizomorphs or from rotting leaves; 3) pileipellis elements usually semi-gelatinized; and 4) distribution in southern South America (Chilé). Type: Chilé, Chiloé, Isla Grande de Chiloé, Ancud, 41°52’18”S 73°49’06”W, 9.V.1995, coll. RHP, TFB 7363 (Holotype, TENN-F-054432). EryMo_ocy: Referring to the Patagonian origin of the collections. BASIDIOMATA (Fic. 444) marasmielloid, short-stipitate, similar to those of Ps. nidus-avis. Piteus <11 mm broad, campanulate with downturned margin becoming more or less applanate, often abruptly umbilicate, subtly striate, somewhat pulvinate, matte, from uniformly 6C6 (“tawny”) to disc 6B5 (“cinnamon”) when young, paler in age; outward “pinkish buff” (6A3) to off-white. LAMELLAE adnate to shortly decurrent, distant, ranging from well-defined to reduced, usually as shallow (<1 mm broad) pleats, <20 total folds, 5-8 through folds, 5A2 (“pale cinnamon pink”) to off-white (now 4A2 “pale ochraceous buff”). Stripe 2-8 x 1.5-2.5 mm, terete, equal, straight to usually strongly curved, black, upward glabrous shining, downward minutely pruinose, non-insititious with minimal basal tuft. RHIZOMORPHS rare, slender, black, of unknown length. Opor and TasTE negligible. HABITAT & PHENOLOGY: A poorly understood taxon, erumpent on woody substrate, noted as “on hardwood boughs;” Chilean winter. PILEIPELLIS comprising the following: 1) at pileus margin, repent diverticulate hyphae 2.5-5.5 um diam, firm-walled, not gelatinized, hyaline; diverticula stout, <7 x 1-2.5 um at base, gnarled-pyramidal, often dichotomous; 2) at pileus margin, diverticulate hyphal termini (hardly broom cell-like) (Fic. 45), resembling repent diverticulate hyphae, perhaps sometimes erect; 3) over disc and limb, occasional pileal hairs <75 x 3-4.5 um, cylindrical, subtly capitulate, arising as an erect branch of the repent hypha; 4) unorganized hyphae 3-6.5 um diam, thin-walled, variously ornamented (Fic. 46) with crust material often coarsely in scabs <2 um thick (Fic. 468), often appearing annular (Fic. 464,c), easily disarticulated; 5) hyphae 2.5—4.5 um, not easily crushed, thick-walled (wall <0.7 um thick), Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 69 Fic. 44, Pseudomarasmius patagonianus (TENN-F-054432, holotype). A. Basidiomata (left) habit; (right) longitudinal section. B. Basidiospores. Scale bars: A = 5 mm; B = 5 um. TFB 7363 often with thin individual slime sheath with flake-like ornamentation (Fic. 46D). Diverticulate hyphae or hyphal termini not observed over disc or limb. Lamellar trama loosely interwoven; hyphae 2-5.5 um diam, firm- to thick-walled (wall<1 um thick), often gelatinizing or with individualthin slime sheath, without clamp connections. PLEUROCYSTIDIA (Fic. 47) uncommon or poorly defined, 22-25 x 5-6 um, fusiform, without clamp connections. Basip1A (Fic. 48) 28-37 x10-12 um, clavate, (2—)4-sterigmate, without clamp connections; contents heterogeneous when mature, emptying but not collapsing (“husking”) when effete. BAstprosporEs (F1G.44B) 9-9.5 x 4-5 um (Q = 1.60-2.25; E™ = 2.07), ellipsoid, thin-walled, not tapered proximally, inamyloid. CHEILOCysTIDIA absent or undifferentiated. STIPE MEDULLARY 70 ... Petersen & Hughes Fic. 45. Pseudomarasmius patagonianus (TENN-F-054432, holotype). Pileipellis diverticulate hyphal termini. Scale bars: 10 um. HYPHAE 2.55 um diam, thick-walled (wall <1 um thick), strictly parallel in slime matrix, hyaline, without clamp connections. STIPE CORTICAL HYPHAE 35 um diam, thick-walled, repent in thin slime matrix, roughened outward, without clamp connections, weakly dextrinoid (reddish tan Melzer’s reagent + BF; reddish brown Melzer’s reagent + PhC). CAuLocystTipia <65 x 3.55 um, cylindrical, firm-walled, gnarled and often with rudimentary lobes, hyaline singly, dull brown in mass, without clamp connections. COMMENTARY: Collection TFB7363 (TENN-F-054432) comprises several basidiomata on hardwood substrate. TENN-F-054424, collected in the same Paramycetinis & Pseudomarasmius gen. & spp. nov.... 71 Fic. 46. Pseudomarasmius patagonianus (TENN-F-054432, holotype). Pileipellis hyphae. A, B. Encrusted hyphae. A, C. Ornamentation in stripes. B. Ornamentation of scabs -2 um thick. D. Hypha with individual slime sheath and flake-like encrustation. Scale bars: 10 um. location at the same time, is a mixed collection composed of several mycenoid basidiomata and a single marasmielloid basidiome. The ITS-based sequence deposited in GenBank (KY352649; MF978330, rbp2) derived under this accession number must have come from the latter basidiome which is 72. ... Petersen & Hughes ( hag | Fic. 47. Pseudomarasmius patagonianus (TENN-F-054432, holotype). Pleurocystidia. Scale bars: 10 um. Fic. 48. Pseudomarasmius patagonianus (TENN-F-054432, holotype). Basidia. Scale bars: 10 um. Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 73 morphologically identical to basidiomata in TFB7363: the sequence places the collection in Pseudomarasmius and the single basidiome matches the generic diagnosis. The basidiome has been segregated within the specimen. As surveys of herbarium material and phylogenetic analysis of such collections are processed, there appears to be a convergent, relatively common basidiome architecture generally regarded as “marasmielloid,” in which basidiomata conform to the following: 1) pileus small, rarely exceeding 15 mm broad, with thin, tough consistency; 2) lamellae usually distant, often reduced and with no anastomoses; 3) stipe central, usually strongly curved, rarely exceeding 10 x 2 mm, black or nearly so, usually glabrous, 4) fruiting on woody substrate often in vertical position; and 5) rhizomorphs usually present and sometimes dominant, slender, black. This architecture might be epitomized by Gymnopus neobrevipes (= Marasmius brevipes), almost indistinguishable from Ps. nidus-avis and Ps. patagonianus but distinguishable by presence of clamp connections, dominant rhizomorphs, more temperate distribution and unique ITS sequence. ADDITIONAL SPECIMEN EXAMINED: CHILE, Curiof, Isla Grande de Chiloé, 20 km E Aucud, 41°53’10”S 73°38’52”W, 9.V1.1995, coll. RHP, TFB 7364 (TENN-F-054424). Pseudomarasmius quercophiloides R.H. Petersen, sp. nov. Figs 49-59 IF 555732 Differs from Gymnopus quercophilus by its 1) lack of clamp connections; 2) abundant rhizomorphs that are resupinate on sclerophyllous leaves; 3) sulcate-striate pileus with low umbo; and 4) distribution in interior or subtropical China. Type: China, Yunnan Prov., Anning Pref., grounds of Southwest Forestry University, 25°03’51"N 102°45'16”E, 10.VHI.1990, coll. RHP, Q. Wu, Li, TFB 3162 (Holotype, TENN-F-049177). EryMoLocy: Resemblance to Marasmius quercophilus Pouzar. BASIDIOMATA (Figs. 494, 50) diminutive. Prteus (Fic. 498) 25 mm broad, downturned, sulcate-striate with small umbo, matte, “vinaceous buff” (9B2). LAMELLAE (Fic. 498) not collariate, 19-26 total lamellae, 8-11 through lamellae, shallow (<1 mm broad), thick with rounded edge, not marginate, “tilleul buff” (7B2) to “pinkish buff” (6A3). StrPE 8-14 x 0.6-0.9 mm, terete, dull brown apically and there minutely decorated with hyaline caulocystidia, downward appearing glabrous-shining but microscopically pruinose, black, insititious. Rhizomorphs plentiful, resupinate on sclerophyllous hardwood leaves (Fic. 51), dark copper color with minute fringe of ochraceous rust color, rarely becoming aerial but then never pileate. 74 ... Petersen & Hughes Fic. 49. Pseudomarasmius quercophiloides (TENN-F-049179, holotype). A. Basidioma. B. Pileus, exterior + cross-section. C. Basidiospores. Scale bars: A, B= 5 mm; C= 5 um. DISTRIBUTION & PHENOLOGY: On at least Castanopsis leaves in southwestern, subtropical China; summer. PILEIPELLIS composed of three hyphal types with no evidence of gelatinization: 1) repent hyphae (Fic. 52a) 3-6.5 um diam, thin-walled, Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 75 bi ee Pe ak Fic. 50. Pseudomarasmius quercophiloides (TENN-F-049179, holotype). Basidiomata on leaf surface. Scale bar = 10 mm. 3148 Fic. 51. Pseudomarasmius quercophiloides (TENN-F-049179, holotype). Resupinate rhizomorphs on sclerophyllous leaves. Scale bar = 10 mm. encrusted in scabs not in annular or striped configuration; profile scabs <1 um thick; clamp connections not observed; 2) diverticulate hyphae (Fic. 52B,c) 3-6.5 um diam, thin-walled, beset with numerous diverticula; 76 ... Petersen & Hughes Fic. 52. Pseudomarasmius quercophiloides (TENN-F-049179, holotype). Pileipellis elements. A. Repent, encrusted hypha. B,C. Diverticulate hyphae. D,E. Broom cell-like hyphal termini. Scale bars: 10 um. diverticula digitate (not conical), 1-8 x 0.7-1.1 um, often slightly gnarled, dense (PhC); and 3) diverticulate broom cell-like hyphal termini (Fic. 52D,.), Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 77 Fic. 53. Pseudomarasmius quercophiloides (TENN-F-049179, holotype). Pleurocystidia. Scale bars: 10 um. Fic. 54. Pseudomarasmius quercophiloides (TENN-F-049179, holotype). A. Basidiole. B—D. Basidia. Scale bars: 10 um. 78 ... Petersen & Hughes Fic. 55. Pseudomarasmius quercophiloides (TENN-F-049179, holotype). Clusters of upper stipe caulocystidia. Scale bars: 10 um. similar to diverticulate hyphae, without clamp connections. No evidence of gelatinization observed in pileipellis. Pileus trama loosely interwoven; hyphae 2-5.5 um diam, thin-walled, hyaline, without clamp connections. Hymenium composed of three elements with no evidence of gelatinization: 1) PLEUROCYSTIDIA (Fic. 53) occasional (not abundant), 19-24 x 5-6 um, fusiform to narrow-fusiform, often with small, subglobose, apical Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 79 Fic. 56. Pseudomarasmius quercophiloides (TENN-F-049179, holotype). Individual upper stipe caulocystidia. Note caulocystidial origins in D. Scale bars: 10 um. protuberance (appearing mammilate), without clamp connections; 2) basidioles (Fic. 544) clavate to subcapitulate, without clamp connections; BASIDIA (FIG. 54B—D) (22—)27-30 x 6-7 um, 4-sterigmate (sterigmata slender, easily collapsed), without clamp connections; and 3) CHEILOCYSTIDIA not observed. BAsipIospoREs (FIG. 49c) 6-7 x 3-4(-5) um (Q = 1.40-1.86; 80 ... Petersen & Hughes A ma Fic. 57. Pseudomarasmius quercophiloides (TENN-F-049179, holotype). Lower stipe medullary hyphae. A. Stout, thick-walled hypha. B. Gelatinized or shredded stout hypha. C. Slender hypha. D. Serpentine slender hypha. Scale bars: 10 um. E™ = 1.53; L™ = 6.60 um), ellipsoid, not tapering proximally, thin-walled, hyaline, inamyloid. Upper stipe medullary hyphae <12 um diam, strictly parallel, free (no evidence of gelatinization), hyaline, with no clamp connections, thick-walled (wall <1.5 um thick). Upper stipe cortical hyphae 4-6 um diam, thick-walled (wall <1 um thick), strongly pigmented (olive brown, PhC), without clamp connections, decorated with numerous caulocystidia (Fic. 55); CAuLocysTip1A (Fic. 56) 3-30(-40) x 7-13 um, Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 81 Fic. 58. Pseudomarasmius quercophiloides (TENN-F-049179, holotype). Individual lower stipe caulocystidia. Scale bars: 10 um. lobate to thumb-shaped, usually gnarled or constricted, firm- to thick-walled (wall <1 um thick), weakly pigmented, arising as constricted side branches of surface cortical hyphae. Lower stipe medullary hyphae strictly parallel, of two types: 1) 5-8 um diam, thick-walled (wall <2 um thick), occasionally gelatinized (Fic. 574) or shredded (Fic. 578); and 2) slender, 2-2.5 um diam, thin-walled, straight (Fic. 57c) to serpentine (Fic. 57p), with dense contents (PhC). Lower stipe cortical hyphae 3.5-6 um diam, thick-walled (wall <2 um thick, often obscuring cell lumen), densely pigmented (dark olive brown, PhC), beset with crowded caulocystidia; CAULOCYSTIDIA (Fic. 58) 5-30 x 8-13 um, crowded, stout-vermiform, usually gnarled and/ or constricted, thin- to thick-walled (wall <1 um thick), hyaline to weakly pigmented proximally. Resupinate rhizomorph anatomy similar to that of lower stipe. Cortical hyphae with superficial film of gelatinous material (Fic 59a) acting as adhesive to the leaf surface. Rhizocystidia (Fic. 59B-E) widely scattered, similar to caulocystidia but thick-walled (wall <1.5 um thick) and strongly pigmented. 82 ... Petersen & Hughes Fic. 59. Pseudomarasmius quercophiloides (TENN-F-049179, holotype). Rhizomorph anatomy. A. Surface of cortex with suggestion of superficial gel. B-E. Rhizocystidia. Scale bars: 10 um. COMMENTARY: Similarities with G. quercophilus: 1) abundant rhizomorphs, not black (in G. quercophilus chestnut brown to red- brown), dominantly resupinate on sclerophyllous hardwood leaves; 2) pleurocystidial general shape and size; 3) pileus sulcate-striate with small umbo; and 4) basidiomata arising from leaf surface not from rhizomorphs. Dissimilarities to G. quercophilus include: 1) clamp connections lacking; 2) absence of cheilocystidia; 3) presence of pruinose caulocystidia. Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 83 The ITS sequence of Ps. quercophiloides is largely identical to GenBank sequence KF251072, deposited as Gymnopus glabrocystidiatus (not holotype) from South Korea (2 bp difference in 636bp). Geographical proximity and sequence homology suggest that these likely represent the same taxon. ADDITIONAL SPECIMEN EXAMINED: CHINA, YUNNAN PrRov., Jinhong Pref., vic. Menghai, Xishuangbanna Nature Reserve, 8.VIII.990, coll. RHP, Q Wu, Li, TFB 3148 (TENN-F-049179) Pseudomarasmius straminipes (Peck) R.H. Petersen, comb. nov. FiGs 60-64 IF 555734 = Marasmius straminipes Peck, Bull. Buffalo Soc. Nat. Sci. 1:59. = Chamaeceras straminipes (Peck) Kuntze, Revis. Gen. P. 3(2): 457. Ho.ortyPe (implicit, Peck): United States, New York, Albany Co, Center (= Kamer), 42°39'10”"N 73°45'52”W, Oct. 1872. C.H. Peck (NYS!). Diagnostic characters include: 1) stipe capillary, buff above, ochraceous downward, glabrous-shining; 2) rhizomorphs usually common, pallid; 3) fruiting on fallen conifer needles, usually spruce/fir; 6) distribution in eastern North America. BASIDIOMATA (Fic. 60A) diminutive, marcescent, reviving. PILEuS (1.5-) 3-7 mm broad, hemispherical or convex, expanding to plano-convex, occasionally with suggestion of low umbo, dull, opaque, glabrous or minutely suede-like, light brown (7D4-5) or pale greyish brown (7D3) overall when young, soon fading to pale brownish orange (7C3) or pale brownish grey (6B2, 6C3); disc smooth or weakly rugulose; margin rugulose-striate or rugulose but not striate, soon fading to greyish orange (6C2) or pale greyish orange (5B2), in age with a hint of grey and buff; context thin, buff, often fading to whitish, buff or pinkish buff overall. LAMELLAE adnate, subdistant, without anastomoses or interveining, narrow, whitish or buff when young, becoming greyish buff or pale brownish grey (6C3) in age; edges even or crystalline-fimbriate; lamellulae in 1-2 series. Stipe 10-35 x 0.2-0.5 mm, terete, equal, tough, rigid or wiry, glabrous-shining, sometimes tacky, hollow, instititious, brownish orange (5C4, especially downward) to yellow (4A5-6) or straw-colored overall when young or with a slightly paler apex, darkening overall with age to stramineous, becoming slightly more brownish at maturity, never dark brown or black. RH1IZoMoRPHs (FIG. 60A) extensive, with short resupinate lengths on needle surface, then loosely curly, <20 x 0.1-0.3 mm, repeatedly branched with branches often long and flagelliform, glabrous, dull yellow-brown in basal parts, champagne colored upward and finally off-white. Opor and TasTE negligible. 84 ... Petersen & Hughes Fic. 60. Pseudomarasmius straminipes (TENN-F-026266). A. Basidiomata and rhizomorphs. B. Basidiospores. Scale bars: A = 10 mm; B = 5 um. HABITAT & PHENOLOGY: Gregarious to scattered to dead conifer needles including Picea/Abies and Pinus (especially P strobus); eastern North America [reported by Desjardin & Petersen (1989a) from Alabama, New Jersey, New York, North Carolina, Ohio and Tennessee]; summer—autumn. Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 85 Fic. 61. Pseudomarasmius straminipes (TENN-F-047638). Pileipellis hairs. Note production of abortive side branch. Scale bars: 10 um. PILEIPELLIS without evidence of slime matrix, a well-developed Rameales-structure, of the following elements: 1) pileal hairs (Fic. 61) densely scattered, <80 x 4-6 um, firm-walled, cylindrical, without clamp connections, often with aborted side branch, hyaline, usually subcapitulate; 2) repent hyphae 4-8.5(-15) um diam, thin- to firm-walled, without clamp 86 ... Petersen & Hughes Fic. 62. Pseudomarasmius straminipes (TENN-F-047642). Pileipellis elements. A-C. Diverticulate hyphae. D-K. Broom cell-like hyphal termini. Scale bars: 10 um. connections, strongly to weakly encrusted (crust material in scabs with vaguely annular to spiral patterning but occasionally protruding and then resembling diverticula); 2) scattered repent diverticulate hyphae (Fic. 62A-c) irregular in outline, often lobed, 2.5-8(-12) um diam, hyaline to pale brownish, smooth or weakly encrusted, inamyloid; diverticula 1-8 x 1-3 Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 87 ~~ * Fic. 63. Pseudomarasmius straminipes (TENN-F-047638). Hymenial structures. A-D. Pleurocystidia. E. Basidiole. F-H. Basidia. Scale bars: 10 um. um, knob-like, often dichotomous and in fascicles, obtuse, thin-walled; and 3) broom cell-like hyphal termini (Fic. 62p-K) common, stalked (stalk 4-8 x 3.5-5.5 um, without clamp connections), usually branched, distally variously lobed to diverticulate; diverticula 1-5 x 1-1.5 um, nodulose, subrefringent, often appearing dichotomous. Pileus trama interwoven; hyphae 28-11) 88 ... Petersen & Hughes Fic. 64. Pseudomarasmius straminipes (TENN-F-047638). Cheilocystidia. Note suggestion of dichotomous setulae. Scale bars: um. um diam, inflated or not, hyaline, inamyloid, thin-walled, smooth or weakly incrusted; incrustations granular or often helical and yellowish to pale brownish. Lamellar tramal hyphae similar to pileus tramal hyphae, but hyaline and non-encrusted throughout, without clamp connections. PLEUROCYSTIDIA (FIG. 63A4-D) abundant, 21-26(-29) x 5-8 um, fusiform to fusiform-rostrate, without clamp connections; contents more or less homogeneous. Basidioles (Fic. 63E) clavate; BASIDIA (FIG. 63F-H) 19-28 x 6.4-10 um, clavate, (2—)4-sterigmate, without clamp connections; contents more or less homogeneous; effete basidia emptying but not collapsing (“husking”). BASIDIOSPORES (FIG. 60B) 6.5-9.5 x 3.2-4.5 um (Q = 1.70-2.40; E™ = 2.00; L™ = 8.00 um), ellipsoid, slightly tapered proximally, smooth, hyaline, inamyloid; spore deposit whitish. CHEILOCysTIDIA (Fic. 64) scattered to abundant, 13-—23(-33) x 10-15 um 4.5-8(-11) um, stalked (stalk <2-6 x 3.5-8 um, thin-walled, without clamp connections), unbranched Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 89 or once-branched (and then branches short, stout), often lobed, setulose, hyaline, firm-walled; setulae 1-4 x 0.5-2 um, sub-refringent, often saddle- shaped to dichotomous). STIPE MEDULLARY HYPHAE 3.5—7(-10) um diam, strictly parallel, firm- to thick-walled (wall <0.5 um thick, hyaline, Melzer’s reagent) without evidence of slime matrix (Melzer’s reagent, PhC), without clamp connections. inamyloid. STIPE CORTICAL HYPHAE 1.5-6.5 um diam, strictly parallel, thick-walled (walls <2.5 um thick, non-encrusted), smooth, yellowish or pale brownish orange, moderately dextrinoid (Melzer’s reagent + BF). RHIZOMORPH tissue similar to that of the stipe, with dextrinoid cortical hyphae. Clamp connections absent on all tissues. COMMENTARY: Protologue (Peck 1873 Bull. Buffalo Soc. Nat. Sci. 1(2): 59.): “Pileus membranaceous, hemispherical or convex, smooth, striate, whitish; lamellae distant, unequal, white; stem corneous, smooth, shining, filiform, inserted, pale straw colored. Plant 1”—2” [inches] high, pileus 1”—3” [lines] broad. Fallen leaves of pitch pine, Pinus rigida. Center [N.Y.] October.” After examination of the type specimen of M. straminipes, Gilliam (1976: 136) opined that: “The type specimen is closest to Marasmiellus, and reviewed some putative diagnostic characters. In the same publication, Gilliam (1976: 131) reported on another Peck taxon, M. filopes Peck, basidiomata of which are similar to those of M. straminipes, and stated: “There is too little left of the specimen to section,’ but concluded that “M. filopes is nearest Marasmiellus’ Transfers of neither species (M. straminipes, M. filopes) to Marasmiellus, however, were formally accomplished. Redhead (1980), in his treatment of Marasmiellus filopes, did not mention or compare M. straminipes (validly published by Peck 1873, repeated in 1874), but, like Gilliam (1976), considered M. filopes, M. thujinus Peck, and M. piceina Kauffman as taxonomic synonyms. In a later discussion of clampless Marasmius taxa, Redhead (1984) rejected Gilliam’s (1976) opinion of M. straminipes as Marasmiellus, retaining the species in Marasmius sect. Androsacei, but without mention of Marasmiellus filopes and its synonyms. Redhead (1984) mentioned the clampless state of M. straminipesby way ofcomparison with M. pallidocephalus. Desjardin (1989) examined Peck’s type specimens of M. straminipes and M. filopes. Still later, in a paper redescribing Marasmius straminipes (Desjardin & Petersen 1989b), M. straminipes was again reportedasclampless and Redhead’s (1984) retention of M. straminipes in Marasmius sect. Androsacei was accepted, but M. filopes was not mentioned. In addition to the characters dictating placement of M. straminipes in Marasmius for Redhead (1984), Desjardin & Petersen 90 ... Petersen & Hughes (1989b) added the dextrinoid reaction on stipe medullary hyphae in M. straminipes, theretofore unknown in Marasmiellus. Description of basidiomata exhibiting clamp connections and those lacking clamps as forms or varieties of the same species is commonly found in the literature. Often, the clampless state is found accompanied by 2-sterigmate basidia and somewhat larger basidiospores than expected (see such forms in Hymenopellis, Petersen & Hughes 2010; and in Mycena, Kithner 1938, Lamoure 1957a,b, 1959, 1960). Upon further study, such forms may be found to be technically anamorphic (asexual) structures reproducing without meiosis and existing in a haploid state. In clampless forms in Gymnopus subg. Gymnopus, however, basidia appear to be 4-sterigmate and basidiospore metrics conform to the norms expected. Causes for such correlation remain opaque. In background research resulting in this paper, Ps. efibulatus was first noted as a clampless form of an undescribed Marasmius species. Marasmius straminipes (clampless) was thought to have a variety with clamp connections (M. straminipes var. fibulatus Desjardin & R.H. Petersen 1989. Bull. Torrey Bot. Club 49: 184.) With the proposal of Pseudomarasmius, a different avenue is offered, with molecular analyses important. DNA sequences of the analogous forms might clarify taxonomy, but so far such sequences have not been produced. The proposed transfer of M. straminipes to Pseudomarasmius is based on morphological characters: unfortunately, sequences derived from M. straminipes basidiomata have consistently been “dirty,” with more than one simultaneous sequence overlapping one another. The close resemblance to Ps. glabrocystidiatus in form, ecology, and microstructure, however, makes our transfer relatively comfortable. SPECIMENS EXAMINED: UNITED STATES, NorTH CaRo.Lina, Macon Co., vic. Otto, Coweeta Hydrologic Laboratory, Ball Creek area, 35°02’58”N 83°26’20’W, 13.VIII.1987, coll. D.E. Desjardin, DED 4458 (TENN 47642); vic. Highlands, Blue Valley, ca 3 mi up Clear Creek Rd., 9.VII.1991, coll. D.E. Desjardin, DED 5149 (TENN-F-050018, SFSU); Swain Co., vic. Bryson City, GSMNP, Clingman’s Dome, 1.VIII.1989, coll. S.A. Gordon, TFB 2125 (TENN-F-049143). TENNESSEE, Sevier Co., GSMNP, Old Indian Gap Road off Clingman’s Dome Road, 11.VI.1986, coll. D.E. Desjardin, DED 3578 (TENN-F-047638); Indian Gap, 35°36’34”N,83°26'47” W, 12.VI.1952, coll. L.R. Hesler (as Marasmius sp.), det. D.E. Desjardin (TENN-F-026266). General discussion The clampless state has always raised questions about nuclear behavior in the basidial primordium and basidiospores (see Petersen 1995). Often, the clampless state is accompanied by 2-sterigmate basidia, with the ploidy of Paramycetinis & Pseudomarasmius gen. & spp. nov. ... 91 parent mycelium, dikaryotization, meiosis and subsequent mitosis unclear (Petersen 1978). In Pseudomarasmius, however, the clampless state seems coupled with 4-sterigmate basidia (rarely 2-sterigmate in Ps. pallidocephalus and Ps. nidus-avis). This allows some conjecture that parent mycelium was dikaryotic, with diploidization in the basidial primordium probable, although nuclear segregation in the basidium and nuclear number in the basidiospores remains unknown. Clarifying experiments in this situation cannot be assumed by culturing single basidiospores and pairing resultant cultures, for the nuclear condition of the “monokaryons” is unknown until observed Cytogenetic evidence must be gathered directly, usually with the aid of DAPI staining and phase contrast microscopy or some more cumbersome stains (Restivo & Petersen 1976, Hubbard & Petersen 1979). In an entirely different direction, if any doubt remains, morphological identification and especially generic (or infrageneric) placement has become almost impossible in large groups of marasmioid fungi. Gymnopus in the sense of Wilson & Desjardin (2005), especially, has been repeatedly shown to form a monophyletic clade but inclusive of disparate basidiomatal morphology, ranging from G. androsaceus (traditionally type of Marasmius sect. Androsacei) and G. perforans (traditionally type of Micromphale sect. Perforantia) rather typical of marasmioid stature, to G. fusipes (typus generis) forming much larger, coarser and fasciculate basidiomata, and Caripia which forms only a subclavarioid hymenophore. Typical Micromphale (M. venosum typus generis), with gelatinized tissue layer in the pileus trama, also is now found in the Gymnopus clade (see Fie. 1). In another different taxonomic direction, Mycetinis (and Paramycetinis, see above), Rhodocollybia, Lentinula, Connopus, and Pseudomarasmius (see above), all traditionally within larger genera, are now known to resolve as monophyletic clades (see Fie. 1). Acknowledgments We acknowledge NSF DEB-1354802 awarded to M.E. Smith and P.B. Matheny, through which sequence data was obtained for Ps. patagonianus. The authors recognize and appreciate several National Science grants (especially two PEET grants) which supported fieldwork and provided experience basic to this effort. Equally, the Hesler Endowment Fund of the University of Tennessee provided funds for travel. Thanks are extended to Dr. Scott Redhead and Dr. Else Vellinga for help with nomenclatural issues. Dr. Victor Bandala provided valuable information on Ps. nidus-avis. Jerry Cooper (New Zealand), Genevieve Gates (Tasmania, Australia), and Tom May (Melbourne, Australia) pre-reviewed materials on Paramycetinis 92 ... Petersen & Hughes for possible nomenclatural repetition. David Lewis contributed photos. Vladimir Antonin and Clark Ovrebo are thanked for providing presubmission reviews. The editors of Mycotaxon have been particularly helpful in seeing this paper through the press. Bibliography Aldrovandi MSP, Johnson JE, O’Meara BC, Petersen RH, Hughes KW. 2015. The Xeromphalina campanella/kauffmanii complex: species delineation and biogeographical patterns of speciation. Mycologia 107: 1270-1284. https://doi.org/10.3852/15-087 Antonin V, Halling RE, Noordeloos ME. 1997. 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Mycologia 97: 667-679. https://doi.org/10.1080/15572536.2006.11832797 MY COTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 January-March 2020—Volume 135, pp. 97-102 https://doi.org/10.5248/135.97 Caliciopsis sambaibae sp. nov. from the Brazilian Cerrado JosE Luiz BEZERRA*, MARUZANETE PEREIRA MELO’, JosE EVANDO AGUIAR BESERRA JR”, ELLIOT WATANABE KITAJIMA3, SAMARA RAQUEL SOUSA’, CRISTIANE DUARTE SANTOS? "Centro de Ciéncias Agrarias, Ambientais e Bioldgicas, Universidade Federal do Reconcavo da Bahia, Cruz das Almas BA, 44380-000, Brazil ? Departamento de Fitotecnia, Universidade Federal do Piaui, Teresina PI, 64049-550, Brazil ° Departamento de Fitopatologia, Universidade de Sado Paulo, Escola Superior de Agricultura Luiz de Queiroz/USP Piracicaba SP, 13418-900, Brazil “CORRESPONDENCE TO: evando@ufpi.edu.br ABSTRACT—A new species, Caliciopsis sambaibae, is described associated with Davilla elliptica plants. The collections were made in the Cerrado of Sete Cidades National Park, in the municipality of Piracuruca, Piaui State, Brazil. Key worps—Ascomycota, Coryneliaceae, Coryneliales, Eurotiomycetes, sambaiba Introduction The Cerrado, designated as a neotropical plant diversity hotspot (Simon & al. 2009), is the main vegetation type of five Brazilian states— Minas Gerais, Goias, Mato Grosso do Sul, Maranhdo, and Tocantins—and also occurs in six other states—Bahia, Mato Grosso, Piaui, Sao Paulo, Parana, and Rond6énia (Beuchle & al. 2015). This vegetation covers about 23% of Brazil and incorporates c. 12,000 species of angiosperms (Zappi & al. 2015). Davilla Vand., one of the most diverse genera within Dilleniaceae Salisb., comprises about 30 neotropical species of lianas and erect or climbing shrubs (Fraga & Stehmann 2010). Plants of this genus are locally known as “sambaiba’ and are commonly used in traditional medicine in anti-inflammatory treatments (Lima & al. 2014). 98 ... Bezerra & al. The survey of fungi associated with plants from the Cerrado has revealed many new fungi (Dornelo-Silva & Dianese 2004, Dornelo-Silva & al. 2007)— c. 100 new species and 20 new genera of fungi (mainly ascomycetes) have been described (Dianese 2000, Hernandez-Gutiérrez & Dianese 2008, Pereira- Carvalho & al. 2009, Armando &al. 2015, Firmino & al. 2016, Melo & al. 2017). Caliciopsis (Coryneliaceae), represented by c. 30 species and found in temperate and tropical regions of North and South America, Eurasia, and Australia (Crous & al. 2016, Pratibha & al. 2010, Fitzpatrick 1920), is characterized by long black perithecia lacking paraphyses, evanescent asci, and brown ascospores. Some species produce an asexual morph, which is rarely observed because the conidia are produced within stromatic pycnidia (Garrido-Benavent & Pérez-Ortega 2015). Some species are associated with tree cankers or leaf spots (Jordal & al. 2004, Pratibha & al. 2010); severe cankering is caused by the type species C. pinea Peck (on Pinus), C. calicioides (Fr.) Fitzp. (on Populus), and C. pleomorpha P.A. McGee & Pascoe (on Eucalyptus) (Benny & al. 1985, Fitzpatrick 1920, 1942; Munck & al. 2015, Pascoe & al. 2018). The symptoms on our collections of Davilla elliptica leaves began with yellow spots, which turned necrotic and brown. In the lesions, we noted numerous fungal structures similar to black spines corresponding to the rostrate perithecial necks characteristic of a Caliciopsis fungus. As the morphology and biometric data of our specimens do not correspond with any described Caliciopsis, we propose a new species, Caliciopsis sambaibae. Materials & methods During 2014-16, Davilla elliptica leaves bearing signs of fungal infection were collected in Sete Cidades National Park in the municipality of Piracuruca, Piaui State, Brazil. The leaves were examined under a stereoscopic microscope and fragments and hand sections of the ascomata were mounted between slide and coverslip using Amann’ lactophenol. Leaf samples were submitted to scanning electron microscopy (SEM) in the Electron Microscopy laboratory of the Escola Superior de Agricultura Luiz de Queiroz, Universidade de Sao Paulo, as follows: leaf fragments containing the fungal structures were placed on filter paper moistened in a Petri dish, along with a small plastic container containing ca. 1 mL of 2% OsO, in aqueous solution. The container was wrapped with Parafilm and foil and left overnight (Kitajima & Leite 1999). The plate was then opened, and the leaf fragment was removed, air dried, mounted in the scanning electron microscope stub port with carbon double-sided adhesive tape, and covered with a thin layer of gold on a Bal-tec SCD050 sputter coater. The sample was examined and digitally recorded with a LEO 435 VP scanning electron microscope at 20 kV and a 10-mm working distance. Caliciopsis sambaibae sp. nov. (Brazil) ... 99 Specimens were conserved in the herbarium, Universidade Federal de Vicosa, Minas Gerais, Brazil (VIC). Taxonomy PxaTE 1. Caliciopsis sambaibae (holotype, VIC 44133) on Davilla elliptica. A-C. leaf symptoms; D-F: perithecia on leaves (LM). Scale bars: D = 30 um, E, F = 100 um. Caliciopsis sambaibae J.L. Bezerra, M.P. Melo & Beserra, sp. nov. Figs 1, 2 MB 819221 Differs from other Caliciopsis spp. by its perithecia having scars on their middle third and a basal dilation where ascospores are stored, and by its dilleniaceous host. Type: Brazil, Piaui, Piracuruca 4°02’08”S 41°40’45’”W, on living leaves of Davilla elliptica A.St.-Hil. (Dilleniaceae), 20 May 2016, M.P. Melo (Holotype, VIC 44133). Erymo ocy: Referring to “sambaiba’, the common name of the host. Spots yellowish, becoming brown and necrotic with age. COLONIES amphigenous, rarely epiphyllous. Srromata irregular to circular in outline, superficial mycelium absent; internal mycelium intercellular; stromata black, formed on the leaf surface from which the perithecia and pycnidia are inserted. SEXUAL MORPH: PERITHECIA ellipsoid, with long necks, simple or forked, straight or curved, 550—730 x 30-60 um, with a swollen ascogenous region near the base; ring-like scars and apical filaments on the necks of mature perithecia; ASCI bitunicate with eight ascospores, formed in the ascogenous swellings, evanescent, 17-20 x 5—7 um (n = 20); ascospores 1-celled, smooth-walled, 100 ... Bezerra & al. PLATE 2. Caliciopsis sambaibae (holotype, VIC 44133) on Davilla elliptica. A, B. perithecia (SEM); C. asci and ascospores (LM). D, E. scars (red arrows) on face of perithecia (SEM); FE apical filaments (red arrow) on the neck of mature perithecium (SEM); G. neck of immature perithecium (SEM); H. pycnidia in the basal stroma (SEM); I. cross sections of pycnidia (SEM). Scale bars: A, B = 1 mm; C = 10 um; D, E = 200 um; F, G = 20 um; H = 200 um; I = 10 um. globose to lenticular, brown, 3-6 x 3—5 um. pycnidia cespitose, developed at the perithecial base, irregular, stromatic, 23-20 um diam.; CONIDIOGENOUS CELLS holoblastic, nearly inconspicuous; conrp1A 1-celled, smooth, hyaline, ellipsoid, 3-4 x 2 um. ADDITIONAL SPECIMEN EXAMINED: BRAZIL, Prauf, Piracuruca, on living leaves of Davilla elliptica, 11 October 2014, J.E.A. Beserra Jr. (VIC 44132). Discussion Caliciopsis sambaibae produces ellipsoid, spine-shaped perithecia with ascogenous swellings near the bases where the asci are formed. Sections in this region revealed asci in formation, and ascogenous swellings were not observed in immature perithecia. The apex of the perithecial ostiole is somewhat dilated, and hyphal filaments may assist in the transfer of ascospores to this region. Due to their evanescent nature, only a few intact asci could be observed. Possibly for this reason, asci have been observed only in C. indica J. Pratibha & Bhat, C. valentina Garrido-Ben. & Pérez-Ort., Caliciopsis sambaibae sp. nov. (Brazil) ... 101 C. calicioides, and C. beckhausii (Korb.) Garrido-Ben. & Pérez-Ort. (Garrido- Benavent & Pérez-Ortega 2015, Jordal & al. 2014, Pratibha & al. 2010). After disintegration of the asci, passive ascospore liberation and agglomeration occur at the extremities of the perithecial necks. The presence of pycnidia immersed in plant tissue separates C. arrhiza (Pat. & Gaillard) Bat. & J.L. Bezerra and C. confusa Bat. (Batista & Maia 1964, Batista 1956) from C. sambaibae. The presence of scars in the middle or near the apex of the ostiole, as well as bifurcated ostioles distinguish C. sambaibae from other Caliciopsis species. The pycnidia of C. sambaibae are generally produced in groups inserted in the basal stroma, a morphological feature shared with C. beckhausii. Although most Caliciopsis species are associated with tree cankers (Jordal & al. 2004, Benny & al. 1985, Fitzpatrick 1942), we did not observe cankers in Davilla elliptica plants. Caliciopsis indica is the only species associated with leaves (Pratibha & al. 2010). Acknowledgments The authors thank CAPES (Coordenacao de Aperfeigoamento de Pessoal de Nivel Superior) and CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnoldgico) for funding through grants to authors. They are also grateful to Roger Fagner Ribeiro Melo (Universidade Federal de Pernambuco, Brazil) and Jadson Diogo Pereira Bezerra (Universidade Federal de Goias, Brazil) for helpful comments and review. 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(Ascomycota: Coryneliales) causing a severe canker disease of Eucalyptus cladocalyx and other eucalypt species in Australia. Fungal Systematics and Evolution 2: 45-56. https://doi.org/10.3114/fuse.2018.02.04 Pereira-Carvalho RC, Sepulveda-Chavera G, Armando EAS, Inacio CA, Dianese JC. 2009. An overlooked source of fungal diversity: novel hyphomycete genera on trichomes of cerrado plants. Mycological Research 113: 261-274. https://doi.org/10.1016/j.mycres.2008.11.005 Pratibha J, Amandeep K, Shenoy BD, Bhat DJ. 2010. Caliciopsis indica sp. nov. from India. Mycosphere 1: 65-72. Simon MF, Grether R, Queiroz LP de, Skema C, Pennington RT, Hughes CE. 2009. Recent assembly of the Cerrado, a neotropical plant diversity hotspot, by in situ evolution of adaptations to fire. PNAS [Proceedings of the National Academy of Sciences of the United States of America] 106: 20359-20364. https://doi.org/10.1073/pnas.0903410106 Zappi DC, Filardi FLR, Leitman P, Souza VC, Walter BMT, Pirani JR, Morim MP & al. 2015. Growing knowledge: an overview of seed plant diversity in Brazil. Rodriguésia 66: 1085-1113. https://doi.org/10.1590/2175-7860201566411 MYCOTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 January-March 2020—Volume 135, pp. 103-117 https://doi.org/10.5248/135.103 Morphological and molecular identification of Phlebia wuliangshanensis sp. nov. in China Ruo-X1A HUANG?”, KAI-YUE Luo?, RuI-XIN MA3, CHANG-LIN ZHAO??? ' Key Laboratory of State Forestry Administration for Highly Efficient Utilization of Forestry Biomass Resources in Southwest China, ’ Key Laboratory for Forest Resources Conservation and Utilization in the Southwest Mountains of China, Ministry of Education,and * College of Biodiversity Conservation, '23Southwest Forestry University, Kunming 650224, PR. China " CORRESPONDENCE TO: fungichanglinz@163.com ABSTRACT —A new white-rot fungus, Phlebia wuliangshanensis, is proposed based on a combination of morphological features and molecular evidence. The species is characterized by an annual growth habit, resupinate basidiocarps with a smooth to tuberculate hymenial surface, a monomitic hyphal system with thin- to thick-walled generative hyphae bearing simple septa, presence of cystidia, and narrow ellipsoid to ellipsoid basidiospores (5-6 x 3-3.7 um). Our phylogenetic analyses of ITS and LSU nrRNA sequences performed with maximum likelihood, maximum parsimony, and Bayesian inference methods support P. wuliangshanensis within a phlebioid clade in Meruliaceae (Polyporales). ITS+nLSU sequence analyses of additional Phlebia taxa strongly support P. wuliangshanensis within a monophyletic lineage grouped with P. chrysocreas and P. uda. Key worps—Basidiomycota, Ceriporiopsis, taxonomy, wood-inhabiting fungi, Yunnan Province Introduction Phlebia Fr. (Meruliaceae, Polyporales) is typified by P. radiata Fr. (Fries 1821). Basidiocarps are resupinate or rarely pileate with a subceraceous to subgelatinous consistency when fresh and membranaceous to coriaceous when dry. The hymenophore may be smooth, tuberculate, phlebioid, 104 ... Huang & al. odontoid, merulioid, or poroid. Microscopic characters include a monomitic (rarely dimitic) hyphal structure with clamp connections or simple-septa, narrowly clavate basidia, and hyaline basidiospores that are thin-walled, smooth, allantoid to ellipsoid, acyanophilous, and negative in Melzer’s reagent (Bernicchia & Gorjon 2010). So far about 100 species have been accepted in the genus worldwide (Fries 1821; Ginns 1969; Nakasone & Burdsall 1984, 1995; Dhingra 1989; Nakasone 1997, 2002, 2003, 2009; Roberts 2000; Gilbertson & Hemmes 2004; Duhem & Michel 2007; Duhem 2009, 2013; Bernicchia & Gorjon 2010; Singh & al. 2010; Westphalen & al. 2018; Gorjon & Greslebin 2012; Kaur & al. 2017; Shen & al. 2018). Recent, molecular studies have elucidated the classification of Phlebia among corticioid homobasidiomycetes (Larsson & al. 2004; Larsson 2007; TomSsovsky & al. 2010; Binder & al. 2013, Justo & al. 2017; Shen & al. 2018). Larsson & al. (2004) showed that Phlebia clustered into a phlebioid clade and grouped with Ceriporia Donk and Gloeoporus Mont. Larsson (2007) emended part of Polyporales and demonstrated that Phlebia was polyphyletic and nested within Meruliaceae. A phylogenetic study of European taxa of Ceriporiopsis Domanski showed that Phlebia radiata and the generic type of Ceriporiopsis, C. gilvescens (Bres.) Domanski, grouped closely on the basis of combined nuclear ribosomal large subunit RNA (nLSU) and mitochondrial ribosomal small subunit rRNA (mtSSU) gene sequences (TomSovsky & al. 2010). Binder & al. (2013) multi-gene sequence analyses placed P. radiata within a phlebioid clade and apparently grouped with Ceriporiopsis and Climacodon P. Karst. Also using multi-gene datasets, Justo & al. (2017) revised the family-level classification of Polyporales, including eighteen families. They showed that P. radiata belonged to Meruliaceae and grouped with Aurantiporus Murrill and Ceriporiopsis gilvescens. Shen & al. (2018) described a new Phlebia species based on morphological characters and rDNA sequences. This species belonged within the phlebioid clade and was related to P. radiata. During our investigations of wood-inhabiting fungi in southern China, we found an additional taxon that could not be assigned to any described species. In examining the taxonomy and phylogeny of this new species, we employed a two-gene molecular phylogenetic approach using internal transcribed spacer (ITS) and long subunit (nLSU) plus an expanded sampling of Phlebia isolates. Materials & methods The specimens studied are deposited at the herbarium of Southwest Forestry University (SWFC). Macro-morphological descriptions are based on field notes. Colour terms follow Petersen (1996). Micro-morphological data were obtained TABLE 1. Species, specimens, and sequence data used in this study. New sequences in bold. SPECIES NAME Abortiporus biennis Antrodia albida A. heteromorpha Antrodiella americana A. semisupina Ceriporiopsis gilvescens Climacocystis borealis Coriolopsis caperata Dacryobolus karstenii Daedalea quercina Earliella scabrosa Fomitopsis pinicola E rosea Fragiliporia fragilis Ganoderma lingzhi Gelatoporia subvermispora Gloeoporus dichrous G. pannocinctus Grammothelopsis subtropica Heterobasidion annosum Hornodermoporus martius Hydnophlebia chrysorhiza Hypochnicium lyndoniae Junghuhnia nitida Obba rivulosa O. valdiviana Phanerochaete chrysosporium P. velutina Perenniporia medulla-panis Perenniporiella neofulva Phlebia acanthocystis P. acerina SAMPLE NO. TFRI 274 CBS 308.82 CBS 200.91 Gothenburg 3161 FCUG 960 BRNM 710166 KH 13318 LE(BIN)-0677 KHL 11162 HHB 8735 PR 1209 CCBAS 536 ATCC 76767 Dai 13080 Dai 13559 Dai 13561 Wu 1006-38 BRNU 592909 KHL 11173 BRNM 709972 Cui 9041 PFC 5252 MUCL 41677 FD-282 NL 041031 KHL 11903 KCTC 6892 FF 503 BKM-F-1767 HHB-15343 MUCL 49581 MUCL 45091 FP 150571 FD-301 HHB-11146 FP-135252 DR-60 Phlebia wuliangshanensis sp. nov. (China) ... 105 GENBANK ACCESSION NO. ITS EU232187 DQ491414 DQ491415 JN710509 EU232182 FJ496684 JQ031126 AB158316 EU118624 FJ403214 JN165009 FJ608588 DQ491410 KJ734260 KJ734261 KJ734262 JQ781858 FJ496694 EU118627 EU546099 JQ845094 KC492906 FJ411092 KP135338 JX124704 EU118638 FJ496693 HQ659235 HQ188436 KP135184 FJ411087 FJ411080 KY948767 KP135378 KP135372 KP135371 KP135375 LSU EU232277 AY515348 AY515350 JN710509 EU232266 FJ496720 JQ031126 AB158316 EU118624 FJ403214 JN164793 DQ491410 KJ734264 KJ734265 KJ734266 FJ496706 EU118627 FJ496708 JQ845099 KC492906 FJ393859 KP135216 JX124704 EU118638 FJ496710 HQ659235 GQ470643 FJ393875 FJ393852 KY948844 KP135378 KF691615 REFERENCES Unpublished Kim & al. 2007 Kim & al. 2007 Binder & al. 2013 Binder & al. 2005 Tomsovsky & al. 2010 Binder & al. 2013 Tomsovsky & al. 2010 Binder & al. 2005 Lindner & al. 2011 Binder & al. 2005 Unpublished Kim & al. 2007 Zhao & al. 2015a Zhao & al. 2015a Zhao & al. 2015a Zhao & al. 2013 Tomsovsky & al. 2010 Binder & al. 2005 Tomsovsky & al. 2010 Zhao & al. 2013 Binder & al. 2013 Robledo & al. 2009 Floudas & Hibbett 2015 Unpublished Binder & al. 2005 Miettinen & Rajchenberg 2012 Miettinen & Rajchenberg 2012 Wu & al. 2010 Floudas & Hibbett 2015 Robledo & al. 2009 Robledo & al. 2009 Justo & al. 2017 Justo & al. 2017 Floudas & Hibbett 2015 Floudas & Hibbett 2015 Floudas & Hibbett 2015 Table 1, continued on next page 106 ... Huang & al. Table 1, continued SPECIES NAME P. ailaoshanensis P aurea P. centrifuga P. chrysocreas P floridensis P. fuscoatra = Mycoacia fuscoatra P. hydnoidea P. lindtneri P livida P. ludoviciana P. nantahaliensis P. nothofagi = Mycoacia nothofagi P radiata P rufa P. setulosa P. subochracea P. subserialis P. uda P. wuliangshanensis SAMPLE NO. CLZhao 3996 CLZhao 4036 DLL 2011-100 FCUG 2767 NH-14434 RLG-5075 HHB-9239 L-15541 GB-1013 HHB-6333 HHB 3946 FP-102161 KUC 20121123-24 HHB-9905 HHB-6466 HHB-7175 FP-102562-T HHB-10782 HHB 15354T HHB 18642 FP-102173 KHL 13275 HHB-1993 GB-501 FCUG 2189 FD-427 HHB-2816 HHB-4273 HHB-6906 HHB- 12067 KHL 13750 UBCF 19726 AFTOL-ID 484 FD-85 FD-121 HHB-14924 HHB-6891 AH 31879 PH 11749 HHB 8715 FCUG 1434 FP-101544 FCUG 2452 USDA Kropp-1 CLZhao 3475 T CLZhao 3639 GENBANK ACCESSION NO. ITS MH784926 MH784927 KJ140614 HQ153409 AY463445 KY948759 KP135380 KP135381 KP135379 KP135358 KP135357 AY219367 KJ668482 KP135383 KP135385 KP135384 KP135386 KP135364 KP135367 KP135366 KP135364 JN649352 KY948778 KY948772 AF141624 KP135342 KY948777 KP135369 KP135368 KP135370 GU480000 HQ604797 AY854087 KP135377 KP135376 KP135374 KP135382 GQ259417 GU461312 KY948770 AF141631 KP135361 AF141614 AB084621 MK881787 MK881788 LSU MH784936 MH784937 HQ153409 AY586691 KY948918 KP135262 KP135263 AY586695 KJ668335 KP135264 KP135265 KP135363 JN649352 KY948853 KY948847 AF141624 KY948852 KP135266 GU480000 HQ604797 AF287885 KP135377 KX065989 KP135267 GQ259417 KY948846 AF141631 KP135232 MK881897 MK881898 REFERENCES Shen & al. 2018 Shen & al. 2018 Unpublished Binder & al. 2013 Larsson & al. 2004 Justo & al. 2017 Floudas & Hibbett 2015 Floudas & Hibbett 2015 Floudas & Hibbett 2015 Floudas & Hibbett 2015 Floudas & Hibbett 2015 Binder & al. 2005 Floudas & Hibbett 2015 Justo & al. 2017 Floudas & Hibbett 2015 Floudas & Hibbett 2015 Floudas & Hibbett 2015 Floudas & Hibbett 2015 Floudas & Hibbett 2015 Floudas & Hibbett 2015 Floudas & Hibbett 2015 Tomsovsky & al. 2010 Justo & al. 2017 Justo & al. 2017 Tomsovsky & al. 2010 Floudas & Hibbett 2015 Justo & al. 2017 Floudas & Hibbett 2015 Floudas & Hibbett 2015 Floudas & Hibbett 2015 Tomsovsky & al. 2010 Binder & al. 2013 Binder & al. 2005 Justo & al. 2017 Floudas & Hibbett 2015 Floudas & Hibbett 2015 Justo & al. 2017 Binder & al. 2005 Binder & al. 2005 Floudas & Hibbett 2015 Tomsovsky & al. 2010 Floudas & Hibbett 2015 Unpublished Suhara & al. 2002 Present study Present study CLZhao 3645 MK881789 MK881899 Present study Phlebia wuliangshanensis sp. nov. (China) ... 107 Table 1, concluded GENBANK ACCESSION NO. SPECIES NAME SAMPLE NO. Torr REFERENCES ITS LSU Piloporia sajanensis Mannine 2733a HQ659239 HQ659239 TomSovsky & al. 2010 Podoscypha venustula CBS 65684 JN649367 JN649367 Sjoekvist & al. 2012 Polyporus tuberaster CulTENN 10197 AF516596 AJ488116 Binder & al. 2013 Postia guttulata KHL 11739 EU11865 EU11865 Kim & al. 2007 Pouzaroporia subrufa BRNM 710164 FJ496661 FJ496723 TomSsovsky & al. 2010 Sebipora aquosa Miettinen 8680 HQ659240 HQ659240 Miettinen & Rajchenberg 2012 Skeletocutis amorpha Miettinen 11038 FN907913 FN907913 TomSovsky & al. 2010 S. jelicii H 6002113 FJ496690 FJ496727 Tomsovsky & al. 2010 S. portcrosensis LY 3493 FJ496689 FJ496689 TomSovsky & al. 2010 S. subsphaerospora Rivoire 1048 FJ496688 FJ496688 TomSsovsky & al. 2010 Steccherinum KHL 11905 EU118668 EU118668 TomSovsky & al. 2010 fimbriatum S. ochraceum KHL 11902 JQ031130 JQ031130 Tomsovsky & al. 2010 Stereum hirsutum NBRC 6520 AB733150 AB733325 TomSovsky & al. 2010 Truncospora ochroleuca MUCL 39726 FJ411098 FJ393865 Robledo & al. 2009 Tyromyces chioneus Cui 10225 KF698745 KF698756 Zhao & al. 2013 Xanthoporus syringae Gothenburg 1488 JN710607 JN710607 Tomsovsky & al. 2010 from the dried specimens and observed under light microscopy following Dai (2012). Abbreviations are: KOH = 5% potassium hydroxide, CB = Cotton Blue, CB- = acyanophilous, L = mean spore length (arithmetic average of all spores), W = mean spore width (arithmetic average of all spores), Q = variation in the L/W ratios among the specimens studied, n (a/b) = number of spores (a) measured from given number (b) of specimens. We extracted genomic DNA from dried specimens using HiPure Fungal DNA Mini Kit II (Magen Biotech Co.) according to the manufacturer's instructions with some modifications. A small piece (c. 30 mg) of dried fungal material was ground to powder with liquid nitrogen. The powder was transferred to a 1.5 ml centrifuge tube, suspended in 0.4 ml of lysis buffer, and incubated in a 65 °C water bath for 60 min, after which 0.4 ml phenol-chloroform (24:1) was added to each tube and the suspension was shaken vigorously. After centrifugation at 13,000 rpm for 5 min, 0.3 ml supernatant was transferred to a new tube and mixed with 0.45 ml binding buffer. The mixture was then transferred to an adsorbing column (AC) for centrifugation at 13,000 rpm for 0.5 min. Then, 0.5 ml inhibitor removal fluid was added in AC for centrifugation at 12,000 rpm for 0.5 min. After washing twice with 0.5 ml washing buffer, the AC was transferred to a clean centrifuge tube, and 100 ml elution buffer was added to the middle of adsorbed film to elute the genomic DNA. The ITS region was amplified with primer pairs ITS5 and ITS4 (White & al. 1990). Nuclear LSU region was amplified with primer pairs LROR and LR7 (Vilgalys 2018). The ITS was amplified by initial denaturation at 95 °C for 3 min, followed by 35 cycles at 94 °C for 40 s, 58 °C for 45 s, and 72 °C for 1 min, and a final extension of 72 °C for 10 min. The nLSU was amplified by initial denaturation at 94 °C for 1 min, followed by 35 cycles at 94 °C for 108 ... Huang & al. 30 s, 48 °C 1 min, and 72 °C for 1.5 min, and a final extension of 72 °C for 10 min. The PCR products were purified and directly sequenced at Kunming Tsingke Biological Technology Limited Company. All newly generated sequences were deposited in GenBank (TABLE 1). DNA sequences were edited using Sequencher 4.6. Sequences were aligned in MAFFT 7 (Katoh & Toh, 2008) using the “G-INS-I” strategy and manually adjusted in BioEdit (Hall 1999). The sequence alignment (ID 24249) was deposited in TreeBase. In Fic. 1, Heterobasidion annosum (Fr.) Bref. and Stereum hirsutum (Willd.) Pers. were used as outgroup to root the tree following Binder & al. (2013). In Fic. 2, Hydnophlebia chrysorhiza (Eaton) Parmasto and Phanerochaete velutina (DC.) P. Karst. were used as outgroup to root the tree following Floudas & Hibbett (2015). Phylogenetic analyses of the ITS+nLSU sequences were performed using maximum parsimony, maximum likelihood, and Bayesian inference methods. Maximum parsimony (MP) analyses followed Zhao & Wu (2017), and tree construction was performed in PAUP* version 4.0b10 (Swofford 2002). All characters were equally weighted and gaps were treated as missing data. Trees were inferred using the heuristic search option with TBR branch swapping and 1000 random sequence additions. Max- trees were set to 5000, branches of zero length were collapsed and all parsimonious trees were saved. Clade robustness was assessed using bootstrap (BP) analysis with 1,000 replicates (Felsenstein 1985). Tree statistics tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC), and homoplasy index (HI) were calculated for each Maximum Parsimonious Tree generated. Sequences were analyzed using Maximum Likelihood (ML) with RAxML-HPC2 through the Cipres Science Gateway (Miller & al. 2009). Branch support (BS) for ML analysis was determined by 1000 bootstrap replicates. MrModeltest 2.3 (Posada & Crandall 1998; Nylander 2004) was used to determine the best-fit evolution model for each data set for Bayesian inference (BI). Bayesian inference was calculated with MrBayes_3.1.2 using a general time reversible (GTR+G) model of DNA substitution and a gamma distribution rate variation across sites (Ronquist & Huelsenbeck 2003). Four Markov chains were run for 2 runs from random starting trees for 8 million generations (ITS+nLSU) in Fie. 1, for 5 million generations (ITS+LSU) in Fic. 2 and trees were sampled every 100 generations. The first 25% of the generations were discarded as burn-in. A majority rule consensus tree of all remaining trees was calculated. Branches that received bootstrap support for maximum likelihood (BS), maximum parsimony (BP) and Bayesian posterior probabilities (BPP) greater than or equal to 75 % (BP) and 0.95 (BPP) were considered significantly supported, respectively. Molecular phylogeny The first ITS+nLSU dataset (Fic. 1) included sequences from 57 fungal specimens representing 49 species of Polyporales plus the outgroup had an aligned length of 2104 characters, of which 1236 characters were constant, 248 variable and parsimony-uninformative, and 620 parsimony-informative. Phlebia wuliangshanensis sp. nov. (China) ... 109 ofulve Gecaoed ochroleuca MUCL 39726 Grammothelopsis subtropica Cui 9041 { sani ai i Wu 1006-38 core polyporiod ‘Hornodermoporus martius MUCL 41677 lad “Perenniporia Sella ATM MUCL 49581 clade Earliellascabrosa PR 1209 CECE caperata LE(BIN)-0677 orus tuberaster CulTENN 8976 _ Outgroup Fic. 1. Maximum Parsimony strict consensus tree illustrating the phylogeny of Phlebia wuliangshanensis and related species in Polyporales based on ITS+nLSU sequences. Branches are labeled with maximum likelihood bootstrap >70%, parsimony bootstrap proportions >50% and Bayesian posterior probabilities >0.95. Clade names follow Binder & al. (2013). Maximum parsimony analysis yielded 3 equally parsimonious trees (TL = 4783, CI = 0.307, HI = 0.693, RI = 0.526, RC = 0.162). Best model for this dataset estimated and applied in the Bayesian analysis: GTR+I+G, lset nst = 6, rates = invgamma; prset statefreqpr = dirichlet (1,1,1,1). Bayesian analysis and ML analysis produced a similar topology as MP analysis (average standard deviation of split frequencies = 0.003241 (BI). The tree clustered the 57 polypore species into seven major clades, placing our new species, Phlebia wuliangshanensis, in the phlebioid clade (Fie. 1). The second ITS+nLSU dataset (Fic. 2) comprising sequences from 51 fungal specimens representing 22 Phlebia species plus the outgroup had an aligned length of 2102 characters, of which 1588 characters were constant, 142 variable 110... Huang & al. 100/100/1.00 90/89/0.99 99/100/1.00 98/100/1.00. -/6510.98 81/100/1.00 84/78/0.99 -/65/0.99 Phlebiafloridensis HHB-6466 Phlebia floridensis HHB-7175 Phlebiafloridensis HHB-9905 Phlebiafloridensis FP-102562-T Phlebia acerina FD-301 Phlebia acerina HHB-11146 Phlebia acerina FP-135252 Phlebia acerina DR-60 Phlebiaradiata AFTOL 484 Phlebiaradiata F 19726 Phiebia radiata FD-85 Phlebia radiata FD-121 Phlebia rufa HHB-14924 Phlebia lindtneri GB-501 100/99/1.00 |Phlebia setulosa HHB-6891 -/79/- 91/90/1.00_| 'Phiebiasetulosa AH 31879 Phlebiasetulosa PH 11749 Phlebia centrifuga L-15541 -/65/0.99 99/100/1.00 | ' Phlebia centrifuga HHB-9239 Phlebia centrifuga GB-1013 93/90/1.00 Phlebiahydnoidea HHB-1993 Phlebianantahaliensis HHB-2816 Phlebia fuscoatra HHB-10782 g99/100/1.00 -PAlebia fuscoatra HHB-15354 Phlebia fuscoatra HHB-18642 Phlebia fuscoatra FP-102173 Phlebia nothofagi HHB-12067 Phlebianothofagi HHB-6906 Phlebia nothofagi HHB-4273 Phlebia aurea RLG-5075 94ni00.00| 'Phlebia aurea NH-14434 Phlebia aurea FCUG 2767 Phlebia aurea DLL2011-100 90/82/- 98/100/1.00 -/70/- -/69/- Phlebiasubserialis FCUG 1434 Phlebiauda FP-101544 Phlebiauda FCUG 2452 Phlebiauda USDA Kropp-1 Phlebia chrysocreas HHB-6333 'Phlebia chrysocreas HB-3946 100/100/1.00 -Phlebia chrysocreas FP-102161 Phlebia chrysocreas KUC 20121123-24 Phlebia wuliangshanensis CLZhao 3639 Phlebia wuliangshanensis CLZhao 3475 Phlebia wuliangshanensis CLZhao 3645 Phlebia ailaoshanensis CLZhao 4036 Phlebia ailaoshanensis CLZhao 3996 Phlebialudoviciana FD-427 Phlebia subochracea HHB-8715 Phlebia acanthocystis FP 150571 Hydnophlebia chrysorhiza FD-282 Phanerochaete velutina HHB-15343 100/100/1.00 100/100/1.00 -/6910.99 100/100/1.00 100/100/1.00 100/100/1.00 /100/1.00 99/100/1.00 85/81/1.00 Fic. 2. Maximum parsimony strict consensus tree illustrating the phylogeny of Phlebia wuliangshanensis and related species in Phlebia based on ITS+nLSU sequences. Branches are labeled with maximum likelihood bootstrap >70%, parsimony bootstrap proportions >50% and Bayesian posterior probabilities >0.95. and parsimony-uninformative, and 372 parsimony-informative. Maximum parsimony analysis yielded 10 equally parsimonious trees (TL = 1113, CI = 0.518, HI = 0.482, RI = 0.873, RC = 0.403). Best model for the ITS+nLSU dataset estimated and applied in the Bayesian analysis: GTR+I+G, lset nst = 6, Phlebia wuliangshanensis sp. nov. (China) ... 111 rates = invgamma; prset statefreqpr = dirichlet (1,1,1,1). Here also Bayesian analysis and ML analysis generated a similar topology as MP analysis (average standard deviation of split frequencies = 0.002221 (BI). The phylogeny (Fic. 2) inferred from the combined ITS+nLSU sequences from 49 Phlebia isolates grouped the new species with P chrysocreas (Berk. & M.A. Curtis) Burds. and P. uda (Fr.) Nakasone. Taxonomy Fic. 3. Phlebia wuliangshanensis (holotype, SWFC 003475). Scale bar = 1 cm. Phlebia wuliangshanensis C.L. Zhao, sp. nov. Figs 3, 4 MB 830801 Differs from Phlebia chrysocreas by its narrower ellipsoid basidiospores and from P. uda by its smooth to tuberculate hymenium and generative hyphae with simple septa. Type: China. Yunnan Province: Puer, Jingdong County, Wuliangshan National Nature Reserve, on angiosperm trunk, 2 October 2017, CLZhao 3475 (Holotype, SWFC 003475; GenBank MK881787, MK881897). Erymo.ocy: ‘The specific epithet wuliangshanensis (Lat.) refers to the locality (Wuliangshan) of the type specimen. BASIDIOMATA annual, resupinate, easily separable from the substratum, ceraceous to gelatinous, without odor or taste when fresh, becoming membranaceous upon drying, <12 cm long, 200-700 um thick. Hymenial 112... Huang & al. gts ee = es <> A B 20,4 Ot Ok o) 0 £03 : +l y tN C : es, | : i | is Oper i aaa Paley) (| py Sor se \/ vu pro VP >Q } i, \2\ i? .. “aff a, | Losey] fa fe (SS, J) ge is i % jp ? es Ret) | bes Ip S p ! tS Fic. 4. Microscopic structures of Phlebia wuliangshanensis (drawn from the holotype, SWFC 003475). A. Basidiospores; B. Basidia and basidioles; C. Cystidia; D. Basidiocarp section; E. Hymenium section; F Hyphae from subiculum. Scale bars: A = 5 um; B-F = 10 um. Phlebia wuliangshanensis sp. nov. (China) ... 113 surface smooth to tuberculate, white to cream to pale brown when fresh, cream to pale brown upon drying. Sterile margin distinct, white. HyYPHAL STRUCTURE monomitic; generative hyphae with simple septa, negative in Melzer’s reagent, CB-; tissues unchanged in KOH. SUBICULUM generative hyphae hyaline, thin- to thick-walled, branched, 3-5.5 um in diam. HyYMENIUM cystidia of two kinds: 1) hyaline, cylindrical, numerous, thick- walled, strongly encrusted, 20-55 x 6-10.5 um; 2) hyaline, lanceolate, few, thin-walled, 12-18 x 3-4.5 um; basidia barrel-shaped, with four sterigmata and a simple basal septum, 10-16 x 4-5 um; basidioles dominant, in shape similar to basidia, but slightly smaller. BASIDIOSPORES narrowly ellipsoid to ellipsoid, more or less curved, hyaline, thin-walled, smooth, negative in Melzer’s reagent, CB-, 5-6(-6.5) x 3-3.7 um, L = 5.42 um, W = 3.32 um, Q = 1.64-1.73 (n = 180/3). TYPE OF ROT: white. ADDITIONAL SPECIMENS EXAMINED: CHINA. YUNNAN PROVINCE. Puer: Jingdong County, Wuliangshan National Nature Reserve, on angiosperm trunk, 2 October 2017, CLZhao 3639 (SWFC 003639; GenBank MK881788, MK881898), CLZhao 3645 (SWFC 003645; GenBank MK881789, MK881899). Discussion We describe a new species, Phlebia wuliangshanensis, based on phylogenetic analyses and morphological characters. Previously, seven clades were found in Polyporales: /antrodia, /core polyporoid, /fragiliporia, /gelatoporia, /phlebioid, /residual polyporoid, and /tyromyces (Binder & al. 2013, Zhao & al. 2015). Our combined ITS+nLSU sequence analysis (Fic. 1) strongly supports Phlebia wuliangshanensis (100% BS, 100% BP, 1.00 BPP) within the phlebioid clade and related to the type species, P. radiata. Phlebia wuliangshanensis is closely related to P. chrysocreas and P. uda in the ITS+nLSU phylogenetic tree (Fic. 2). Morphologically, P chrysocreas differs from P. wuliangshanensis by having ochraceous-buff to yellow ochre color hymenophore and narrowly ovoid basidiospores (4-6 x 2-2.5 um, Lombard & al. 1975), while P uda presents an odontoid hymenophore, generative hyphae bearing clamp connections, and smaller basidiospores (5-5.5 x 2-2.5 um, Bernicchia & Gorjon 2010). Phlebia wuliangshanensis morphologically resembles other Phlebia species: P. bispora (Stalpers) Nakasone, P capitata Bernicchia & Gorjon, P. coccineofulva Schwein., P. femsjoeensis (Litsch. & S. Lundell) J. Erikss. 114... Huang & al. & Hjortstam, P. livida (Pers.) Bres., P. nothofagi (G. Cunn.) Nakasone, P. radiata, P. rufa (Pers.) M.P. Christ., P segregata (Bourdot & Galzin) Parmasto, and P. subserialis (Bourdot & Galzin) Donk. These species differ from P. wuliangshanensis as follows: P. bispora by a hydnoid hymenophore, dimitic hyphal system, and smaller basidiospores (4—5 x 2.5-3 um, Nakasone 2002); P. capitata by an odontoid hymenophore, generative hyphae with clamps, and the presence of capitate cystidia (Bernicchia & Gorjén 2010); P. coccineofulva by a granular hymenophore with vivid yellow radiating margin and metuloid cystidia (Schweinitz 1832); P femsjoeensis by orange to violaceous basidiocarps (Eriksson & al. 1981); P livida by its reddish hymenophore and a monomitic hyphal system with generative hyphae with clamps (Bernicchia & Gorjén 2010); P nothofagi by effused basidiocarps with a hydnoid hymenophore, a monomitic hyphal system with generative hyphae with clamps, and narrower basidiospores (4—6 x 2-3 um, Nakasone 1997); P. rufa by its effused basidiocarps with the reticulate or subporoid hymenophore (Christiansen 1960); P segregata by its smooth hymenophore and cylindrical basidiospores (6-7 x 2-2.5 um, Parmasto 1967); and P. subserialis by a white hymenium, clamped generative hyphae, and allantoid basidiospores (6-7 x 1.5-2 um, Bernicchia & Gorjon 2010). Although wood-rotting fungi are an extensively studied group in Basidiomycota (Gilbertson & Ryvarden 1987, Nufiez & Ryvarden 2001, Bernicchia & Gorjoén 2010, Dai 2012, Ryvarden & Melo 2014, Dai & al. 2015), Chinese wood-rotting fungi diversity is still not well known, especially in the subtropics and tropics. The new species Phlebia wuliangshanensis is from the Chinese subtropics, where many new taxa in the Polyporales have been described (Cui & al. 2007, 2009, 2010, 2011; Cui & Dai 2008; Du & Cui 2009; Li & Cui 2010; He & Li 2011; Jia & Cui 2011; Yu & al. 2013; Yang & He 2014; Chen & al. 2015; Zhao & Wu 2017; Zhao & Ma 2019). We anticipate that more new polypore taxa will be found in China after further investigations and molecular analyses. Acknowledgments Special thanks are due to Jason Karakehian (Harvard University, USA) and Dr. Mei-Ling Han (Langfang Normal University, P.R. China) who reviewed the manuscript. 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Mycotaxon 134: 125-137. https://doi.org/10.5248/134.125 MY COTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 January-March 2020—Volume 135, pp. 119-130 https://doi.org/10.5248/135.119 Pseudocercospora seropedicensis & P. solani-cernui spp. nov. on Solanum from Brazil KERLY M. ANDRADE, PETER S. MEDEIROS, JESSICA REMBINSKI, JUCIMAR M. OLIvErRA, Cartos A. INACIO Plant Pathology Sector, Dept? de Entomologia e Fitopatologia, Inst. Ciéncias Biologicas e da Saude, Universidade Federal Rural do Rio de Janeiro, BR 465 - Km 7 - Campus, Rural Zone, 23851-970, Seropédica, Rio de Janeiro, Brazil * CORRESPONDENCE TO: inacio@ufrrj.br ABSTRACT— Using stereo- and light microscopy, two leaf-spotting cercosporoid fungi, collected from areas of the Brazilian Atlantic Forest in the states of Minas Gerais and Rio de Janeiro, were shown to represent two new species (Pseudocercospora seropedicensis on Solanum asperum and P. solani-cernui on S. cernuum), which are herein described and illustrated. Key worps—cercosporoid fungi, hyphomycetes, morphology, mycodiversity, Solanaceae Introduction Cercosporoid fungi, which are found worldwide, are especially abundant and diverse in tropical and subtropical areas of Africa, Asia, Australia, and Central and South America (Beilharz 2002; Braun & Freire 2003, 2004, 2006; Castaneda & Braun 1989; Hernandez-Gutiérrez & Dianese 2008, 2009; Inacio & al. 1996; Silva & al. 2016). These fungi have been studied by several authors in Brazil (Batista & al., cf. Silva & Minter 1995; Braun & Freire 2004; Chupp 1954; Crous & Braun 2003; Crous & Wingfield 1997; Crous & al. 1999; Fernandes & al. 2013; Hernandez-Gutiérrez & Dianese 2008, 2009; Hernandez-Gutiérrez & al. 2014, 2015; Inacio & Dianese 1998, 1999, 2006; Inacio & al. 1996; Pereira & Barreto 2006; Silva & al. 2016; Viégas 1945). They have been reported as plant pathogens on several hosts including different economically important crops, 120 ... Andrade & al. and some cercosporoid fungi are also used for biological control of weeds (Farr & Rossman 2019, Groenewald & al. 2013, Hanada & Gasparotto 2002, Mendes & Urben 2019, Mendes & al. 1998, Pereira & Barreto 2006, Pons 1987, Spegazzini 1910). Pseudocercospora Speg. (introduced by Spegazzini 1910 for the type species P. vitis (Lév.) Speg.) contains endophytic, pathogenic, or saprotrophic species, some of which are used for weed control (Breeyen & al. 2006). The genus now comprises synnematal fungi previously assigned to Phaeoisariopsis Ferraris (Ferraris 1909) and fungi with slightly pigmented conidiogenous structures and with paracercospora-like loci, although most Pseudocercospora species are characterized by unthickened, non-pigmented conidiogenous loci and conidial hila (Braun 1995, Deighton 1976). Sexual morphs are unknown for most species, but when present they are mycosphaerella-like, previously allocated in Mycosphaerella, now a heterotypic synonym of Ramularia Unger (Braun & al. 2013, Crous & al. 2013). Solanaceae comprises about 3000 species in 150 genera, distributed primarily in tropical and subtropical South America. In Brazil there are 350 species in 32 genera (Souza & Lorenzi 2008). Solanum, the most abundant genus, encompasses about 1500 species and occurs in tropical and subtropical areas worldwide (Sarmento-Silva & al. 2003). Several economically important species also belong in Solanaceae: pepper (Capsicum), potato (Solanum tuberosum), tomato (S. lycopersicum), and tobacco (Nicotiana tabacum) (Martins & Costa 1999). Cercosporoid fungi occur on Solanum species (Silva & al. 2016), and recently Braun (2017) published an identification key for Pseudocercospora species on Solanum hosts with descriptions and illustrations for some species. Here we describe and illustrate two new Pseudocercospora species found on leaves of Solanum asperum and S. cernuum. Materials & methods Plant material showing necrotic leaf spots was collected during July 2013-October 2017 in the States of Rio de Janeiro (UFRRJ campus in Paracambi Natural Park and the National Forest in Seropédica) and Minas Gerais (town of Liberdade). Fresh and dried symptomatic leaves were examined with a dissecting microscope to select fungal structures for mounting on slides with Cotton Blue / lactoglycerol or phloxine / KOH glycerol. Material was also hand-sectioned and mounted on slides. Tissues were microscopically observed and photographed using an Olympus BX41 light microscope coupled to a Canon Power Shot ELPH 100 HS camera. Structures were measured using a calibrated ocular micrometer. The exsiccates were registered and deposited in the Plant Pathological Herbarium “Verlande Duarte Silveira’ at the Rural University of Rio de Janeiro (UFRJ). TABLE 1. Selected Pseudocercospora species on Solanum SPECIES P. atromarginalis Pseudocercospora seropedicensis & P. solani-cernui spp. nov. (Brazil) ... 121 Hosts Solanum spp.; S. americanum, S. lycopersicum DISTRIBUTION Cuba, Taiwan, Korea, USA REFERENCES Atkinson (1892), Castafieda & Braun (1989), Choi et al. (2015), Deighton (1976), Guo & Hsieh (1995) P. marcelliana S. nudum, Spain, Venezuela Chupp (1954), S. torvum var. Crous & Braun (2003), hartwegianum, Braun (2017) S. micranthum P. seropedicensis S. asperum Brazil This work P. solani-cernui S. cernuum Brazil This work P. solani- S. pseudocapsicum Brazil Silva et al. (2016) pseudocapsicicola P. solani-torvicula S. torvum Taiwan Guo & Hsieh (1995) P. trichophila Solanum spp. Asia, Central and Stevens (1917), var. trichophila South America, Chupp (1954), North America, Braun (2017) Oceania P. trichophila S. hirtum Venezuela Braun & Urtiaga (2012) var. punctata P. venezuelae Solanum sp., Brazil, Venezuela, Chupp (1954), S. argenteum, South America Crous & Braun (2003), S. “aculeatum’, Braun & Freire (2003; 2006), S. aethiopicum Braun (2017) [= S. gilo] Taxonomy Pseudocercospora seropedicensis Andrade, Medeiros & Inacio, sp. nov. PL. i MB 819383 Differs from Pseudocercospora solani-asperi |= P. trichophila var. trichophila] by its much longer conidia and conidiophores and its larger, amphigenous. stromata. Type: Brazil, Rio de Janeiro, Seropédica, road to cattle farm, near Instituto de Florestas, 22°45'31”S 43°41'57”W, on leaves of Solanum asperum Rich. (Solanaceae), 07 May 2016, C. A. Inacio 122, (Holotype, UFRJ 12.390). ErymMoLocy: honoring the municipality, Seropédica, where the species was first discovered. LESIONS <50 mm diam., epiphyllous, sparse, sometimes gregarious, confluent, circular to irregular, reddish brown to light brown in the center, later becoming necrotic, grayish, with dark brown border. CoLoNiIEs amphigenous, mainly hypophyllous, caespitose, sparse. SUPERFICIAL MYCELIUM colorless to pale brown, septate, branched, amphigenous, mostly hypophyllous, sometimes emerging through the stomata, bearing conidiophores; hyphae 2-4(-7) um 122 ... Andrade & al. diam.; CONIDIOPHORES 5-42(-45) x 3-4 um, 1-septate, sometimes curved, slightly geniculate due to sympodial proliferation, not branched, borne terminally or (usually) laterally on superficial hyphae, olive to olive brown, rounded at apex. INTERNAL MYCELIUM colorless to olivaceous brown or pale brown, septate, branched, penetrating deep into the mesophyll and spreading through leaf tissue; hyphae 2-5(-6) um diam. STROMATA 25-80(-85) x 20-140(-160) um, brown, amphigenous, subepidermal, erumpent, textura angularis; cells 4-7 um wide, giving rise to a fascicle of conidiophores; CONIDIOPHORES 20-70 x 1-4 um, 1—-5-septate, numerous, macronematous, mononematous, olive to olive brownish, smooth, densely caespitose, arising from stromata, straight or slightly sinuous. CONIDIOGENOUS CELLS integrated, polyblastic, usually monoblastic and terminal, sympodial, geniculate, with inconspicuous conidiogenous loci. Conrp1A solitary, 20-145(-226) x 2-6 um, 1-16-septate, straight, sometimes sinuous to curved, subcylindrical to obclavate, smooth, hyaline, subhyaline to olivaceous brown, attenuated at apex, obconically truncate at base, hila neither thickened not darkened, 1-2 um wide. COMMENTS—Several Pseudocercospora species on solanaceous hosts are morphologically close to P. seropedicensis. Pseudocercospora atromarginalis (G.F. Atk.) Deighton (Deighton 1976), P marcelliana (Chupp) U. Braun & Crous (Crous & Braun 2003; Braun 2017), P. solani-torvicola Goh & W.H. Hsieh, P. trichophila var. trichophila (F. Stevens) U. Braun [as P. solani-asperi (R.E.D. Baker & & W.T. Dale) Deighton] (Stevens 1917; Baker & Dale 1951; Chupp 1954; Deighton 1976; Crous & Braun 2003; Braun 2017), P. trichophila var. punctata U. Braun & Urtiaga, P. venezuelae (Chupp) Deighton, and P solani-pseudocapsicicola Meir. Silva & al. are compared in TaBLEs 1-2. Pseudocercospora atromarginalis lacks superficial mycelia with secondary conidiophores; collections from Taiwan with conidiophores (10-65 x 3-5 um/0-4-septa) and close to our specimen differed in lacking stromata (Guo & Hsieh 1995, Choi & al. 2015). Pseudocercospora marcelliana differs by shorter conidia (15-70 um) and conidiophores (5-25 um), an absence of superficial mycelium, and occurrence on other hosts or in different locations (Braun 2017; Crous & Braun 2003; Chupp 1954, as Cercospora). Pseudocercospora venezuelae differs by shorter conidiophores (10-70 um), conidia (15—)30-65 wm), and lacks superficial mycelium; and P solani- torvicola differs by smaller (<30 um) stromata, shorter and wider primary conidiophores (35-60 x 4.0-5.5 um), shorter secondary conidiophores (10-25 um), and shorter conidia (20-115 um) with fewer septa (1-11). Pseudocercospora seropedicensis & P. solani-cernui spp. nov. (Brazil) ... 123 PLATE 1. Pseudocercospora seropedicensis (holotype, UFRJ 12.390) on leaves of Solanum asperum. A. Branch of S. asperum. B. Leaf with lesions. C. Detail of fructifications on lesions. D-EF Conidiophores and conidia as seen under dissecting microscope. G-I. Stroma (vertical section). J. Secondary mycelia on leaf undersurface. K-M. Conidia. Scale bars: A= 5 cm; B= 1 cm; C= 1 um; D-I = 20 um; J, K= 10 um; L, M=5 um. 124 ... Andrade & al. Pseudocercospora trichophila var. punctata has smaller dark brown to blackish stromata (10-60 um diam) (Braun 2017). Finally, although P solani-asperi [= PB. trichophila var. trichophila] (Braun 2017) has been reported in Brazil on the same host species (Chupp 1954 as Cercospora solani-asperi, Crous & Braun 2003, Deighton 1976 as Pseudocercospora solani-asperi, Fernandes & al. 2013), it produces shorter 20-110(165) conidia with fewer 1-10(15) septa; P. solani-asperi also usually shows hypophyllous caespituli and lacks a stroma, clearly distinguishing it from the new species found now in the Atlantic Forest. Pseudocercospora solani-pseudocapsicicola, a new species recently reported on leaves of Solanum pseudocapsicum from Brazil (Silva & al. 2016), clearly differs in lacking stromata, shorter (42-128 um) conidia with fewer (2-6) septa, and hypophyllous conidiophores. Pseudocercospora seropedicensis is the first report of a Pseudocercospora species on leaves of Solanum asperum in the State of Rio de Janeiro and the second in Brazil. ADDITIONAL SPECIMENS EXAMINED—BRAZIL. RIO DE JANEIRO: Paracambi, Parque Natural Municipal do Curid, 22°35’44”S 43°42’18”W, 9.XII.2013, C. A. Inacio 61, (UFRJ 12015); 19.III.2014, C. A. Inacio 74 (UFRJ 12050). Seropédica, Campus UFRRJ, Jardim Botanico near the road to Agricultural Institute, 22°45’53”S 43°41’35”W, 11.VHI.2017, J. M. Oliveira 32 (UFRJ 12.495); 11.VIII.2017, J. D. Almeida 03 (UFRJ 12.496); near Bus Station in front of Female Students Dormitory, 22°45’59”S 43°41’24”W, 22.1X.2017, J. Rembinski 35, J. (UFRJ 12.594); National Forest (Fiona), 22°43’46”S 43°42’36”W, 29.V1.2013, C. A. Inacio 79 (UFRJ 12064). Pseudocercospora solani-cernui Rembinski, Oliveira & Inacio, sp. nov. Pl. 2 MB 824359 Differs from P. seropedicensis by its narrower conidiophores, its larger stromata, and its lack of secondary mycelium. Type: Brazil, Minas Gerais, Liberdade, Sitio Carcara, Zona Rural, Bairro Taquarucu- Rodovia Joanito Balieiro Km 9, 22°04’26”S 44°23’48”W, on leaves of Solanum cernuum Vell. (Solanaceae); 14.Oct. 2016, J.C. Oliveira 29 (Holotype, UFRJ 12.422). EryMoLoey: referring to the host species, Solanum cernuum. LESIONS <25 mm diam., amphigenous, mainly epiphyllous, sparse, sometimes confluent, circular, later irregular, initially visible as yellowish spots becoming reddish brown to light brown or grayish brown in the center with yellowish margin at the adaxial face and shown as pale areas PLATE 2. Pseudocercospora solani-cernui (holotype, UFRJ 12.286) onleaves of Solanum cernuum. A, B. Affected leaf (A: upper surface; B: under surface). C. Leaf spots on upper surface. D. Fructifications on upper surface. E. Conidia and conidiophores seen under stereomicroscope. Pseudocercospora seropedicensis & P. solani-cernui spp. nov. (Brazil) ... 125 FE. Stroma with conidiophores showing textura angularis (vertical section). G. Stromata (vertical section). H. Stroma and conidiophores. I-K. Conidia. Scale bars: A, B = 5 cm; C=10mm; D =5 mm; E£, F = 20 um; G = 50 um; H = 5 um; I, J =10 um; K = 5 um. 126 ... Andrade & al. at the abaxial face of the leaf, becoming slightly brownish at the center, corresponding to the upper leaf spots. CoLonigs epiphyllous, caespitose, sparse. MycELium: internal, colorless to pale brown, septate, branched, penetrating the mesophyll; hyphae 2-4 um diam. Stromata 10-70 x 30-90(-150) um, brown, epiphyllous, subepidermal, erumpent, textura angularis, with brownish angular cells; cells 4-7 um wide, forming a fascicle of conidiophores. CONIDIOPHORES 20-46 x 3-6 um, 1-3-septate, numerous, macronematous, mononematous, brown, smooth, densely caespitose, arising from stromata, straight or slightly sinuous. CONIDIOGENOUS CELLS integrated, polyblastic, mostly monoblastic and usually terminal, sympodial, geniculate, with inconspicuous conidiogenous loci. CONIDIA solitary, 30-145 x 2-5 um, 2-12-septate, straight, sometimes sinuous to curved, subcylindrical to obclavate, smooth, subhyaline to brown olivaceous, attenuated at the apex, obconically truncate at the base, hila neither thickened nor darkened, 1-2 um diam. TABLE 2. Morphological comparison of selected Pseudocercospora species on Solanum SPECIES STROMATA (um) CONIDIOPHORE/ —- CONIDIA (tm) / REFERENCES SEPTA (um) SEPTA P. atromarginalis Absent or only a 15-65(100) x 20-95 x Guo & Hsieh (1995) few brown cells 3-5 / 0-4 3.5-5 / 3-11 P. marcelliana 15-75 5-30 x 15-110 x Braun (2017) 1.5-4/ 1-2 2-357 (3-14 P. seropedicensis 25-80 (-85) x 1’—20-70 x 20-140(226) x This work 20-140 (-160) 1-4/ 1-5; 2-6 / 1-16 2’—5-42(45) x 3-5/ 0-1 P. solani-cernui 10-60 x (15)30-46 x 30-145 x This work 30-90 (-150) 2-4 / <3 2-5 / 2-12 P. solani- Lacking 10-35 x 42-128 x Silva et al. (2016) pseudocapsicicola 3-5 / 0-3 2-3.5 / 2-6 P solani-torviculaz <30 1’—35-60 x 20-115 x Guo & Hsieh (1995) 4.0-5.5 / 1-3 4.5-6/ 1-11 2’—10-25 x 4-5/ 0-1 P. trichophila 10-60 10-70 x (10)20-90(110) x Braun & Urtiaga var. punctata 3-7 / 0-4 (2.5)3-5 (6) / (2012) (1)2-10(12) P. trichophila Lacking to few 5-80 x 20-110(165) x Braun (2017) var. trichophila swollen hyphal 2.5-5 / 0-4 3-5(7) / 1-10(15) cells P. venezuelae 15-40 10-70 x (15-)30-65 Braun (2017) 2-4 / 0-1(2) x 3-5 / (0-)3-6 Pseudocercospora seropedicensis & P. solani-cernui spp. nov. (Brazil) ... 127 CoMMENTS—Pseudocercospora solani-cernui is closely related to P. trichophila var. trichophila, P. solani-pseudocapsicicola, and P. seropedicensis (TABLES 1-2). Although showing similar conidial dimensions (20-110(165) x 3-5(-7) um) and occurring on various Solanum species, P. trichophila var. trichophila is distinguished by much smaller (<5 mm) leaf spots, absence of a stroma, and longer (45-80 um) conidiophores (Deighton 1976). Pseudocercospora solani- pseudocapsicicola (reported on S. pseudocapsicum in Brazil) differs in lacking stromata and narrower conidia (42-128 x 2-3.5 um) with fewer (2-6) septa (Silva & al. 2016); P. seropedicensis exhibits similar sized stromata ((20-)140 (-160) um) but is clearly distinguished from the new species on S. cernuum by its much longer (20-60(-70) um) conidiophores, conidia with more (1-16) septa, and superficial mycelium bearing secondary conidiophores. The above differences morphologically support the Pseudocercospora specimen on S. cernuum as a new species, here designated as P. solani-cernui. ADDITIONAL SPECIMENS EXAMINED—BRAZIL. RIO DE JANEIRO: Paracambi, Parque Natural Municipal do Curid, 22°35’44’S 43°42’18”W, 05.11.2016, J.M. Oliveira 14 (UFRJ 12.271); 10.VI.2016, J.M. Oliveira 17 (UFRJ 12.290). Seropédica, Campus UFRRJ, reserve in front of the Instituto de Florestas UFRRJ, 22°45’30”S 43°41'54” W, 26.X.2017, C.A. Inacio 160 (UFRJ 12.553). Minas GERAIS, Liberdade: Sitio Carcara, Zona Rural, Bairro Taquarugu—Rodovia Joanito Balieiro Km 9, 22°04’26”S 44°23’48”W, 28.X.Oct. 2015, J.C. Oliveira 5 (UFRJ 12172). Key to Pseudocercospora species on Solanum from Brazil IMyceliuni dntennalarid external «x! «Sica! 9 sits wi ioltia wiseltia wibeltia weenie w podea Wied a Wie 2 DALI isCelititia COMSIStEIIU Yat RL Ra i eg Mase seo UMS nee EASE ogo VASE ogo VA go Ve ng Gea Ona 4 2 Leaf spots lacking or indistinct, stroma absent ........ P. trichophila var. trichophila 2’ Leaf spots amphigenous, stroma well developed................. P. seropedicensis 3 Stromata lacking or small as small substomatal clumps of swollen hyphae ......... 4 3° Stromata well-developed; often forming sporodochial conidiomata .............. 5 4 Conidia 42-128 um long; on S. pseudocapsicum ........ P. solani-pseudocapsicicola 4’ Conidia shorter (<110(-120) um); on Solanum spp. ............ P. atromarginalis 5 On Solanum cernuum; conidia 30-145 um long, <12-septate ...... P. solani-cernui 5° On Solanum aculeatum; conidia (15-)30-65 um long, <6-septate.... P. venezuelae Acknowledgments The authors acknowledge CAPES, CNPq, FAPERJ for financial support and EMBRAPA and UFRRJ for facilities. Thanks are also given to Dr. Uwe Braun (Martin Luther University, Germany), Dr. Paul Kirk (Royal Botanical Garden Kew, UK), and Prof. José Dianese (Universidade de Brasilia, Brazil) for reviewing our manuscript and to Jonas Dias de Almeida (UFRRJ) and Ernandes Silva Barbosa (UFRRJ) for technical assistance. 128 ... Andrade & al. Literature cited Baker RED, Dale WT. 1951. Fungi of Trinidad and Tobago. Mycological Papers 33:1-123. Beilharz V. 2002. Two additional species of Verrucisporota, one with a Mycosphaerella teleomorph, from Australia. Mycotaxon 82: 357-365. Braun U. 1995. A monograph of Cercosporella, Ramularia and allied genera (phytopathogenic hyphomycetes) 1. 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Boletim da Sociedade Brasileira de Agronomia 8: 1-160. MYCOTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 January-March 2020—Volume 135, pp. 131-141 https://doi.org/10.5248/135.131 The nomenclatural history of Umbilicaria spodochroa and nomenclatural corrections in Umbilicariaceae Evceny A. Davybov’, TEUvo AHTI’, ALEXANDER N. SENNIKOV”? ‘Altai State University, Lenin Avenue, 61, Barnaul, 656049, Russia *Botanical Museum, Finnish Museum of Natural History, PO. Box 7, 00014 University of Helsinki, Finland *Herbarium, Komarov Botanical Institute of Russian Academy of Sciences, Prof. Popov str. 2, 197376 St. Petersburg, Russia * CORRESPONDENCE TO: eadavydov@yandex.ru ABSTRACT— The name Umbilicaria spodochroa is currently applied to a species with an oceanic distribution in Europe and East Asia. The upper surface of its thallus is grey to dark brown, apothecia are omphalodiscs with prominent central umbilicus-like buttons. Its designated type and other original material are referred to U. hirsuta. Conservation is required to retain this name in current use. The nomenclatural history of U. spodochroa is presented to serve as the background for its conservation. The subgeneric nomenclature of Umbilicaria is revised and one new name (U. subg. Papillophora) is proposed to replace the illegitimate U. subg. Gyrophora. The status of many new names published by G.E. Hoffmann in his “Deutschlands Flora” (1796) is discussed and their nomenclatural validity is supported. Key worps—conservation, Ehrhart, historical collections, Hoffmann, typification Introduction Species of Umbilicariaceae Chevall. are predominantly saxicolous lichens mostly found in regions of higher latitudes or altitudes worldwide. Multilocus phylogenies resulted in a new generic concept of the family, which currently includes three genera comprising together about one hundred species with mostly umbilicate growth habit (Bendiksby & Timdal 2013, Davydov & al. 2017). 132 ... Davydov, Ahti, Sennikov Morphological details of the upper and lower thallus surfaces as well as traits of the rhizinomorphs were mostly taken into consideration by earlier lichenologists to distinguish species of Umbilicaria Hoftm. However, these characters have not always been easily recognizable. For this reason, the circumscriptions of some of the early described species have been controversially discussed. One species with a rather complicated taxonomic and nomenclatural history is Umbilicaria spodochroa which, in the 18" and 19" centuries, was often confused with another early described species, U. hirsuta (Sw.) Ach. Umbilicaria spodochroa, with an oceanic distribution in Europe and East Asia (Wei & Jiang 1993), is characterized by a large thallus with a grey to dark brown upper surface and pale brown to black papillate lower surface with abundant rhizinomorphs and characteristic omphalodisc-type apothecia with prominent central umbilicus-like buttons. By such characterization, this species is easily recognizable, and specimens thereof were frequently distributed in exsiccatal series (see the list in Llano 1950). Umbilicaria hirsuta, a Holarctic species, is characterized by producing parasoredia on the marginal part of the upper thallus surface, unlike the closely related U. grisea Hoffm. in which the parasoredia clump and become farinose with variously sized granules (Codogno & al. 1989). The lower surface of U. hirsuta is beige to almost black with sparse to abundant rhizinomorphs. Apothecia, which are rarely produced, belong to the gyrodisc type. As currently defined (Frey 1933, Llano 1950, Codogno &al. 1989, Wei & Jiang 1993), Umbilicaria hirsuta and U. grisea can be unambiguously distinguished by their diagnostic characters as stated above. Both species names are currently accepted (Poelt & Vézda 1981, Hitch & Purvis 2009, Stenroos & al. 2016), and the species were assigned to the recently resurrected “Umbilicaria subg. Gyrophora (Ach.) Frey” (Davydov & al. 2017), which corresponds to a rather large clade of 13 phylogenetically analysed species. The core of this clade is constituted by the Umbilicaria vellea group, which includes U. spodochroa and U. hirsuta. In spite of the current advances in the taxonomy of Umbilicariaceae, the nomenclature of several species is still to be clarified. In the present contribution we aim at unravelling the old confusion concerning the species name Umbilicaria spodochroa. Figure 1. Type collection of Umbilicaria spodochroa, voucher from Gottingen (GOET 019934). A. printed label of Ehrhart’s exsiccata; B. upper surface of thallus; C. lower surface of thallus. Photo: Marc Appelhans. Umbilicari i ia subg. Papillophora subg. nov. & U. . NOV. . spodochroa . oettingen j | Herb. Goett wna Mte © GOERS oe 316. Lichen fpadochrous Ehrh. j Upfalia 320. Spherocephalus { 7 Herrenhaufit. ee — a Wn jplitarra Pinsnda (Fe 7 133 134 ... Davydov, Ahti, Sennikov Materials & methods The protologue of Umbilicaria spodochroa and the relevant historical literature was examined to uncover the history and the original material of the name. Herbarium collections and high resolution digital photographs of Umbilicaria from GOET, H, LE, LINN, and MW were studied de visu, via online portals (https://plants.jstor.org), or provided by curators. Taxonomic literature was screened for relevant treatments. Historical background A species of Umbilicaria with the specific epithet “spodochrous” was first introduced by Ehrhart (1793), who distributed a specimen named “Lichen spadochrous Ehrh?” among his exsiccatae (Fic. 1). The specimen was accompanied with a printed label, but no description or diagnosis of the species was provided. The species epithet is controversial because in its original spelling it is meaningless and has long been considered linguistically erroneous (e.g., Schade 1955). Nylander (1861: 115; 1869: 11) stated that the epithet “spadochrous” seems to be a misspelling because it is apparently derived from the Greek word omodoc (cinder), meaning “ashes” Since the species epithet was incorrect, either as a typographic or orthographic error, under ICN (Shenzhen Code) Art. 60.1 its spelling may be corrected to “spodochrous,’ which is currently in common use. Acharius (1794) referred Ehrhart’s specimen of Lichen “spadochrous” to L. polyrrhizos L. This was done under a broad species concept and had no practical influence on the further taxonomy or nomenclature. Valid publication and protologue Hoffmann (1796) revised species of cryptogams (ferns, mosses, and lichens) known from Germany. Since the available knowledge was very uneven and several taxa were not sufficiently understood at that time, he treated the taxa differently as he explained in the Preface: “Varietaten, Halbarten (Subspecies), auch Arten, welche ich als solche aufzufihren noch unentschlossen war, findet man entweder in Klammern .. den Anmerkungen, oder ohne Bezifferung der nachstverwandten Art beigestellt” [= Varieties, subspecies, also species that I was still undecided to list as such, are to be found either in parentheses ( ) placed with the annotations, or, without numeration, with the most closely related species] (Hoffmann 1796: [Vorbericht: 4]). In the Index, according to our interpretation of this work, Hoffmann (1796) implicitly listed accepted names and their basionyms in italics and Umbilicaria subg. Papillophora subg. nov. & U. spodochroa ... 135 synonyms in the regular font. In the taxonomic part of his work, he listed many species without numbers, yet with binomial names in the accepted genus. Several of such species were new to science. In annotations placed in brackets, Hoffmann mentioned quite a number of species names published by previous authors, with their original generic assignments. He also mentioned several varieties in these annotations, usually unnamed or under old polynomials, sometimes with previously published species names; according to the Index, such species names were listed as synonyms. Hoffmann’s use of subspecies was very sparse, and we are not aware of any name that he may have applied at this rank. Isoviita (1966) considered new names in Hoffmann’s treatments of “undecided” taxa invalidly published because of the presumed absence of explicit acceptance by the author [ICN (Shenzhen Code) Art. 36.1]. Contrary to his opinion, we consider the internal evidence in Hoffmann (1796) (typesetting of the Index and explanations in the Preface) to be sufficient to dispel doubts about Hoffmann’s acceptance of such taxa. Hoffmann (1796) was the first to provide a description for Ehrhart’s lichen under the name Umbilicaria “spadochroa”; although this species name was left unnumbered in the synopsis, it was listed as accepted in the Index and therefore was validly published in spite of any doubts that Hoffmann may have had at that time. The species description provided by Hoffmann inadequately distinguishes between the Umbilicaria species in their current circumscription. Hoffmann distinguished U. spodochroa from its presumed closest relative U. hirsuta mostly by the colour of the upper thallus surface (bluish grey vs. grey) and the lower thallus surface (light brown vs. brownish grey), and also by rhizinomorph density (scarce vs. abundant). These characters are variable in both species in that the colour of the lower thallus surface varies from beige to black-brown and the rhizinomorphs are scarce to abundant. According to the current species concept, Hoffmann’s description fits both taxa, U. spodochroa and U. hirsuta, so that it can be applicable either to a species different from U. hirsuta or it may indicate a phenotypic variation within the same species. Further treatments A later author who treated this lichen species was Acharius (1799), who validly published the combination Lichen spodochrous (as “spadochrous’), which he explicitly accepted and accompanied by a species description. 136 ... Davydov, Ahti, Sennikov Acharius broadened the limits of this taxon and, disregarding priority, included one previously described species, Umbilicaria cirrosa Hoftm.; this name has not yet been typified but the figures accompanying the description by Hoffmann (1789) suggest that it may be a synonym of U. vellea. Acharius (1799, 1803) specified the character of the lower surface of the thallus as dark hirsute (“subtus ater hirsutus”). Later, Acharius (1810: 673) emphasized that his Gyrophora vellea (L.) Ach., G. spodochroa, and G. crustulosa Ach. can be optimally distinguished by their apothecial morphologies. In this work, Acharius was the first to mention one of the most important diagnostic characters of Umbilicaria spodochroa, i.e., the prominent central button on the apothecia. Finally Acharius (1814) lowered the rank of the taxon to the varietal level but maintained its diagnostic characters. It is rather obvious that the works of Acharius were essential in establishing the current concept of U. spodochroa. Nevertheless, despite the characters indicated in his descriptions, the specimens labelled by Acharius as “Gyrophora vellea B G. spadochroa” belong to U. vellea (H-ACH 581) or U. vellea and U. cinereorufescens (Schaer.) Frey (H-ACH 580). The specimens referable to U. spodochroa were identified by Acharius as “Gyrophora vellea” (H-ACH 576), in accordance with the illustration of the latter species in Acharius (1794: Tab. II], fig. 3). Stenhammar (1825) described the same type of apothecia, with thick margin and a central verruca, for Umbilicaria vellea var. spodochroa, although the respective herbarium specimen was referred to U. vellea (Merrill 1906). The other important diagnostic characters of the species were unknown at that time. Nylander (1861) established an additional character that can diagnose Umbilicaria spodochroa: ascospores that are simple and colourless to submuriform and brown. However, he treated U. spodochroa broadly and included other Umbilicaria taxa with submuriform ascospores: U. cinereorufescens, U. cirrosa, U. crustulosa, and U. depressa (Ach.) Duby (Nylander 1869). This broad concept of Umbilicaria spodochroa prevailed until Frey (1933) recognized the four aforementioned species as separate and provided an artificial key and detailed descriptions, which are still accepted. Frey’s treatment became the basis for all subsequent interpretations of U. spodochroa and established the current application of its name. Umbilicaria subg. Papillophora subg. nov. & U. spodochroa ... 137 Type designation Hoffmann (1796) cited two collections in the protologue, which are therefore syntypes. The first collection is Lichen “spadochrous” of Ehrhart (1793), which was erroneously cited under no. 317 instead of no. 316. The distribution of Ehrhart’s exsiccatae was so limited (Gubanov & Balandina 2000) that this incorrect citation was reproduced in the great majority of subsequent taxonomic publications. We were able to locate three specimens of Ehrhart’s “Lichen spadochrous” at GOET, LINN-HS, and MW. In addition we checked B, BM, G, HAL, LE, M, and UPS (the herbaria in which some Ehrhart’s collections are known to be located), but without success. The specimen at GOET (barcode 019934) was studied by Arnold (1880) and Schade (1955), who referred it to Umbilicaria hirsuta. We agree with this identification because of its prominent marginal farinose-granular parasoredia. The specimen at LINN-HS (1703.19.3) belongs to the Herbarium of Sir James Edward Smith, to which it went through the collection of Edmund Davall in 1802 (Beer 1947). According to the annotations, Smith referred it to “Lichen polyrrhizos,” although its correct identity is Umbilicaria hirsuta. The specimen at MW belongs to the personal collection of F. Ehrhart, which was owned by Hoffmann who left its part, including sets of the exsiccatae, to the Moscow Branch of the Military Medical-Surgical Academy, from which the collections were transferred to the Moscow University after the Academy was closed in 1842 (Sokoloff & al. 2002). Some specimens of Ehrhart’s cryptogams were purchased by the Botanical Museum of the St. Petersburg Academy of Sciences (now the Komarov Botanical Institute) (Karavaev & Barsukova 1968) but the specimen of Lichen “spadochrous” was left in Moscow (Gubanov & Balandina 2000). This specimen was certainly examined by Hoffmann and apparently was the main basis for the original description and his concept of Umbilicaria spodochroa. The traits of his specimen do match the original description well, but it evidently also belongs to U. hirsuta. The second collection mentioned by Hoffmann (1796) is a specimen of “Lichen polyrrhizos,” which was communicated by Smith. We cannot recognise this specimen among the lichen collections of Hoffmann, which were purchased from him by Moscow University (Hoffmann 1825). However, a suitable specimen in Smith’s herbarium (LINN-HS 1273.212), identified as “Lichen polyrrhizos” and originating from Ehrhart’s collection, matches the 138 ... Davydov, Ahti, Sennikov protologue of Umbilicaria spodochroa. This specimen, which was received by Smith most likely in 1793 (as evident from annotations on other similar specimens in this collection, e.g. LINN-HS 39.34) and may have been shared with Hoffmann prior to 1796, could be part of the gathering mentioned by Hoffmann in the protologue. This specimen also belongs to U. hirsuta. The original description of Umbilicaria spodochroa and the relevant herbarium material convincingly demonstrate that this name was applied by Hoffmann to a variant of U. hirsuta, not to the species known as U. spodochroa in the current use. Arnold (1880) studied Ehrhart’s lichen collections and stated that Ehrhart’s specimen of Lichen “spadochrous” was mixed and the typical U. spodochroa was also present under no. 316; however, we have found no evidence for this statement. Moreover, U. hirsuta is common in the vicinity of Uppsala (Shah & Coulson 2018), the type locality of U. spodochroa, whereas U. spodochroa in its current concept is absent from Uppsala proper, although it occurs fairly close to the town, mostly along the coast and to some extent along the shores of Lake Malaren (S. Ekman, pers. comm.). Llano (1950: 101) rather mechanically cited “Ehrhart ... Crypt. Exs. 317” as the type of Umbilicaria spodochroa, thus fulfilling conditions for effective type designation [ICN (Shenzhen Code) Art. 7.11, 9.17]. This typification is formally correct but has an undesirable effect that a familiar species name would change its application because the type collection belongs to U. hirsuta although the species name has been widely and persistently used in the sense of U. spodochroa since Frey (1933). Llanos type designation is referable to a gathering rather than a specimen, since he failed to specify the herbarium in which the type is housed, but we refrain from the second-step typification of the name as unnecessary in view of a conservation proposal currently under review (Hestmark, submitted). Umbilicaria spodochroa Hoftm., Deutschl. Fl. 2: 113. 1796, [as “spadochroa”| = Lichen spodochrous (Hoffm.) Ach., Lichenogr. Suec. Prodr.: 149. 1799 [“1798”; as “spadochrous”] = Gyrophora spodochroa (Hoftm.) Ach., Methodus: 108. 1803 [as “spadochroa” | = Gyrophora vellea var. spodochroa (Hoffm.) Ach., Syn. Meth. Lich.: 68. 1814 [as “spadochroa”] = Umbilicaria vellea var. spodochroa (Hoftm.) Stenh., Sched. Crit. Lichen. Suec. 5-6: 4. 1825 [as “spadochroa” | = Omphalodiscus spodochrous (Hoftm.) Schol., Nyt Mag. Naturvid. 75: 26. 1934 LECTOTYPE (designated by Llano 1950: 101)—Sweden. Uppsala, F. Ehrhart in Plantae Cryptogamae Linn. no. 316 (GOET [image!], LINN-HS [image!], MW [image!]). Umbilicaria subg. Papillophora subg. nov. & U. spodochroa ... 139 Subgeneric nomenclature Gyrophora Ach. was published as an explicit substitute [replacement name, ICN (Shenzhen Code) Art. 6.11] for the illegitimate Umbilicaria Hoftm. 1789 (non Fabr. 1759), even though the illegitimacy of Hoffmann’s genus was not realized at that time (e.g., Leighton 1856). Acharius (1803: 100) cited Hoffmann’s name in synonymy and stated that he changed the latter because he considered it “not optimal’, thus making both names homotypic [ICN (Shenzhen Code) Art. 7.4]. Since the generic name Umbilicaria Hoffm. was illegitimate prior to its conservation in 1996 [ICN (Shenzhen Code) Art. 14.15], the autonym “Umbilicaria subg. Umbilicaria” cannot be established [ICN (Shenzhen Code) Art. 22.5]; because of the illegitimacy of the generic name, ICN (Shenzhen Code) Art. 22.2 does not apply and the combination Umbilicaria subg. Gyrophora (Ach.) Frey was validly published for a subdivision of the genus that includes the type of the generic name. When Davydov & al. (2017) accepted U. subg. Gyrophora but excluded from its circumscription the type of Umbilicaria (which is also the type of U. subg. Gyrophora (Ach.) Frey), also providing a description of this subgenus and a type designation, they created an illegitimate later homonym [ICN (Shenzhen Code) Art. 48.1] which, however, was not validly published under ICN (Shenzhen Code) Art. E5.1. This subgenus is formally named here with the same type and a reference to the validating description in Davydov & al. (2017). Two other subgeneric names accepted by Davydov & al. (2017) are revised with their corrected nomenclature as follows. Umbilicaria subg. Agyrophora Nyl. [Flora 61: 247. 1878, nom. nud.] ex Cromb., Monogr. Lich. Britain 1: 323. 1894 TyPE (designated by Llano 1950: 49)—Umbilicaria atropruinosa Schaer. [= Umbilicaria leiocarpa DC.] Umbilicaria subg. Lasallia (Mérat) Frey, Hedwigia 71: 106. 1931 = Lasallia Mérat, Nouv. Fl. Env. Paris, ed. 2, 1: 202. 1821 TypeE—Unmbilicaria pustulata (L.) Hoftm. Umbilicaria subg. Papillophora Davydov, Ahti & Sennikov, subg. nov. MB 830067 = “Umbilicaria subg. Gyrophora” sensu Davydov & al., Taxon 66: 1297. 2017. TypeE—Unmbilicaria vellea (L.) Ach. DESCRIPTION—see Taxon 66: 1297. 2017, under Umbilicaria subg. Gyrophora “(Ach.) Frey”. EryMoLoGy—The name refers to the papillose lower surface and rhizinomorphs, the characteristic trait for the majority of species in the subgenus. 140 ... Davydov, Ahti, Sennikov Acknowledgments Norbert Kilian (Berlin) kindly commented on the complicated taxonomy and nomenclature of Hoffmann (1796) and provided his English translation of the quoted passage. We are grateful to Marc Appelhans (Gottingen) for scanned images and a photocopy of the printed matter of Ehrhart’s exsiccatae from GOET. Mikhail Kozhin (Moscow) supplied a photograph from Ehrhart’s collection at MW. The following curators kindly reported the absence of Ehrhart’s specimens: Stefan Ekman (UPS), Uwe Braun (HAL), Andreas Beck (M), Robert Lticking (B), Philippe Clerc (G), Len Ellis (BM). Stefan Ekman is also thanked for information on the occurrence of the species of Umbilicaria around Uppsala. A photograph of Umbilicaria spodochroa from GOET is reproduced with kind permission from the Georg-August- Universitat Gottingen. We thank Christian Printzen (Senckenberg Research Institute Frankfurt, Germany) and Gerhard Rambold (University of Bayreuth, Germany) for expert presubmission review. Literature cited Acharius E. 1794. Nya och mindre kanda Svenska Laf-Arter. Kongl. Vetensk. Acad. Nya Hand. (ser. 2) 15: 81-103, 176-194. Acharius E. 1799 (“1798”). Lichenographiae suecicae prodromus. Link6ping: D.G.Bjérn. 264 p. https://doi.org/10.5962/bh1.title.79420 Acharius E. 1803. Methodus qua omnes detectos lichens secundum organa carpomorpha ad Genera, Species et Varietates redigere atque observationibus illustrare. Stockholm: ED.D.Ulrich. 393 p. https://doi.org/10.5962/bh1.title.79411 Acharius E. 1810. Lichenographia universalis. 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MY COTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 January-March 2020—Volume 135, pp. 143-150 https://doi.org/10.5248/135.143 Ochroconis terricola sp. nov. from China XIN ZHANG, KUN- YING WANG, PENG-PENG REN, YU-LAN JIANG Department of Plant Pathology, Agriculture College, Guizhou University, Guiyang, 550025, China *CORRESPONDENCE TO: yljchsd@163.com ABSTRACT—A new species, Ochroconis terricola, was isolated from soil in Guizhou Province, China. Morphology and phylogenetic analyses of the combined sequence data of nuclear ribosomal DNA genes (ITS, LSU, SSU) revealed the strain as different from other Ochroconis species. Key worps—Dothideomycetes, hyphomycetes, Sympoventuriaceae, taxonomy, Venturiales Introduction Ochroconis, typified by O. constricta (E.V. Abbott) de Hoog & Arx, was morphologically separated from Scolecobasidium E.V. Abbott (Abbott 1927) by de Hoog & von Arx (1974), for species with unbranched, ellipsoidal to cylindrical conidia, while Scolecobasidium was retained for species having T- or Y-shaped conidia. However, the taxonomic status of Scolecobasidium was questioned due to ambiguity of its type species, S. terreum E.V. Abbott, and species with lobed conidia similar to S. terreum in Scolecobasidium have been combined in Ochroconis on phylogenetic grounds (Samerpitak & al. 2014). Historically, Ochroconis was characterized by sympodial conidiogenesis and septate, mostly rough-walled conidia that are liberated rhexolytically (Samerpitak & al. 2015, Ellis 1971). A recent multigene (nSSU, nLSU, mtSSU, RPB2) phylogenetic analysis places Ochroconis in Sympoventuriaceae (Venturiales, Dothideomycetes) (Machouart & al. 2014). By combining molecular phylogeny, morphology, and ecology, Samerpitak & al. (2014) revised the taxonomy of the Ochroconis lineage, introducing 144 ... Zhang & al. Verruconis Samerp. & al. as a new genus for a group of thermophilic species around O. gallopava (W.B. Cooke) de Hoog [= Verruconis gallopava (W.B. Cooke) Samerp. & de Hoog]. (Ochroconis species are mesophilic.) ITS and LSU nuclear ribosomal sequences as well as the conserved SSU gene are all suitable for identifying species in Ochroconis and Verruconis, but the ITS and LSU regions are recommended as the best DNA barcoding candidates, due to the high variability found even in conserved markers (Samerpitak & al. 2014, 2015). Presently, 31 species are accepted in Ochroconis (http://www.mycobank.org). During our 2018 investigation of dematiaceous hyphomycete diversity, we isolated one Ochroconis-like strain from soil in Guizhou Province, China. Now we propose a novel species, Ochroconis terricola, based on morphological characters and phylogenetic analyses of ITS, LSU, and SSU nuclear ribosomal sequences. Materials & methods Fungal isolates and morphological studies Soil samples were collected from Guizhou Province, China, in 2018 and transported to the laboratory in sterilized, zip lock polyethylene bags. All isolates are conserved in the Herbarium of Department of Plant Pathology, Guizhou University (HGUP). The taxon is described from cultures grown for 2 weeks at 25°C on potato dextrose agar (PDA). Conidia and conidiophores were placed in a drop of 60% lactic acid, examined, and photographed at 100x magnification using a Nikon 90i microscope. DNA extraction, amplification, sequencing Genomic DNA was extracted from colonies grown on PDA using the Fungal gDNA Kit GD2416 following the manufacturer's instructions. ITS, LSU and SSU genes were amplified via PCR using primers ITS4/ITS5 (White & al. 1990), LROR/LR5 (Rehner & Samuels 1994, Vilgalys & Hester 1990), and NS1/NS4 (White & al. 1990), respectively. The PCR products were purified and sequenced by Sangon Biotech. The new sequences were deposited in GenBank, and other sequences were obtained from GenBank (TABLE 1). Phylogenetic analyses Sequences were aligned using ClustalX 1.81 (Thompson & al. 1997) and edited manually using BioEdit (Hall 1999). The alignments were concatenated as a Fasta document using MEGA 6.0 (Tamura & al. 2011). Phylogenetic analyses of combined ITS, LSU, and SSU rDNA sequences were computed using maximum likelihood (ML) analysis and Bayesian Inference (BI). The ML tree was generated using RAxML- HPC2 on XSEDE v.8.2.8 (Stamatakis 2014) via the CIPRES Science Gateway platform (Miller & al. 2010) with 1000 bootstrap replicates. Bayesian analysis was performed via MrBayes v3.0b4 (Huelsenbeck & Ronquist 2001) using Markov Chain Monte Ochroconis terricola sp. nov. (China) ... 145 TABLE 1. Sequences used in phylogenetic analyses. GENBANK ACCESSION NO. SPECIES STRAIN ITS SSU LSU Mycosisymbrium cirrhosum GUFCC 18012 KR259883 KR259885 KR259884 Ochroconis bacilliformis CBS 100442 KP798632 KP798638 KP798635 O. constricta CBS 202.27(T) MH854929 KF156072 KF282652 CBS 211.53 HQ667519 KF282671 KF282653 NH 1234 —_ = LC187202 FMR 3906 LM644509 — LM644552 NBRC 9375 DQ307327 AB564608 AB564619 O. cordanae CBS 475.80(T) KF156022 KF282672 KF282654 O. gamsii CBS 239.78(T) KF156019 KF156088 KF156150 O. icarus CBS 536.69(T) HQ667524 KF156084 KF156132 O. macrozamiae CBS 101179 KF156020 KF156091 KF156151 CBS 102491 KF156021 KF156092 KF156152 O. minima CBS 423.64 = KF282680 KF282666 CBS 119792 KF156027 KF156086 KF156133 CBS 510.71(T) HQ667522 KF156087 KF156134 O. phaeophora CBS 206.96 KP798631 KF282675 KF282660 O. ramosa UTHSC 121082(T) LM644524 LM644551 LM644567 O. robusta CBS 112.97(T) KP798633 KP798639 KP798636 O. sexualis PPRI 12991(T) KF156018 KF156089 KF156118 O. terricola HGUP1808(T) MK377301 MK377071 MK377073 Pleospora herbarum CBS 191.86 KC584239 GU238232 GU238160 Scolecobasidium cateniphorum CBS 769.83 KF156013 KF156044 KF156153 S. excentricum CBS 469.95(T) HQ667543 KF156096 KF156105 S. fusarioideum CBS 210.95 — KF156043 KF156154 S. fusiforme CBS 586.82 KF156012 KF156101 KF156155 Scolecobasidium sp. NH503 — AB564606 AB564617 S. terreum PO43 — EU107356 EU107306 S. tricladiatum POS1 — EU107354 EU107286 S. tropicum CBS 380.87 ard KF156042 KF156102 Venturia asperata ATCC 34052 — EF114736 EF114711 V. inaequalis CBS 594.70 KF156040 GU296205 GU301879 V. populina CBS 256.38 MH855959 GU296206 GU323212 V. pyrina ATCC 38995 — EF114739 EF114714 Veronaeopsis simplex CBS 588.66(T) KF156041 KF156095 KF156103 Verruconis calidifluminalis CBS 125818(T) MH875239 KF156046 KF156108 V. gallopava CBS 437.64(T) HQ667553 KF282674 KF282656 V. verruculosa CBS 119775 KF156014 KF156055 KF156106 New sequence is set in bold font. Type strains are marked by T. Carlo method. Pleospora herbarum (CBS 191.86) was used as an outgroup. The phylogenetic tree was viewed in Treeview v. 1.6.6 and the layout was completed in Adobe Illustrator CS5. 146 ... Zhang & al. Taxonomy Ochroconis terricola Xin Zhang & YL. Jiang, sp. nov. Fia. 1 MB 831137 Differs from Ochroconis macrozamiae by its dark brown conidiophores and subhyaline to pale brown conidia, from O. gamsii by its flexuous conidiophores and bigger conidia, and from O. sexualis by its 1-septate and much smaller conidia. Type: China, Guizhou Province, Leishan County, from forest soil, March 2018, X. Zhang (Holotype, HGUP1808; ex-type culture, HGUP1808; GenBank MK377301, MK377071, MK377073). Erymo oey: referring to living on the soil. Cotoniges on PDA effuse, flat, velvety, olivaceous, reverse dark brown, growing slowly, attaining 9 cm diam. at 25° after 4 weeks. MycELIUM immersed and superficial, hyphae hyaline to pale brown, smooth. CONIDIOPHORES unbranched, 1-6 septa, flexuous, thick-walled, dark brown, 2-4 um diam., with sympodially proliferating conidiogenous cells bearing one or more denticles in the apical region. CONIDIOGENOUS CELLS terminal, integrated, 1-3 um long, polyblastic, cylindrical, subhyaline to medium brown. Conipia rhexolytic secession from conidiophores, solitary, subhyaline to pale brown, coarsely verrucose, 1-septate, sometimes slightly constricted at the septum, ellipsoidal to cylindrical or fusiform, apex mostly rounded, base narrowly truncated with minute marginal frills, 7-12 x 2—4 um. Phylogenetic analysis The final alignment contained three genes for 36 isolates and 2568 (765 ITS + 827 LSU + 976 SSU) characters with Pleospora herbarum (CBS 191.86) as outgroup. The ML phylogenetic tree is shown (Fic. 2). Empirical base frequencies with 1,000 bootstrap inferences are 0.249211 (pi A), 0.221282 (pi C), 0.295026 (pi G), and 0.234480 (pi T). Bayesian analysis generated a similar phylogenetic tree (one million generations; average standard deviation of split frequencies = 0.009724). The Ochroconis terricola (HGUP 1808) sequences cluster together in a clade with O. macrozamiae Crous & R.G. Shivas, O. gamsii de Hoog, and O. sexualis Samerp. & al. (Fic. 2) and form a single branch sister to the other three species with high bootstrap support (ML/BI = 84/1). Discussion In this study, one Ochroconis strain (HGUP1808) isolated from soil was identified based on morphological characters and molecular data. Ochroconis terricola sp. nov. (China) ... 147 Fic. 1. Ochroconis terricola (holotype, HGUP1808). A, B: Colony on PDA at 14 days; C: Mycelia on the colony; D-F: Conidiophores and conidia; G-M: Conidia. Scale bars = 5 um. 148 ... Zhang & al. 100/1 Scolecobasidium cateniphorum CBS 769.83 84/1 Scolecobasidium fusarioideum CBS 210.95 Scolecobasidium fusiforme CBS 586.82 Scolecobasidium tropicum CBS 380.87 Venturia asperata ATCC 34052 Venturia pyrina ATCC 38995 3/1 100/1 100/1 | - Venturia populina CBS 256.38 oB/t Venturia inaequalis CBS 594.70 88/1 Veronaeopsis simplex CBS 588.66 98/1 Scolecobasidium excentricum CBS 469.95 100/1 Mycosisymbrium cirrhosum GUFCC 18012 Scolecobasidium tricladiatum P051 1o0/1 100/1 Verruconis gallopava CBS 437.64 Verruconis calidifluminalis CBS 125818 96/1 971-- Ochroconis constricta NH1234 99/1 Ochroconis constricta NBRC 9375 Verruconis verruculosa CBS 119775 100/1 Ochroconis cordanae CBS 475.80 Ochroconis phaeophora CBS 206.96 79/1 Ochroconis sexualis PPRI 12991 100/1 Ochroconis gamsii CBS 239.78 a 100/1- Ochroconis macrozamiae CBS 101179 a Ochroconis macrozamiae CBS.102491 Ochroconis terricola HGUP1808 Ochroconis constricta CBS 202.27 ma toon | 2Colecobasidium sp. NH503 Ochroconis constricta CBS 211.53 98/1 Ochroconis constricta FMR 3906 Scolecobasidium terreum P043 1001 ) Ochroconis icarus CBS 536.69 89/1 Ochroconis minima CBS 423.64 oo, Ochroconis minima CBS 510.71 Ochroconis minima CBS 119792 Ochroconis ramosa UTHSC 121082 100/1 Ochroconis bacilliformis CBS 100442 Ochroconis robusta CBS 112.97 Pleospora herbarum CBS 191.86 0.2 Fic. 2. Maximum Likelihood (ML) tree based on combined LSU, ITS, and SSU data of Ochroconis species and other related genera species, with Pleospora herbarum (CBS 191.86) as outgroup. Branch support is shown as: Maximum Likelihood bootstrap support values >75% / Bayesian posterior probabilities >0.95. The new species is in bold. SSU, ITS, and LSU multi-gene analyses support phylogenetic separation of O. terricola from known Ochroconis species within a single clade in the ML tree. Although Ochroconis terricola is phylogenetically close to O. macrozamiae, O. gamsii, and O. sexualis in the phylogram (Fic. 2), there are obvious morphological differences separating O. terricola and the three species. Ochroconis macrozamiae is distinguished by its red-brown conidiophores and conidia that constrict at the middle septum (Crous & al. 2014); O. gamsii differs by its erect conidiophores and curved or unilaterally flattened, smaller conidia (6-9 x 2.4-2.8 um; de Hoog 1985); and O. sexualis differs by its 1-3-septate, larger conidia (15-22 x 3.5-5.0 um; Samerpitak & al. 2014). The combined morphological and phylogenetic analysis supports O. terricola as a new taxon. Ochroconis terricola sp. nov. (China) ... 149 Acknowledgments The authors are grateful for pre-submission comments and suggestions provided by Drs. Hui Deng (Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing), Wen-Xiu Sun (Bioengineering, College of Life Sciences, Changjiang University), and Shaun Pennycook (Nomenclature Editor, Mycotaxon). Yun Chen is thanked for valuable help. This project was supported by the National Natural Science Foundation of China (no. 31660006). Literature cited Abbott EV. 1927. Scolecobasidium, a new genus of soil fungi. Mycologia 19(1): 29-31. https://doi.org/10.2307/3753662 Crous PW, Shivas RG, Quaedvlieg W, van der Bank M, Zhang Y, Summerell BA & al. 2014. Fungal Planet Description Sheets: 214-280. Persoonia 32: 184-306. https://doi.org/10.3767/003158514X682395 de Hoog GS, von Arx JA. 1974 [“1973”]. Revision of Scolecobasidium and Pleurophragmium. Kavaka 1: 55-60. de Hoog GS. 1985. Taxonomy of the Dactylaria complex, IV. Dactylaria, Neta, Subulispora and Scolecobasidium. Studies in Mycology 26: 1-60. Ellis MB. 1971. Dematiaceous hyphomycetes. Commonwealth Mycological Institute, Kew, Surrey, England. 608 p. Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/ 98/ NT. Nucleic Acids Symposium Series 41: 95-98. Huelsenbeck JP, Ronquist F 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17: 754-755. https://doi.org/10.1093/bioinformatics/17.8.754 Machouart M, Samerpitak K, de Hoog GS, Gueidan C. 2014. A multigene phylogeny reveals that Ochroconis belongs to the family Sympoventuriaceae (Venturiales, Dothideomycetes). Fungal Diversity 65: 77-88. https://doi.org/10.1007/s13225-013-0252-7 Miller MA, Pfeiffer W, Schwartz T. 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE), 14 Nov. 2010, New Orleans, LA, p. 1-8. https://doi.org/10.1109/GCE.2010.5676129 Rehner SA, Samuels GJ. 1994. Taxonomy and phylogeny of Gliocladium analysed from nuclear large subunit ribosomal DNA sequences. Mycological Research 98: 625-634. https://doi.org/10.1016/S0953-7562(09)80409-7 Samerpitak K, Van der Linde E, Choi HJ, Gerrits van den Ende AHG, Machouart M, Gueidan C, Hoog GS de. 2014. Taxonomy of Ochroconis, genus including opportunistic pathogens on humans and animals. Fungal Diversity 65: 89-126. https://doi.org/10.1007/s13225-013-0253-6 Samerpitak K, Duarte APM, Attili-Angelis D, Pagnocca FC, Heinrichs G, Rijs AJMM, Alfjorden A, Gerrits van den Ende AHG, MenKen SBJ, de Hoog GS. 2015. A new species of the oligotrophic genus Ochroconis (Sympoventuriaceae). Mycological Progress 14, Article number 6: 1-10. https://doi.org/10.1007/s11557-015-1023-5 Stamatakis A. 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30(9): 1312-1313. https://doi.org/10.1093/bioinformatics/btu033 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGAS: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and 150 ... Zhang & al. maximum parsimony methods. Molecular Biology and Evolution 28(10): 2731-2739. https://doi.org/10.1093/molbev/msr121 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997. The CLUSTAL-X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25: 4876-4882. https://doi.org/10.1093/nar/25.24.4876 Vilgalys R, Hester M. 1990. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: 4238-4246. https://doi.org/10.1128/jb.172.8.4238-4246.1990 White TJ, Bruns T, Lee SB, Taylor JW. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. 315-322, in: MA Innis MA & al. (eds). PCR protocols, a guide to methods and applications. San Diego: Academic Press. https://doi.org/10.1016/B978-0-12-372180-8.50042-1 MY COTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 January-March 2020—Volume 135, pp. 151-165 https://doi.org/10.5248/135.151 Neomyrmecridium asymmetricum sp. nov. from Ecuador LIZETTE SERRANO’, DAYNET SOSA’?’, FREDDY MAGDAMA', FERNANDO ESPINOZA’, ADELA QUEVEDO’', MARCOS VERA’, MIRIAM VILLAVICENCIO’, GABRIELA MARIDUENA’, SIMON PEREZ-MARTINEZ’, ELAINE MALOSSO?, BEATRIZ RAMOS-GARCIA4, RAFAEL F. CASTANEDA-RUuIz? ' Escuela Superior Politécnica del Litoral, ESPOL, Centro de Investigaciones Biotecnologicas del Ecuador, Campus Gustavo Galindo, Km. 30.5 Via Perimetral, PO. Box 09-01-5863, Guayaquil, Ecuador ? Universidad Estatal de Milagro (UNEMI), Facultad de Ingenieria, Cdla. Universitaria Km. 1.5 via Milagro-Km26. Milagro 091706, Guayas, Ecuador > Centro de Biociéncias, Departamento de Micologia, Universidade Federal de Pernambuco, Avenida da Engenharia, s/n Cidade Universitaria, Recife, PE, 50.740-600, Brazil ‘Instituto de Investigaciones Fundamentales en Agricultura (INIFAT), Tropical Alejandro de Humboldt, OSDE, Grupo Agricola, Calle 1 Esq. 2, Santiago de Las Vegas, C. Habana, Cuba, C.P. 17200 * CORRESPONDENCE TO: dasosa@espol.edu.ec ABSTRACT—A new species Neomyrmecridium asymmetricum, found on decaying leaves of Theobroma cacao, is distinguished by grouped conidiophores and polyblastic production of narrow clavate to subclavate, 1-septate, asymmetrical, and yellowish or subhyaline conidia. An ITS- and LSU-based phylogenetic analysis, description, and illustrations are provided. A key and illustrations to Neomyrmecridium species are also presented. Key worps—asexual fungi, Myrmecridiaceae, taxonomy, tropics Introduction Crous & al. (2018a) introduced Neomyrmecridium Crous for three species: N. septatum Crous (type species), N. asiaticum Crous, and N. sorbicola (Crous & R.K. Schumach.) Crous. Neomyrmecridium is distinguished by macronematous, unbranched, subcylindrical, multiseptate, smooth, brown 152 ... Serrano & al. conidiophores with polyblastic, terminal, subcylindrical, denticulate, pale brown conidiogenous cells. The conidia are solitary, fusoid-ellipsoid, obovoid, hyaline or subhyaline (becoming pale brown with age), septate, smooth, and sometimes encased in mucoid tunica (Crous & al. 2018a). The diversity of microfungi in the Ecuadorian rainforests has received little attention, especially in cacao plantations. During a survey of hyphomycetes associated with plant litter in the Balao cacao plantation, Guayas province, Ecuador (Fic. 1), we collected a Neomyrmecridium specimen that differs remarkably from all previously described taxa (Crous & al. 2018a) and for which we propose a new species. Materials & methods Collections Samples of decaying plant materials were collected and placed in plastic bags for transport to the laboratory, where they were washed, treated according to Castaneda- Ruiz & al. (2016), and placed in moist chambers. Pure cultures were obtained by transferring single conidium using a flamed needle to solidified media (with pH adjusted to 6.3) containing corn meal extract mixed 1:1 with carrot extract plus 15 g agar (CMC) or V8 according to Crous & al. (2009). Plates were incubated at 25 °C. Color notations in parentheses are from Kornerup & Wanscher (1984). Mounts were prepared in PVL (polyvinyl alcohol, lactic acid) and measurements made at 1000x magnification. Microphotographs were obtained with an Olympus BX51 microscope equipped with bright field and Nomarski interference optics. The type specimen was deposited in the Herbarium of Universidade Federal de Pernambuco, Recife, Brazil (URM) and cultures obtained from the type specimen were deposited in the Culture collections of Microorganism CIBE (CCM-CIBE), Guayaquil, Ecuador. DNA extraction, sequencing, and phylogenetic analysis Isolates CCMCIBE-H304 and CCMCIBE-H304-A were cultured on PDA in darkness for 7 days at 25 °C. DNA was extracted using a modified protocol from Cenis (1992). The primers ITS1/ITS4 were used to amplify ITS regions, including the 5.8S gene (Manter & Vivanco 2007), and LROR/LRS5 to amplify the D1/D3 domain of the LSU nrDNA (White & al. 1990). PCR products were sent to Macrogen Inc. (South Korea) for purification and sequencing. Consensus sequences assembled and edited using Geneious (ver. 10.1.2) were later compared with those of the National Center for Biotechnology Information (NCBI) using the Basic Local Alignment Search Tool (BLAST). Each data set was aligned in MEGA 6.0 (Tamura & al. 2013) using ClustalW (Thompson & al. 1994) and refined with MUSCLE (Edgar 2004). The alignment included our strain sequences and those from different genera in the Myrmecridiaceae obtained from NCBI (TABLE 1). ITS- and LSU-based Neomyrmecridium asymmetricum sp. nov. (Ecuador) ... 153 “ti Fee hod - ll “3 a: ,: Pe Be ’ sa ir I a B » mah Fic. 1. Balao cacao plantation, Guayas province, Ecuador. 154 ... Serrano & al. 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Myrmecndium flexuosum CBS 398.76 Myrmecridium fluviae CNUFC-YR61-2 Myrmécridium phragmitis CBS 131311 100 Myrmecnidum schulzeri CBS 100.54 u Myrmecnidium banksiae CBS 132536 93 ' Myrmecndum sparti CPC 24953 Ed Myrmecridaum montsegurinum JF 13180 Myrmecridiaceae Neomyrmecnoum sorbdicola CBS:143433 700 Neomyrmecndium asiatcumn CBS:145080 Neomyrmecridium septatum CBS 145073 Neomyrmecridium asymmetrycum CCMCIBE H304 10+ Neomyrmecridium asymmetrycum CCMCIBE H304-A mn | Woswasia atropurpurea WU 32007 Pynculariomyces asan CPC 27444 Pynculana urashimae CPC 29414 Neopyncularia commelimicola CBS 128303 4 Pseudopyricularia iraniana [RAN 2761C Pyricula “ Pseudopyrculana bothnochioae CPC 21650 Pseudopyncularia hagahagae CPC 26635 - siosphacria ruguios Vermiculanopsielia acaciae CPC 26291 Vermxulanopsiella dichapetal CBS: 143440 Vermicutariopsicla immersa MUCL39135 TCC MYA-257! Vermiculariopsieila eucalypticola CBS. 143442 Vermiculariopsiellaceae Vermiculanopsielia eucalypti CPC 25525 Vermiculariopsieia lauracearum CBS:145055 Vermiculanopseia pedculsta CBS 132484 a portoric: NFCC Castanedelia acacwse CPC 24969 03 - Castanediela cagnizani CBS 101043 7 Castanediella tereticomis CBS:145088 Castanediells eucalypti CPC 24746 Castanediellacea Castanediella couratani CBS 579.71 85 — Castanediella malaysiana CPC 24918 — Saccharata proteae CBS:119218 Fic. 2. The tree derived from the phylogenetic analysis using concatenated sequences of the LSU and ITS of Myrmecridiales revealed that Neomyrmecridium asymmetricum and N. septatum CBS:145073 were nested in a well-supported subclade (bootstrap value of 79). phylogenies were generated using Maximum Likelihood (ML) with the best nucleotide substitution model in MEGA 6.0 (Tamura & al. 2013). Best models used were (for LSU) Tamura-Nei with Gamma distribution and (for ITS) Kimura 2-parameter with Gamma distribution and Invariant sites (G+I). The best nucleotide substitution model for the combined LSU + ITS analysis was the General Time Reversible with Gamma distribution and Invariant sites (G+I). Bootstrap analysis of 1,000 replicates was used to assess the reliability Neomyrmecridium asymmetricum sp. nov. (Ecuador) ... 157 of the reconstructed phylogenies. ML bootstrap values =70% were considered significant. DNA sequences generated in this study were deposited in GenBank (TABLE 1). Phylogeny The BLAST query revealed that the CIBE H304 and CIBE H304-A LSU sequences of N. asymmetricum showed a 97% similarity with N. septatum CBS145073 and N. asiaticum CBS145080. However, they showed sequence identity of 94-96% with LSU sequences of Myrmecridium species, also belonging to the Myrmecridiaceae. The CIBE H304 and CIBE H304-A ITS sequences showed a 90 % similarity with N. sorbicola CBS143433, the highest value matched after blast search. We carried out individual and combined analyses of the LSU and ITS loci to assess relationships with members of the Myrmecridiales (Sordariomycetes) (Fic. 2). The final concatenated analysis encompassed 48 sequences and comprised 1118 bp (ITS 555 bp, LSU 563 bp). The ML tree nested both N. asymmetricum isolates (CIBE H304 and CIBE H304-A) and N. septatum CBS145073 in a well-supported subclade (bs = 79%), with N. asiaticum CBS 145080 as the closest sister species (FIG. 2). Taxonomy Neomyrmecridium asymmetricum R.F. Castafieda, Serrano & D. Sosa, sp. nov. Fries 3-5 MycoBank MB 831330 Differs from Neomyrmecridium sorbicola by its narrow clavate to subclavate, 1-septate, asymmetrical conidia. Type: Ecuador, Guayas Province, Guayaquil, Balao, 2°48 S 79°40 W, on decaying leaves of Theobroma cacao L. (Malvaceae), 8 July 2017, F. Espinoza & S. Pérez-Martinez (Holotype, URM 90896; ex-type cultures, CCMCIBE-H304, GenBank MN014057, MN014055; and CCMCIBE-H304-A, GenBank MN014058, MN014056). ETYMOLOGY: asymmetricum- (Latin), meaning asymmetric. CoLonigs on the natural substrate hairy, effuse, amphigenous, yellowish brown. Mycelium superficial and immersed, composed of branched, 1-2.5 um diam, smooth, brown hyphae. CoNIDIOPHORES macronematous, mononematous, grouped, slightly fasciculate, erect, straight, cylindrical, unbranched, 3-15-septate, brown, pale brown toward the apex, 40-210 x 3.5-9 um, smooth. CONIDIOGENOUS CELLS polyblastic, terminal, integrated, cylindrical or subcylindrical, indeterminate, with several sympodial extensions, denticulate, with tiny cylindrical denticles, pale brown, 8-35 x 158 ... Serrano & al. Fic. 3. Neomyrmecridium asymmetricum (cultures ex holotype, URM 90896). A-D. Colonies and reverses on CMC (A-B) and V8 agar (C-D). 3.5-5 um. Conidial secession schizolytic. Conrp1 solitary, acropleurogenous, narrow clavate or subclavate, 1-euseptate, asymmetrical, 12-15 x 2-3 um, basal cell 7-10 um long, apical cell 4-6 um long, yellowish or subhyaline, smooth-walled. CULTURE CHARACTERISTICS: Colonies on CMC attaining 22 mm in 7 days at 25°C, flat, subfelted, olive (3/E5). Mycelium mostly immersed, scarcely aerial toward the entire margin. Reverse dark green (30/F6). Hyphae septate, Neomyrmecridium asymmetricum sp. nov. (Ecuador) ... 159 Fic. 4. Neomyrmecridium asymmetricum (holotype, URM 90896). A-C. Conidiophores and conidiogenous cells. D. Conidiogenous cell. 160 ... Serrano & al. 2 @ #~-es & @ ' ry in a a : o ‘ . ' . _ a 7 . . i) Fic. 5. Neomyrmecridium asymmetricum (holotype, URM 90896). A-C. Conidia. D. Conidiogenous cell and conidia. E-F. Conidiogenous cells. Neomyrmecridium asymmetricum sp. nov. (Ecuador) ... 161 Fic. 6. Representative conidia of Neomyrmecridium spp. (re-drawn from the literature). A. N. asiaticum (Crous & al. 2018a). B. N. septatum (Crous & al. 2018a). C. N. sorbicola (Crous & al. 2018b). Scale bars = 10 um. subhyaline to pale olivaceous-brown, smooth, 1.5-2 um diam. Sporulation occurred after 4 days, producing conidia similar to those observed from nature. Colonies on V8 agar attaining 12 mm in 7 days at 25°C, flat concentric near cottony center, filamentous-filiform toward margin, yellowish orange (4/A7). 162 ... Serrano & al. Reverse deep orange (5/A8) at the center, light orange (5/A6) toward the margin. Sporulation poor and sparsely after 5 days, conidia similar to those observed from nature. Note: Neomyrmecridium sorbicola (Crous & al. 2018a,b) is superficially similar to N. asymmetricum in producing 1-septate conidia, but N. sorbicola differs in its pale brown obovoid conidia with median regions surrounded by a mucoid tunica (Fic. 6). Neomyrmecridium septatum is separated by its conidia, which are fusoid-ellipsoid, mostly 3-septate, guttulate, pale brown, and with a mucoid tunica encasing the upper two thirds (Crous & al. 2018a) (FIG. 6). Morphological and phylogenetic analyses support N. asymmetricum as a new species in Myrmecridiaceae. Key to Neomyrmecridium species WeOnidta AOS tvs Sep CALC sna hoainanr arte eyettns e eyttente Sache atcha eee hp tie etal nar 2 1. Conidiaanostly more than 1-sepfates< ¢ int. os aed oben} Mine et ote net 3 2. Conidia clavate or subclavate, 12-15 x 2-3 um, asymmetrical, basal cell 7-10 um long, apical cell 4-6 um long, vellowishvorsubhyaline ty. 5 awed 56, gb wn ute ary ale coded 08 go N. asymmetricum 2. Conidia obovoid, (7—)8-10(-15) x 4(-5) um, initially hyaline (pale brown in age), (0O-)1(-3)-septate, with a 1-2 um thick mucoid tunica surrounding the medial region ................ N. sorbicola 3. Conidia fusoid-ellipsoid, (12—)14—16(-20) x (3.5-)4(-5) um, hyaline (pale brown in age), (1-)3-septate, guttulate, with a 1-2 um thick mucoid tunica encasing the upper two thirds ................. N. septatum 3. Conidia ellipsoid to obovoid, (13—)15-17 x 4-5 um, pale brown, (2—)3-septate, guttulate, surrounded by a 0.5 um thick DelANOUS HUMIC Ags file, 52. che 5 Mec ene iene oh aM SM acs maa acto ee N. asiaticum Acknowledgments We are indebted to Dr. Josiane S. Monteiro (Museu Paraense Emilio Goeldi, Belém, Brazil) and Dr. De-Wei Li (The Connecticut Agricultural Experiment Station Valley Laboratory, Windsor CT, USA for their critical reviews. The authors are grateful to Escuela Superior Politécnica del Litoral (ESPOL), CIBE for financial support and the International Society for Fungal Conservation for facilities. RFCR is grateful to the Cuban Ministry of Agriculture. We acknowledge the websites provided by Dr. Paul Kirk (Index Fungorum) and Dr. Konstanze Bensch (MycoBank). 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San Diego, Academic Press. https://doi.org/10.1016/B978-0-12-372180-8.50042-1 Zelski SE, Balto JA, Do C, Raja HA, Miller AN, Shearer CA. 2014. Phylogeny and morphology of dematiaceous freshwater microfungi from Peru. IMA Fungus 5: 425-438. https://doi.org/10.5598/imafungus.2014.05.02.07 MY COTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 January-March 2020—Volume 135, pp. 167-182 https://doi.org/10.5248/135.167 A comparison of anamorphs of some Pachyphlodes species and the type of Chromelosporium: are they congeneric? GREGOIRE L. HENNEBERT! & CONY DECOCK? 'Rue de l'Elevage 32, B 1340 Ottignies-Louvain-la-Neuve, Belgium ?MUCL, Place Croix du Sud, 1348 Louvain-la-Neuve, Belgium CORRESPONDENCE TO: hennebertg@scarlet.be ABSTRACT— There is a question of whether or not the name Chromelosporium competes with the name Pachyphlodes given that both genera exhibit similar conidiogenesis. We address here the question through a comparative study of their anamorphs. The type specimen of C. ochraceum (generic type of Chromelosporium), has been studied and compared to newly collected specimens of two Pachyphlodes species, P. nemoralis and P. citrina (whose identity was confirmed through comparison of ITS DNA sequences) and an unidentified species from the Pachyphlodes—Plicariella lineage. Comparison of the morphology of the Pachyphlodes— Plicariella lineage anamorphs with Chromelosporium ochraceum reveals discriminating characters that may support a generic distinction. Keyworps—Ascomycota, Glischroderma, Pezizaceae, molecular analyses Introduction Corda (1833) described Chromelosporium in Sturm’s DEUTSCHLANDS Fiora fora fungus collected in 1827 on dead stems of Allium (Amaryllidaceae) and Hemerocallis (Xanthorrhoeaceae) and characterized by ochre spores and hyphae immersed in a gel. Subsequent authors, however, overlooked the presence of the gelatinous matrix reported by Corda (1833). Chromelosporium has had a confusing history due to similarities of conidiogenesis found in Hyphelia Fr., Ostracoderma Fr., and Glischroderma Fuckel. Juel (1920) noted that the conidiogenesis in collections of Hyphelia terrestris Fr. and in a fungus that he collected on soil in Mésseberg in western 168 ... Hennebert & Decock Gotland, Sweden, considered to represent Ostracoderma pulvinatum Frt., were similar. Consequently, Juel reduced Ostracoderma to a synonym of Hyphelia. No original specimen of Ostracoderma pulvinatum, from Fries or Juel, were found in the S herbarium, impeding attempts to confirm whether the peridium described by Fries for Ostracoderma as “peridio rotundato minime villoso sed crustaceo” and the one supposedly observed by Juel (1920) are made of a definite pseudotissue of dense intricate hyphal cells enveloping the totality of the conidioma as in Glischroderma cinctum Fuckel or comprises just a superficial agglutination of hyphae embedded in a gel that upon drying becomes a crust-like thin layer, covering only parts of the hyphal mat—a “peridium indeterminatum e villo in pelliculam cohaerentem context,’ as observed by Fries in Hyphelia and by Korf (1994). Because Fries based his generic type, Hyphelia rosea, on “Trichoderma roseum Pers. (non Trichothecium Link), Hughes (1958) [having identified Trichoderma roseum Pers. and Trichothecium roseum (Pers.) Link as homotypic] considered the name Hyphelia no longer available. Therefore, Hughes (1958) rejected Juel’s synonymy and, following Juel’s observations, adopted Ostracoderma for species with similar conidiogenesis, such as Hyphelia terrestris and Chromelosporium ochraceum, making Chromelosporium a synonym of Ostracoderma. Hennebert (1973) segregated the non-peridiate Chromelosporium (with C. ochraceum and other species) from the peridiate Ostracoderma and Glischroderma. Korf (1994) reported several Chromelosporium-like specimens from soil and litter in New York State with a superficial gelatinous mass, which he placed in Glischroderma, a genus described by Fuckel in 1870 and characterized by a superficial sticky gel (glischros = viscous). Norman and Egger (1999) sequenced one of Korf’s anamorphs and demonstrated that it was affiliated with the Pachyphlodes-Plicariella |as Scabropezia] lineage. In examining Korf’s specimens identified as Glischroderma, Healy (2015) recognized additional epigeous anamorphs of Pachyphlodes species and molecularly linked Pachyphlodes pfisteri, a truffle with a Chromelosporium-like anamorph. Hennebert (2017) subsequently distinguished Chromelosporium from Glischroderma”. “) The list of specimens examined under Pachyphlodes pfisteri in Hennebert (2017) erroneously cites specimens received on loan from CUP instead of citing specimens identified as Pachyphlodes pfisteri (represented by CUP 62646, 62650, 62651, 62653, 63540, 63648) based on conidial size and ornamentation and hyphal width; CUP 62652, 62654, 63647, 63695 represent undefined species. Are Chromelosporium & Pachyphlodes congeneric? ... 169 The presence of gel embedding the conidiogenous cells in Chromelosporium incited the senior author to search for fresh samples for further investigations. Fourteen samples of Chromelosporium-like spore mats were then collected on bare or mossy soils or mixed with organic debris in forests in Belgium, some of them bearing a few gel droplets. These Chromelosporium-like specimens were identified by ITS barcoding as two species of Pachyphlodes and an undetermined species of the Pachyphlodes- Plicariella lineage. Their anamorphs are described below and compared to the type specimen and original description of Chromelosporium ochraceum. This comparative morphological study revealed clear distinctions from Chromelosporium ochraceum. Materials & methods SPECIMENS. Fourteen specimens were collected in the forest, accessioned, and deposited in MUCL. The type specimen of Chromelosporium ochraceum (A.C,]. Corda 155414) was borrowed from the Herbarium Kryptogamologicum Musei Nationalis Pragae in Prague (PRM). Microscopical examinations and drawings were carried out using a 1960 phase contrast Olympus FH microscope equipped with a Wild drawing tube. The photographs used in figure 6 were made with an Olympus BX50 microscope equipped with Olympus SC100 numerical camera and Olympus screen. All drawings and photographs are made from samples mounted in lactic acid with Cotton blue. SEQUENCING. DNA was extracted from fragments of five freshly collected samples in lysis buffer, using innuPREP Plant DNA kit following the manufacturer’s recommendations. Sequences were determined for the nuc rDNA ITS1-5.8S-ITS2 region (ITS) with the primer pair ITS5 and ITS4 (White & al. 1990). PCR conditions are as described in Gordillo & Decock (2018). Amplicons were sequenced in both directions by Macrogen Inc. (Korea) using the same primers. MK prefixes designate sequences deposited in GenBank. Taxonomy Anamorph of Pachyphlodes nemoralis Hobart, Bona & A. Paz, Ascomycetes.org. 7(6):363, 2015 FIGS 1, 6A HypHAL MATS forming cushion-shaped or flattened patches, irregular in outline, grouped, 3-23 mm in diam., 1-3 mm high, white when young discoloring to pale yellow ochre when mature, the surface appearing fluffy to compacted, with a few superficial, small droplets of translucent, glutinous, liquid gel, drying orange and flattened, small, brittle crust-like, the margin remaining fluffy and powdery. 170 ... Hennebert & Decock Fic. 1. Pachyphlodes nemoralis anamorph (MUCL 56524). A. Synnema. B. Young branching. C. Open branching. D. Compact terminal cells. E. Conidiogenous branches. F. Conidia (Scale bars = 10 um). Are Chromelosporium & Pachyphlodes congeneric? ... 171 HypuHae at first loosely interwoven, smooth, hyaline, 4-8 um diam., becoming fasciculate, forming hyaline, erect synnemata <35 um diam., made of hyphae with 2—20 septa, with lateral branches at wide angles, all hyphae turning into apically branched conidiogenous cells, with a repeated asymmetrical bifurcating pattern, coralloid, at variable angles, openly disposed to very compact, sparsely septate, each branch 15-50 um long, and <10 um diam. at the inflated apical section, covered at maturity along their entire length with conidia. CONIDIOGENEsIS holoblastic, more or less synchronous with minute denticles, 1-2 um long. Conrp1A globose to subglobose, hyaline, 4-6.5 um, averaging ~5 um diam. excluding warts, rarely ovoid or pyriform in moist conditions, 4 x 6.5(—8) um, the wall cyanophilic, 0.5 um thick, warted or tuberculate all over, warts, <0.3 um high, evenly-spaced, 12—14 in median section. Hasirat. On soil, moss, and organic debris along paths under Quercus and Fagus sylvatica trees in forest. SPECIMENS EXAMINED: BELGIUM, WALLOON BRABANT, Ottignies-LLN, Lauzelle forest, on soil, moss, and organic debris along paths under Quercus and Fagus sylvatica, October 2017, coll. GL Hennebert (MUCL 56520, GenBank MK714923; MUCL 56524, GenBank MK7 14924; MUCL 56529, GenBank MK71492). Anamorph of Pachyphlodes citrina (Berk. & Broome) Doweld, Index Fungorum 31: 1, 2013 FIGS 2, 6B HyYpPHAL MATS forming cushion-shaped or flattened patches, irregular in outline, somewhat in groups, 2-25 mm in diam., 1-2 mm high, white when young, discoloring to pale yellow ochre when mature, with a dense surface and fluffy margin, the largest patches bearing superficially droplets of translucent, glutinous, liquid gel, becoming orange flattened crust-like and brittle when dried. HypHAe at first loosely interwoven and branched, smooth, hyaline, 3—6(—8) um diam., with 2-15 septa, becoming fasciculate in hyaline, erect synnemata, <25 um wide, the hyphae frequently anastomosed, laterally and apically branched, furcating repeatedly and asymmetrically, sparsely septate, each part 10-25 um long and terminally inflated <8 um wide, covered at maturity all along their length with conidia. CoNIDIOGENEsIS holoblastic, more or less synchronous with minute, denticles, 1 um long. Conrp1A4 globose to subglobose, hyaline, pale yellow ochre in mass, 4.5—7 um, averaging ~5.2 um diam., excluding warts, with a cyanophilic 172 ... Hennebert & Decock Fic. 2. Pachyphlodes citrina anamorph (MUCL 56652). A. Synnema. B. Anastomosis. C. Young branching. D. Developed branches. E. Conidiogenous branches. F. Conidia (Scale bars = 10 um). Are Chromelosporium & Pachyphlodes congeneric? ... 173 wall 0.5 um thick, coarsely warted or baculate all over, warts cylindrical, apically blunt, <0.8 um high, evenly spaced, 12—14 in median view. Hapsirat. On soil along path near Castanea and Larix in forest. SPECIMEN EXAMINED: BELGIUM, WALLOON BRABANT, Ottignies-LLN, Lauzelle Forest, on soil along path near Castanea and Larix in Lauzelle forest, October 2017, coll. GL Hennebert (MUCL 56652, GenBank MK714926). Anamorph of undetermined Plicariella sp. or Pachyphlodes sp. FIGS 3, 6C HypHAL mats forming cushion-shaped patches, determinate, irregular in outline, grouped, 1-5 mm in diam., 1-2 mm high, white when young to yellow ochre in color when mature, with surface fluffy to compacted and granulose, a few fresh mature patches bearing a droplet of translucent glutinous gel, forming a local orange flattened brittle crust when dried, the margin, if not the entire patch, remaining fluffy and granulose. HypuHaeE at first loosely interwoven, smooth, hyaline, 3-7 um diam., 2—10-septate, becoming fasciculate in hyaline erect synnemata <20 um wide, becoming terminally branched through repeated and asymmetric bifurcations to form coralloid branch tips that are openly disposed or compacted and sparsely septate. Each part of branch is 10—70 um long and inflated <8 um, covered at maturity all along their length with conidia. CONIDIOGENESIS holoblastic more or less synchronous with minute denticles 1—1.5 um long. Conip1A globose, hyaline, (3-)3.5-4.5 um, most 4 um diam., with a finely punctate wall. Hasitart. Soil, moss, and organic debris along path under mixed trees in forest. SPECIMEN EXAMINED: BELGIUM, WALLOON BRABANT, Ottignies-LLN, Lauzelle forest, on soil along path in, October 2017, coll. GL Hennebert (MUCL 56522, GenBank MK714927). Original descriptions by Corda (1833) Chromelosporium Corda and C. ochraceum Corda, Sturm’s Deutschlands Flora. Abbildungen nach Natur., Abt. III Die Pilze, Bd. 3, p. 81, Tab. 41, 1833. “CHROMELOSPORIUM Corda Char. Gen. Sporae continuae coloratae, in gelatinae nidulantes, floccis heterogeneis destructis ramosis, articulatis, hyalinis inspersae. Acervuli effuse colorati. [Gen. char. Spores simple, colored, embedded in gel, hyphae dispersed, hyaline, heterogeneous, articulate, branches fragmented; mats extended, colored. ] 174 ... Hennebert & Decock Fic. 3. Plicariella sp. or Pachyphlodes sp. anamorph (MUCL 56522). A. Tip of synnema, B. Branching, C-E Conidiogenous hyphae. F. Conidia. (Scale bars = 10 um). Are Chromelosporium & Pachyphlodes congeneric? ... 175 CHROMELOSPORIUM OCHRACEUM Corda Ocherfarbige Chromelosporie. Tab. 41 Ch. acervulis effusis ochraceis pulverulentis; sporis copiosissimis globosis ochraceis, gelatinae coloratae immersis; floccis hyalinis grosse-articulatis albis. [Fungus cushion-like, effuse, ochre in color, pulverulent; spores very abundant, globose, ochre in color, immersed in a colored gel, dispersed in hyphae hyaline, white, branched and broadly articulate. ] Chr. mit verbreiteten ocherfarbigen, bestaubten Haufchen, sehr haufigen ocherfarbigen, mit gefarbten Schleim eingehiillten, kuglichen Sporen, und durchsichtigen, weissen, grossgegliederten Faden. Wohnt auf faulenden Stengeln der Liliaceen, verziiglich der Hemerocallis und Allium, 1827. Die grossgegliederten, und nicht durch Scheidewande (septa) getheilten Faden unterscheiden diese Gattung von Sporotrichum, so wie auch die gefarten Sporen, da Sporotrichum flocci septati (non articulati) und sporae homogeneae besitzt. Die glashellen, weissen, grossgliedrigen, niederliegenden und verworrenen Faden, sind wenig 4stig, und im Vergliech zu ihren Sporen sehr gross und sparsam. Die Sporen selbst sind nicht aus den Faden entstanden, daher heterogen, sind kuglich, klein, sehr zahlreich, intensive ocherfarb, fast ziegelroth gefarbt. Die sind einfach, und nicht getheilt, auch sind sie durch einen im Wasser leicht ldsslichen und gefarbten Schleim Haufchenartig an die Faden befestigt. [Ch. in extended, ochre, powdery mats, very often covered by a colored gel, spores globose, hyphae hyaline, in broad segments. Habitat on rotten stems of Liliaceae, such as Hemerocallis and Allium. 1827. The articulate and not septate hyphae distinguish this genus from Sporotrichum, which also has colored spores, septate hyphae and homogeneous spores. The hyphae are hyaline, articulate, prostrate, interwoven, sparsely branched and, compared to the spores, broad and sparse. The spores themselves are not formed from the hyphae, thus heterogeneous. They are globose, small, very abundant, deep ochre, near reddish colored. They are simple, not septate; they are also fixed to the hyphae forming a kind of head by the colored gel slightly dissolved in water] Tab. 41. A. Nattirliche Grosse. B. Faden mit Sporen. C. Sporen noch starker vergrossert. A. J. Corda.” CORDA’S SECOND GENERIC DESCRIPTION (1842) In ICONES FUNGORUM HUJUSQUE COGNITORUM, V, p. 8, Prague 1842, Corda gives a shortened description of Chromelosporium with a new comment: 176 ... Hennebert & Decock tf. . a ’ a : Chrmeley ava vtec € PLATE 4. Chromelosporium ochraceum Corda, Sturm’s Deutschl. FI., III (Pilze), 3(13): 81, tab. 41, 1833 Are Chromelosporium & Pachyphlodes congeneric? ... 177 “CHROMELOSPORIUM Corda, Sturm FI. III, 13, p. 81. Sporae continuae, coloratae, gelatinae immersae, floccis heterogeneis, articulatis, repentibus, ramosis, hyalinis inspersae. K. Wir haben eine deutliche Schleimmasse gefunden, welche die Sporen und Flocken einhiillte” [Spores simple, colored, embedded in gel, among hyphae heterogeneous, articulate, prostrate, branched, hyaline. CoMMENTS. We have found a conspicuous mass of gel covering the spores and hyphae. | Chromelosporium ochraceum re-examined Chromelosporium ochraceum Corda, Sturm’s Deutschl. FI., III (Pilze), 3(13): 81, tab. 41, 1833. FIGS 5, 6D Ho.otype: Czechoslovakia, Prague, “Chromelotrichum|(crossed out|sporium ochraceum Ca. ramulis furcis, Myrinema generis” [scr. & del. A.C.J. Corda]. On Allium dead stem. (Herb. Corda in PRM 155414). COLONIES in small patches <10 mm, velvety, at first white then ochraceous when sporulating, made of two layers, one of conidiophores under one of terminal conidiogenous branches. CONIDIOPHORES erect, mononematous, emerging from creeping hyphae, 3-5 um diam. in the substratum, made of a bulbous basal cell, 28-35 x 12-15 wm, ochraceous, extending into a cylindrical, septate stipe, 100-400 um long, light to ochraceous, the individual cells 40-95 x 10-14 um, repeatedly branched, terminating with up to five dichotomous branches, each branch most often with a basal septum, and a second one next to the subsequent dichotomous branches, their length decreasing from one dichotomy to the next, usually the first basal dichotomy 40-90 x 9-10 um, the second 30-70 x 6-10 um, the third 30-50 x 6-9 um, but in some cases dichotomies very short, decreasing from 20-10 um, the range of angles in the dichotomies from 35-45°. CONIDIOGENOUS CELLS comprising up to four terminal dichotomies, together 70-100 x 6-9 um, often apically clavate to 11 um diam., bearing conidia all along with small, denticles 2 x 1 um, collapsing after conidial release, seceding at their basal septum, rarely leaving a frill on the last conidiophore cell. Conip1A holoblastic, borne singly on denticles, at maturity globose or subglobose, thick-walled, cyanophilic, verrucose, ochraceous to salmon in mass, 4—5.5 um diam., <6 um including warts, warts prominent and blunt, 12-18 in median view. 178 ... Hennebert & Decock . tt= tt re —c— | eae 9 =f Sines Tq Fic. 5. Chromelosporium ochraceum Corda (Holotype PR 155414). A. Conidiophores stipes with basal cell and dichotomous branching. B. Terminal conidiogenous dichotomies. C. conidia (Scale bars = 10 um). Are Chromelosporium & Pachyphlodes congeneric? ... 179 ComMMENTS— The terms used by Corda interpreting his observations illustrated in Fic. 4 (Corda’s fig. 41b) may raise some questions. The hyphae described as broadly articulate (“grosse-articulatis, grossegegliederten”) appear in the figure to be hyphae distantly septate in long cells, drawn in their natural dichotomous position. Also the “ramis destructis” might refer to collapsed and disappeared conidiogenous branches after conidial release That would explain why Corda did not report attachments of the conidia to hyphae, which he said were heterogeneous (“floccis heterogeneis, sporen nicht aus den Faden entstanden, daher heterogen’). The gel reported as colored (“gelatinae coloratae, gefarbten Schleim”’), is translucent and may appear colored because of the embedded ochre spores. Our observations suggest that Corda noted those characters from an over-mature collection. Discussion Corda (1833, 1842) described Chromelosporium ochraceum with conidia embedded in a gel. However, this character was not considered significant by Hughes (1958) or Hennebert (1973) until Korf (1994) collected Chromelosporium-like specimens with a superficial gel and Healy (2015) noticed gel on anamorphic growth of Pachyphlodes spore-mats when crushed. The gel, if noticed on fresh anamorphs in humid conditions, may appear on dried herbarium specimens as an orange spot, membranous and brittle, variable in size (as small as 2 mm) or may leave no traces, as is the case for the type specimen of Chromelosporium ochraceum. After the very dry summer of 2017, we observed gel droplets on only a few samples in shady places. In our opinion, the presence of gel is not a reliable, stable character, and its degree of development is probably circumstantial and dependent on environmental conditions. The anamorphs of P nemoralis and P. citrina are not yet described (R. Healy, pers. comm.). The anamorph of Pachyphlodes pfisteri Tocci & al., an endemic North American taxon, was described and illustrated with macro- and micro-photographs by Healy & al. (2015) and drawn by Hennebert (2017). Its conidiogenesis is in all respects identical to that of P. nemoralis and P. citrina (Fics 1, 2). The anamorphs of the two Pachyphlodes and one undetermined Pachyphlodes-Plicariella species here described show distinctive features, particularly in hyphal width, conidiophore branching, and conidial size and ornamentation (Fic. 6). These characters should be examined in more Pachyphlodes and Plicariella species to evaluate their diagnostic value. 180 ... Hennebert & Decock Although they have the same type of conidiogenesis, Chromelosporium ochraceum, the generic type species, differs significantly from Pachyphlodes and Plicariella in its anamorphic morphology. Chromelosporium, as demonstrated by C. ochraceum, has mononematous, erect apically branched conidiophores with regular dichotomies, each branch delimited by a septum, with up to four terminal dichotomies bearing holoblastic, synchronous conidia. Pachyphlodes and Pachyphlodes—Plicariella as shown here differ from Chromelosporium by creeping and erect, intermixed synnematous conidiophores, with lateral and apical coralloid branches that are sparsely septate and bearing holoblastic synchronous conidia along the entire length. Conclusion Differences found here between the anamorphs of Pachyphlodes species and the Pachyphlodes-—Plicariella lineage and the anamorphs and the type species of Chromelosporium include synnematous versus mononematous conidiophores, apical and lateral branching versus solely apical branching, and spore production along most branches of the spore mat versus being limited to terminal hyphal branches. These differences might provide morphological evidence for maintaining Chromelosporium as a distinct genus from Pachyphlodes. It remains to compare the morphologies of the still unknown anamorphs of P ligerica (Tul. & C. Tul) Zobel, the type species of Pachyphlodes Zobel, and of Plicariella radula (Berk. & Broome) Rehm, the type species of Plicariella (Sacc.) Rehm, with the description provided here for C. ochraceum to confirm the morphological distinctiveness of the anamorphs of these lineages. The most expedient solution to answering the question of relatedness would be molecular analyses of C. ochraceum in its fresh anamorphic and/or sexual phases (yet to be discovered) and of the types of Pachyphlodes and Plicariella. Another group of species presently named in Chromelosporium, C. coerulescens (Bonord.) Hennebert and C. carneum (Pers.) Hennebert, do not produce gel but form erect synnematous conidiophores, coralloid branching, and similar conidiogenesis (Hennebert 1973). These appear to be morphologically closer to the anamorphs of Pachyphlodes-Plicariella than to Chromelosporium. Genetic analysis should decide their taxonomic position in the Pezizaceae. Are Chromelosporium & Pachyphlodes congeneric? ... 181 PiaTE. 6. Conidial ornamentation: A. Pachyphlodes nemoralis (MUCL 56524). B. P. citrina (MUCL 56652). C. Plicariella sp. or Pachyphlodes sp. (MUCL 56522). D. Chromelosporium ochraceum (Type PR 155414). Acknowledgments We are very thankful to the revisers of the manuscript, Dr Rosaria Ann Healy (Department of Plant Pathology, Florida University) and Dr Keith Seifert (Agriculture and Agri-Food Canada, Ottawa Research Center) for their very appreciated annotations. Dr Jan Holec (Director, Mycological Herbarium, Prague) is greatly thanked for the loan of the precious holotype of Corda’s Chromelosporium ochraceum, which allowed us to fulfill the purpose of this paper. We thank Stephanie Huret (MUCL genetic service) who conducted the sequence analyses. Also we are thankful to the online Biodiversity Heritage Library for providing with color reproduction of Sturm’s Deutschlands Flora as sent by Prof Kathie Hodge, Cornell University. Cony Decock gratefully acknowledges financial support received from courtesy of the Belgian Federal Science Policy and the BCCM™ research program). 182 ... Hennebert & Decock Literature cited Corda ACJ. 1854. Icones Fungorum hucusque cognitorum. 6: 55. Prague, Ehrlich. Gordillo A., Decock C. 2018. Myrothecium-like (Ascomycota, Hypocreales) species from tropical areas: Digitiseta gen. nov. and additions to Inaequalispora and Parvothecium. Mycological Progress, 2018: 179-180. https://doi.org/10.1007/s11557-017-1302-4 Healy RA, Hobart C, Tocci GE, Béna L, Merényi Z, Paz Conde A, Smith ME. 2015. Fun with the discomycetes: revisiting collections of Korf’s anamorphic Pezizales and Thaxter’s New England truffles leads to a connection between forms and the description of two new truffle species: Pachyphlodes pfisteri and P nemoralis. Ascomycetes.org. 7(6):357-366. Hennebert GL. 1973. Botrytis and Botrytis-like genera. Persoonia 7(2): 183-204. Hennebert GL. 2017. Glischroderma Fuckel. Mycotaxon 132: 745-757. https://doi.org/10.5248/132.745 Hughes SJ. 1958. Revisiones hyphomycetum aliquot cum appendice de nominibus rejiciendis. Can. J. Bot. 36: 727-836. https://doi.org/10.1139/b58-067 Juel HO. 1920. Uber Hyphelia und Ostracoderma, zwei von Fries aufgestellte Pilzgattungen. Svensk. Bot. Tidskr. 14: 212-222. Korf RP. 1994. Fifty years of fun with the discomycetes and what's left to do. Opening lecture of the First Whetzel-Wescott-Dimock Lectureship, Cornell Univ. Oct. 1994, 19 p. Norman JE, Egger KN 1999. Molecular phylogenetic analysis of Peziza and related genera. Mycologia 91: 820-829. https://doi.org/10.1080/00275514.1999.12061087 White TJ, Bruns TD, Lee SB, Taylor JW. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, 315-322, in MA Innis & al. (eds). PCR protocols: a guide to methods and applications. San Diego: Academic Press. https://doi.org/10.1016/B978-0-12-372180-8.50042-1 MY COTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 January-March 2020—Volume 135, pp. 183-193 https://doi.org/10.5248/135.183 Pseudosperma flavorimosum sp. nov. from Pakistan SANA JABEEN? & ABDUL NASIR KHALID? ‘Department of Botany, Division of Science and Technology, University of Education, Township, Lahore, Punjab, Pakistan *Department of Botany, University of the Punjab, Quaid-e-Azam Campus, Lahore, Punjab, Pakistan “ CORRESPONDENCE TO: sanajabeenue@gmail.com; sanajabeen@ue.edu.pk ABSTRACT—Pseudosperma flavorimosum is a new species described from Khyber Pakhtunkhwa province, Pakistan. It is delimited based on morphological characters combined with a molecular phylogeny inferred from nuclear ribosomal DNA internal transcribed spacer (ITS) sequence analyses. The ITS-based phylogeny supports the independence of the new species, which is also is morphologically distinct from closely related taxa. Key worps—Agaricales, Basidiomycota, Inocybaceae, taxonomy Introduction Inocybaceae Julich (Basidiomycota, Agaricales) is one of the larger families of agaric fungi with more than 700 species distributed worldwide (Matheny & al. 2009; Kropp & al. 2010; Bougher & Matheny 2011; Bougher & al. 2012; Fan & Bau 2013, 2014; Braaten & al. 2014; Esteve-Raventos & al. 2015; Jabeen & al. 2016; Farooqi & al. 2017; Matheny & Bougher 2017; Latha & Manimohan 2017; Ullah & al. 2018; Matheny & al. 2019). The number of species has increased considerably with the exploration of tropical and southern temperate areas. Many representatives of genera of Inocybaceae have been reported from Asia (Kobayashi & Onishi 2010; Fan & Bau 2013, 2014; Horak & al. 2015; Latha & Manimohan 2015, 2016, 2017; Saba & al. 2015; Jabeen & al. 2016; Pradeep & al. 2016; Farooqi & al. 2017; Naseer & 184 ... Jabeen & Khalid al. 2017; Liu & al. 2018; Ullah & al. 2018), with 24 species reported from Pakistan (Ahmad & al. 1997, Ilyas & al. 2013, Saba & al. 2015, Jabeen & al. 2016, Faroogi & al. 2017, Naseer & al. 2017, Liu & al. 2018, Ullah & al. 2018). All these species were previously placed in Inocybe (Fr.) Fr., but the recent classification by Matheny & al. (2019) distributes these taxa among separate genera. Infrageneric classifications of Inocybe have been based on the morphology of the basidiospores, cystidia, and stipe (Kuyper 1986), with various morphological classifications proposed by different workers. Traditionally, I. sect. Rimosae was placed in I. subg. Inosperma (Kithner 1980, Kuyper 1986, Stangl 1989, Larsson & al. 2009). Larsson & al. (2009), who studied the phylogeny within I. sect. Rimosae using multigene DNA sequence analyses, found that I. sect. Rimosae comprised two strongly supported clades: /maculata and I. sect. Rimosae s. str. (sensu Larsson & al. 2009) The species in /maculata clustered with those in I. sect. Cervicolores and these two groups collectively represent I. subg. Inosperma s. str. Inocybe sect. Rimosae s. str. [corresponding with /pseudosperma of Matheny (2005)] has emerged as an independent clade, well distinguished from I. subg. Inosperma s. str. species of I. sect. Rimosae s. str. are characterized by the lack of metuloids and pleurocystidia and the presence of cylindrical to clavate cheilocystidia; smooth, radially appressed-fibrillose to rimose pilei; and smooth, elliptical to indistinctly phaseoliform basidiospores. In their recent revised classification, Matheny & al. (2019) recognize I. sect. Rimosae s. str. as an independent genus, Pseudosperma Matheny & Esteve-Rav., which we recognize here. During 2012-2014 we wished to analyze fungal communities in coniferous forests of Khyber Pakhtunkhwa, Pakistan, that host a highly diverse mycota including mycorrhizal and non-mycorrhizal species. Here we identify one species in Inocybaceae based on morphological and molecular analyses and reveal the phylogenetic relationships among taxa. Materials & methods Morphological analysis Basidiomata were collected from two administrative divisions of Pakistan. The first sampling site—Khanian, District Mansehra, Hazara Division, Khyber Pakhtunkhwa province—lies immediately south of the main Himalayan range with typical moist temperate climate and is dominated by Cedrus deodara along Pseudosperma flavorimosum sp. nov. (Pakistan) ... 185 with Abies pindrow (Royle ex D. Don) Royle, and Pinus wallichiana. (Siddiqui & al. 2013). The second site is Mashkun, a high mountainous region in the Swat district of Malakand division, Khyber Pakhtunkhwa province. The climate is dry temperate, and forests are dominated by C. deodara and P. wallichiana, with some A. pindrow also present (Champion & al. 1965). Basidiomata were collected and photographed in their natural habitat. Morphological characters were recorded from fresh specimens. Color codes follow Munsell (1975). Each collection was preserved using hot air dryers. Tissues from dried basidiomata were rehydrated in 5% KOH prior to anatomical observation under a Meiji MX4300h light microscope. Crush mounts were prepared and stained with Congo red. The abbreviation (n/m/p) represents n number of basidiospores measured from m number of fruit bodies and p number of collections. Basidiospores were measured in lateral view. The dimensions were recorded as (a—)b-—c(—d), where (a) = extreme minimum value, range b-c contains minimum of 90% of the calculated values and (d) = extreme maximum value, Q indicates |/w ratio of the spores and avQ = average Q of all spores. Other hyphal measurements are given as ranges. The examined specimens are deposited in the herbarium (LAH), Department of Botany, University of the Punjab, Quaid-e-Azam Campus, Lahore, Pakistan. Molecular analysis Genomic DNA was extracted and amplified according to White & al. (1990), Gardes & Bruns (1993), and Bruns (1995). The PCR products were purified and sequenced by Macrogen Inc. (Korea). The newly generated sequence was deposited in GenBank. Sequences generated using forward and reverse primers were combined in the BioEdit software version 7.2.5 (Hall 1999) to obtain a consensus sequence. A sequence homology search was conducted using the BLAST algorithm (http://www.ncbi.nlm.nih.gov) on 25 May 2019. Complete ITS sequences that showed the maximum similarity and sequences of the putative closest relatives of our species according to the published literature were included in the final matrix (Larsson & al. 2009, Kropp & al. 2013, Latha & Manimohan 2017, Liu & al. 2018, Matheny & al. 2019) to reconstruct phylogeny. Auritella foveata C.K. Pradeep & Matheny (GU062740) served as outgroup following Larsson & al. (2009) and Matheny & al. (2019). Multiple sequences were aligned using the online webPRANK at EMBL-EBI (https://www.ebi.ac.uk/goldman-srv/webprank/). Maximum likelihood analysis was performed using General Time Reversible model (Nei & Kumar 2000) in MEGA version 6 (Tamura & al. 2013) at 1000 bootstrap pseudoreplicates by finding best-fit substitution model. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.9359)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 0.0000% sites). 186 ... Jabeen & Khalid shit 52 KJ546158 Pseudosperma mimicum 400 tk cntion Pseudosperma mimicum KF056319 Pseudosperma mimicum FJ904124 Pseudosperma mimicum 96 -—— FJ904134 Pseudosperma arenicola Ge | FJ904133 Pseudosperma arenicola ido FJ904126 Pseudosperma ‘dulcamaroides' 66 KJ726737 Pseudosperma mimicum 60 JX630909 Pseudosperma ‘dulcamaroides' FJ904127 Pseudosperma '‘dulcamaroides' JQ408750 Pseudosperma breviterincamatum 100 JQ408753 Pseudosperma breviterincarnatum JQ408754 Pseudospermma breviterincarnatum JQ408751 Pseudosperma breviterincarnatum 78, JF908162 Pseudosperma squamatum 100 |' AM882780 Pseudosperma squamatum 56 | FJ904136 Pseudosperma squamatum FJ904132 Pseudosperma squamatum 56, JF908260 Pseudosperma spurium 100 | Fj904139 Pseudosperma spurium JQ408794 Pseudosperma spurium 1 Er Pseudosperma flavellum 80 JQ724026 Pseudospema flavellum JQ724025 Pseudosperma flavellum JQ724027 Pseudospema flavellum 100 , MF588965 Pseudospemma pakistanense '— MF575849 Pseudosperma pakistanense 100 , AM882769 Pseudosperma obsoletum 98 AM882770 Pseudosperma obsoletum 58 400 , AM882772 Pseudosperma perlatum AM882771 Pseudosperma perlatum 100; NR 153171 Pseudosperma luteobrunneum KX073581 Pseudospemma luteobrunneum 78 96> NR 153172 Pseudosperma brunneosquamulosum ge): NR 153173 Inocybe rubrobrunnea* | NR 152346 Pseudosperma araneosum Pseudosperma 100 | KJ729878 Pseudosperma araneosum 100 | KY440094 Pseudosperma keralense 96 NR 160442 Pseudosperma keralense — & NR 153169 Pseudosperma griseorubidum , MH216093 Pseudosperma sp. u 00! 1216091 Pseudosperma sp. MH216090 Pseudosperma sp. 100 ,MH578031 Pseudosperma sp. 7 98 MH212073 Pseudospema sp. 32 isl + KP636864 Pseudosperma sp. | _—— @ MG495391 Pseudosperma flavorimosum MH578027 Pseudosperma sp. 60 ; FJ904166 Pseudosperma umbrinellum 100 || FJ904163 Pseudosperma umbrinellum FJ904165 Pseudosperma umbrinellum oe Pseudosperma bulbosissimum 100 76 |'FJ904160 Pseudosperma bulbosissimum FJ904158 Pseudosperma bulbosissimum 66! 99 |) _FJ904159 Pseudosperma bulbosissimum | AM882777 Inocybe rimosa* JF908172 Inocybe rimosa* -- MH734760 Pseudosperma himalayense 88 + HG796995 Pseudosperma himalayense MH745138 Pseudosperma himalayense MH745140 Pseudosperma himalayense HQ604610 Pseudosperma sororium 100 |HQ604617 Pseudosperma sororium HQ604618 Pseudosperma sororium HQ604607 Pseudosperma sororium AM882844 Inocybe rimosa* 94 | |! AM882761 Inocybe rimosa* 6 AM882762 Inocybe rimosa* FJ904147 Inocybe rimosa* 92, FJ904148 Pseudosperma melliolens | FJ904149 Pseudosperma melliolens | 100 FJ936169 Inosperma quietiodor | 90 —___— KY616964 /nosperma shawarensis ol JQ408762 Inosperma lanatodiscum 100 ; MH578017 Inosperma maculatum 'MH578013 Inosperma maculatum GU062740 Auritella foveata ] 72 Inosperma Out group '———— 0.05 Fic. 1. Molecular phylogenetic analysis of Pseudosperma flavorimosum based on ITS sequences. Sequence generated during this study is marked by @. Scale bar = nucleotide substitutions per site. The names of the taxa are written according to Matheny & al (2019). Those represented in quotes lack type specimens. * represents taxa that are not discussed in Matheny & al (2019) and given names according to their publication source and GenBank record. Pseudosperma flavorimosum sp. nov. (Pakistan) ... 187 Phylogeny The NCBI BLAST search revealed that the Pseudosperma flavorimosum sequence (MG495391) shared 89-90% identity with unidentified “Inocybe sp. sequences (MH578031, MH578027, MH212073) from USA. Closely related ITS sequences were retrieved from GenBank for generating a phylogeny for the Pakistani taxon based on 77 nucleotide sequences. The final ITS matrix comprised a total of 1403 positions of which 734 were conserved, 442 were variable, 337 were parsimony informative, and 99 were singletons. The P. flavorimosum sequence forms a sister clade with unidentified Inocybe sequence KP636864, and both sequences clustered on the same branch with two more unidentified sequences (MH212073, MH578031) in the same clade as unidentified Pseudosperma sequence MH578027 with 82% bootstrap support and were separated from the clade containing P bulbosissimum (Kihner) Matheny & Esteve-Rav., P. himalayense (Razaq & al.) Matheny & Esteve-Rav., P. melliolens (Kihner) Matheny & Esteve-Rav., P. rimosum (Bull.) Matheny & Esteve-Rav., P. sororium (Kauffman) Matheny & Esteve- Rav., and P. umbrinellum (Bres.) Matheny & Esteve-Rav. Our phylogenetic tree (Fic. 1) places the above Pseudosperma species in the same relationships as shown in Larsson & al. (2009, there as Inocybe) and Matheny & al. (2019). Pseudosperma flavorimosum Jabeen & Khalid, sp. nov. FIGs. 2, 3 MB 823494 Differs from Pseudosperma sororium by its smooth ellipsoidal to slightly amygdaliform basidiospores with a broad base and narrow apex. Type: Pakistan. Khyber Pakhtunkhwa province: Hazara division, Mansehra district, Kaghan valley (Khanian), 2500 m a.s.l., on soil under Pinus wallichiana A.B. Jacks., 3 Aug 2014, Muhammad Burhan SJ103 (Holotype, LAH35042; GenBank: MG495391). EryMmo.ocy: The specific epithet (Lat.) refers to the yellowish and rimose pileus. Piteus 45-5.1 mm wide, initially conico-convex with a prominent umbo, becoming broadly conico-convex with a prominent umbo at maturity, umbo conical when young, becoming more obtuse in maturity, surface yellowish brown (10YR8/8) with dark brown (10R4/12) fibrillose streaks, dry, fibrillose, radially rimose towards the margin, margin incurved. LAMELLAE adnate to adnexed, moderately close, <2.5 mm deep, narrow, initially yellowish brown (10YR7/8), becoming dark brown (10R6/14) at maturity, edges fimbriate. LAMELLULAE in different lengths. Stripe 75 x 7 mm, central, cylindrical, narrower towards the apex and wider towards the base, apex furfuraceous, base splitting, surface dry, context off white under a yellowish (10YR8/8) to dark brown (5YR2/6) cuticle. 188 ... Jabeen & Khalid BASIDIOSPORES [40/2/2] (7.8—)9.5-12.8(-13.31) x (5.7—)6.3-7.8(-8.9) um, Q = (1.21-)1.39-1.91(-1.98), avQ = 1.62, elliptical, amygdaliform with broad apex and narrow base, smooth, uniguttulate. Basrp1a (14.8-) 26.4-31.2(-36.7) x (10.0-)10.5-10.8(-11.8) um, clavate, 4-spored, thin- walled, guttulate. CHEILOCYSTIDIA (18.5—)19.2—21.7(-25.3) x (7.5-)7.7-8.6 (-9.1) um, broadly clavate, thin-walled, frequently arranged on lamellar edges. PLEUROCYSTIDIA absent. PILEIPELLIS hyphae (3.8—)4.9-6.3(-6.6) um diam., intricate trichoderm, non-encrusted, frequently septate, filamentous, fusoid terminals, clamped septa common, hyaline. StiprriPELLis hyphae (4.5-)4.7-8.7(-9.2) um diam., trichoderm, septate, filamentous, rarely branched, clamp connections not observed. ECOLOGY & DISTRIBUTION—among conifers in Himalayan moist and dry temperate forests; known only from Pakistan. ADDITIONAL SPECIMEN EXAMINED—PAKISTAN. KHYBER PAKHTUNKHWA PROVINCE: Malakand division, Swat district, Mashkun, 2500 m a.s.l., on soil under Cedrus deodara (Roxb. ex D. Don) G. Don, 5 Sep 2013, Sana Jabeen MTI (LAH35043). Discussion Pseudosperma flavorimosum is characterized by its yellowish brown pileus with dark brown striations and radially rimose margins and a stipe with a rimose base and losing its stipitipellis over time. It is characterized with smooth ellipsoidal to slightly amygdaliform basidiospores with a broad base and narrow apex and broadly clavate cheilocystidia. ITS sequence analysis clusters P flavorimosum with other Pseudosperma species. Taxa within the genus separated into several clades, with P flavorimosum clustering in subclade A. Pseudosperma melliolens, described from France, also looks like a typical P. rimosum (Bull.) Matheny & Esteve-Rav. (Bon 1997). Pseudosperma flavorimosum shares morphological similarities with P. sororium, as originally described by Kauffman (1924). But the elliptical or elongate-ellipsoid basidiospores of P. sororium are not truly subreniform, sub-inequilateral and are obtuse at both ends, distinguishing from P. flavorimosum. Pseudosperma himalayense, a recently described taxon from Pakistan, differs morphologically from P. flavorimosum by its paler pileus (varying from white to cream, pale yellow, olive yellow, and light brown to camel brown) with dentate margins, ellipsoid to slightly amygdaliform basidiospores, and clavate to cylindrical cheilocystidia (Liu & al. 2018). Pseudosperma umbrinellum bears yellowish to reddish brown pileus with a dark centre and strongly rimose and lighter periphery while P. flavorimosum Pseudosperma flavorimosum sp. nov. (Pakistan) ... 189 os \ bie = + sate Fic. 2. Pseudosperma flavorimosum (LAH35042, holotype). Basidiomata. Scale Photos by Sana Jabeen bars = 1 cm. 190 ... Jabeen & Khalid 0 i A Q Ds —_— Le Fic. 3. Pseudosperma flavorimosum (LAH35042, holotype). A. Basidiospores; B. Cheilocystidia; C. Basidia; D. Stipitipellis; E. Pileipellis. Scale bars = 10 um. Drawings by Sana Jabeen. Pseudosperma flavorimosum sp. nov. (Pakistan) ... 191 bears yellowish pileus with brown striations, but the anatomical features are identical to P. rimosum. The taxa adjacent to P. flavorimosum in the phylogenetic tree are undescribed morphologically but form separate lineages within subclade A of Pseudosperma with a strong bootstrap value supporting P. flavorimosum as a distinct taxon. Acknowledgments This work was financially supported by Higher Education Commission (HEC)- Pakistan under Indigenous PhD Fellowship. Sincere thanks to Dr. K.P. Deepna Latha (Fungal Diversity Division, Department of Botany, University of Calicut, Kerala, India) for valuable comments and suggestions, which greatly improved the manuscript. Thanks are also due to Dr. Arooj Naseer (Centre for Undergraduate Studies, University of the Punjab, Lahore, Pakistan) for also making corrections as presubmission reviewer. We are also grateful to Dr. Abdul Rehman Khan Niazi and Dr. Najam ul Sehar Afshan, Muhammadah Khalid, Abdul Rehman, Muhammad Burhan, Fatima-tu-Zahra, Muhammad Umar Khan Niazi, and all laboratory fellows for accompanying the field trips to different areas of Pakistan. Literature cited Ahmad §S, Iqbal SH, Khalid AN. 1997. Fungi of Pakistan. Sultan Ahmad Mycological Society of Pakistan, Lahore. 1-248 pp. Bon M. 1997. Clé monographique du genre Inocybe (Fr.) Fr. Documents Mycologique 27: 1-51. Bougher NL, Matheny PB. 2011. Two species of Inocybe (Fungi) introduced into Western Australia. Nuytsia 21: 139-148. Bougher NL, Matheny PB, Gates GM. 2012. Five new species and records of Inocybe (Agaricales) from temperate and tropical Australia. Nuytsia 22: 57-74. Braaten CC, Matheny PB, Viess DL, Wood MG, Williams JH, Bougher NL. 2014. Two new species of Inocybe from Australia and North America that include novel secotioid forms. 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MY COTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 January-March 2020— Volume 135, pp. 195-201 https://doi.org/10.5248/135.195 Three Rinodina species new to China X1A0-JIA ZHENG & QIANG REN* College of Life Sciences, Shandong Normal University, Jinan 250014, China " CORRESPONDENCE TO: rendagiang@hotmail.com ABSTRACT—Rinodina ascociscana, R. subminuta, and R. trevisanii are reported as new to China. A detailed description of each species is provided, and comparisons with related species are made. Key worps—Ascomycota, lichenized fungi, Physciaceae Introduction Rinodina (Physciaceae, Caliciales), which comprises more than 300 species worldwide (Sheard & al. 2017), occurs on rock, bark, mosses, soil, or debris and is characterized by a crustose thallus, mostly lecanorine apothecia with brown to black discs, a hyaline hymenium, a red-brown or (rarely) blue-grey epihymenium, clavate asci, and brown, primarily 1-septate (rarely 3-septate) to submuriform ascospores with + unequally thickened walls (Mayrhofer & Moberg 2002; Sheard 2004, 2010). Although many have recently contributed to crustose lichen diversity in China (e.g., Kou & al. 2013, Ren & Zhao 2014, Ren 2017, Ren & al. 2018, Zhou & Ren 2018), knowledge of Rinodina species remains poor in this country. Forty-five species and three forms have been reported from China (e.g., Mayrhofer 1984; Wei 1991; Abdulla & Wu 1994, 1998; Aptroot & Seaward 1999; Aptroot & Sipman 2001; Aptroot & Sparrius 2003; Joshi & al. 2014; Kondratyuk & al. 2016; Sheard & al. 2017). During a survey of Rinodina specimens, we detected three species new for China: Rinodina ascociscana, R. subminuta, and R. trevisanii. 196 ... Zheng & Ren Materials & methods The specimens examined in this study were housed in SDNU (the Plant Herbarium of Shandong Normal University, Jinan, China) and HMAS-L (Herbarium of Mycology, Chinese Academy of Sciences, Beijing, China). Morphological and anatomical characters were observed using an Olympus SZ51 stereo microscope and an Olympus CX21 light microscope. Lichen substances were detected by thin layer chromatography (TLC) with solvent systems A, B,, and C (Elix 2014). Photos were taken using Olympus SZX16 and BX61 microscopes with a DP72 digital camera. We identified the specimens primarily from Sheard & al. (2017). Species Rinodina ascociscana (Tuck.) Tuck., Gen. Lich.: 124 (1872). FIG: THALLUS crustose, thick, well-developed, surface continuous, ochraceous to brownish; prothallus absent; vegetative propagules absent. APOTHECIA lecanorine, not erumpent, 0.5-1.2 mm in diam, scattered, sessile, narrowly attached; pisc brown to black, epruinose, persistently plane, thalline margin concolourous with thallus, often with radially cracked, sometimes becoming flexuous; excipular ring present, confluent, prominent, darker than thalline margin. THALLINE EXCIPLE 50-80 um wide; proper exciple hyaline, 15-25 um wide; HYMENIUM hyaline, 80-110 um high, paraphyses 2-3 um in diam.; EPIHYMENIUM red-brown, 10-20 um high; HYPOTHECIUM hyaline, 20-40 um deep. Asci clavate, 8-spored; ASCOSPORES brown to dark brown, type A development, Physcia-type, ellipsoid, 1-septate, 27.5-35 x 12.5-15 um, torus absent, ornamented walls not evident. PYCNIDIA not seen. CHEMISTRY—No lichen substances detected by TLC. SPECIMENS EXAMINED: CHINA. JiLin: Antu County, c. 30 km from Erdaobaihe town toward Changbaishan mountain and lake, alt. c. 1100 m, on bark of conifer tree, 2011.09, Sohrabi with Ghobad-Nejhad 16644 (HAMS-L 120853); Helong City, Mt. Zengfeng, alt. 1600 m, on bark of deciduous trees, 2011.08.19, Y.L. Cheng 20119406A, 20119440B, 20119844 (SDNU). SUBSTRATE & DISTRIBUTION—Rinodina ascociscana occurs on bark of conifers or deciduous trees in mixed forests with Acer, Abies, and Larix. It was previously known from North America (Sheard 2010) and north-eastern Asia (Japan, Siberia, and South Korea; Sheard & al. 2017); new from China. CoMMENTS—Our material closely matched the descriptions of Sheard (2010) and Sheard & al. (2017). Rinodina ascociscana is characterized by the often radially cracked thalline margin, the large Physcia-type ascospores Rinodina spp. new to China... 197 Fic. 1. Rinodina ascociscana (SDNU, Cheng 20119844). A. Thallus with substratum; B. Apothecia showing radially cracked thalline margin; C. Section of apothecium; D. Immature Physcia-type ascospore; E. Physcia-type ascospore mature. Scale bars: A = 1 mm; B = 0.5 mm; C = 100 um; D, E= 10 pm. with type A development, and the absence of lichen substances. Rinodina dolichospora Malme differs by its thalline margin that is not radially cracked and Pachysporaria-type I ascospores (Sheard 2010). Rinodina subminuta H. Magn., Bot. Not. 1947: 44 (1947). FIG. 2 THALLUS crustose, thin to endophloeodal, continuous to rimose, surface matte or glossy, yellowish or light to dark grey; prothallus absent; vegetative propagules absent. APOTHECIA lecanorine, erumpent; pisc black, epruinose, plane, sometimes convex, 0.4-0.65 mm in diam; thalline margin concolourous with thallus, excipular ring absent. THALLINE EXCIPLE 50-80 um wide; proper exciple hyaline, 15-25 um wide; HYMENIUM hyaline, 60-80 um high, paraphyses 2.5-4.5 um in diam.; EPIHYMENIUM red-brown; HYPOTHECIUM hyaline, 30-60 um deep. Asci clavate, 8-spored, 45-70 x 20-30 um; 198 ... Zheng & Ren Fic. 2. Rinodina subminuta (HMAS-L 073131). A. Thallus with substratum; B. Section of apothecium; C. Mature ascospore. Scale bars: A = 0.5 mm; B = 50 um; C = 10 um. ASCOSPORES brown, type A development, ellipsoid, 1-septate, Physcia- type, 17-20(-22.5) x 8-9.5(-10) um, torus present, ascospore walls not ornamented. PYCNIDIA not seen. CHEMISTRY—Zeorin, + one unknown substance with UV+ blue (TLC: A=f, B=7,.C=7): SPECIMENS EXAMINED: CHINA. JILIN: Mt. Changbai, Tianchi, alt. 1850 m, on Betula ermanii Cham. (Betulaceae), 1984.08.14, X.D. Lu 848410-1 (HMAS-L 0141286); Hot spring, on Betula ermanii, 1994.08.06, Wei & al. 94423 (HMAS-L 073131); South Slope, alt. 1900 m, on Betula ermanii, 1983.08.03, J.C. Wei & J.B. Chen 6586-1 (HMAS-L 0141288); on Betula ermanii, 2003.8.20, H.M. LiCBS114-1 (HMAS-L 0141273); North Slope, alt. 1810 m. on Sorbus sp. (Rosaceae), 2018.08.19, Q. Ren 7119, 7120 (SDNU); on Betula ermanii, 2018.08.19, Q. Ren 7029, 7040 (SDNU). SUBSTRATE & DISTRIBUTION— Rinodina subminuta occurs on the bark of Betula ermanii and Sorbus sp. in open sites of deciduous forests. It was previously known from North America and Siberia (Sheard 2010), and from north-eastern Asia (Japan, Siberia, and South Korea; Sheard & al. 2017); new from China. Rinodina spp. new to China... 199 COMMENTS—Otur material closely matches the description in Sheard (2010). Rinodina subminuta is characterized by a thin thallus, erumpent apothecia, Physcia-type ascospores with type A development, and the presence of zeorin. Rinodina orientalis Sheard differs by strongly convex discs of mature apothecia, smaller ascospores (16-18 x 7-8.5 um), and the absence of lichen substances (Sheard & al. 2017). Rinodina trevisanii (Hepp) K6rb., Parerga Lichenol.: 70 (1859) FIG. 3 THALLUS crustose, very thin, surface continuous to rimose, brownish; prothallus absent; vegetative propagules absent. APOTHECIA lecanorine, not erumpent, 0.4-0.6 mm in diam, sessile, broadly or narrowly attached; pisc brown to black, epruinose, plane, frequently becoming convex, thalline margin concolourous with thallus, often becoming excluded or margin biatorine, excipular ring not prominent. THALLINE EXCIPLE 50-70 um wide; proper exciple hyaline, c. 15 um wide; HYMENIUM hyaline, 65-80 um high, paraphyses 1.5-2.5 um in diam.; EPIHYMENIUM red-brown, 10-30 um high; HyPOTHECIUM hyaline, 30-110 um deep. Asc clavate, 8-spored, 50-60 x 17.5-30 um; ASCOSPORES brown, type A development, Physconia-type, ellipsoid, 1-septate, 18-—22.5(-23.5) x 8-9.5(-10) um, torus sometimes present but not prominent, ascospore walls not ornamented. PYcNIDIA not seen. CHEMISTRY—+Zeorin. SPECIMENS EXAMINED: CHINA. HEILONGJIANG: Heihe City, Mt. Xifeng, alt. 220 m, on bark, 2009.08.17, Q. Ren 1192 (SDNU); Mohe City, Mt. Guanyinshan, alt. 554 m, on bark, 2009.08.13, Q. Ren 1179 (SDNU); Tahe City, Mt. Baikalu, alt. 800 m, on bark, 2011.08.25, Y.L. Cheng 20125237B (SDNU); Daxing/anling, alt. 800 m, on tree trunk, 1984.08.07, X.Q. Gao 105 (HMAS-L 072400). Jrt1n: Antu City, Mt. Changbai, alt. 1750 m, on Larix sp. (Pinaceae), 1984.08.28, X.D. Lu 848421-5 (HMAS-L 076349); North Slope of Mt. Changbai, alt. 1810 m. on Betula ermanii, 2018.08.19, Q. Ren 7118 (SDNU). SUBSTRATE & DISTRIBUTION— Rinodina trevisanii occurs on bark of Larix sp., Betula ermanii, and other trees in mixed forests. It was previously known from Europe, Asia (Caucasus, Turkey, Siberia), western North America (Mayrhofer & Sheard 2007, Sheard 2010), Kazakhstan (Hauck & al. 2013a), and western Mongolia (Hauck & al. 2013b); new from China. COMMENTS—Our material closely corresponds with the description of Sheard (2010). Rinodina trevisanii is characterized by a thin thallus, mostly scattered apothecia with frequently convex discs and margins often biatorine, and Physconia-type ascospores with type A development. Rinodina archaea 200 ... Zheng & Ren Fic. 3. Rinodina trevisanii (HMAS-L 076349). A, B. Thallus with substratum; C. Immature ascospores; D. Mature ascospore. Scale bars: A = 1 mm; B = 0.5 mm; C, D = 10 um. (Ach.) Arnold differs by its relatively thick, typically areolate thallus, often compressed (hence angular) apothecia, persistently plane discs of mature apothecia, and larger ascospores (Mayrhofer & Sheard 2007). Acknowledgments HMAS-L is thanked for loan of specimens. We are very grateful to Prof. H. Mayrhofer (Institute of Biology, University of Graz, Austria) and Dr. S.Y. Guo (State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China) for reviewing the manuscript. 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Mycotaxon 133: 71-77. https://doi.org/10.5248/133.71 MY COTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 January-March 2020—Volume 135, pp. 203-212 https://doi.org/10.5248/135.203 Gymnopus barbipes and G. dysodes, new records for Pakistan MALKA SABA", JUNAID KHAN’, SAMINA SARWAR}, HASSAN SHER’, ABDUL NASIR KHALID‘ “Department of Plant Sciences, Quaid-i-Azam University, Islamabad, 45320, Pakistan *Center for Plant Sciences and Biodiversity, University of Swat, Swat, Pakistan *Department of Botany, Lahore College for Women University, Lahore, Pakistan ‘Department of Botany, University of the Punjab, Quaid-e-Azam Campus, Lahore, 54590, Pakistan *CORRESPONDENCE TO: rustflora@gmail.com; msaba@qau.edu.pk AsBstTRACT— Morphological and phylogenetic analyses were conducted to identify Gymnopus species collected in Pakistan during 2013-14. Phylogenetic analysis was performed using internal transcribed spacer region (ITS) of ribosomal DNA sequences. Among the collected taxa, we identified Gymnopus barbipes and G. dysodes, represent new records for Pakistan. Their detailed descriptions and illustrations are also provided. Key worps —Khyber Pakhtunkhwa, Omphalotaceae, pine forests Introduction Gymnopus (Pers.) Roussel (Omphalotaceae, Agaricales) is a large fungal genus, which consists of c. 300 species distributed worldwide (Jang & al. 2016). It is characterized by a collybioid basidioma; a convex to applanate or slightly concave pileus; free, emarginate or adnate and crowded to fairly distant lamellae; a central stipe; a white spore print; inamyloid or non- dextrinoid hyphae with clamp connections; ellipsoid to oblong, thin-walled, hyaline, inamyloid basidiospores. Cheilocystidia are often present while pleurocystidia are usually absent (Antonin & al. 2013, Jang & al. 2016, Saba & Khalid 2014). Seven species of Gymnopus—worldwide a relatively common 204 ... Saba & al. saprobe on leaf litter and dead wood—have been reported in Pakistan (Ahmad 1980, Iqbal & Khalid 1996, Shibata 1992, Sultana & al. 2011, Saba & Khalid 2014). In view of the high worldwide diversity, we would expect the diversity of Pakistani Gymnopus to be much greater than currently reported. To explore the diversity of indigenous Gymnopus spp., we collected specimens in Pakistan, which we identified through morphological and phylogenetic analyses. Here we report Gymnopus barbipes and G. dysodes as new records for Pakistan. Materials & methods Morphological evaluation Basidiomata were collected, photographed, characterized morphologically, dried, and deposited in the University of Swat Herbarium (SWAT) in Pakistan and Farlow Herbarium of Harvard University in USA (FH). Color designations are from Munsell (1975). Microscopical observations were made from slide preparations from dried specimens mounted in 5% potassium hydroxide (KOH) or Melzer’s reagent under a Meiji Techno MX4300H biological microscope. Dimensions of anatomical features (basidiospores, basidia, cystidia, hyphae) were calculated from at least 25 measurements using an ocular micrometer and 100x oil-immersion objective with x = arithmetic means of spore lengths and widths for all spores measured. Line drawings were made with a camera lucida. DNA extraction, PCR amplification, DNA sequencing Genomic DNA was extracted from a small piece of basidioma by a modified CTAB method (Lee & al. 1988) using the Qiagen DNeasy Plant Mini Kit (cat. no. 69104). The entire rDNA internal transcribed spacer region (ITS = ITS1+5.8S+ITS2) was targeted with primer pair ITS1F/ITS4 (White & al. 1990, Gardes & Bruns 1993) and then amplified using Econo Taq DNA Polymerase following the PCR cycling parameters: initial denaturation (94 °C for 1 min), 35 cycles (94 °C for 1 min, 53 °C for 1 min, 72 °C for 1 min), and final extension (72 °C for 8 min). The PCR amplicons were sent to Macrogen (Republic of Korea) for purification and bidirectional sequencing. Sequence alignment & phylogenetic analysis The ITS sequence of MSM0034 comprised 700 base pairs and that of ING-21 comprised 691 base pairs. We retrieved closely related ITS sequences via a BLAST search of GenBank for subsequent phylogenetic analysis, according to Saba & Khalid (2014). Sequences used in phylogenetic construction are shown in TABLE 1. Sequences were manually edited and assembled using BioEdit (www.mbio. ncsu.edu/bioedit/bioedit.html). Following Dentinger & al. (2011) for complete ITS sequences, all sequences were trimmed with the conserved motifs 5’-(...GAT) CATTA- and -GAccT (CAAA...)-3’ with the enclosed aligned portions analysed. Sequences of Gymnopus spp. new for Pakistan ... 205 TABLE 1: Gymnopus and Omphalotus ITS sequences analyzed. New sequences in bold. TAXON COUNTRY GENBANK # COLLECTION/ VOUCHER # G. barbipes Pakistan MK450334 MSM 0034 USA KJ416265 TENN 69173 USA KJ416266 TENN 69173 USA KJ416269 TENN 67858-T G. biformis Costa Rica DQ450064 TFB7843 Costa Rica DQ450063 TFB7820 G. confluens Russia AY256697 TENNS58242 — HM240527 UBC F19677 G. cylindricus Costa Rica AY256696 TENNS58024 Costa Rica DQ450057 TFB10091 G. dryophilus Italy JX536157 BRNM707149 G. dysodes Pakistan MT114698 SWAT 001355 USA AF505778 TENN 59141 USA KY026666 TENN F-61125 Czech Republic JX536158 BRNM712600 G. erythropus Slovakia DQ449996 SAV X12002 USA DQ449998 WTU JFA12910 G. fibrosipes Costa Rica AF505763 TENNS56660 = AY842953 PR23TN G. fusipes France AY256710 TENNS59217 Austria AF505777 TENN59300 G. gibbosus — AY 263437 AWW66 — AY 263438 AWW95 G. impudicus Russia KJ416263 TENN 60094 Russia KJ416264 Culture G. luxurians Pakistan KF803760 MSM 001 Pakistan KF803761 MSM 002 USA AY256709 TENN57910 Switzerland DQ450022 TENNS50619 G. menehune Pakistan KF803762 MSM 003 _— JN182864 _— — AY263426 DED5866 G. mesoamericanus Costa Rica DQ450036 TENN58106 Costa Rica AF505768 NYBG REH7379 G. polygrammus Puerto Rico DQ450028 TENN56589 = AY 842954 PR 2542TN G. readiae New Zealand DQ450034 TENN53687 New Zealand HQ533036 PDD95844 G. subcyathiformis Puerto Rico DQ450041 TENNS58130 Dominican Republic DQ450042 TENN59550 G. subpruinosus USA DQ450026 TENNS6242 USA DQ450027 TENN59477 O. illudens USA AY313271 TENN54507 O. olearius France AY313277 Culture 9061b O. olivascens USA AY313281 TENN55337 206 ... Saba & al. Omphalotus spp. were used as outgroup based on results reported by Moncalvo & al. (2002) and Mata & al. (2004). We aligned our new sequences with GenBank sequences from related taxa using ClustalX (Thompson & al. 1997) and manually edited where necessary using BioEdit (Hall 1999). A maximum likelihood analysis was performed via CIPRES Science Gateway (Miller & al. 2010) employing RAxML-HPC v.8 and Rapid bootstrap analysis/search for best scoring, with the topology assessed by 1000 bootstrap replicates. Taxonomy Gymnopus barbipes R.H. Petersen & K.W. Hughes, N. Amer. Fung. 9(3): 2 (2014). Fic. 1 Pileus 25 mm diam., broadly convex, expanding with age to plane, flat, thin; margin straight; surface matte, smooth; central disc brownish orange (5YR5/8) to strong brown (5YR4/6) or light brown (5YR8/8). Lamellae adnexed or free, subdistant, light orange (2.5YR8/8), edges even, concolorous. Lamellulae 1-2 tiered. Stipe 40 x 5 mm, central, equal, cylindrical, hollow, sheathed in a thin, off-white, felty covering, brownish orange (5YR5/8). Odor & taste not distinctive. Basidiospores 5-7 x 2.3-4 um [x = 5.6 x 3.0 um, Q = 1.2-1.6], ellipsoid in profile view, slightly flattened adaxially, rarely ovoid, smooth, thin- walled, aguttulate, hyaline in KOH, inamyloid. Basidia 23-28 x 5-6 um, clavate, 4-spored (rarely 2-spored) basidia, thin-walled, hyaline in KOH; sterigmata 3-3.7 um. Basidioles subclavate, abundant. Pleurocystidia absent. Cheilocystidia abundant, sometimes constricted at neck. Pileipellis a cutis, repent hyphae cylindrical, 5-14 um, thin-walled, hyaline in KOH. Stipe hyphae cylindrical, 5-9 um, non-encrusted, hyaline in KOH. Caulocystidia cylindrical, knobby, abundant, hyaline, thin-walled, 9-14 um in diam. Clamp connections present in all tissues. MATERIAL STUDIED: PAKISTAN, KHYBER PAKHTUNKHWA, Shangla, Yakh Tangay, under Pinus wallichiana A.B. Jacks., 2 September 2013, Malka Saba & Abdul Nasir Khalid, MSM#0034 (FH00304574; GenBank MK450334). CoMMENTS—Our Pakistani specimen agrees morphologically with the original protologue of G. barbipes except for its broader pileus (cited as 5-15 mm diam. in Petersen & Hughes 2014), robust stipe, and presence of ovoid basidiospores in addition to ellipsoid basidiospores. Gymnopus barbipes was described from southeastern North America and characterized by its occurrence on leaf litter and absence of well-developed pleurocystidia and cheilocystidia (Petersen & Hughes 2014). Our Pakistani collection Gymnopus spp. new for Pakistan ... 207 Fic. 1. Gymnopus barbipes (MSM 0034). A, B. Basidiomata; C. Basidia; D. Cheilocystidia; E. Basidiospores; F. Caulocystidia; G. Pileipellis. Scale bars: A, B = 10 mm; C, D = 10 um; E=5 um; KR G= 25 um. 208 ... Saba & al. represents the first occurrence of G. barbipes reported since the Tennessee collection was described. Gymnopus dysodes (Halling) Halling, Mycotaxon 63: 364 (1997). Fia. 2 = Collybia dysodes Halling, Mycol. Mem. 8: 79 (1983). Pileus 15-30 mm across, conic to campanulate at first, later plano-convex to plane, color light pinkish brown (5YR 6/4) to pale reddish brown (SYR 4/4) with a darker (7.5YR 1/2) disc, surface dry, dull, disc smooth, otherwise sulcate, pileus margin crenate or eroded in some specimens, context thin, 1-2 mm at the disc,