MYCOTAXON THE INTERNATIONAL JOURNAL OF FUNGAL TAXONOMY & NOMENCLATURE VOLUME 137 (1) JANUARY-MARCH 2022 \oy Abas ~~ t 4 te 1 Na q % ” 2 Tet Ty "> Wats g as Fae 2 Claviceps bavariensis sp. nov. (Liu, Tanaka, Kolarik— Fie. 3, p. 81) ISSN (PRINT) 0093-4666 https://doi.org/10.5248/137-1 ISSN (ONLINE) 2154-8889 MYXNAE 137(1): 1-172 (2022) EDITORIAL ADVISORY BOARD ELSE C. VELLINGA (2019-2022), Chair Berkeley, California, U.S.A. KAREN HANSEN (2014-2021), Past Chair Stockholm, Sweden XINLI WEI (2019-2023) Beijing, China Topp W. 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 2022 VOLUME 137 (1) http://dx.doi.org/10.5248/137-1 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 MyYcoTAxoONn, LTD. © 2022 www.mycotaxon.com & www.ingentaconnect.com/content/mtax/mt P.O. BOX 264, ITHACA, NY 14581-0264, USA IV ... MYCOTAXON 137(1) MY COTAXON VOLUME ONE HUNDRED THIRTY-SEVEN (1) — TABLE OF CONTENTS ROVIC WEISS 5 sick ite hn ale OE 6 OEP hte note FORE wards ob obi ehete » vi Nomenclatural novelties Oty pificanens sch bey nin soo ten pie has She vii COPFICON OG gu arte chert ithe potter ce cap shee te ob cae SEES kg bel ateak ene ee viii PROMI UOEAIOF va Gi & cooks 32 Soke SARL Bath Bee oR PPE EE cok bos ix BOD NSO MISSTONEDTOCR AINE: price pes oe sack cee PAC ACR OM GER EL xi NEW GENERA & SPECIES Phaeocollybia chefensis sp. nov. and new synonyms for P rifflipes, P. rufotubulina, and P. tibiikauffmanii LORELEI L. NORVELL, RONALD L. EXETER, MATTHEW GORDON, SAHRA- TAYLOR MULLINEUX, SCOTT A. REDHEAD Beltrania shenzhenica sp. nov. from Guangdong Province, China ZHAO-XUE ZHANG, TAI-CHANG Mu, ZHUANG LI, XI1U-GUO ZHANG, JI-WEN XIA Termitomyces cryptogamus sp. nov. associated with Macrotermes natalensis in Africa LENNART J.J. VAN DE PEPPEL, Z. WILHELM DE BEER, Duur K. AANEN, BEN AUXIER Clitopiloides prati and Trichopilus lecythiformis spp. nov. from Australia Davip L. LARGENT & MOLLy B. CRIBARI Neoacrodictys elegans gen. & sp. nov. from Hainan Province, China JI-WEN X14, TaI-CHANG Mu, ZHAO-XUE ZHANG, ZHUANG LI, XIU-GUO ZHANG Neotypification of Claviceps humidiphila and recognition of C. bavariensis sp. nov. M1ao Liu, Ey1 TANAKA, MIROSLAV KOLARIK NEW COMBINATION Passalora golaghati comb. nov. from India GARGEE SINGH, SANJAY YADAV, RAGHVENDRA SINGH, SHAMBHU KUMAR 31 4] 51 63 73 89 JANUARY-MARCH 2022... V NEW RANGES/HOSTS Stigmatomyces aff. limnophorae on dipteran hosts in Peninsular Malaysia NATASHA AZMI Nour ALIAH, JINGYU LIv, NuRUL AZMIERA, CHONG CHIN HEO 95 Pluteus variabilicolor and Volvopluteus earlei, new records for Pakistan JUNAID KHAN, HASSAN SHER, AIMAN IZHAR, MUHAMMAD HAQNAWAZ, ABDUL NASIR KHALID 109 New Turkish records of Hebeloma excedens and H. geminatum, and confirmation of H. celatum AYTEN D1zkIRICI, AYSENUR KALMER, ISMAIL ACAR 123 Ganoderma multipileum and Tomophagus cattienensis— new records from Pakistan AISHA UMAR, SHAKIL AHMED, LauRA GUZMAN-DAVALOS, MILAY CABARROI-HERNANDEZ 135 Marasmius tageticolor and M. tucumanus from the Dominican Republic Nico.tAs NIVEIRo, NATALIA A. RAM{REZ, CLAUDIO ANGELINI 153 MycosBiotTa (FUNGA) NEW TO THE MYCOTAXON WEBSITE New records of Chaetomium and Chaetomium-like species (Ascomycota, Chaetomiaceae) on Syagrus coronata from the Raso da Catarina Ecological Station (ESEC), Caatinga, Bahia, Brazil (SUMMARY) NILO GABRIEL SOARES FORTES & NADJA SANTOS VITORIA 171 VI ... MYCOTAXON 137(1) REVIEWERS — VOLUME ONE HUNDRED THIRTY-SEVEN (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. Mustafa Emre Akcay Joe Ammirati Flavia Rodrigues Barbosa Timothy J. Baroni Sarah Bergemann R.E Castaneda Ruiz Tobias Guldberg Froslev Danny Haelewaters Egon Horak Alfredo Justo Abdullah Kaya Rachel A. Koch N’golo Abdoulaye Koné Jian Ma Li-Guo Ma Brandon Matheny Roger Fagner Ribeiro Melo Nelson Menolli Jr. Lorelei L. Norvell Jadson José Souza de Oliveira Shaun R. Pennycook Walter P. Pfliegler Mario Rajchenberg Scott A. Redhead Gerardo Robledo Michelline Lins Silvério Adna Cristina Barbosa de Sousa Joey B. Tanney JANUARY-MARCH 2022... VII NOMENCLATURAL NOVELTIES AND TYPIFICATIONS PROPOSED IN MYCOTAXON 137(1) Beltrania shenzhenica Z.X Zhang, J.W. Xia & X.G. Zhang [MB 839268], p. 36 Claviceps bavariensis M. Kolarik, E. Tanaka & M. Liu [MB 838352] p. 80 Claviceps humidiphila Pazoutova & M. Kolarik 2015 (neotypified) [MBT 395372], p. 78 Clitopiloides prati Largent [IF 558177], p. 53 Neoacrodictys J.W. Xia & X.G. Zhang [MB 816515], p. 66 Neoacrodictys elegans J.W. Xia & X.G. Zhang [MB 816516], p. 66 Passalora golaghati (Saikia & Sarbhoy) Gargee Singh, Sanj. Yadav, Raghv. Singh & Sh. Kumar [MB 835579], p. 90 Phaeocollybia chefensis Norvell & Exeter [IF 559441], p. 12 Termitomyces cryptogamus van de Peppel [MB 838129], p. 44 Trichopilus lecythiformis Largent [IF 558178], p. 56 vill ... MYCOTAXON 137(1) CORRIGENDA MYCOTAXON 136(4) p.v,line9 FOR: Five new foliicolous micromycete records from Turkey READ: Additions to the knowledge of foliicolous micromycetes in Turkey JANUARY-MARCH 2022... IX FROM THE EDITOR-IN-CHIEE LATE AGAIN! — Unseasonal snowfalls, power & computer outages at critical times, the Covid Omicron variant in New Zealand, time-consuming medical treatments in Oregon, research obligations, and fewer final submissions (despite a last-minute 25-day extended deadline) have all conspired to delay our 2022 January-March MycotTaxon. Your editors regret the unavoidable delay and heartily thank all authors for their extreme patience. The GoopD news is that nomenclatural reviews are now being returned very quickly by a recovered Nomenclature Editor and soon-to-be acknowledged final submissions are arriving at an equally brisk pace. Being realistic, we suspect the April-June issue will probably go out in early (if lucky) July rather than late June, but your two volunteer editors are valiantly trying to bring back the journal to the quick turn-around its founders intended. Welcome to the slim—but mighty—MycotTaxon 137(1). Our 2022 January-March MycoTaxon may indeed be slender, but it offers a wide variety of truly fascinating papers. There are 13 contributions by 49 authors (representing 16 countries) as revised by 28 expert reviewers and the editors. The NEW GENERA & SPECIES Section proposes ONE new genus (Neoacrodictys from China) and SEVEN species new to science representing Beltrania & Neoacrodictys from Cuina; Claviceps from GERMANY; Clitopiloides & Trichopilus from AUSTRALIA; Phaeocollybia from the U.S.A.; and Termitomyces from SOUTH AFRICA. We also offer one new combination in Passalora from India, and one newly registered neotypification for Claviceps humidiphila from its type locality in Japan. The NEW RANGES/HOSTS section contains five titles. Species range extensions are reported for [basidiomycetes] Ganoderma & Tomophagus and Pluteus & Volvopluteus from PaxisTan; Hebeloma from Turkey; Marasmius from the Dominican Republic; and [laboulbeniomycetes] a possible new Stigmatomyces species from MataysiaA. New dipteran hosts (Boettcherisca and Hypopygiopsis [the first for any laboulbenialean species] are cited for Stigmatomyces. MycoTAxON 137(1) also provides keys to species in Neoacrodictys, Passalora, and Phaeocollybia (the last also proposing three long-awaited synonymies). And a first: papers providing conclusions supported by sequence analyses include all seven newly described species (including one cryptospecies diagnosed by sequence data alone—for good reason) and all eleven range extensions. Our issue concludes with the announcement of a new annotated species list on our MYCOBIOTA website, which describes and illustrates six new records and includes a key to Chaetomium and Chaetomium-like species in one Caatinga ecological research station in BRAZIL Warm regards, Lorelei L. Norvell (Editor-in-Chief) 20 April 2022 x ... MYCOTAXON 137(1) PUBLICATION DATE FOR VOLUME ONE HUNDRED THIRTY-SIX (4) MYCOTAXON for OCTOBER-DECEMBER 2021 (I-XLVIII + 693-880) was issued on February 8, 2022 JANUARY-MARCH 2022... XI 2022 MyCOTAXON SUBMISSION PROCEDURE Prospective MycotTaxon authors should download the MycoTaxon 2022 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. 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MyYcOTAXON LTD— www.mycotaxon.com The Mycotaxon Webmaster <mycotaxon@gmail.com> posts announcements, subscription & publication information, and author forms & templates on the official MycoTAXxon 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. ©2022 January-March 2022— Volume 137, pp. 1-30 https://doi.org/10.5248/137.1 Phaeocollybia chefensis sp. nov. and new synonyms for P. rifflipes, P. rufotubulina, and P. tibiikauffmanii LORELEI L. NORVELL’, RONALD L. EXETER?, MATTHEW GORDON’, SAHRA- TAYLOR MULLINEUX‘, SCOTT A. REDHEAD‘ ' Pacific Northwest Mycology Service, 6720 NW Skyline, Portland OR 97229 USA 72477 SW Maplewood Drive, Dallas OR 97338 USA > Molecular Solutions, 4216 N. Castle Ave., Portland OR 97217 USA ‘ National Mycological Herbarium (DAOM), Agriculture & Agri-Food Canada, 960 Carling Avenue, Ottawa ON KIA 0C6 Canada *CORRESPONDENCE TO: I/norvell@pnw-ms.com ABSTRACT—Phylogenetic analyses of ITS and RBP2 sequence data from Phaeocollybia collections made at Cascade Head Experimental Forest in Oregon support recognition of a new species, P. chefensis. Collections of the new species were previously referred to P. tibiikauffmanii. Sequence analyses also establish that P tibiikauffmanii is a synonym of P. spadicea, P. rifflipes is a synonym of P lilacifolia, and P. rufotubulina is a synonym of P. californica. A revised general key to Pacific Northwest Phaeocollybia species is provided. KEY worps—Basidiomycota, Hymenogastraceae, nomenclature, Northwest Forest Plan, taxonomy Introduction Phaeocollybia R. Heim (Agaricomycetes, Hymenogastraceae) is a genus of brown-spored agarics characterized by conic-campanulate pilei, cartilaginous stipes and pseudorhizae, ornamented “beaked’ basidiospores, sarcodimitic tissue, monovelangiocarpy, and tibiiform diverticula that arise from the mycelium and primordial pellicular remnants (Norvell 1998a,b; Norvell & Exeter 2009). The forests of western North America have provided an exceptionally large number of species in this genus (Murrill 1911; Smith 1937, 1957a,b; Smith & Trappe 1972; Redhead & Norvell 1993; Norvell 1998a,b, 2 ... Norvell & al. 2000, 2002, 2004; Norvell & Redhead 2000, Norvell & Exeter 2007, 2009; Norvell & al. 2010). In 1995 the first author collected a solitary tawny colored Phaeocollybia from Oregon's Cascade Head Experimental Forest (CHEF). Although the specimen’ general morphology resembled that of P kauffmanii (A.H. Sm.) Singer, it differed by possessing abundant refractive tibiiform cheilocystidia, prompting application of the provisional name P. tibiikauffmanii to the Oregon coast taxon in Norvell’s 1998 doctoral dissertation. The author’s research on Phaeocollybia coincided with establishment of the Northwest Forest Plan (NWFP; USDA-USDI 1994), which listed 14 phaeocollybias among its 234 fungal species of concern (Castellano & al. 1999, 2003; ORBIC 2021). In 1998 transects were established for surveying epigeous ectomycorrhizal basidiomycetes in Benton County’s Bureau of Land Management (BLM) Green Peak Density Management Study and Polk County’s BLM Fungal Chronosequence Study (Norvell & Exeter 2004). Numerous gregarious to caespitose clusters of P kauffmanii-like specimens with tibiiform cheilocystidia were collected that matched the solitary 1995 specimen from Lincoln County. After morphological comparison with other specimens from Washington, Oregon, and California, a particularly well-documented collection from the chronosequence ‘old growth’ study transect (approximately 64 km from the CHEF site) was selected as type for P. tibiikauffmanii (Norvell 2004). In 2008, the U.S. Forest Service (FS) & BLM Interagency Special Status/ Sensitive Species Program initiated a project to generate DNA sequence data for Phaeocollybia specimens (Gordon 2009). Since then, 350 sequences for the internal transcribed spacer region (ITS1-5.8S rRNA-ITS2) in the nuclear ribosomal RNA gene cassette and 46 sequences of a portion of the gene encoding the second largest subunit in the DNA-directed RNA polymerase (RPB2) were deposited in GenBank. These data, along with NWFP surveys, have provided several hundred Phaeocollybia collections for taxonomic and genetic analysis, and serve as an excellent resource for phylogenetic analyses and specific primer design. Initial molecular analyses that clustered P. tibiikauffmanii and P. spadicea sequences within one intermixed clade were received on the same day as the arrival of the galley proofs for PHAEOCOLLYBIA OF PACIFIC NORTHWEST NortTH AMERICA (Norvell & Exeter 2009). Although the vital data arrived too late for a thorough publication revision, the authors annotated their keys and commentaries in anticipation of subsequent taxonomic changes. Phaeocollybia chefensis sp. nov. (U.S.A.) ... 3 Additional ITS sequence analyses confirmed the synonymy of P. spadicea and P. tibiikauffmanii but also supported newly collected material from the original Lincoln County ‘tibiikauffmanii site as a distinct species, independent of P. spadicea. A revised ITS-based phylogeny of Phaeocollybia was presented in the Norvell & al. (2010) poster at the International Mycological Congress in Edinburgh. In this paper we provide additional ITS and RPB2 sequence data that establish P chefensis as a new species and formally publish synonymies for P. californica (= P. rufotubulina), P lilacifolia (= P. rifflipes), and P. spadicea (= P. tibiikauffmanii). Materials & methods Collections Specimen collection and examination, ultraviolet inspection, and syringaldazine spot tests of fresh material followed procedures outlined in Norvell & Exeter (2009), where morphotaxonomic terms (e.g., tibiiform diverticula, sarcodimitic tissues, pellicular veil, vertical-monopodial, and rhizomorphic pseudorhizae) are also defined. Parenthesized color references from Ridgway (1912: e.g., “Pale Pinkish Cinnamon”) accompany colors of fresh specimens described in general non-standardized color names in lower case. Microscopical examinations were made of fresh tissues in H,O or (for the type material) dried tissues rehydrated in 6% aqueous KOH. Basidiospores were taken from the stipe apex. Dimensions of all anatomical cells follow the format n (number measured): (outlier)low-average-high(outlier) [e.g., basidia n = 19: 25-34.2-41(43) x 6.5-8.1-9.4(9.9) um]. Collector abbreviations include LLN (Norvell), RLE (Exeter), and sAR (Redhead). Latitude/longitude geographic coordinates were converted and rounded to four decimal places from surveyor input using [for TRS] https://www.earthpoint.us/ TownshipsSearchByDescription.aspx and [for UTM] https://www.ngs.noaa.gov/ NCATY/. Vegetation abbreviations follow those used during Northwest Forest Plan surveys and include (in all caps for overstory) ABPR (Abies procera Rehder), PISI (Picea sitchensis (Bong.) Carr.), PSME (Pseudotsuga menziesii (Mirbel) Franco), and TSHE (Isuga heterophylla (Raf.) Sarg.) and (in small caps for understory) BENE (Berberis nervosa Pursh = Mahonia nervosa (Pursh) Nutt., the preferred synonym), GASH (Gaultheria shallon Pursh), oxor (Oxalis oregana Nutt.), Pomu (Polystichum munitum (Kaulf.) C. Presl), vaov (Vaccinium ovatum Pursh), and vapa (Vaccinium parviflorum Andrews = Gaylussacia baccata (Wangenh.) K. Koch, the preferred synonym). Material cited in the SPECIMENS EXAMINED Sections is housed (unless otherwise specified) in Norvell’s Pacific Northwest Mycology Service fungarium (PNW) and will be distributed among DAOM, NY, OSC, PNW, and WTU after publication. Herbarium acronyms follow Thiers (2021). 4 ... Norvell & al. TABLE 1: PNW Phaeocollybia ITS & RPB2 sequence data. (Type data in bold). SPECIES ammiratii attenuata benzokauffmanii californica # # chefensis dissiliens fallax COLL./FUNG. NUMBER LLN1941028-10-T LLN2051018-01 OSC 155802 RLE2007-103 RLE2007-105 RLE2008-068 RLE2009-19 RLE2009-31 RLE2009-41 LLN2071029-51 RLE2007-026 RLE2007-090 RLE2007-176 LLN1921120-01-T RLE2007-035 RLE2009-15 MICH 11607-T OSC 109290 OSC 109332 RLE2007-177 RLE2010-05 LLN1921116-1 ruf-T SAR7500 RLE2009-04-T LLN2091026cc6a OSC 155803 RLE2007-074 RLE2007-129 OSC 114217 RLE2008-138 LLN1921007-01 RLE2004-01 RLE2007-140 RLE2007-159 RLE2007-175 ITS JN102495 GQ165629 KJ450913 (GQ165627) GQ165624 KX574502 JN102493 JN102494 KX574503 JN102501 MZ352106 JN102498 (GQ165632) GQ165636 GQ165636 (JN102502) JN102503 EU669240 EU846292 GQ165647 JN102504 GQ165707 GQ165708 MZ352102 MZ352101 MK326851 MZ352097 MZ352096 EU846271 KE219569 JN102512 JN102511 MZ352103 JN102516 JN102518 RGB2 KU574760 KU574759 KU574787 KU574790 KU574794 KU574761 KU574785 KU574764 KU57484 KU574779 KU574767 KU574791 KU574795 KU574796 CouNTY, STATE/PROV; PRIMARY COLLECTOR Clackamas, OR; Norvell Skamania, WA; Norvell Coos, OR; Rodenkirk Benton, OR; Exeter Polk, OR; Exeter Benton, OR; Exeter Benton, OR; Exeter Benton, OR; Exeter Benton, OR; Exeter Benton, OR; Norvell Lincoln, OR; Exeter Benton, OR; Exeter Benton, OR; Exeter Mendocino, CA; Norvell Benton, OR; Exeter Benton, OR; Exeter Del Norte, CA; Smith Linn, OR; Smith Josephine, OR; Friend Benton, OR; Exeter Benton, OR; Exeter Mendocino, CA; Norvell Mendocino, CA; Redhead Lincoln, OR; Exeter Benton, OR; Norvell Polk, OR; Christensen Lincoln, OR; Exeter Lane, OR; Exeter Coos, OR; Sperling Benton, OR; Exeter Vancouver Isl. BC; Redhead Benton, OR; Exeter Benton, OR; Exeter Benton, OR; Exeter Benton, OR; Exeter SPECIES gregaria kauffmanii lilacifolia A A luteosquamulosa ochraceocana olivacea oregonensis COLL./FUNG. NUMBER LLN2091026cg8 RLE2007-061 RLE2008-026 RLE2013-07b LLN1931015-02 RLE2006-10 RLE2006-13 RLE2007-095 RLE2008-027 RLE2008-045 LLN1921111-06 LLN2071018-12 RLE2006-43 RLE2013-44 LLN2071029-33 LLN2071029-43 RLE2007-016 WTU-F-003084 RLE2007-033-T OSC 134678 OSC 134679 RLE2007-009 LLN1921015-03 LLN1921122-04 LLN1941128-01 OSC 109501 OSC 113875 RLE2007-122 RLE2007-133 RLE2008-050 RLE2008-051 RLE2008-129 LLN2001105-01 OSC 67425 RLE2006-16 RLE2013-42 ITS JN102520 GQ165652 GQ165654 KJ450915 JN102542 KE219573 JN102547 JN102524 KE219572 JN102526 KE219576 GQ165657 KE219580 GQ165668 GQ165667 GQ165663 JN102528 GQ165674 GQ165672 GQ165671 GQ165673 JN102533 JN102530 JN102532 EU846281 EU846282 JN102534 JN102535 MZ352094 JN102536 GQ165678 GQ165681 EU846273 GQ165685 Phaeocollybia chefensis sp. nov. (U.S.A.) ... RGB2 KU574772 KU574782 KU574781 KU574763 KU574803 KU574774 KU574775 KU574798 KU574799 KU574766 KU574776 KU574804 CouNTtTY, STATE/PROV; PRIMARY COLLECTOR Benton, OR; Norvell Lincoln, OR; Exeter Polk, OR; Exeter Polk, OR; Exeter Clallam, WA; Leuthy Benton, OR; Exeter Benton, OR; Exeter Polk, OR; Exeter Polk, OR; Exeter Lane, OR; Exeter Clackamas, OR; Norvell Lincoln, OR; Exeter Polk, OR; Exeter Lane, OR; Exeter Benton, OR; Norvell Benton, OR; Norvell Lincoln, OR; Exeter Snohomish, WA; Ammirati Polk, OR; Exeter Benton, OR; Villella Tillamook, OR; Paque Polk, OR; Exeter Jefferson, WA; Norvell Mendocino, CA; Norvell Clackamas, OR; Norvell Coos, OR; Sperling Douglas, OR; Kersens Benton, OR; Exeter Lane, OR; Exeter Lane, OR; Exeter Benton, OR; Exeter Benton, OR; Exeter Multnomah, OR; Norvell Douglas, OR; Goldenberg Lane, OR; Exeter Lane, OR; Exeter 6 ... Norvell & al. SPECIES phaeogaleroides piceae pleurocystidiata pseudofestiva radicata redheadii scatesiae sipei spadicea P sp. 1 COLL./FUNG. NUMBER RLE199-064a-T RLE2009-29a RLE2010-086 MICH 11629-T RLE2007-178 RLE2009-12 LLN1940330-02-isoT LLN1930516-01 RLE2008-002 LLN1921104-10 RLE2007-069 RLE2007-070 OSC 112980 RLE2006-19 RLE2008-131 LLN2071018-17 LLN2091026cg18 RLE2007-106 RLE2008-038 LLN1921015-19 LLN1931104-09 LLN2071029-02 RLE2007-151 LLN1971023-69 OSC 96908 RLE2007-123 OSC 112482 OSC 113791 OSC 134542 RLE2009-18 RLE2009-20 A2011031o0x1 tib-T A206111402-O RLE2007-165 OSC 155805 RLE2010-05 RLE2014-01 ITS OM065392 ME737171 KX574499 ME737169 (MZ352093) KE219583 GQ165688 GQ165686 GQ165687 KE219588 KE219589 KE219590 EU846275 GQ165696 GQ165695 JN102544 JN102541 JN102546 MZ352098 GQ165701 GQ165703 GQ165699 GQ165700 EU644706 EU644707 GQ165704 EU697252 EU669364 MZ352099 JN102550 JN102551 KF219597 KEF219596 (KE219594) JN102507 JN102504 KU574726 RGB2 KU57489 KU574762 KU574765 KU574797 KU574771 KU574783 KU574768 KU574769 KU574773 KU574786 KU574788 KU574780 KU574778 KU574777 KU574793 KU574801 County, STATE/PROV; PRIMARY COLLECTOR Benton, OR; Exeter Benton, OR; Exeter Benton, OR; Exeter Tillamook, OR; Smith Mendocino CA; Bojantchev Benton, OR; Exeter Clallam WA; Norvell Linn, OR; Bailey Benton, OR; Exeter Multnomah, OR; Norvell Lincoln, OR; Exeter Lincoln, OR; Exeter Coos, OR; Rodenkirk Benton, OR; Exeter Benton, OR; Exeter Lincoln, OR; Norvell Benton, OR; Norvell Polk, OR; Exeter Lincoln, OR; Exeter Jefferson, WA; McClenaghan Lincoln, OR; Norvell Benton, OR; Norvell Benton, OR; Exeter Benton, OR; Norvell Linn, OR; Bacheller Benton, OR; Exeter Benton, OR; Giachini Douglas, OR; Wetzel Douglas, OR; Sperling Benton, OR; Exeter Benton, OR; Exeter Polk, OR; Norvell Polk, OR; Norvell Benton, OR; Exeter Lane, OR; Loring Benton, OR; Exeter Benton, OR; Exeter Phaeocollybia chefensis sp. nov. (U.S.A.) ... ey eee COLL./FUNG. ITS RGB2 CounrTY, STATE/PROV; NUMBER PRIMARY COLLECTOR P sp. 2 RLE2009-09 KX574498 _ Benton, OR; Exeter RLE2010-109 KX575400 — Benton, OR; Exeter P. sp. 3 LLN2071029-53 (KF219598) | KU574770 Benton, OR; Norvell RLE2007-100 KX574493 aan Polk, OR; Exeter RLE2007-152 KF219600 _ Benton, OR; Exeter RLE2015-35 KU574725 KU574800 — Benton, OR; Exeter P sp. 4 RLE2010-009 MZ352104 KU574792 —_ Benton, OR; Exeter RLE2015-01 KX574505 _ Benton, OR; Exeter RLE2015-06 KU574727 KU574802 Benton, OR; Exeter Psp. 5 OSC 155806 MF957115 = Josephine, OR; Scelza RLE2013-14 KJ450918 — Benton, OR; Exeter P. sp. 6 OSC 134575 GQ165677 — Linn, OR; Nakvasil OSC 151647 MH819350 MH823881 Douglas, OR; Scelza # = P. rufotubulina, = P rifflipes, and * = tibiikauffmanii in Norvell (1998ab, 2002, 2004) and Norvell & Exeter (2004, 2007, 2009). (T = type); ITS sequences enclosed in parentheses not included in Fie. 1. DNA amplification & sequencing Standard protocols and published primers were followed for DNA extraction, PCR amplification, and sequencing (White & al 1990, Gardes & Bruns 1993, Liu & al. 1999, Matheny 2005). Amplifications of the ITS region from 350 Phaeocollybia collections representing 34 putative species utilized fungal primers ITS1 and ITS4b (Gardes & Bruns 1993). Amplifications of the RPB2 gene region from 46 collections representing 26 putative species utilized the degenerate basidiomycete specific primers bRPB2-6F and bRPB2-7.1R (Matheny 2005). Additionally, a new RPB2 forward primer, RPB2Phf (5’-GcAGAAACACCYGAGGGC-3’, slightly downstream from the bRPB2-6F primer) was designed based on existing Phaeocollybia RPB2 sequences and used successfully where the original primer pair failed after multiple attempts. In total 396 (350 ITS + 46 RPB2) new sequences were generated. Additional ITS and RPB2 sequences were added from GenBank to provide a comprehensive phylogeny for Phaeocollybia (Norvell & al. 2010, this publication). TABLE 1 provides collection and sequence data for PNW specimens, and TABLE 2 provides sequencing information for extralimital taxa and the outgroup cited in Fics 1 and 2. Separate gene regions were initially aligned with Clustal X (Thompson & al. 1997). Subsequent new sequences were aligned manually in MacClade 4.0 (Maddison & Maddison 2000). Taxa for which gene regions were not sequenced were coded as missing. Sequences were aligned using MAFFT ver. 7 (Katoh & al. 2019) and manually corrected using MEGA X 10.0.0 (Kumar & al. 2018, Stecher & al. 2020). All new sequences were deposited in GenBank. 8 ... Norvell & al. (90298r04) (NLM) 6881 WEd ByeaqUENWAS euUE!eD {2 ‘PlOq UI payedpuT are suTeIys adAT, “anyq Ul payedIpuUT aie Y6'O> sanfea AyTIqeqoid sJoTIa}sod YIM SapoN ‘e]yINO snsuasuos uetsadeg oy} SUIsN poUTUrJajap oiam sy\suay youerg ‘dds viqdjos0avyg JO sutesjs UI UOISII WNCI 7ZSLI-S8'S-IS.LI 24} Jo sisdjeue s9uUa80[Ayg “] “DIA Phaeocollybia chefensis sp. nov. (U.S.A.) .. S00 _(uobao) posrZsxOr = - {ubn) cores 7 (uob210) BEPPLSXO | oo} saplosjeBooeyd (uoB2io) b2LLELAWpgry (uoBa10) z6cs90W0 go0 jouer sezavena) PIPPI ; uobald) 96959109 DoT re ~— Z (uobai9) Sane! su d (pugjeaz MeN) ELELZBAM - ene eS 7 eiodsojeBaw J (i228) 9112999% . (pusjeaz maN) BLE/ZBAM : (eyeasny) 2951054 oor (wo5210) 11998100") (uofiaig) ose6r@HW” 90 P30 920 (panuquoy™) 10 ... Norvell & al. TABLE 2: Sequences from extralimital Phaeocollybia spp. and outgroup used in phylogenetic analyses SPECIES FUNGARIUM # GENBANK # COUNTRY christinae MCVE 3539 JF908573 Italy cidaris O-F-252891 UDB036543 Norway elegans PDD 72723 KY827313 New Zealand festiva (ITS) WTU-F-053245 DQ494682 Norway — (RPB2) WTU-F-053245 AY509118 Norway gracilis PDD 88665 KY827314 New Zealand graveolens PERTH 5311586 AF501567 Australia lugubris O-F-253794 UDB037370 Norway MCVE 14619 JF908574 Italy megalospora SP 445402 KC662116 Brazil pakistanica-T SWAT 15-1560 KY007615 Pakistan SWAT 15-1561 KY007616 Pakistan ratticauda PDD 72678 KY827316 New Zealand PDD 72544 KY827315 New Zealand tenuis PDD 72672 KY827318 New Zealand Phaeocollybia sp. TRTC 157723 BOLD CRA031-09 Costa Rica AAM A1064 KF041417 Mexico PDD 71198 KY827317 New Zealand Galerina WTU-F-007080 DQ486706 Washington USA semilanceata (RPB2 gene) WTU-F-007080 AY337357 Washington USA All sequences represent the ITS region unless otherwise indicated. Sequence analyses Sequences were analyzed using programs available in Geneious Prime (version 2021.2.2). Sequence data were obtained from NCBI and compiled into a multiple sequence alignment. The original ITS dataset contained 296 sequences identified as Phaeocollybia spp. Duplicate sequences were removed. Representative sequences for each taxon were retained and the final dataset, including the outgroup, comprised 129 unique sequences. Sequences were trimmed to remove the CATTA and GACCT motifs. For the analysis of partial RPB2 gene sequences, the dataset was composed of 49 unique sequences. Sequences were aligned automatically using the Geneious alignment tool, and the alignment was refined manually. Phylogenetic analyses were carried out using the MrBayes 3.2.6 (Huelsenbeck & Ronquist 2001) plugin developed in Geneious. The following parameters were used: substitution model GTR; rate variation gamma; gamma categories 4; 5 million generations; subsampling frequency 1000; burnin Phaeocollybia chefensis sp. nov. (U.S.A.) ... 11 Galerina semilanceata PBM 1398 (WTU) (AY337357) KUS574789 (Oregon) P. phaeogaleroides P. olivacea P. gregaria P. olivacea Phaeocollybia sp. 6 Avs00118 (Nowy) festival 1.90(KU574782 (Oregon) 1,00{ (LKUS74781 (Oregon) P. ammiratii Poregonensis -P. benzokauffmanii 00} (00 | | KU574778 (Oregon) Pana KUS574788 (Oregon) KU574786 (Oregon) oo(-KU574724 (Oregon) KUS574761 (Oregon) P. attenuata "Hg XU574802 (Oregon) 1.01 eee (Oregon) Phaeocollybia sp. 4 5 KU57484 (Oregon) "i P. chefensis Y KU574779 (Oregon) KU574771 (Oregon) P. radicata Fic. 2. Phylogenetic analysis of the partial RPB2 gene region in strains of Phaeocollybia spp. Branch lengths were determined using the Bayesian consensus outfile. Nodes with posterior probability values <0.90 are indicated in blue. Type strains are indicated in bold. frequency 50%; heated chains 4; heated chain temperature 0.2; unconstrained branch lengths GammaDir (1, 0.1, 1, 1); and shape parameter exponential (10). The outgroup was Galerina semilanceata PBM 1398 (WTU); DQ486706 for the ITS analysis and AY 337357 for the RPB2 analysis, respectively). Phylogenetic trees were drawn using the Bayesian consensus outfile and annotations were added using Inkscape 0.92.4. Phylogenetic results FIGs 1-2 Norvell & al. (2010) presented the first comprehensive ITS phylogeny for the genus based on 300 Phaeocollybia sequences (including two from Australia— P. graveolens B.J. Rees & K. Syme, P. ratticauda E. Horak—and P. festiva (Fr.) 12 ... Norvell & al. R. Heim from Norway) and the outgroup—Hebeloma radicosum (Bull.) Ricken, Psilocybe semilanceata (Fr.) P. Kumm., Galerina marginata (Batsch) Kuhner—revealing a cohesive clade of 69 putative taxa with the Galerina- like P phaeogaleroides clade in the basal-most position within Phaeocollybia. The current ITS tree (pruned to eliminate duplicates) confirms the existence of three synonymies, several difficult species complexes, and six unnamed species (Fic. 1). The three synonymies supported are P rifflipes = P. lilacifolia (>99.7% similarity), P rufotubulina = P. californica (>99.5% similarity), and P. tibiikauffmanii = P. spadicea (>99.8% similarity (Fics 1, 2). The comprehensive ITS phylogeny (not shown) generated from 296 sequences (plus the outgroup Inocybe pallidicremea Grund & D.E. Stuntz [as I. lilacina (Peck) Kauffman)] and Galerina semilanceata (Peck) A.H. Sm. & Singer) and the trimmed 129-sequence ITS tree (Fic. 1) both support 29 PNW species and 14 extralimital putative taxa. A familial relationship between Phaeocollybia and Galerina is also supported, with both genera now classified in Hymenogastraceae (Matheny & al. 2006, Kalichman et al. 2020). The successful amplification of far fewer (46) RPB2 sequences prohibits generating a robust multigene consensus tree. Nevertheless, both ITS (Fic. 1) and RPB2 (Fic. 2) trees support the same clades. With insufficient coverage across the genus for 28S sequence data and with the two-gene phylogeny well supported by convincing morphological characters, we do not include a 28S-based phylogeny at this time. Taxonomy Phaeocollybia chefensis Norvell & Exeter, sp. nov. Fics 3-6 IF 559441 Differs from Phaeocollybia kauffmanii by its tibiiform cheilocystidia and from P. spadicea by its uniformly carrot orange to orange-tawny coloration, larger basidiospores, and solitary to scattered (never gregarious or cespitose) habit. TyPeE: Oregon, Lincoln County, Cascade Head Experimental Forest, W of Hwy 12, 30m S Tillamook Co. line, 45.0447°N 123.9153°W, 245 m asl, 27.x.2009, Tsuga heterophylla (old and young), Picea sitchensis, Vaccinium parviflorum, Polystichum munitum, RLE2009-04 (holotype OSC 155804, isotype PNW; GenBank MZ352102, KU574784). EryMo.oey: derived from the acronym (CHEF) for the type locality, Cascade Head Experimental Forest. PitEus 70-80 mm diam, broadly conic-campanulate with acute papillate umbo, upturned inner margin, down-turned outer margin, and straight edge; glabrous, glutinous in rain with the gluten at times somewhat milky, non-striate, overall brownish orange or foxy brown (Tawny, Orange Cinnamon); dried Ron Exeter Phaeocollybia chefensis sp. nov. (U.S.A.) ... 13 SS — | ? ! Le, Fic. 3. Phaeocollybia chefensis (Holotype, RLE2009-04). Top: excavated specimens with intact vertical monopodial pseudorhizae. Bottom: Two views of the type collection in situ in Cascade Head Experimental Forest, Lincoln County, Oregon. Five collections of 1-2 specimens were collected from this same site during 1995-2021. pileus uniformly copper metallic. CONTEXT ~6 mm at the disc and confluent with stipitipith, pale orangish white. Opor faintly floral with farinaceous overtones; TASTE mild, not distinctive. LAMELLAE nearly free, ventricose, thin with + even edges, polydymous with 3-7 irregularly interspersed tiers of lamellulae, narrow (4-5 mm, with average length/width ratio 5.5), close, pale orangish buff (Warm Buff) when young, developing darker spots in maturity. VEIL sparse (when present, evident only as occasional darker fibrillose 14 ... Norvell & al. remnants on stipe apex). STIPE slightly eccentric, terete, 80-90 mm above ground level, overall length including pseudorhiza <200 mm, apex 10-12 mm diam, + equal above, gradually narrowing below toward pseudorhiza; glabrous except for occasional fibrils, moist, finely longitudinally lined, apex pale to deep pinkish orange (Pinkish Cinnamon, Orange Cinnamon) below grading to dull pinkish brown (Fawn Color), 2 mm thick cartilaginous rind surrounding compact fibrillose orangish white stipitipith, rind sometimes with small perpendicular separations producing ~5 mm long recurved rind patches. PSEUDORHIZAL FORM vertical-monopodial, <2/3 overall stipe length, gradually tapering to pale salmon-colored pointed to blunt origin, firm pith brown where water-soaked, otherwise concolorous with stipitipith. SPORES IN MASS dull pinkish brown (Fawn Color). BASIDIOSPORES (n = 64): 8-8.9-10 x (5)5-5.6-5.8 um, Q = (1.45)1.52- 1.65-1.81(1.84), limoniform with a protruding beaked apex in profile, fusoid-elliptical to amygdaliform in face view, apical callus 0.5-1 um long (occasionally abrupt or more often tapering to end), ornamentation verruculose to verrucose except on smooth apical callus and eccentric apiculus, suprahilar plage an indistinctly bordered area of lowered ornamentation (oil immersion); orangish amber in KOH (ochraceous in H,O, based on examinations of paratypes). Basip1A (n = 19): 4-spored, curved; broadly clavate above narrower base, 25-34.2-41(43) x 6.5-8.1- 9.4(9.9) um, sterigmata 2.0-3.0-3.7 um long, curved; hyaline to dull orangish brown, guttulate, granular, or uniformly oily. CHEILOCYSTIDIA 24.2-28.7-34 um long, diameters (basal septum) 2-2.6-3 x (stomach) 4.9-5.7-6.9 x (neck) 1-1.3-1.6 x (capitulum) 1.5-2.0-2.6 um (n = 13), abundant, intermixed with basidia, secretory; capitulate tibiiform (primarily) and lageniform, (only occasionally intermixed with thin-walled clavate elements), bases hyaline, necks and capitula refractive, thick-walled, pale amber. PLEUROCYSTIDIA absent except for isolated cheilocystidia scattered occasionally on the gill face. LAMELLAR TRAMA hyphae parallel, 65-80 x 3-6 um, thin-walled, hyaline inflated, subgelatinized, narrowing toward gill edges to 2-3 um diam and giving rise to the subhymenial layer. SUBHYMENTIUM c. 25 um thick, hyphae 2-4 um diam, tightly packed, parallel, cylindrical, hyaline, gelatinized. PELLICULAR HYPHAE: PILEIPELLIS a bilaminate ixocutis with a >300 um thick SUPRAPELLIS with elements tightly compact in freshly dried material (but readily separating in gel matrix after 11 years in fungarium), hyphae >40 x 1.5-5.0 um, radially aligned, sinuous, long-branching, gelatinized, frequently (spirally) gel encrusted, hyaline; sUBPELLIS c. 500 um thick, gelatinized, vessel A-C, E-G: Lorelei Norvell. b, 4,1. Ron Exeter 1: Heidi Christensen. Phaeocollybia chefensis sp. nov. (U.S.A.) ... 15 Fic. 4. Phaeocollybia chefensis paratypes. [Lincoln Co.: LLN1951109-16 (A-C), RLE2007-074 (F), RLE2015-21 (H), RLE2021-1 (J); Lane Co.: RLE2007-129 (D, E); Polk Co.: HC2018-LB4 (I); Benton Co.: LLN2091026cc6 (G)]. A-C. Three views of the first specimen from CHEF that fostered the concept for P. tibiikauffmanii in 1995. D. Young robust specimens excavated from Lobster Creek. E. Pristine pseudorhizal tissues display strong magenta reaction in syringaldazine after 15 minutes. F. Second specimen retrieved from CHEF in 2007. G. Solitary specimen retrieved from Conner’s Camp on Marys Peak. H. Basidiome at the original CHEF site prior to excavation in 2015. I. The first primordium excavated with its more mature partner in 2018 at Boulder Creek. J. Exeter’s 2021 negative syringaldazine reaction from this waterlogged solitaire cautions that adverse environmental conditions do disable this usually reliable diagnostic tool. 16 ... Norvell & al. hyphae with thinner walls; hyphae long, 4-12 um diam at septa and inflating to <25 um; pigments orange to dark brownish orange, diffuse to occasionally encrusting, soluble in KOH (forming droplets in mountant); small hyaline crystals scattered infrequently throughout. STIPITIPELLIs hyphae 75-100 x 1.5-3(6) um, parallel aligned, gelatinized, pigments diffuse, pale amber to reddish in places. RHIZOPELLIS cells long x 4-10 um, pale to dark amber, heavily gel- and/or red-brown pigment-encrusted. TRAMAL TISsUEs: lightly (pileus) to noticeably (stipe, pseudorhiza) sarcodimitic, gelatinized, +hyaline except darkening in pseudorhiza; VESSEL HYPHAE fusoid (sometimes tapering to obtuse end), 25-100 x 3-13 (at septa), rigid walls 1-2 um thick; FLExUOUS HYPHAE generally winding or curving around vessels, more or less cylindrical but sometimes appearing flattened, thin-walled, <50 um long, diameters narrowest (1-2 um) in pileus and broadest (3-5 um) in stipe; OLEIFEROUS HYPHAE occasional to frequent in tramal tissues throughout, sinuous, aseptate, 3-10 um diam, thin-walled, contents dull brown, oily. Trs11FoRM DIVERTICULA infrequent on aerial stipe apex, lageniform to (more often) tibiiform, frequent to abundant on rhizopellis, 8-20 um long, 1-2 at base, narrowing in refractive neck area before expanding when capitula (1-2 um diam) present; arising directly from hypha and lacking basal septum, secretory, hyaline, refractive to very pale amber. CLAMP CONNECTIONS absent in all tissues. SYRINGALDAZINE SPOT TEST: strongly positive (pseudorhizal origin dark burgundy in five minutes; stipe (cross-section) and pileus and lamellae leaching magenta after ten minutes). FLUORESCENCE fresh material (of holotype) not tested; dried lamellae dull yellow orange (one small area of one specimen a brilliant orange yellow). ADDITIONAL SPECIMENS EXAMINED—UNITED STATES, OREGON, Benton Co. Conner’s Camp, 44.5060°N 123.5565°W, 762 m asl, 200yo PSME TSHE 26.x.2009 LLN2091026cc6 (MZ352101). Lane Co. Lobster Valley, 44.2268°N, 123.6148°W, 427 m asl, 200yo PSME young TSHE Gasu, 6.xi.2007 RLE2007-129 (MZ352096). Lincoln Co. Cascade Head EF, 30 m S Tillamook Co. Line, 45.0447°N 123.9153°W, 245 m asl, ~120yo PISI TSHE vapa POMU OxOR: 9.xi.1995 LLN1951109-19; 18.x.2007 RLE2007-074 Fic. 5. Phaeocollybia chefensis (Holotype, RLE2009-04): A. Colorless suprapellis in gelatinous matrix overlying a pigmented subpellis [400x]. B. Pileipellis under oil [1000x]. C, D. Suprapellis hyphae (gel encrustations detailed in C). E. Lightly sarcodimitic pileus tramal tissues with relatively thin-walled vessel hyphae. F. Oleiferous hyphae in stipititrama (just below pellis hyphae). G. Strongly sarcodimitic pseudorhizal trama with thick-walled vessel hyphae and thin- walled flexuous (arrow) hyphae. Fe Phaeocollybia chefensis sp. nov. (U.S.A.) ... [[2410N 1a[al0] 18 ... Norvell & al. (MZ352097, KU574779); 2.xi.2015 RLE2015-21 (KX574495); 14.x.2021 RLE2021-01. Polk Co. Boulder Creek 44.8983°N 123.4995°W, 823 m asl, ~80yo ABPR PSME TSHE POMU VAPA 10.x.2018 HChristensen HC2018-LB4 (OSC 155803; MK326851). ECOLOGY & DISTRIBUTION: autumn (October-November); basidiomes solitary or in pairs in needle duff under mature Abies procera, Tsuga heterophylla, Picea sitchensis and/or Pseudotsuga menziesii with understory of Gaultheria shallon, Oxalis oregana, Polystichum munitum, Vaccinium ovatum, and/or V. parviflorum. Known thus far from only five sites along the Oregon coast and in the Oregon coast range. Discussion Phaeocollybia chefensis Diagnostic characters include a bright orange to brownish orange viscid conic-campanulate pileus, seemingly robust stature that in age becomes limp or flaccid, stuffed stipe, vertical monopodial pseudorhiza, medium-sized limoniform verrucose basidiospores, thick-walled tibiiform cheilocystidia, and lack of clamp connections. ITS and RPB2 sequence analyses strongly support P. chefensis as an independent species (Fics 1, 2). Morphologically, all paratypes match the type, except for the smaller (7.5- 8-9 x 4—-4.5-5.2 um) basidiospores measured in the immature 1995 specimen (LLN1951109-19), which in part explains why Norvell (2004) did not detect the selection of a P spadicea collection as type until after sequence analyses. The 1995 collection has not been sequenced, but sequences from the four subsequent collections from the identical site strongly support the earlier collection as P. chefensis. The molecularly confirmed collections imply Tsuga heterophylla as the primary ectomycorrhizal associate; possible secondary associates include Picea sitchensis at the type locality (245 m asl), Pseudotsuga menziesii at the Lobster Creek (427 m asl) and Conner’s Camp (762 m asl) sites, and Abies procera and Pseudotsuga menziesii at Boulder Creek (823 m asl). Lamellae of fresh specimens examined under UV exhibited the strong yellow fluorescence characteristic of all PNW phaeocollybias except for the uniquely non-fluorescing P gregaria A.H. Sm. & Trappe. As noted above, Phaeocollybia chefensis closely resembles P. kauffmanii macroscopically, while microscopically it is quite similar to P spadicea and P. pseudofestiva A.H. Sm., Phaeocollybia chefensis shares with P. kauffmanii a brownish orange viscid pileus, stuffed stipe, vertical-monopodial pseudorhiza, magenta syringaldazine reactivity, and morphologically similar basidiospores, but the presence of refractive thick-walled tibiiform cheilocystidia and Phaeocollybia chefensis sp. nov. (U.S.A.) .. “Se. Sr en) 3 ~ ee Lorelei Norvell Fig. 6 Phaeocollybia chefensis (Holotype, RLE2009-04 (A,D,E); RLE2007-074 (B,C); RLE2007-129 (4,B): A, B. Cheilocystidia. C. Granular basidia, sterigmata, and orangish brown basidiospores with smooth apical beaks (arrow). D. Basidiospores with older basidia lacking guttules or granules. E. Comparison of verrucose spore ornamentation and less ornamented plage regions (arrows). FE Abundant tibiiform diverticula on pseudorhizal pellis. “ | kd 20 ... Norvell & al. the absence of heavily gelatinized, strongly sarcodimitic elements in the stipe and less extensive gelatinous matrix in the suprapellis clearly separate P. chefensis from P. kauffmanii. In the field the much longer-lived P kauffmanii can be distinguished by its larger (at times massive) and more robust stature, sharp farinaceous odor and taste, strongly inrolled mature pileus edge, and frequently gregarious habit. Phaeocollybia spadicea, sharing many morphological features that led to the selection of an orange form as type for P. tibiikauffmanii, differs in its darker tawny (“date-colored”) to frequently blackish brown pileus, abundant rough fibrillose patches (invariably covered with tibiiform diverticula) on the stipe apex, closely gregarious habit, and (often) negative reactivity of the pileus and lamellar tissues to syringaldazine. Specimen age and condition may explain the variable syringaldazine reactivity in both P chefensis and P. spadicea. Phaeocollybia pseudofestiva, which shares general basidiospore and cheilocystidial morphology, syringaldazine reactivity, and odor and taste is easily separated from P. chefensis by its dark green pileus, smaller size, more prominently beaked basidiospores, cord-like pseudorhiza, and closely gregarious habit. Phaeocollybia tibiikauffmanii and P. spadicea As noted above, comparisons of ITS and RPB2 sequences from collections designated as either P spadicea or P. tibiikauffmanii based on the presence/ absence of orange coloration (Norvell 1998a,b,c, 2004; Norvell & Exeter 2009) revealed that all (including the holotype) except one (Fic. 3a) P. tibiikauffmanii collection represented P spadicea. Subsequent morphological comparisons have shown that P. spadicea pileus colors range along a foxy orange to tawny to deep brown continuum. Additionally, all seven P. chefensis collections occurred singly or in pairs in contrast to P. spadicea, which typically occurs in closely gregarious to cespitose clusters; Alexander Smith's notes archived in MICH describe an “orange” spadicea that he provisionally named P. “caespitosa” (a name he also provisionally applied to what was to become P. scatesiae A.H. Sm. & Trappe; Norvell 1995). Thus, we propose the synonymy: Phaeocollybia spadicea A.H. Sm., Brittonia 9: 215, 1957. FIG. 5 = Phaeocollybia tibiikauffmanii Norvell, Mycotaxon 90: 248, 2004. SPECIMENS CONFIRMED AS Phaeocollybia spadicea (listing those previously cited as P. tibiikauffmanii in Norvell (2004) and/or Norvell & Exeter (2004, 2009)—UNITED STATES. OREGON: Benton Co. Conner’s Camp 44.5060°N 123.5565°W, 762 m asl, Phaeocollybia chefensis sp. nov. (U.S.A.) ... 21 =~ v 5 =, ~ v ™ vB i an ~~ x ® Ry S Re < CG Elixabeth Fox. Fic. 7. Phaeocollybia spadicea [RLE208-55 (A); A201103102-1 (B); LLNI1921015-10 (c)]. A. Gregarious cluster on Oregon’s Marys Peak (as P. tibiikauffmanii in Norvell & Exeter 2009). B. Excavated P. tibiikauffmanii holotype from the Polk County Chronosequence Study. C. “Typical” brown-capped P. spadicea specimens in Hoh Valley, Olympic National Park. 200yo PSME TSHE, 5.xi.2005 RLE2008-55 (KF219592); Ernest Creek 44.4185°N 123.5165°W, 610m asl, 200yo PSME TSHE 15.xi.2007 RLE2007-165 (KU574777, KF219594); Running Bear 44.4774°N 123.5769°W, 696 m asl, 60yo PSME TSHE, 6.xi.2000 DCalver RLE200-103 (KF219595). Polk Co. Pedee Chronosequence Study 44.8113°N 123.5212°W, 460 m asl, 150yo PSME TSHE pomu 4.xi.1998 (all by LLN & RLE) A198110401-01, A198110402-02-04 (with CHibbler); 5.x.2000 RLE200- 045; 18.x.2000 A200101801-15,02-25; 1.xi.2000 RLE200-091, A200110102-30; 31.xi.2001 A2011031lox1 (P_ tibiikauffmanii holotype; KF219597, KU574780), A201103102-03,04,05,06,08, A2011031ox-02; 14.xi.2011 «a201111401-01,02,03, A20111140x-01; 15.x.2002 =a202101502-01; = 13.xi.2002 = =a202111302-01,02,03; 14.xi.2006 A206111402-O (KF219596, KU574778), RLE2006-47, 16.x.2007 rle2007- 060 (KF219593). WASHINGTON: Jefferson Co. Hoh Valley @ Twin Creek 47.8307°N 124.0012°W, 180 m asl, ~400yo PISI TSHE pomMu oxor 15.x.1992 LLN1921015-10 . (w sAR) (WTU-F-003698; previously cited and RFLP-confirmed as P. spadicea). dsl ADDITIONAL EXAMINATION NEEDED BUT PROBABLY ALSO Phaeocollybia P. -UNITED STATES. OREGON: Benton Co. Green Peak BLM Density Management Study 44.366°N 123.455°W, (all Gp collections by LLN & RLE): Clear-cut transect (pre-treatment)— 610m asl, 65yo PSME TSHE pomu 24.xi.1998 GP1981124c1-04 (w TFennell); High Retention transect 579m asl, 65yo PSME TSHE pomu— 8.xii.1999 GP1991208hx.03 (w sAR) (PNW, DAOM), 27.xi.2000 GP2001127hx3; Klickitat BLM Unit-3 44.4406°N 123.5673°W, 60yo PSME TSHE GasH 30.x.2000 RLE200-065; 16.xi.2000 RLE200-252; Klickitat Unit-6 T.S. 44.4548°N 123.5473°W, 50yo PSME TSHE pomu: 30.x.2000 RLE200-082,83,90, 14.xi.2000 RLE200-187b; 44.4260°N 123.5472°W, 60yo PSME TSHE GasuH: 30.x.2000 RLE200-070; RUNNING BEAR TS. 44.4774°N 123.5770°W, 200yo PSME TSHE pomu 9.xi.1998 LLN1981109.108e w RLE, 6.xi.2000 RLE200-103; 28.x.2002 RLE2002-16. Linn Co. Keel Flats T.S. 44.5133°N 122.6518°W, PSME BENE POMU 6.xii.1999 KScott RLE199-BK. Polk Co. Pedee Crk head waters near Cold Springs 44.8233°N 123.4862°W, 610m asl, 200yo PSME TSHE Gasu 26.xi.2001 RLE 2001- 112,115. WASHINGTON: Clallam Co. Olympic NF Klahanie Campground 47.9657°N 124.3057°W, 250yo PISI TSHE 18.x.1992 STrudell LLN1921018-4 __, (WTU-F-003646). usps3 22 ... Norvell & al. Phaeocollybia rufotubulina and P. californica On November 16, 1992, numerous densely gregarious orange phaeocollybias in Jackson State Forest (Mendocino County, California) were collected and referred to P californica. This first highly informative collection contained numerous pin-head primordia that helped establish the presence of a universal veil for all Phaeocollybia species and introduced the ‘sequential- racemose’ pseudorhiza arising from a horizontal ‘mother rhizomorph within the soil (Norvell 1998b, 2004). Restriction Fragment Length Polymorphism (RFLP) analyses of the ITS region from type materials supported separation of P rufotubulina from P. californica and P. scatesiae (Norvell 1998a), indicating a new species and supporting the synonymy of P. scatesiae under P. californica proposed by Horak (1977). The 2009 ITS sequence analyses, however, clustered P californica and P. rufotubulina within one intermixed clade well-separated from P. scatesiae, a brownish species macroscopically easily distinguishable in the field from the two orange phaeocollybias. Phaeocollybia scatesiae is now supported as independent from P. californica (Fics 1,2). Given the difficulty encountered in isolating DNA from the P. californica holotype (obtained in 2011), it is probable that the 58-year-old P californica DNA isolated in 1994 was contaminated by DNA isolated in the same run from the much younger P scatesiae isotype. We hereby propose the synonymy: Phaeocollybia californica A.H. Sm., Brittonia 9: 216, 1957. = Phaeocollybia rufotubulina Norvell, Mycotaxon 90: 243, 2004. Norvell & Exeter (2009) presented P. californica (pp. 63-70) and P. rufotubulina (pp. 175-180) as separate species; all material related to P rufotubulina throughout that monograph as well as those in Norvell (1998a,b, 2004) should now be referred to P. californica. Phaeocollybia rifflipes and P. lilacifolia During a two-month long collecting expedition through western North American coastal rainforests 1992, several small lilac-gilled tawny to dark brown-capped phaeocollybias were collected and provisionally determined as P. lilacifolia. Except for flaccid small basidiomes and unusually small basidiospores, the specimens morphologically matched Smith's P. lilacifolia type from Washington’s Mt. Rainier Park (Smith 1957a). However, 1994 restriction digests produced a RFLP profile unique to these collections. Unable to extract DNA from the Washington P /ilacifolia holotype or Smith's Phaeocollybia chefensis sp. nov. (U.S.A.) ... 23 Cascade Head Experimental Forest paratype, Norvell (1998a, 2002) proposed the name P rifflipes (honoring its unique RFLP fingerprint) for the specimens that were morphologically separated by unusually small basidiospores and stature. During 2001-2013, numerous small P. rifflipes specimens were collected from the Polk County old growth chronosequence transect; not until 2007, however, was the first recognizable P. lilacifolia collected from Lincoln County's Saddle Bag Mountain, c. 25 km southeast of Cascade Head Experimental Forest. The Saddle Bag collections comprised robust healthy specimens with normal-sized basidiospores. In 2009, ITS phylogenetic analyses clustered P lilacifolia sequences and all but one P rifflipes sequences within one clade, a clade supported by our current ITS and RPB2 phylogenies. Disposition of 4592sl (MZ352100), a Loring collection from Josephine County (initially identified as P. rifflipes), remains unresolved at this time. Although we have not yet successfully isolated DNA from Smith's P. lilacifolia holotype or paratypes, the small “limp” stature, poor condition, and age of the 1992-1994 collections suggest that their peculiar RFLP profiles resulted from an unknown contaminant. The following synonymy is proposed: Phaeocollybia lilacifolia A.H. Sm., Sydowia Beih. 1: 59, 1957. = Phaeocollybia rifflipes Norvell, Mycotaxon 81: 102, 2002. All keys, descriptions, and photos of P. rifflipes in Norvell (1998a, 2002) and Norvell & Exeter (2009) should be referred to P. lilacifolia. Revised general key to Pacific Northwest Phaeocollybia species Basidiospore size ranges of measurements taken from holotype specimens are cited for species; means of measurements taken from molecularly confirmed specimens are cited for complexes. 1. Basidiomes small: pileus <50 mm diam, and never green or drab; stipe apex usually <4 mm diam and never drab....................2200005 2 1. Basidiomes medium to large: pileus usually [50 mm diam; stipeapex tsttally. SSimummsdiamy 4h. -aaths.- 2aihaegdige+adthar 2 4thernw A Qaenuld haceuls 5 2. Basidiospores ellipsoid, ornamentation minutely punctate and small apical callus detectable in 1000x oil immersion; Clainp-CcONMECtONS PrESENL, 52. os see < +a als alka sew polled ON gle OR Ga OE 3 2. Basidiospores limoniform, ornamentation verrucose and protruding apical beak visible without oil immersion lens; ClaniIp,COnNeEChOnis-ADSCM ts ys, Beet Re rc ty Raney Muay Bendy Menten tenet cathy wate 4 24 ... Norvell & al. 3. Spores small (5-6 x 3-3.5 um); cheilocystidia lageni-/tibiiform, necks narrow and thick-walled, abundant but inconspicuous, difficult to see in dense gelatinous matrix; basidiome-collybicid, stipeplaable-. cis T icons rs 20h. ype ne ape ere eee P. radicata 3. Spores larger (~10 x 6 tum); cheilocystidia thin walled, narrowly clavate, extending well beyond hymenium; basidiome mycenoid, stipe fragile and easily broken . . . P. phaeogaleroides complex 4. Stipe and pseudorhiza fleshy, not shiny or brittle; cheilocystidia tibiiform, necks and capitula refractive and thick-walled; pleurocystidia present and, frequent on gill faces; spores 8-11 x 5-7 um, tapering to straight beaks; Phenology: Veriiah F.-:o0 vos yaa oa: Monies Nowute Homie Howse He P. pleurocystidiata 4. Stipe polished, corneous, soon hollow; pseudorhiza lateral monopodial, brittle and wire-like (criniform); cheilocystidia clavate, thin-walled; pleurocystidia absent; spores 7-8.5 x 5-5.5 um, big-bellied with abrupt tilted beaks; prenologscatiturinmale st cit tn shot hin tg hle ty phat oiahed Cee P. attenuata 5. Young pileus green, rapidly aging to brown or brownish olive .................. 6 5. Young pileus ochre, orange, tawny, brownish, or drab (never green) ............. 8 ON . Young lamellae violet; spores ~9 x 5.3 um, moderately beaked, in face view fusoid or naviculate, verrucose to marbled; cheilocystidia clavate, thin-walled, apices swollen to subcapitate, only rarely forming filiform apical outgrowths .............. P. fallax complex 6. Young lamellae creamy to yellowish buff; spores limoniform with pronounced beak, +/- ovate in face view, rugulose to warty roughened; chéilocystidiasclavate or Tibiitoritit. 4.2.0.3 ela ete lends sate ee alas vi 7. Spores 7.5-8 x 4.5-5 um, with long, projecting beaks; cheilocystidia tibiiform with narrow refractive necks ......... P. pseudofestiva 7. Spores ~10 x 6 um, ‘turtle-backed’ with abruptly protruding eccentric refractive beaks; cheilocystidia thin-walled, clavate, filiform apical outgrowths frequent in age ................ P. olivacea complex 8. Spores ellipsoid, short (<7.5 um), punctate-roughened; cheiloeystidid-clavate, thin-walled: . ota. ete sla poled a tends atta sigan ntar sian 9 8. Spores limoniform, verruculose to warty; cheilocystidia tibittorimiorelavater oe. a6. le donated set ae karst sleas ecigs 11 9. Basidiome drab to gray, robust; stipe <20 mm diam, stout, firmly stuffed; pseudorhiza fleshy; taste and odor cucumber-farinaceous; spores 6-7.5 x 4-4.5 um; all tissues soon deep magenta in SVE AMAZING: \ 0M et esc eee hee Ng EE Teh Ae Ue et Ee P. oregonensis 9. Basidiome orange to auburn colored, fragile; stipe <13 mm diam, slender, hollow; pseudorhiza cord-like; taste and odor mild; pilets:cclamellacsyringaldazine negative c.% ae aid Sh We ls hon wheal ances vices 10 Phaeocollybia chefensis sp. nov. (U.S.A.) ... 25 10. Clamp connections abundant; pileus orange with yellowish margin, viscid, conic-campanulate; cheilocystidia filamentous to narrowly clavate; SPORES515= O15 9CB, GS SMe fast yd pestis ¢ otis se wha siiee etal iay tor alats ee BS P. dissiliens 10. Clamp connections absent; pileus uniformly auburn colored, subviscid, campanulate with a raised blunt umbo; cheilocystidia narrowly clavate with long pedicels and subcapitate apices; SPOTLESS. Or oN aa aa ha 8a a tal ae P. sipei 1 — . Pileipellis 3-layered with colorless gelatinized middle layer between yellow to yellow-orange top and bottom layers; pileus minutely scaly (appressed), dry to subviscid (never glutinous) ........ 12 1 _ . Pileipellis 2-layered, with colorless top layer and variously pigmented lower layer; pileus bald, smooth (never appressed scaly), subviscid to glutinous ......... 13 12. Spores large, 9-11 x 5-6.8 um; pileus dry to greasy, yellowish ochre to ochraceous gold; frequently associated with Abies ................. 0.0 eee P. luteosquamulosa 12. Spores smaller, 7.2-8.7 x 4.4-5.5 um; pileus greasy to subviscid, tawny ochraceous or tawny; associated with Pseudotsuga or Tsugd ........ cece eee eee P. ochraceocana 13. Cheilocystidia tibiiform, broad bases thin-walled, capitula and narrow necks refractive and thick-walled; Stipeliollowrors tuted hs oso s.iha 290s ge spares Sasa nt Crh eee nN altel 14 13. Cheilocystidia variably cylindrical to clavate, filamentous apical secondary growth occasional in older specimens but always lacking refractive thick-walled necks; stipes. stubbed th: frm Pithy 5.2 eaee eae a Pee Pays eae Coe Pale es Le. ae 17 14. Mature stipe tubular and hollow; basidiomes closely gregarious to fasciculate, arising from a branching rhizomorphic cord ............. 00. e cece eee eee ies 14. Mature stipe stuffed with firm compact pith; solitary to closely gregarious, arising from unbranched fleshy (not cord-like) pseudorhiza ................. 16 15. Pileus heavily glutinous, acutely conic-campanulate, yellowish to dark brown; habit in dense fasciculate mounds; suprapellis thick, colorless, hyphae kinked, not spirally pigment-encrusted and with septa obscured by thick gel matrix; spores 8.5-9.5 x 4.5-5.5 UM ...... eee eee eee ee P. scatesiae 15. Pileus moist to viscid, obtusely convex-campanulate, red orange to tawny; habit in troops, arcs, or (rarely) fasciculate mounds; suprapellis compact, amber to orange, hyphae spirally pigment-encrusted with refractive septa; SPOTeS:8-a=1 OMG tiie |. hese he OP RN ose tas ree hy P. californica 16. Pileus foxy orange to orangish brown; stipe apex orange cinnamon colored; habit solitary or paired, never gregarious; all tissues soon magenta in syringaldazine; spores 8-10 x 5-5.8 UM... 6. eee eee eee eee P. chefensis 16. Pileus tawny to dark brown; stipe apex pallid to drab; habit gregarious to fasciculate; only pseudorhiza magenta in syringaldazine; spores 7.5-9 X 4.5-5 UM ....... 0 ce eee eee eee P. spadicea 26 ... Norvell & al. 17. Clamp connections frequent throughout, most easily seen in pileus suprapellis and on cheilocystidia; pileus campanulate with often papillate umbo, tawny ochraceous to tawny; cheilocystidia cylindrical to narrowly clavate; S/SLOS REIN: Mz laay Wie Ge eae BIEN 101 Mix i ie PO MOE Py fe Pi ee io ek Pio tt P. ammiratii 17. Clamp connections lacking (rarely in stipitipellis); 18 —_— 9 — 9 pileus shape & color, cheilocystidial shape, and spore size varied ........... 18 . Young lamellae whitish, smoky gray, or deep violet; young pileus colors tawny, brownish-pink or drab; pileus and lamellae magenta in syringaldazine .......................004, 19 . Young lamellae pinkish, orangish, or yellowish; young pileus colors rarely pink or drab; syrnealdavineweactivityevatted «o,os dase dada tes deca dandea geld cd pondis 4 ehahdio ee 20 . Spores 7.5-9.4 x 4.5-5.5 um; young lamellae intensely bluish lilac to violet; pileus tawny to deep brown; taste not distinctive; psetidorhiza syringaldazine tegative::. oiwios es of sont nea cte non ft nen P. lilacifolia . Spores 9 X 5.5 um; young lamellae white (pinkish) or ash gray, never lilac or violet; pileus pinkish, drab, or purplish brown; taste “bitter-cucumber’ farinaceous; all tissues soon deep magenta in syringaldazine ........... P. benzokauffmanii 20. Young lamellae never fluorescing under UV; pileus yellow tan, soon darker (cocoa brown); young stipe ivory tan, lower stipe staining orange or orange banded at ground level; habit densely gregarious; syringaldazine negative; Spores Da A SSG MNT ein 2 New 23 ahs inge Sis inge 8 elena Nein eS Sts P. gregaria 20. Young lamellae fluorescing under UV; 21 21 pileus orange or tawny; young stipe apex buff, orange, or pale cinnamon, lower stipe colors similar to apex, habit scattered to gregarious, syringaldazein negative or positive; spores 8-12 umlong ................. 21 . Basidiome small to moderately large; stipe apex <12 mm diam (slender), stipe stuffed but larval infested, leaving a hollow rind at ground level; pileus conic umbonate, subviscid, uniformly bright apricot-/peach-colored; all tissues negative in syringaldazine; pileipellis hyphae colorless, spirally gel-incrusted, subpellis pigments KOH soluble, diffuse; spores 8-9.5 x 5-6 um, beaks short (<5 pm), straight ............... P. piceae . Basidiome large to massive; stipe apex 10-25 mm diam (robust), stipe long-lived, stuffed with firm, insect-free pith; pileus campanulate with obtuse umbo, viscid to glutinous, orange to tawny; all tissues strongly magenta in syringaldazine; pileus subpellis pigment-encrusted, pigments not KOH soluble; spores with slightly tilted long (<1.5 um) beaks ....................0.0005. 22 Phaeocollybia chefensis sp. nov. (U.S.A.) ... 27 22. Spores 7.5-10 x 4-6 um, limoniform, beak forward-tilted; pileus +/- uniformly orange, tawny, or orangish brown, typically drying with a burgundy-colored peaked umbo; lower stipe and pith staining first orange, then orangish brown; cheilocystidia cylindrical or narrowly to broadly clavate, rarely pedicellateand ‘subicapitate i. etch tind ees eee oe P. kauffmanii 22. Spores 8.5-12 x 5-7 um, amygdaliform with long ‘belly; beak back-tilted; young pileus with reddish tawny umbo, amber margin, and tan edge, eventually uniformly mahogany or dark reddish brown; lower stipe and pith staining reddish brown; cheilocystidia clavate to subcapitate with subglobose apices, pedivellate;-catermulate, Sune ews et. eis ects weed ores P. redheadii Research on Pacific Northwest phaeocollybias is ongoing; a fully revised second edition of PHAEOCOLLYBIA OF PACIFIC NORTHWEST NORTH AMERICA will be released after several additional taxa have been published. Acknowledgments The Pacific Northwest Phaeocollybia consortium wishes to acknowledge earlier assistance given by individual collectors, government surveyors and agencies, fungarium curators (BPI, DAOM, F, HSC, MICH, O, OSC, NY, SFSU, TENN, UBC, UC, WTU), technicians, and others cited in full on p. 205 of Norvell & Exeter (2009). We also acknowledge the excellent molecular work by M.N. Louise Lefebvre in 2010 at Agriculture & Agri-Food Canada in Ottawa. Kelli Van Norman (Interagency Special Status/Sensitive Species Program of the Forest Service (Region 6) PNW Region and Oregon/Washington Bureau of Land Management) is thanked for facilitating molecular sampling of government Phaeocollybia collections; particular thanks are due Darci Rivers-Pankratz (also of ISSSP) for providing data on specimens inventoried for the Northwest Forest Plan, facilitating microscopical reevaluation, and forwarding Heidi Christensen’s Boulder Creek collection for examination. We also thank Dr. Joe Ammirati (University of Washington, Seattle, USA), Dr. Egon Horak (Innsbruck, Austria), and Dr. P. Brandon Matheny (University of Tennessee, Knoxville, USA) for their greatly appreciated expert presubmission reviews. Egon Horak is further thanked for sharing his SEM-based basidiospore measurements for selected type specimens. 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Academic Press, New York. https://doi.org/10.1016/B978-0-12-372180-8.50042-1 MYCOTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2022 January-March 2022— Volume 137, pp. 31-39 https://doi.org/10.5248/137.31 Beltrania shenzhenica sp. nov. from Guangdong, China ZHAO-XUE ZHANG, TAI-CHANG Mu, ZHUANG LI, X1u-Guo ZHANG, JI- WEN XIA* Shandong Provincial Key Laboratory for Biology of Vegetable Diseases and Insect Pests, College of Plant Protection, Shandong Agricultural University, Taian, Shandong 271018, China “CORRESPONDENCE TO: xiajiwen1@126.com; xiajw@sdau.edu.cn ABSTRACT— During a survey of saprophytic microfungi on dead leaves from Futian Mangrove Nature Reserve in Guangdong Province, China, a new species of asexual ascomycota, Beltrania shenzhenica, was identified based on morphology and phylogenetic analyses of partial gene sequences of ITS and LSU. The detailed morphological description, phylogenetic tree, and photographs are provided. Key worps—Amphisphaeriales, anamorphic fungi, Beltraniaceae, phylogeny, taxonomy, Introduction Beltrania Penz., typified by Beltrania rhombica Penz. (Penzig 1882), is currently classified in Beltraniaceae, Amphisphaeriales (Wijayawardene & al. 2020, Zheng & al. 2020). The genus was mainly characterized by mostly unbranched dark setae with radially lobed basal cells, macronematous conidiophores arising from basal cells of setae or from separate radially lobed basal cells, and polyblastic, integrated, terminal, sympodial and denticulate conidiogenous cells that produce solitary acropleurogenous swollen separating cells and biconic, spicate or apiculate conidia with a hyaline equatorial band (Ellis 1971, Seifert & al. 2011, Lin & al. 2017, Hyde & al. 2020). Beltrania contains twenty species, of which eight are represented by molecular data (Harkness 1884, Wakefield 1931, Hughes 1951, Pirozynski & Patil 1970, Matsushima 1975, Rao & Varghese 1978, Zhang & Zhang 2003, 32 ... Zhang & al. Crous & al. 2014, Tibpromma & al. 2018, Bandgar & Patil 2019, Hyde & al. 2020, Zheng & al 2020). Futian Mangrove Nature Reserve, located northeast of Shenzhen Bay, was officially created in 1984 and designated as a national nature reserve in 1988. It belongs to the East Asian monsoon region with humid subtropical climate. The annual average temperature of 22.4 °C and average annual rainfall of 1700-1900 mm are conducive to the development of various microbial species (Meng & al. 2013). During our ongoing survey of anamorphic fungi associated with mangroves in China, we collected a new species representing Beltrania in the reserve. Materials & methods Isolation & morphological analysis Specimens of dead leaves were collected from Futian Mangrove Nature Reserve, Shenzhen, Guangdong Province, China, and returned to the laboratory in plastic bags. The samples were incubated in plastic boxes lined with moistened tissue paper at room temperature for one week. Tissue pieces (5 x 5 mm) were randomly taken from the leaf and surface-sterilized by consecutively immersing in 75% ethanol solution for 1 min, 5% sodium hypochlorite solution for 30 s, and then rinsing three times in sterile distilled water for 1 min. The pieces were dried with sterilized paper towels and then placed on potato dextrose agar (PDA) (Cai & al. 2009). All the PDA plates were incubated at 25 °C for 2-4 d, and subcultures from the colony margins were inoculated TABLE 1. Details of strains and sequences of Beltrania and related genera included in the phylogenetic analyses. New sequences are in bold; ex-(epi)type isolates are marked with ". GENBANK ACCESSION NUMBERS SPECIES VOUCHER ITS LSU Beltrania dushanensis GZCC18-0020 MN252875 MN252882 Beltrania krabiensis MFLUCC 16-0257" MH275048 MH260280 Beltrania pseudorhombica CPC 23656! KJ869158 KJ869215 Beltrania querna CBS 126097 MH864016 MH875474 CBS 122.51 MH856775 MH868293 Beltrania rhombica CPC 27482 KX519515 KX519521 CBS 123.58 MH857718 MH869260 strain 10353 — AB496423 CBS 121.50 _ MH868082 Beltrania shenzhenica SAUCC 00617 MW784619 MW784621 SAUCC 0065 MW784620 MW784622 SPECIES Beltrania sinensis Beltraniella acaciae Beltraniella botryospora Beltraniella brevis Beltraniella carolinensis Beltraniella endiandrae Beltraniella fertilis Beltraniella humicola Beltraniella pandanicola Beltraniella portoricensis Beltraniella pseudoportoricensis Beltraniella ramosiphora Beltraniella thailandica Beltraniopsis longiconidiophora Beltraniopsis neolitseae Beltraniopsis sp. Castanediella couratarii Hemibeltrania cinnamomi Porobeltraniella porosa Pseudobeltrania cedrelae Pseudobeltrania ocoteae Subramaniomyces fusisaprophyticus Subsessila turbinata VOUCHER JS43 JS42 JS260 JS101 CPC 294987 TUFC 100837 GZCC 18-0081 GZCC 18-0082 IFO 9502 CPC 221937 MFLUCC 17-2137 MFLUCC 17-2138 CBS 203.64 MFLUCC 18-0121" NECCI 3993 CBS 856.70 CBS 145547 MFLUCC 17-2582" MFLUCC 16-03777 MFLUCC 17-2139? MFLUCC 17-2140 CPC 221687 TUFC 10081 CBS 579.71 NECCI 3695 NECCI 3997 MFLUCC 17-2141 NECCI 3994 NECCI 3995 NECCI 3996 COAD 2098' PF9 CPC 262197 CBS 418.95 MFLUCC 15-0831 Beltrania shenzhenica sp. nov. (China) ... 33 GENBANK ACCESSION NUMBERS ITS MNO077366 MNO077365 MNO077364 MN077363 KY173389 MN252877 MN252876 KJ869128 MF580247 MF580248 MH858416 MH275049 KX519516 MH859981 MK876377 MG717500 MH275050 MF580249 MF580250 KJ869126 MH860269 KT119564 KX519517 MF580251 KX519518 KX519519 KX519520 MG559548 MG559552 KT950856 EU040241 KX762288 LSU MNO077266 MNO077265 MN077264 MNO077263 KY173483 AB496426 MN252884 MN252883 DQ810233 KJ869185 MF580254 MF580255 MH870044 MH260281 KX519522 MH871777 MK876416 MG717502 MH260282 MF580256 MF580257 KJ869183 AB496424 MH872031 KT119565 KX519523 MF580258 KX519524 KX519525 KX519526 MG559558 MG559562 KT950870 EU040241 KX762289 34 ... Zhang & al. onto new PDA plates. Colonies at 7 d and 15 d were photographed using a PowerShot G7X mark II digital camera. Fungal micromorphological structures were observed and photographed using Olympus SZX10 stereomicroscope and Olympus BX53 microscope, both fitted with Olympus DP80 high definition colour digital cameras. All fungal strains were stored at 4 °C in 10% sterilized glycerin for further studies. The specimens are deposited in the Herbarium of Plant Pathology, Shandong Agricultural University, Taian, Shandong, China (HSAUP). Ex-type cultures are deposited in the Shandong Agricultural University Culture Collection, Taian, Shandong, China (SAUCC). DNA extraction, PCR amplification, sequencing Genomic DNA was extracted from colonies grown on PDA based on a modified CTAB method (Doyle & Doyle 1990). The nuclear ribosomal internal transcribed spacer (ITS) was amplified and sequenced using the primer pair ITS4/ITS5 (White & al. 1990), and the large subunit ribosomal RNA gene (LSU) using the primer pair LROR/LRS (Vilgalys & Hester 1990, Glass & Donaldson 1995). PCR was performed using an Eppendorf Mastercycler Thermocycler. The DNA was amplified in 25 uL reaction volumes containing 12.5 uL Green Taq Mix, 1 uL of each forward and reverse Biosune primer (10 uM), and 1 uL template genomic DNA in amplifier, and adjusted with distilled deionized water to a total 25 uL volume. PCR parameters followed were 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and a 1-min extension at 72 °C and ending with a final 10-min elongation at 72 °C. PCR products were estimated visually by staining with GelRed after 1% agarose gel electrophoresis. Sequencing was performed bi-directionally by BioSune Co. Ltd. (Shanghai). Consensus sequences were obtained using MEGA v. 7 (Kumar & al. 2016). All sequences generated in this study were deposited in GenBank (TABLE 1). Sequence alignment & phylogenetic analysis The quality of our amplified nucleotide sequences was checked and combined by MEGA v. 7 (Kumar & al. 2016), and reference sequences were retrieved from the National Center for Biotechnology Information (NCBI). Sequences were aligned using MAFFT v. 7.310 (Katoh & al. 2019) and manually corrected using MEGA Wi, 2 The combined gene regions were phylogenetically analyzed using Maximum- Likelihood (ML) and Bayesian Inference (BI). RaxML analyses (using RaxML v. 8.2.9) and Bayesian analyses (using MrBayes v. 3.2.6) were run on the CIPRES Science Gateway Portal (Miller & al. 2012). Evolutionary models were calculated using MrModeltest v. 2.3 (Nylander 2004) selecting the best-fit model for each data partition according to the Akaike criterion. For ML analyses the default parameters were used and bootstrap support (BS) was carried out using the rapid bootstrapping algorithm with the automatic halt option. Bayesian analyses included two parallel runs of 5,000,000 generations with the stop rule option and a sampling frequency set to each 1000 generations. The 50% majority rule consensus trees and posterior Beltrania shenzhenica sp. nov. (China) ... 35 Beltrania sinensis Js1 01 “Beltrania sinensis JS42_ 74 “ Beltrania sinensis JS260. 4 Beltrania querna CBS 126097 ~’ Beltrania querna CBS - | Beltrania sinensis JS43° 7 SAUCC0065 ‘SAUCCo061 | « Bel rania : Beltrania rhombica CPC 27482 peltrania Beltrania rhombica CBS 121. 50 Beltrania rhombica strain 10353 y, Pseudobeltrania cedrela COAD Pseudobeltranie cedrelae PFO Beltrania rhombica CBS 123 58 seeltrania dushanensis Gzcct 8-0020 Beltrania krabiensis MFLUCC C 16-( Beltrania pseudorhombica CP Beltraniella botryospo raT Beltraniella endiandrae CPC 22 = Beltraniella brevis GZ oa Beltraniella brevis GZ Beltraniella ramosiphora MFLUCC Beltraniella thailandica MFLUCC 16-02 Beltraniella pseudoportoricensis CBS 14554 Beltraniella humicola CBS 203.64 Tm opeltraniella carolinensis |FO 9502 Beltraniella portoricensis CBS 856.70 Beltraniella portoricensis NFCCI 3993 Beltraniella pandanicola MFLUGC 62. ‘,,Beltraniella fertilis MFLUCC 17-2137 Beltraniella fertilis MFLUCC 17-21 38 Beltraniella acaciae CPC 29498 Forobeltraniella porosa Wen orobeltraniella porosa NFCCI 3995 e20.93t Porobeltraniella porosa NFCCI 996 3X , eZ Pseudobeltrania ocote oat Hemibeltrania cinnamomit Feta | "Femibeltrania cinnamomi_ 82/0.08 Hemibeltrania cinnamomi MF Subsessila turbinata MFLUCC 15-( Beltraniopsis longiconidiophora MFLUCC Beltraniopsis pete er aItranionaie ‘T ,Peltraniopsis sp. TUFC 10081 te ae = Beltraniopsis neolitseae CPC 221 68 Subramaniomyces fusisaprophyticus ¢ CBS. Castanediella couratarii CBS 579.71 “Ei Fic. 1. Phylogram of Beltrania and related genera, generated from Bayesian analysis based on combined ITS and LSU sequence genes. Bootstrap support values are shown as ML >50% first, followed by BI >0.90. Some branches were shortened to fit the page - these are indicated by two diagonal lines and a correction factor indicating the full length. Ex-type/ex-epitype strains are in bold. Newly generated sequences are indicated in red. probability (PP) values were calculated after discarding the first 25% of the samples as burn-in. The resulting trees were plotted using FigTree v. 1.4.2 (http://tree.bio. 36 ... Zhang & al. ed.ac.uk/software/figtree) and edited with Adobe Illustrator CS v. 5. The individual gene datasets were assessed for incongruence before being concatenated by checking their individual phylogenies for conflicts between clades with significant ML and BI support (Mason-Gamer & Kellogg 1996, Wiens 1998). Phylogenetic results The dataset comprised 46 sequences representing 29 species including Castanediella couratarii (CBS 579.71) as the outgroup. The final alignment comprised a total of 1549 characters of the combined ITS and LSU including gaps, ITS: 1-940 and LSU: 941-1549. Of these characters, 1256 were constant, 152 parsimony-uninformative and 141 parsimony-informative. For the BI and ML analyses, the substitution model GTR+I+G for ITS and LSU were selected and incorporated into the analyses. The topology of Bayes tree confirmed the tree topologies obtained from the ML analyses, and therefore, only the Bayes tree is presented (Fic. 1). In this tree, our two sequences formed a distinct clade. Therefore, we determined that our strains belonged to a novel species of Beltrania. Taxonomy Beltrania shenzhenica Z.X Zhang, J.W. Xia & X.G. Zhang, sp. nov. Fic. 2 MB 839268 Differs from Beltrania querna by its wider conidia and from B. rhombica by its longer conidia. Type: China, Guangdong Province, Shenzhen, Futian Mangrove Nature Reserve, on dead leaves of a broadleaf tree, 14 July 2020, Z.X Zhang (Holotype, HSAUP 0061; ex- type living culture SAUCC 0061; GenBank MW784619, MW784621). EryMo_oey: in reference to the city where the type was found. CoLonies on PDA at 25 °C in darkness, increasing in diameter by 11-15 mm/d, surface greyish white to black, flat, dense, reverse black to dark black. Mycelium partly superficial and partly immersed. Stroma absent. Setae erect, brown, thick-walled, septate, straight to flexuous, conical at the apex, slightly swollen at basal cell. Conidiophores 58-88 x 3-5 um, aggregated in dense fascicles, pale brown, cylindrical, septate, unbranched, straight to variously curved, proliferating sympodially at apex. Conidiogenous cells terminal, integrated, subhyaline, smooth, holoblastic, polyblastic, with several flat tipped denticles. Separating cells 8-16 x 5-7 um, subhyaline, finely roughened, with several apical, flat-tipped denticles. Conidia 28- 33 x 8.5-12 um (including apical appendage), biconic, aseptate, solitary, acropleurogenous, subhyaline to pale brown, with distinct granules, Beltrania shenzhenica sp. nov. (China) ... 37 Fic. 2. Beltrania shenzhenica (holotype, HSAUP 0061). a, b. Surface and reverse of colony on PDA; c. Mycelium on PDA; d. Setae; e, f. Conidiophores, separating cells, conidiogenous cells, and conidia; g—i. Separating cells; j-1. Conidia. Scale bars: d-] = 10 um. without median transverse band, apical appendage 5-9 um long, tapering to an acutely rounded tip, smooth, without a mucilaginous sheath. ADDITIONAL SPECIMEN EXAMINED: CHINA, GUANGDONG PROVINCE, Shenzhen, Futian Mangrove Nature Reserve, on dead leaves of a broadleaf tree, 14 July 2020, Z.X. Zhang (HSAUP 0065; living culture SAUCC 0065; GenBank MW784620, MW784622). ComMENTs - Based on phylogenetic analysis, our Beltrania shenzhenica sequences grouped together with B. querna and B. rhombica, but they formed a distinct clade. Morphologically, B. shenzhenica is most similar to B. querna 38 ... Zhang & al. and B. rhombica in conidial shape, but B. querna differs by its narrower conidia (6-8 um wide; Harkness 1884), and B. rhombica differs by its shorter conidia (25-26 um long; Penzig 1882). Acknowledgments The authors express gratitude to Dr. Jian Ma (College of Agronomy, Jiangxi Agricultural University, Nanchang, China) and Dr. Li-Guo Ma (Institute of Plant Protection, Shandong Academy of Agricultural Sciences, Jinan, China) for serving as pre-submission reviewers and to Dr. Shaun Pennycook for nomenclatural review and Dr. Lorelei L. Norvell for editorial review. This work was jointly supported by the National Natural Science Foundation of China (Nos. 31900014, U2002203, 31750001) and National Science and Technology Fundamental Resources Investigation Program of China (2019FY100700). Literature cited Bandgar SG, Patil CR. 2019. A new species of Beltrania from Western Ghats, India. International Journal of Life Science 7(2): 325-328. Cai L, Hyde KD, Taylor PWJ, Weir BS, Waller JM, Abang MM & al. 2009. A polyphasic approach for studying Colletotrichum. Fungal Diversity 39: 183-204. Crous PW, Shivas RG, Quaedvlieg W, van der Bank M, Zhang Y, Summerell BA & al. 2014. 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International Journal of Systematic and Evolutionary Microbiology 70: 1178-1185. https://doi.org/10.1099/ijsem.0.003897 MYCOTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2022 January-March 2022— Volume 137, pp. 41-50 https://doi.org/10.5248/137.41 Termitomyces cryptogamus sp. nov. associated with Macrotermes natalensis in Africa LENNART J.J. VAN DE PEPPEL’, Z. WILHELM DE BEER’, DuurR K. AANEN*, BEN AUXIER’ ‘Laboratory of Genetics, Wageningen University, 6700 AA, Wageningen, the Netherlands *Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa CORRESPONDENCE TO: duur.aanen@wutr.nl ABSTRACT—A new species of Termitomyces symbiotic with the termite Macrotermes natalensis is described from Africa. As there are no records of field collected basidiocarps within this lineage, traditional basidiocarp-based morphological taxonomy is not practical. While basidiocarps may be obtained rarely from incubation of fungal comb fragments, their practical use for taxonomical purposes is limited. Therefore, the species is described based on an ITS nucleotide sequence, with comparisons to an asexual culture. Based on samples with similar ITS sequences, this species is likely associated with multiple termite hosts across a large part of Africa. Key worps—Agaricales, Lyophyllaceae, phylogeny Introduction In Africa and Asia, a subfamily of the Termitidae, the Macrotermitinae, live in obligate symbiosis with members of the basidiomycete genus Termitomyces (Lyophyllaceae). The fungus resides inside the termite nest in so-called “fungus gardens,’ with mushroom production being periodically triggered by rain or the potential health of the nest (Koné & al. 2011). The mushrooms vary in size and are generally medium to large, although some species, such as T: microcarpus (Berk. & Broome) R. Heim, produce hundreds of small 42 ... van de Peppel & al. mushrooms. Traditionally, taxonomy in this genus has been based on basidiocarp morphology, with molecular evidence only recently added (Freslev 2003, Mossebo & al. 2017). During research of termites and their associated fungi in Africa, one lineage was found that associates with Macrotermes bellicosus and M. subhyalinus when found in northern Africa, but only with M. natalensis when found in South Africa (Nobre & al. 2011). Surprisingly, sampling over ten years has not resulted in the discovery of mature basidiocarps of this species from any of its termite hosts, frustrating efforts for naming this species. Rarely, while excavating termite mounds, mushroom primordia are recovered (de Fine Licht & al. 2005; Vreeburg & al. 2020) that can occasionally be incubated under laboratory conditions in the absence of termite workers. Presumably due to this laboratory incubation the mushrooms are misshapen, as their appearance is not consistent with other known Termitomyces species. This may be similar to development that occurs in the production of “Enoki” mushroom from Flammulina velutipes which causes long thin stipes, smaller caps, and pale coloration (Kites & Navarro-Gonzalez 2015). Despite the need to provide a name for this well- studied fungus, the lack of basidiocarps has prevented comparisons with published literature descriptions (Botha & Eicker 1991b), leading to an argument for using readily obtained asexual cultures as a stable type against which to compare (Makonde & al. 2013). As extensive comparisons of ITS sequences from herbarium material from South Africa recovered no matches with our M. natalensis symbiont (van de Peppel & al., unpublished data), we provide a description based on sequence identity and asexual morphology. While there is a previous description of asexual characteristics from South African Termitomyces, only mounds producing basidiocarps were used, with M. natalensis mounds apparently not sampled (Botha & Eicker 1991b). As basidiocarp records are lacking, asexual cultures are a logical solution to this taxonomic issue. Three previous publications addressed differences between species in asexual cultures, and while interspecific differences were found, asexual characteristics alone were not considered sufficient to delineate species (Botha & Eicker 1991a,b; Tibuhwa 2012). Recently an ecological study of Macrotermes symbionts in Kenya uncovered results consistent with previous sampling of this lineage (Vesala & al. 2017). Vesala & al. recovered the same fungal species as the one collected from South Africa (Nobre & al. 2011, de Fine Licht & al. 2005; Vreeburg & al. Termitomyces cryptogamus sp. nov. (South Africa) ... 43 2020) from mounds of M. bellicosus, M. herus, M. jeanneli, M. michaelseni, and M. subhyalinus. Vesala & al. (2020), who selected related ITS sequences from GenBank including symbionts of M. natalensis and M. bellicosus, found that these formed a monophyletic clade. These sequences form two well-supported groups with 97% and 98.5% support, with the exception of a single sequence (GenBank AF357024) isolated from an unidentified Macrotermes species. Based on these results we describe the species based on a fungal isolate from South Africa symbiotic with M. natalensis for which the fungal genome has been published (Poulsen & al. 2014). Materials & methods Samples and Isolates A heterokaryotic culture, Mn103, was obtained by opening a termite mound and carefully removing nodules containing asexual spores from the fungal combs without soil contamination. These nodules were placed on agar plates without antibiotics to establish hyphal cultures. A homokaryotic culture, P5, was obtained by protoplasting the Mn103 heterokaryotic culture. Additional details regarding sampling, isolation, and subsequent protoplasting of this culture are found in Poulsen & al. (2014) and Nobre & al. (2014). A dried holotype specimen of the Mn103 heterokaryotic culture and a living ex-holotype culture of the P5 homokaryotic culture are conserved at the Westerdijk Fungal Biodiversity Institute, Utrecht, Netherlands (CBS). DNA extraction, PCR amplification, sequencing Genomic DNA was extracted from the protoplasted P5 homokaryotic culture using the cetyltrimethylammonium bromide (CTAB) protocol using mycelium and spores scraped from a petri dish. The nuclear ribosomal region containing the ITS1 + 5.88 + ITS2 region (ITS) was amplified using a standard PCR reaction using Promega GoTaq polymerase and the fungal specific primer ITS1F and the general reverse primer ITS4 (White & al. 1990, Gardes & Bruns 1993). The 28S region (LSU) was amplified using primers LROR and LR5 (Vilgalys & Hester 1990). As no basidiocarps have been reported from M. natalensis termite mounds, we also surveyed herbarium samples from the South African National Collection of Fungi (PREM) and Schweickerdt Herbarium (PRUM) (van de Peppel & al. unpublished data). Sequence alignment & phylogenetic reconstruction We used a previously published set of ITS sequences (Vesala & al. 2017), six GenBank sequences from Termitomyces symbiotic with M. natalensis, and a sequence generated from the specimen we designate here as the type for the new species. Sequences were aligned using the web software MAFFT v. 7 with default settings (Katoh & Standley 2013). Maximum likelihood trees were reconstructed using IQ-TREE v. 2.0.6 with default settings (Trifinopoulos & al. 2016). 44 ... van de Peppel & al. Culture & microscopy Cultures were maintained on MYA (20g Malt Extract, 2g Yeast Extract, 1 L H,O), and incubated at 25 °C. Microscopical examinations were conducted using a Zeiss Axio Imager Al with 63X objective lens under DIC optics. Taxonomy Termitomyces cryptogamus van de Peppel, sp. nov. Fic. 1 MB 838129 Differs from Termitomyces schimperi by its clearly separated LSU sequence; there are no useful diagnostic differences in the morphology of the asexual morphs of these species. Type—South Africa, Pretoria, Rietfontein 321-Jr, 25.7292°S 28.2347°E, May 2011, DK Aanen, heterokaryotic isolate (Mn103) obtained from asexual nodules on a fungal comb from a mound of Macrotermes natalensis (Holotype, CBS H-24752 [metabolically inactive dried culture]; living ex-type culture CBS 147190]. ETYMOLOGY: cryptogamus, referring to the hidden marriages of a genetically well-mixed species without recorded basidiocarps in vivo. SEXUAL STATE—not observed in vivo. ASEXUAL STATE—Growth of colonies on MYA medium reaching 5-6 cm diam. in 3 weeks at 25 °C (somewhat faster at 30 °C). Growth consisting of white hyphae mostly submerged in agar, with 1-2 mm diam. clusters of asexual spores produced on the agar surface. Conidia highly variable in size (10-100 mm long) and shape, with 2-5 nuclei per spore. Heterokaryotic colonies consistently producing heterokaryotic conidia. ComMMENTS—In the absence of in vivo basidiocarps, T. cryptogamus cannot be distinguished readily from closely related Termitomyces species based on asexual characters. However, ITS and LSU sequence analyses clearly separate a well-supported clade that includes the holotype of T. cryptogamus and several other strains obtained from fungal combs of Macrotermes natalensis in South Africa, as well as from those of M. bellicosus, M. herus, M. jeanneli, M. michaelseni, and M. subhyalinus in Cameroon, Kenya, Senegal, and Ivory Coast. Phylogenetic results NCBI GenBank accession numbers were obtained for the nucleotide sequences generated from the protoplasted homokaryotic culture P5: ITS (MW251838), LSU (MW567773), whole genome (GenBank id: GCA_001263195). The ITS sequence of our isolate P5 places Termitomyces cryptogamus inside Group 1 of Vesala & al. (2017). This group includes fungal individuals symbiotic Termitomyces cryptogamus sp. nov. (South Africa) ... 45 Fic. 1. Termitomyces cryptogamus: morphology. A. Growth on Malt Yeast Agar showing abundant nodule formation of asexual conidia; B. Close up view of A; C-H. Variable morphology of conidia; I, J. Morphology of mushrooms on fragments of excavated fungus garden comb produced after incubation for 5-10 days; note the small caps (<1 cm), which produce viable basidiospores. with Macrotermes natalensis, M. bellicosus, M. subhyalinus, M. michaelseni, M. herus and M. jeanneli (Fic. 2). Except for M. natalensis, these termite species are found with sister species also closely related to Termitomyces cryptogamus. To compare with other common Macrotermes mound symbionts, we also extracted DNA from samples of Termitomyces schimperi (Pat.) R. Heim. However, repeated PCR amplifications of the ITS region were not successful. 46 ... van de Peppel & al. DQ436938 Termitomyces sp. M. natalensis South Africa DQ436958 Termitomyces sp. M. natalensis South Africa DQ436940 Termitomyces sp. M. natalensis South Africa MwW251 T. mus M. natalensi: h Afri DQ494698 Termitomyces sp. M. natalensis South Africa DQ436957 Termitomyces sp. M. natalensis South Africa MG283259 Termitomyces sp. M. natalensis AY764149 Termitomyces sp. M. natalensis South Africa DQ436956 Termitomyces sp. M. natalensis South Africa DQ436964 Termitomyces sp. M. natalensis South Africa GQ383682 Termitomyces sp. M. michaelseni Kenya GQ383679 Termitomyces sp. M. jeanneli Kenya GQ383685 Termitomyces sp. M. michaelseni Kenya AY764150 Termitomyces sp. M. natalensis South Africa GQ383683 Termitomyces sp. M. herus Kenya HQ902219 Termitomyces sp. M. bellicosus \vory Coast HQ902232 Termitomyces sp. M. bellicosus GQ383687 Termitomyces sp. M. michaelseni Kenya HQ902224 Termitomyces sp. M. bellicosus HQ902218 Termitomyces sp. M. bellicosus Cameroon AF357024 Termitomyces sp. GQ922682 Termitomyces sp. M. bellicosus Senegal HQ902227 Termitomyces sp. M. subhyalinus JF302815 Termitomyces sp. M. bellicosus lvory Coast HQ902230 Termitomyces sp. M. bellicosus HQ902229 Termitomyces sp. M. bellicosus GQ383686 Termitomyces sp. M. michaelseni Kenya gg ©Q922681 Termitomyces sp. M. bellicosus Senegal KY197689 Termitomyces sp. M. subhyalinus Kenya GQ922687 Termitomyces sp. M. nobilis Gabon 9g ©Q922688 Termitomyces sp. M. muelleri Gabon AF321368 Termitomyces sp. M. muelleri Gabon GU001666 Termitomyces sp. Macrotermes sp. Malaysia $f GU001670 Termitomyces sp. Macrotermes sp. Malaysia AB051889 Termitomyces sp. M. carbonarius Malaysia AF321362 Termitomyces sp. M. subhyalinus Senegal HQ902240 Termitomyces sp. M. subhyalinus Benin GQ922686 Termitomyces sp. M. subhyalinus Senegal JF302816 Termitomyces sp. M. subhyalinus \vory Coast EU816416 Termitomyces sp. M. subhyalinus Burkina Faso KY197700 Termitomyces sp. M. subhyalinus Kenya JQ088160 Termitomyces sp. Macrotermes sp. Kenya GQ383676 Termitomyces sp. M. michaelseni Kenya GQ383677 Termitomyces sp. M. jeanneli Kenya 82 JQ088143 Termitomyces sp. Macrotermes sp. Kenya GQ383678 Termitomyces sp. M. michaelseni Kenya pAF321371 Termitomyces sp. M. bellicosus Senegal 85 GQ383675 Termitomyces sp. M. bellicosus Kenya [--——— EF091678 Termitomyces sp. Macrotermes sp. Thailand EU816418 Termitomyces sp. M. carbonarius Vietnam 0.05 * Termitomyces cryptogamus Group 1 sensu Vesala & al. Group 3 sensu Vesala & al. Group 4 sensu Vesala & al. Group 2 sensu Vesala & al. Group 5 sensu Vesala & al. Group 6 sensu Vesala & al. Group 7 sensu Vesala & al. Group 9 sensu Vesala & al. Group 8 sensu Vesala & al. 2017 2017 2017 2017 2017 2017 2017 2017 2017 Fic. 2. Maximum likelihood tree based on ITS1-5.8S-ITS2 sequences of Termitomyces cryptogamus and allied Macrotermes symbionts with their host termite species (in bold) and their collection location. Outgroups were removed to increase readability of the tree. Numbers at the nodes indicate ultrafast bootstrap values, only significant node values >95% are displayed. Species delimitation groups with 97% ITS identity (sensu Vesala & al. 2017) are displayed on the right side of the tree. A sequence of the T. cryptogamus ex-type P5 is underlined and indicated by an asterisk. We were able to amplify LSU sequences from both T. cryptogamus and herbarium samples of T’ schimperi (PREM41964), which were then analysed together with other GenBank LSU sequences of T’ schimperi and unidentified M. natalensis symbionts (Fic. 3). The LSU sequence of our P5 isolate was identical to that from a fungal symbiont of a M. natalensis mound. LSU sequences from the commonly recovered Macrotermes symbiont, T. schimperi, were not monophyletic. Termitomyces cryptogamus sp. nov. (South Africa) ... 47 T. schimperi RSK186 KU933614 T. schimperi tgf18 AY232712 T. cryptogamus P5 MW567773 W/V. natalensis * 83 Termitomyces sp. ZA164 DQ110875 M. natalensis Termitomyces sp. EF 667085 MW. natalensis T. schimperi DM24E KY809228 T. schimperi MW567772 M. subhyalinus 100 T. schimperi PREM41964 MW567771 Termitomyces sp. AB073526 Odontotermes sp. 0.006 Fic. 3. Phylogenetic tree based on LSU sequences for Termitomyces cryptogamus. and publicly available LSU sequences of T’ schimperi, acommon Macrotermes symbiont. Sequence of AB073526 used as an outgroup to root the tree. Node values indicate bootstrap support. Microscopical investigations revealed highly variable morphology of conidia harvested from laboratory grown cultures (Fic. 1c-H), which limits morphological comparison with asexual cultures of other Termitomyces species. Discussion There is accumulating evidence, both direct and indirect, that sexual reproduction does occur in T: cryptogamus. First, sexual reproduction between strains associated with M. natalensis was inferred as occurring 48 ... van de Peppel & al. sufficiently frequently (at least 100 sexual events per generation) to explain the observed signature of free recombination (de Fine Licht & al. 2006). Second, mating tests between homokaryons retrieved from heterokaryons demonstrated that the M. natalensis-associated strains represent a single biological species (Nobre & al. 2014). Finally, strains associated with M. natalensis were observed to produce mushrooms and viable basidiospores in vitro (de Fine Licht & al. 2005; Vreeburg & al. 2020). These findings make it all the more surprising that mushrooms have not been found in nature. One hypothesis is that sexual reproduction of this species occurs belowground synchronously with alate dispersal. Nevertheless, recovery of in-vitro basidiocarps only after prolonged laboratory incubation prevents their use as a morphological type specimen, as presumably incubation conditions greatly influence the resulting morphology. Additionally, the infrequency and unpredictability of these mushroom primordia in nests precludes their use for identification. Further, the asexual spores produced are highly polymorphic, and thus cannot be used for reliable identification with this group. As such, although comparisons of asexual cultures remain valuable, molecular markers provide the only reliable way to identify samples (Licking & al. 2020). Based on the work of Makonde & al. 2013, it is likely that other Termitomyces species exist where the only practical identification markers will be molecular. Using the 3% ITS similarity threshold, we find one lineage (group 1, sensu Vesala & al. 2017) that is distributed across Africa, with hosts differing geographically, but always within Macrotermes. The different termite symbionts combined with geographic isolation may indicate barriers to gene flow. Further study should show whether or not geographically separated populations of T: cryptogamus, including populations associated with Macrotermes species other than M. natalensis, all form a single biological species. Although comparisons of ITS sequences from T. cryptogamus and T. schimperi are not currently available, we feel these sequences represent at least two species for two reasons. Firstly, the fact that we are unable to amplify the ITS regions successfully using the ITS1F and ITS4 primers, while we can amplify the LSU sequence indicates that there are likely mutations in the primer binding site not found in T! cryptogamus (for which ITS sequences are readily amplified). This suggests that T. schimperi likely has fixed substitutions not shared with T. cryptogamus. Secondly, the relationships between the LSU sequences generated from T: schimperi and T: cryptogamus Termitomyces cryptogamus sp. nov. (South Africa) ... 49 (Fic. 3) show significant genetic difference, although the backbone nodes of the phylogeny do not receive statistical support of bootstrap values greater than 70. Additionally, the recovery of two clades of T’ schimperi indicates that T: schimperi is potentially paraphyletic and deserves further study. Acknowledgments The authors thank Tobias Guldberg Froslev (Geogenetics, University of Copenhagen, Denmark) and N’golo Abdoulaye Koné (Department of Natural Sciences, Université Nangui Abrogoua, Abidjan, Cote d'Ivoire) for presubmission review. D.K.A., L.J.J.v.d.P., and B.A. were supported by the Netherlands Organization for Scientific Research (D.K.A., L.J.J.v.d.P. by VICIINWO 86514007; D.K.A. and B.A. by ALWGR.2017.010). Literature cited Botha WJ, Eicker A. 1991a. Cultural studies on the genus Termitomyces in South Africa. I. Macro- and microscopic characters of basidiome context cultures. Mycological Research 95(4): 435-443. https://doi.org/10.1016/S0953-7562(09)80843-5 Botha WJ, Eicker A. 1991b. Cultural studies on the genus Termitomyces in South Africa. II. Macro- and micromorphology of comb sporodochia. Mycological Research 95(4): 444-451. https://doi.org/10.1016/S0953-7562(09)80844-7 de Fine Licht HH, Andersen A, Aanen DK. 2005. 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Academic Press, San Diego CA. https://doi.org/10.1016/B978-0-12-372180-8.50042-1 MY COTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2022 January-March 2022— Volume 137, pp. 51-61 https://doi.org/10.5248/137.51 Clitopiloides prati and Trichopilus lecythiformis spp. nov. from Australia Davip L. LARGENT & MOLLy B. CRIBARI Biological Sciences, Humboldt State University, I Harpst St, Arcata CA 95521, USA “CORRESPONDENCE TO: mrp@humboldtl.com ABSTRACT—Clitopiloides prati (from northeastern Queensland) and Trichopilus lecythiformis (from coastal New South Wales) are described as new entolomatoid species based on morphological characters. Morphological similarities are discussed for these and other closely related species. We also provide nuclear ribosomal RNA repeat (nrITS) and large subunit (nrLSU) sequences where obtained Key words—Agaricales, Basidiomycota, Entolomataceae, taxonomy Introduction Agarics are readily recognized as representing Entolomataceae (Agaricales) by their flesh colored basidiospores that are angular at least in end view; the family has a cosmopolitan distribution and iscommon in temperate and tropical ecosystems. Within the family, basidiomes exhibit highly diverse morphologies and ecologies. One consequence of this diversity is the acceptance of 1848 species as valid by Kalichman & al. (2020), the second highest of all families classified in the Agaricales. The multigeneric classification of Entolomataceae utilized in this paper follows Largent (1994). The reasons for adopting this approach are detailed in Largent (2020), but important points are reiterated here: a) the basionym for nearly every genus in Largent (1994) is identical to the infrageneric basionyms in Romagnesi & Gilles (1979), Noordeloos (1992, 2004), and Noordeloos & Gates (2012); (b) it is relatively easy to distinguish the different genera visually 52 ... Largent & Cribari using this classification in the field; c) although a comprehensive classification based on a phylogenetic and morphological approach in Entolomataceae is not yet universally accepted, many segregate entolomatoid genera are supported by phylogenetic analysis (Karstedt & al. 2019, He & al. 2019). Australian field investigations from 2009-12 within northeastern Queensland’s Wet Tropics Bioregion and from 2010-12 in the temperate rainforests of central New South Wales have uncovered several novel species in the Entolomataceae (Largent & Abell-Davis 2011; Largent & al. 2011a,b, 2013a,b, 2014, 2016; Bergemann & al. 2013). Here we describe and illustrate two unusual new species from Australia: Clitopiloides prati and Trichopilus lecythiformis. Materials & methods Macro- & micromorphology Colors were identified subjectively and coded according to Kornerup & Wanscher (1978) with color plates noted in parentheses (E63) in the species description. Common or general names for colors used in this paper are found in the Colour Sample to Colour Name section in the back of the color handbook where each page presents tables flagging technical names for color by * and placing common names in italics; we designate the technical color names using quotation marks (e.g., “Saruk’). Fresh collections were heat dried. Dried basidiomata were sectioned and rehydrated in 3% KOH. A Nikon Eclipse Ci compound microscope with Lumera Infinity 2 imaging software was used to examine and measure microscopic features following Largent (1994). In the taxonomic descriptions x = mean dimensions, Q = range of length/width ratios from individual structures, Q. = mean of all individual length/width ratios, and x/y = the total number of structures measured (x) and the total number of collections examined (y). Specimens were deposited in the Plant Pathology Herbarium, Orange Agricultural Institute, Orange, New South Wales, Australia (DAR); or the Fungarium, Biology Department, Humboldt State University, Arcata, Calfornia, USA (HSC). DNA sequences Dried basidiomata were pulverized and DNA extracted according to Largent & al. (2011a,b). The ITS region (ITS1, 5.88 and ITS2) of the nuclear ribosomal RNA repeat (nrITS) was amplified with primers ITSIF (Gardes & Bruns 1993) and ITS4 (White Q al. 1990) and the nuclear large subunit (nrLSU) was amplified with primers ctb6 and twl3 (White & al. 1990). PCR reactions of the nrITS and nrLSU were performed in 50 uL reactions following the procedure as outlined in Largent & al. (2011b) and PCR cycling followed the protocol detailed in Bergemann & Garbelotto (2006). PCR products were cleaned using 1 uL of ExoSAP-IT (GE Healthcare) and incubated at 37 °C for 15 min followed by 80 °C for 45 min. Cycle sequencing was performed at MCLAB (South San Francisco CA) using an Clitopiloides & Trichopilus spp. nov. (Australia) ... 53 ABI3730XL sequencer to obtain bi-directional sequences. Contigs were assembled and edited with Sequencher 4.8. Taxonomy Clitopiloides prati Largent, sp. nov. FIG. 1 IF 558177 Differs from Entoloma cyathus by its greyish brown, appressed fibrillose pileus, equal stipe, encrusting pigments, pileipellis with erect to suberect distal cells, longer cheilocystidia, ammonia-like odor, and habitat in a lawn. Type: Australia, Queensland, Cook Region, Smithfield, 10 Marcia Close, 16.8261°S 145.6857°E, gregarious in lawn, 27 September 2010, Peter Newling 40a (Holotype HSC A1429); GenBank M“W520128, MW520127) EryMoLoGcy—the genitive of the Latin partum, referring to the habitat in a lawn. BASIDIOMATA Clitocyboid. PrLEus 30-70 mm broad, <25 mm high, plano- convex to plane, deeply depressed to infundibuliform; radially appressed- fibrillose at all times; typically with a metallic luster when fresh; at first greyish brown (6E2, “Saruk”) fading with age to greyish brown to brown (6E3-4) with the disc area remaining greyish brown; not hygrophanous nor translucent-striate; margin incurved becoming plane, entire then eroded; CONTEXT white to off-white, thickness not measured. LAMELLAE <30 mm long, 7-10 mm broad; white at first, flesh colored with spore maturation; uncinate with a decurrent tooth or subdecurrent to decurrent; close; edges smooth then eroded, not denticulate; wavy, concolorous; lamellulae 4-7 mm high, 4-15 mm long. STIPE <60 x <14 mm, equal; white becoming at the base faintly brownish with handling and age; longitudinally appressed fibrillose; BASAL TOMENTUM absent. Opor strong, ammonia-like when crushed. TASTE not taken. BASIDIOSPORES 5 to (rarely) 6 distinct angles in profile and side views, very rarely 4-angled in polar view; in profile view isodiametric to nearly heterodiametric, on average subisodiametric; apex acute, obtuse, or flat; 6.9-8.8 x 5.6-7.6 um (x= 8.0 + 0.34 x 6.8 + 0.4 um; Q =1.04-1.4;Q. = 12 + 0.1; x/y = 64/2). Basrp1a clavate, tapered to a narrow or moderately broad base, granules abundant but obscure, 26.6-39.9 x 9.9-12.0 um (x = 33. + 3.4 x 11.0 + 0.65 um; Q =2.6-3.6;Q. = 2.6 + 0.3; x/y = 22/2); 4-sterigmate, sterigma 1.2-5.8 um long. LAMELLAR EDGE partially sterile. CHEILOCYSsTIDIA abundant, but often not on every lamella; appear as terminal cells of tramal hyphae, versiform in shape (clavate, broadly cylindro-clavate, cylindro-clavate, rarely vesiculose, sometimes fusiform), short to long, at times at the end of a long stalk; colorless; 27.6-104.7 x 6.7-28.6 um (x= 53.8 + 16.2 x 13.7 + 4.0 um; 54 ... Largent & Cribari Q =1.8-9.4;Q. = 4.2 +.1.7; x/y = 61/2). PLEUROCySTIDIA absent. LAMELLAR TRAMA when sectioned distinct and 20-30 um deep, with the mediostratum composed of subparallel, longitudinally entangled, dark colored hyphae bordered by two subhymenia of entangled, colorless, narrow hyphae with non-gelatinized walls; in squash mounts hyphae narrow to very broad and with both ends rounded, 59.8-451.0 x 2.3-23.2 um (x = 202 + 110.6 x 10.3 + 5.1 um; Q =6.1-54.2; Q. = 22.4 + 12.4; x/y = 26/2). PILEIPELLIS ~80 um deep, a tightly entangled layer of narrow hyphae with distal 1-4 cells erect to suberect; PILEOCYSTIDIA narrowly clavate to cylindric, 23.4-79.0 x 3.0-12.8 um (x = 44.6 + 15.7 x 7.6 + 2.3 um; Q =2.0-10.6;Q. = 6.3 + 2.4; x/y = 27/2). PILEAL TRAMAL HYPHAE in squash sections with distal cells with narrow to broadly acuminate ends, 67.8-261.0 x 8.8-30.6 wm (x = 173.8 + 67.4 x 16.3 + 7.2 um; Q. = 12.0 + 5.2; x/y = 11/2). STIPITIPELLIs at the apex 33.0-62.3 um deep, an entangled layer of hyphae with erect, suberect, or prostrate distal cells; CAULOCYSTIDIA clavate to cylindric, 20.5-91.4 x 4.1-10.8 um (x = 49.4 + 18.2 x 6.7 + 1.7 um; Q =4.4-12.7; Q =7.7 + 3.0; x/y = 23/2). STIPE TRAMAL HYPHAE in longitudinal sections subparallel and entangled, 103-405 x 6.9-36.8 um (x = 245.2 + 91.0 x 18.6 + 8.2 um; Q = 6.9-25.9;Q. = 14.4 + 5.2; x/y = 20/2). OLEIFEROUS HYPHAE absent in all tramal tissues. L1IpID GLOBULES absent. PIGMENTATION slightly to definitely encrusting on lamellar and pileus tramal hyphae; uniform, cytoplasmic, and light brownish in pileocystidia. CLAMP CONNECTIONS absent in the pileipellis, typically absent at base of basidia and cheilocystidia, and absent on the hyphae of the pileipellis. ECOLOGY & DISTRIBUTION-Gregarious, broadcast over a 10 x 4 m area in a lawn. Spring; early to late September. Known only from type locality. ADDITIONAL SPECIMEN EXAMINED—-AUSTRALIA, QUEENSLAND, Cook Region, Smithfield, 10 Marcia Close, 16.8261°S 145.6857°E, gregarious in lawn, 9 September 2010, Topotype Peter Newling DISTINCTIVE CHARACTERS-Clitocyboid basidiomata with deeply depressed to infundibuliform radially appressed-fibrillose greyish brown pilei that have a metallic luster when fresh, subdecurrent to decurrent close lamellae, an equal stipe that becomes brownish when handled, a strong ammoniac odor, anda lawn habitat. Microscopically distinguished by subisodiametric distinctly angular basidiospores (x = 9 x 7 um), versiform often long-stalked cheilocystidia, a pileipellis ~80 um deep with a tightly entangled layer of narrow hyphae with the distal 1-4 cells erect to suberect, cytoplasmic pigmentation in the pileocystidia and encrusting pigment on the walls of the pileal and lamellar tramal hyphae and the presence of caulocystidia. Clitopiloides & Trichopilus spp. nov. (Australia) ... 55 Fig. 1. Clitopiloides prati (holotype HSC A1429; except B): A. basidioma with deeply depressed to nearly infundibuliform pileus; B. (Topotype HSC A1430) overly mature basidiomata with infundibuliform pileus; C. 5-angled basidiospores; D. clavate sterigmate basidium tapered to narrow base; E. cluster of broadly clavate to cylindro-clavate cheilocystidia; F. tightly entangled stipitipellis ~2.4 cm down stipe apex, with semi-erect to prostrate caulocystidia; G. pileipellis from disc with loosely entangled hyphal layer with semi-erect cylindro-clavate pileocystidia. Scale bars: A, B = 14mm; C = 8 um; D= 33 um; E = 15 um; F = 50 um; G = 50 um. 56 ... Largent & Cribari CoMMENTS~Clitopiloides prati morphologically resembles C. cyathus (Romagn. & Gilles) Largent from Gabon and the Ivory Coast in its brownish colored basidiomata, deeply depressed to infundibuliform pileus, decurrent lamellae, presence of cheilocystidia, and 5-6-angled basidiospores measuring 7-9 x 5.7-7.6 um. However, C. cyathus is distinguished by its yellowish brown (“bistre”) glabrous, translucent-striate, hygrophanous pileus, denticulated lamellae, clavate stipe, lack of odor or an odor of bitter almonds, cheilocystidia measuring 25-45 x (8.5-)12-16(-24) um, vacuolar pigmentation, and rainforest habitat (Romagn. & Gilles, 1979). Entoloma cuboidosporum (Beeli) E. Horak from Malaysia, Singapore, Zaire, Madagascar, Gabon, and the Congo shares the brownish, deeply depressed to infundibuliform pileus and decurrent lamellae found in C. prati but differs in its glabrous pileus, farinaceous odor, and cuboid basidiospores (Horak, 1980). GenBank’s Blastn tool shows the ITS sequence from C. prati as 88.63% similar to Entoloma subclitocyboides W.M. Zhang (= E. subinfundibuliforme T.H. Li & Chuan H. Li) from China. Entoloma subclitocyboides differs from C. prati in its broader (7.0-8.5 um) isodiametric basidiospores, dirty yellowish to pale yellow brown pileus, adnexed subventricose lamellae, lack of odor, lack of encrusting pigments in the lamellar trama and pileipellis, presence of clamp connections, and habit on soil in mixed forest (He & al. 2014). Trichopilus lecythiformis Largent, sp. nov. Fig, 2 IF 558178 Differs from Trichopilus tibiiformis by its yellowish brown, translucent striate pileus, a stipe that bruises yellow brown, larger basidiospores, and abundant lecythiform cheilocystidia and pleurocystidia. Type: Australia, New South Wales, North Coast, Myall Lakes National Park, Mungo Brush campground 32.5457°S 152.3094°E, 30 April 2012, DL Largent 10482 (Holotype, DAR 81840). EryMoLoGy—lecythiformis (Latin) referring to the lecythiform cheilocystidia. BASIDIOMATA omphalinoid. Prteus 13-14 mm broad x 1.0-1.5 mm tall, convex to plano-convex, shallowly depressed, tomentulose on the disc, glabrous elsewhere; dark yellowish brown (5F4 “sepia brown”) in center at all times, elsewhere light yellowish brown (5E4 “hair brown”) becoming darker with age and drying (5F7 “coffee”), margin decurved then plane, very slightly crenulate, translucent striate to the disc, moist and wet, not hygrophanous. LAMELLAE 6 mm long x 2-2.5 mm high, adnate to short decurrent at first, then long decurrent, at first off-white to orange white (5A4), becoming pale orange (5B3) with sporulation, narrow then distant, edge smooth and concolorous; Clitopiloides & Trichopilus spp. nov. (Australia) ... 57 in profile view; C. clavate sterigmate basidia tapered to narrow bases; D. tibiiform and lecythiform cheilocystidia; E. clavate pileocystidia with plasmatic brown pigments; F. disc pileipellis showing loosely entangled palisadoderm. Scale bars: A = 14 mm; B = 8 um; C = 12 um; D = 14 um; E = 13 um; G = 55 um. 58 ... Largent & Cribari lamellae 2-3 between lamellae, in 2 series (one short, one medium long). STIPE 22-28 x 1.0-1.5 mm, straight, equal, faintly pruinose at the apex, elsewhere glabrous, yellowish white (4A2) to light yellowish grey (5B2 “putty”), bruising yellowish brown (~5E5 “bronze” = “bronze brown’); BASAL TOMENTUM absent. Opor and Taste mild, not distinctive. BASIDIOSPORES 5-6-angled, angles distinct, hilar appendage large and distinct, apex rarely acute, often obtuse, nearly all spores with single globule, on average heterodiametric, 9.8-14.4 x 6.8-9.9 wm (x = 11.3 + 1.0 x 8.3 + 0.67 wm; Q = 1.19-1.55;Q. = 1.35 £ 0.10; x/y = 53/1). Basip1a clavate, narrowly tapered at the very base (7/8 inflated), with granules that are not brilliant; 23.0-36.1 x 11.2-13.8 um (x = 29.3 + 2.9 x 12.4 + 0.85 um; Q =1.80-2.76; Q., = 2.38 + 0.26; x/y = 16/1)); 4-sterigmate, sterigma 2.52-5.79 um. CHEILOCYSTIDIA abundant, lamellar edge mostly sterile, tibiiform and lecythiform, colorless, 29.7-57.0 x 10.2-17.4 um (x = 46.7 + 7.8 x 14.2 + 1.5 pm; Q =2.5-4.0; Q_=3.29+0.45;x/y=19/1);head4.0—-9.0 um wide (x/y21/1);neck2.4—6.2 um wide (x/y = 21/1). PLEUROCysTIDIA rare at the lamellar edge, similar in shape and size to cheilocystidia. LAMELLAR TRAMAL HYPHAE subparallel and in squash mounts rounded at both ends, 55.3-100.9 x 12.2-20.6 wm (x = 78.8 + 13.9 x 15.1 + 3.1 um; Q =3.50-6.75; Q. = 5.35 + 1.13; x/y = 7/1). PILEIPELLIs on the disc a distinct palisadoderm with erect hyphal elements attached laterally, distal 4-5 cells inflated, prostrate from the pileus middle to margin; pileocystidia clavate to cylindro-clavate, 27.1-109.3 x 8.25-18.8 um (x = 56.7 + 20.4 x 13.9 + 2.7 um; Q = 2.62-8.11; Q. = 4.44 + 1.44; x/y = 38/1). PILEAL TRAMAL HYPHAE subparallel and entangled, 30.8-56.0 x 5.7-9.5 um (x = 47.0 + 8.9 X 8.6 + 1.5 um; Q =3.41-8.80; Q =5.66 + 1.78; X/Y = 6/1). STIPITIPELLIS a cutis; CAULOCYSTIDIA absent. STIPE TRAMAL HYPHAE in longitudinal section, subparallel and entangled; 55.6-273.3 x 7.4-28.5 um (x = 155.4 + 59.3 x 15.2 + 5.0 um; Q = 4.83-19.40; Q. = 10.74 + 4.35; x/y = 15/1), OLEIFEROUS HYPHAE absent in all tramal tissues. Lip1D GLOBULES absent. PIGMENTATION cytoplasmic, uniform, brown in apical cells of the pileipellis, encrusted in the basal cells; basidia containing non-brilliant granules. CLAMP CONNECTIONS absent. ECOLOGY & DISTRIBUTION- Scattered in grass along the beach track, Myall Lakes National Park, coastal New South Wales, Australia, in late April (autumn). Known only from the type locality. ComMEnts-Trichopilus lecythiformis is known from a single collection of a single basidiome from sandy soil in Myall Lakes National Park in late April. However, this species is distinct from all other Australian entolomatoid fungi due to its combination of small delicate basidiomata, pileus with a Clitopiloides & Trichopilus spp. nov. (Australia) ... 59 yellowish brown disc, long clavate to cylindro-clavate pileocystidia, 5-6-sided basidiospores measuring ~11.3 x 8.3 um, tibiiform to lecythiform colorless cheilocystidia and pleurocystidia, palisadoderm pileipellis, and the absence of caulocystidia and clamp connections. As far as I know, this the first report of a Trichopilus species in Australia with a small delicate basidiome. Seven other species might be confused with T! lecythiformis due to their small delicate basidiomata and tibiiform/lecythiform cheilocystidia. They can be distinguished from T: lecythiformis as follows: T: tibiiformis Largent & Aime from Guyana has a dark violet brown shaggy, opaque pileus and smaller (7.6-9.8 x 4.5-6.7 um) 6-7-angled basidiospores (Aime & Largent 2010). Entoloma mariae G. Stev. from New Zealand has smaller (9-11 x 7-8.5 um) basidiospores, larger (35-100 x 10-22 um) cheilocystidia, a pruinose stipe, and clamp connections; E. perplexum E. Horak, also from New Zealand, has a brownish grey small (5-10 mm diam) pileus, cinnamon-colored young lamellae, a pileipellis composed of vesiculose or clavate capitate pileocystidia, a weak farinaceous taste and odor, and 5-6-angled basidiospores measuring 10-13 x 7-9 um (Horak 2008). Entoloma festivum Noordel. & al. from the Netherlands has a warm reddish brown hygrophanous pileus with a black disc, smaller (8.5-10.5 x 5.5-7.5 um) 5-8-angled basidiospores, cheilocystidia with a vacuolar pigment, and clamp connections (Noordel. & al. 2010). Rhodophyllus capitatus Romagn. & Gilles has a pinkish white, scaly, translucent striate pileus, pale brownish lamellae, smaller (7-8.5 x 5.7-6.7-4m) 4-5-angled prismatic basidiospores; R. applanatus Romagn. & Gilles has a fairly dark pinkish brown, translucent-striate scaly pileus, smaller (7-8.5 x 5.7-6.7 um) 5-6-angled basidiospores; and R. lepiotoides Romagn. & Gilles has a yellow brown opaque pileus with a brown disc and slight umbo and smaller (6.5-7.5 x 5.7-6.5) 4-5-angled basidiospores (Romagn. & Gilles 1979). In the literature cited above, there has only been one collection studied for five of these species and two collections for R. lepiotoides, suggesting that these species are either rare or the small basidiomata overlooked in the field. Unfortunately, because all attempts at obtaining DNA sequences from DLL 10482 (the holotype of T! lecythiformis) were unsuccessful, this collection is proposed as a Trichopilus species based on morphology alone. However, phylogenetic analyses using four genes demonstrated that Trichopilus is a statistically supported genus with bootstrap values of 100%/1 representing a monophyletic group nested deep within the Inocephalus—Cyanula clade. Sequences for all four genes (mtSSU, nLSU, rrpb2, and tefl) were obtained from T. tibiiformis with delicate basidiomata in this study (Karstedt & al. 2019). 60 ... Largent & Cribari We are confident that if sequences are obtained from future collections of T. lecythiformis, the species will cluster in this well-supported group. Acknowledgments Materials required to complete this manuscript were provided by the Largent family trust. The Australian Tropical Herbarium and the School of Marine and Tropical Biology, James Cook University, provided fieldwork and logistical support. The DNA sequences generated in this study were completed and financed by Dr. Sarah Bergemann. Comments by the two expert reviewers, Dr. Sarah Bergemann (Middle Tennessee State University, Murfreesboro, TN, USA) and Dr. Timothy J. Baroni (New York State University at Cortland, NY, USA), and by the Nomenclature Editor, Dr. Shaun Pennycook, were also helpful. We wish to thank Peter Newling for his assistance in collecting in northeastern Queensland and collecting PN40, the holotype collection for Clitopiloides prati. We also wish to thank Pam O'Sullivan and Skye Moore for their assistance in collecting in New South Wales. Finally, David and Pamela Largent especially wish to thank Dr. Sandra Abell for being an extraordinary research advisor and contact at James Cook University during the five years of research in northeastern Queensland. Literature cited Aime MC, Largent DL, Henkel TW, Baroni TJ. 2010. The Entolomataceae of Pakaraima Mountains of Guyana IV: new species of Calliderma, Paraeccilia and Trichopilus. Mycologia 102: 633-649. https://doi.org/10.3852/09-162 Bergemann SE, Garbelotto M. 2006. High diversity of fungi recovered from the roots of mature tanoak (Lithocarpus densiflorus) in northern California. Canadian Journal of Botany 84:1380-1394. https://doi.org/10.1139/b06-097 Bergemann SE, Largent DL, Abell-Davis SB. 2013. Entocybe haastii from Watagans National Park, New South Wales, Australia. Mycotaxon 126: 61-70. https://doi.org/10.5248/126.61 Gardes M, Bruns TD. 1993. ITS primers with enhanced specificity of basidiomycetes - application to the identification of mycorrhizae and rusts. Molecular Ecology 2: 113-118. https://doi.org/10.1111/j.1365-294X.1993.tb00005.x He XL, Li TH, Peng WH, Gan BC. 2014. A taxonomic revision of Entoloma clitocyboides and E. subinfundibuliforme from Hainan Island, South China. Mycoscience 55: 103-107. https://doi.org/10.1016/j.myc.2013.06.002 He XL, Horak E, Wang D, Li TH, Peng WH, Gan BC. 2019. Descriptions of five new species in Entoloma subgenus Claudopus from China, with molecular phylogeny of Entoloma s.l. MycoKeys 61: 1-26. https:// doi.org/10.3897/mycokeys.61.46446 Horak E. 1980. Entoloma (Agaricales) in Indomalaya and Australasia. Beihefte zur Nova Hedwigia 65. 352p. Horak E. 2008. Agaricales of New Zealand 1: Pluteaceae (Pluteus, Volvariella); Entolomataceae (Claudopus, Clitopilus, Entoloma, Pouzarella, Rhodocybe, Richoniella). Fungal Diversity Research Series 19. 305p. Karstedt F, Capelari M, Baroni TJ, Largent DL, Bergemann SE. 2019. Phylogenetic and morphological analyses of species of the Entolomataceae (Agaricales, Basidiomycota) with cuboid basidiospores. Phytotaxa 391: 1-27. https://doi.org/10.11646/phytotaxa.391.1.1 Clitopiloides & Trichopilus spp. nov. (Australia) ... 61 Kalichman J, Kirk PM, Matheny PB. 2020. A compendium of generic names of agarics and Agaricales. Taxon 69(3): 425-447. https://doi.org/10.1002/taxa.12240 Kornerup A, Wanscher JH. 1978. Methuen handbook of colour 3rd ed. Richard Clay Ltd: Chichester, Sussex. 252 p. Largent DL. 1994. Entolomatoid fungi of the western United States and Alaska. Mad River Press Inc: Eureka, California. 516 p. Largent DL. 2020. Nomenclature changes in the “Entolomatoid fungi of western North America and Alaska” (Largent 1994). Mycotaxon 135: 789-794. https://doi.org/10.5248/135.789 Largent DL, Abell-Davis S. 2011 Observations on Inocephalus virescens comb. nov. and Alboleptonia stylophora from northeastern Queensland. Mycotaxon 116: 231-245. https://doi.org/10.5248/116.231 Largent DL, Abell-Davis SE, Cummings GA, Ryan KL, Bergemann SE. 2011la. Saxicolous species of Claudopus (Agaricales, Entolomataceae) from Australia. Mycotaxon 116: 253-264. https://doi.org/10.5248/116.253 Largent DL, Bergemann SE, Cummings GA, Ryan, KL, Abell-Davis SE, Moore S. 2011b. Pouzarella (Agaricales, Entolomataceae) species from New South Wales (Barrington Tops National Park) and northeastern Queensland, Australia. Mycotaxon 117: 435-483. https://doi.org/10.5248/117.435 Largent DL, Bergemann SE, Abell-Davis SE, Kluting KL, Cummings GA. 2013a. Three new Inocephalus species with cuboid basidiospores from New South Wales and Queensland, Australia. Mycotaxon 123: 301-319. https://doi.org/10.5248/123.301 Largent DL, Bergemann SE, Abell-Davis SE, Kluting KL, Cummings GA. 2013b. Five Leptonia species from New South Wales and Queensland, Australia. Mycotaxon 125: 11-35. https://doi.org/10.5248/125.11 Largent DL, Bergemann SE, Abell-Davis SB. 2014. Entoloma species from New South Wales and northeastern Queensland, Australia. Mycotaxon 129(2): 329-359. http://doi.org/10.5248/129.329 Largent DL, Kluting KL, Anderson NM, Bergemann SE. 2016. New leptonioid species from New South Wales and northeastern Queensland, Australia. Mycotaxon 131: 153-176. https://doi.org/10.5248/131.153 Noordeloos ME. 1992. Entoloma s.l. Fungi Europaei 5: 1-760. Noordeloos ME. 2004. Entoloma s.1. Fungi Europaei 5a: 761-1378. Noordeloos ME, Gates GM. 2012. The Entolomataceae of Tasmania. Fungal Diversity Research Series 22: 400 p. https://doi.org/10.1007/978-94-007-4679-4 Noordeloos ME, Rommelaars LCAF, Gelderblom JNJ. 2010. Entoloma festivum, a new species in subgenus Trichopilus from the Netherlands. Mycotaxon 111: 495-499. https://doi.org/10.5248/111.495 Romagnesi H. 1978. Les fondements de la taxinomie des rhodophylles et leur classification. (Tirage a part de “Beiheft 59 zur Nova Hedwigia’). J. Cramer, Vaduz. 79 p. Romagnesi H, Gilles G. 1979. Les rhodophylles des foréts cétiéres du Gabon et de la Céte dIvoire avec une introduction générale sur la taxinomie du genre. Beihefte zur Nova Hedwigia 59. 649 p. White TJ, Bruns T, Lee S, Taylor JW. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. 315-322, in: MA Innis et al. (eds). PCR protocols: a guide to methods and applications. Academic Press, Inc. New York. http://doi.org/10.1016/B978-0-12-372180-8.50042-1 MYCOTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2022 January-March 2022— Volume 137, pp. 63-71 https://doi.org/10.5248/137.63 Neoacrodictys elegans gen. & sp. nov. from Hainan Province, China JI-WEN X14, TAI-CHANG Mu, ZHAO-XUE ZHANG, ZHUANG Li, XIU-GUO ZHANG* Shandong Provincial Key Laboratory for Biology of Vegetable Diseases and Insect Pests, College of Plant Protection, Shandong Agricultural University, Taian, Shandong 271018, China “CORRESPONDENCE TO: sdau613@163.com ABSTRACT—A new anamorphic genus and species, Neoacrodictys elegans, is illustrated and described from dead branches of an unidentified plant in Hainan Province, China. The genus is characterized by darkly pigmented turbinate to obpyriform muriform conidia produced from monoblastic integrated terminal conidiogenous cells on macronematous unbranched conidiophores. KEY worDS—asexual Ascomycota, hyphomycetes, saprobes, taxonomy Introduction During our continuing surveys of dematiaceous hyphomycetes colonizing diverse plant habitats from the forests of Hainan, China, a fungus that does not match any existing genera was collected growing on unidentified dead twigs. Morphological studies and a literature review (Ellis 1971, 1976; Subramanian 1971; Matsushima 1975, 1983, 1985, 1989, 1993, 1995; Castafteda-Ruiz 1986; Castafeda-Ruiz & Kendrick 1990a,b, 1991; Wu & Zhuang 2005; Seifert & al. 2011; Ma & al. 2016, 2021; Xia & al. 2016, 2017; Xu & al. 2020a,b, 2021; Zhang & al. 2020; Niu & al. 2021) indicated that the fungus represents an undescribed genus. Molecular phylogenetic analysis confirmed its morphological identity, and the fungus is described here as a new genus and species. 64 ... Xia & al. Materials & methods Isolates & morphological analysis Samples of dead branches collected were placed in separate zip-lock plastic bags, taken to the laboratory, and then incubated at 27 °C for more than 2 weeks in an artificial climate box in 9 cm diameter plastic Petri dishes with moistened filter paper. Single-spore cultures of hyphomycetes that could not be identified on natural substrate were isolated from dead branches and incubated on potato-dextrose agar (PDA: 200 g boiled and filtered white potatoes, 20 g dextrose, 15 g agar, 1 L distilled water). All PDA plates were incubated at 25 °C for 1-2 months. The colonies were photographed using a Powershot G7X mark II digital camera. Micromorphological characters were observed using an Olympus SZX10 stereomicroscope and Olympus BX53 microscope, both fitted with Olympus DP80 high definition colour digital cameras. All fungal strains were stored in 10% sterilized glycerin at 4 °C for further studies. The specimens are deposited in the Herbarium of Plant Pathology, Shandong Agricultural University, Taian, Shandong, China (HSAUP). Ex-type cultures are deposited in the Shandong Agricultural University Culture Collection, Taian, Shandong, China (SAUCC). DNA extraction, PCR amplification, sequencing Genomic DNA was extracted from colonies grown on PDA, using the CTAB method (Doyle & Doyle 1990). The large subunit ribosomal RNA gene (LSU) was amplified and sequenced by using primers pairs LROR/LRS (Vilgalys & Hester 1990, Glass & Donaldson 1995). PCR was conducted using an Eppendorf Master Thermocycler. DNA was amplified in 25-uL volumes containing 12.5 uL Vazyme Green Taq Mix, 1 uL of each forward and reverse primer (10 uM) (Biosune), and 1 uL template genomic DNA adjusted to a total volume of 25 uL with distilled deionized water. PCR parameters were 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min, ending with a final elongation step at 72 °C for 10 min. PCR products were estimated visually by staining with GelRed after 1% agarose gel electrophoresis. Sequencing was done bi-directionally by Biosune Co. Ltd. (Shanghai, China). Consensus sequences were obtained using MEGA 7 (Kumar & al. 2016). All sequences generated in this study were deposited in GenBank (TaBLE 1). Sequence alignment and phylogenetic analysis The quality of our amplified nucleotide sequences was checked and combined by MEGA 7 (Kumar & al. 2016), and reference sequences were retrieved from the National Center for Biotechnology Information (NCBI). Sequences were aligned using MAFFT 7.310 (http://mafft.cbrc.jp/alignment/server/index.html) (Katoh & al. 2019), and manually corrected using MEGA 7. The LSU sequences were analyzed phylogenetically using Maximum-Likelihood (ML) and Bayesian Inference (BI) methods. RaxML and Bayesian analyses were run on the CIPRES Science Gateway portal (Miller & al. 2012) using RaxML 8.2.9 and MrBayes 3.2.6. Evolutionary models were calculated using MrModelTest 2.3 (Nylander TABLE 1. Strains and sequences included in the phylogenetic analyses. Neoacrodictys elegans gen. & sp. nov. (China) ... 65 The new sequence is set in bold. TAXON Acrodictys bambusicola Acrodictys elaeidicola Asterina sp. Botryotinia fuckeliana Buelliella physciicola Buelliella poetschii Junewangia lamma Junewangia sphaerospora Karschia cezannei Karschia talcophila Labrocarpon canariense ‘Melaspilea lekae Mycosphaerella pneumatophorae Mycosphaerellaceae sp. Melaspileopsis cf. diplasiospora Melaspileopsis sp. Neoacrodictys elegans Rhexoacrodictys erecta Rhexoacrodictys fimicola Stictographa lentiginosa VOUCHER CGMCC 3.18641 CGMCC 3.18642 CGMCC 3.18643 SH-2014 AFTOL-ID 59 Ertz 18113 (BR) Ertz 19173 (BR) Ertz 18115 (BR) Ertz 18116 (BR) CGMCC 3.18652 CGMCC 3.18653 CGMCC 3.18655 Cezanne-Eichler B26 (hb. Diederich) Cezanne-Eichler 7453 (hb. Diederich) Ertz 19186 (BR) Diederich 16749 (hb. Diederich) Ertz 16308 (BR) Ertz 16907 (BR) Ertz 17325 (BR) JK5253B=AFTOL-ID 762 SD-01 KH00300 Ertz 16247 (BR) Ertz 16624 (BR) Ertz 16625 (BR) Ertz 17904 (BR) Ertz 17913 (BR) SAUCC H4600 CGMCC 3.18656 CGMCC 3.18657 CGMCC 3.18658 CGMCC 3.18660 Ertz 17447 (BR) Ertz 17570 (BR) van den Boom 47621 (hb. v.d. Boom) GENBANK LSU KX033564 KX033568 KX033569 KM386978 AY544651 KP456147 KP456148 KP456149 KP456150 KU751882 KU751883 KX033572 KP456152 KP456153 KP456154 KP456155 KP456157 KP456158 KP456162 FJ176856 JN872645 GU017553 KP456164 KP456165 KP456166 KP456167 KP456168 MW907608 KX033555 KX033556 KX033553 KX033554 KP456169 KP456170 KP456171 66 ... Xia & al. 2004) to select the best-fit model for each data partition according to the Akaike criterion. For ML analyses the default parameters were used and bootstrap support (BS) was calculated using the rapid bootstrapping algorithm with the automatic halt option. Bayesian analyses included two parallel runs of 5,000,000 generations, with the stop rule option and a sampling frequency set to each 1000 generations. The 50% majority rule consensus trees and posterior probability (PP) values were calculated after discarding the first 25% of the samples as burn-in. Trees were plotted in FigTree 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree) and edited with Adobe Illustrator CS 5. Phylogenetic results The dataset comprised 35 taxa representing 17 named species and four undetermined species including Botryotinia fuckeliana (AFTOL-ID 59) as outgroup. The final LSU alignment totaled 1178 characters including gaps. Of these characters, 825 were constant, 89 parsimony-uninformative, and 264 parsimony-informative. For the BI and ML analyses, the substitution model GTR+I+G for LSU was selected and incorporated into the analyses. The topology of the ML tree confirmed the tree topologies obtained from BI analyses, so only the ML tree is presented (Fic. 1). In this tree, our strain formed an independent clade. Taxonomy Neoacrodictys J.W. Xia & X.G. Zhang, gen. nov. MB 816515 Differs from Acrodictys by its turbinate conidia and oblique septa that are often marked by dark bands. TYPE SPECIES: Neoacrodictys elegans J.W. Xia & X.G. Zhang ErymMo.Loecy: Neoacrodictys = “neo-” + “acrodictys” (Lat.), referring to its similarity to the genus Acrodictys. CONIDIOPHORES macronematous, mononematous, unbranched, septate, brown to dark brown, paler towards the apex, indeterminate with flask-shaped percurrent extensions. CONIDIOGENOUS CELLS monoblastic, integrated, terminal, cylindrical, pale brown to brown, smooth. Conidial secession schizolytic. Conrpia solitary, acrogenous, muriform, turbinate to obpyriform, euseptate, with one longitudinal and a few transverse oblique septa usually obscured by dark bands. Neoacrodictys elegans J.W. Xia & X.G. Zhang, sp. nov. FIG. 2 MB 816516 Differs from Acrodictys spp. by its turbinate conidia and oblique septa usually obscured by dark bands. Neoacrodictys elegans gen. & sp. nov. (China) ... 67 Botryotinia fuckeliana 0.07 Fic. 1. Phylogram generated from RaxML analysis based on the LSU gene. Bootstrap support values are shown for ML >70% and BI >0.85. Branches shortened to fit the page are indicated by two diagonal lines accompanied by the length reduction factor. The new sequence is indicated in bold. Type: China, Hainan Province: Ledong, Jianfengling National Forest Park, 18.70°N 108.87°E, on dead branches of an unidentified broadleaf tree, 22 April 2014, Y.R. Ma (Holotype, HSAUP H4600; ex-type culture, SAUCC H4600; GenBank MW907608). EryMo_oey: refers to its conidia that have an elegant appearance. COLONIES effuse, brown, hairy. MycEe.ium partly superficial, partly immersed in the substrate. CONIDIOPHORES macronematous, mononematous, erect, unbranched, straight or flexuous, thick-walled, smooth, dark brown; swollen at the base, narrower and paler toward the apex, 3-7-septate, 54-105 x 4.5-7.5 um at the broadest part, indeterminate with 1-2 flask-shaped percurrent 68 ... Xia & al. extensions. CONIDIOGENOUS CELLS integrated, terminal, cylindrical to doliiform, subhyaline to pale brown, smooth, monoblastic; extending through the scar of the last conidium on the conidiophores. Conip1a solitary, muriform, turbinate to obpyriform, 15.5-22.5 x 12-18 um, with 1 or 2 transverse septa and 1 or 2 longitudinal or oblique septa; septa typically crossing at right angles and usually obscured by a black band. Basal cell funnel-shaped, delimited by a transverse septum, pale brown. Conidial secession schizolytic. Discussion Neoacrodictys demonstrates unique morphological and ontogenetic features. It is distinguished by macronematous, mononematous, cylindrical and unbranched conidiophores with holoblastic conidiogenous cells that produce darkly pigmented, muriform, turbinate to obpyriform conidia. A key to Neoacrodictys and morphologically similar genera is provided. Neoacrodictys elegans resembles Acrodictys bambusicola M.B. Ellis, A. elaeidicola M.B. Ellis, and A. malabarica Subram. & Bhat, which differ by conidial shape (Ellis 1961, Subramanian & Bhat 1989, Xia & al. 2017). Also, the genera Acrodictys and Neoacrodictys belong in different phylogenetic clades (Fic. 1). Key to Neoacrodictys and morphologically similar genera LEG onidtadbicellwlar as Ait.28 Bit.0e Pee BEN WEE LE Wet ee Ityorhoptrum Ie@Conidia MUPTOCM: 5... gdb adis gdh edd needs neegd badd oeaw bow dibeeie 4d heres Z 2} CONIA WIEN APPCAGASES ssliez sh ires-z chipsen-agub shea gih sete gr eemengh idea gst Pseudoacrodictys 2 AOhidia Without ap PelidaGes wr. c.s sso. rates sta ssi stadt ora cet aed aed Bas 3 3-Conidial secession thexOlytie wh cede eontegt 8 mcwitege B noeceye B heenegd # nutes Rhexoacrodictys SECOnidial secessign scMiZ@lylie WF 1089 Feats F188 UBB ok WiaBDeh aA COL Pagel SS 4 4, Genidia Inaturing ahter SCCESSION f.i.5.. Wheaton ea iewilerd ste are edd areas Acrodictyella 4, Conidia becoming pigmented and septate prior to secession ................44. 5 5. Conidial midpoint attached to the conidiogenous cell ........... Coleodictyospora 5. Conidial base attached to the conidiogenous cell ............ 0... cee eee eee eee 6 6. Conidiophore aseptate, comprising a single conidiogenous cell that extends percurrently after each conidial dehiscence .......... Junewangia 6. Conidiophores conspicuously septate, a new conidiogenous cell forming: atter each conidial dehiscence 2. fh. wiucbes wboade x wautbes aude ante lt 7 7. Conidial basal cell inconspicuous, reduced to-a:short;-cylindricalsdisk § sav. qay uw See ewe u eee ve Acrodictys 7. Conidial basal cell conspicuous, distinctly funnel-shaped and flat at the base.................... Neoacrodictys Neoacrodictys elegans gen. & sp. nov. (China) ... 69 Fic. 2. Neoacrodictys elegans (holotype, HSAUP H4600): a. Colony on PDA (surface and reverse); b. Colony on MEA (surface and reverse); c. Conidia; d—f. Conidiophores, conidiogenous cells, and conidia. Scale bars: c—f = 20 um. Acknowledgments The authors express gratitude to Dr. Jian Ma (College of Agronomy, Jiangxi Agricultural University, Nanchang, Jiangxi, China) and Dr. Li-Guo Ma (Institute of Plant Protection, Shandong Academy of Agricultural Sciences, Jinan, Shandong, 70 ... Xia & al. China) for serving as pre-submission reviewers and to Dr. Shaun Pennycook for nomenclatural review and Dr. Lorelei L. Norvell for editorial review. This work was jointly supported by the National Natural Science Foundation of China (Nos. 31900014, U2002203, 31750001) and National Science and Technology Fundamental Resources Investigation Program of China (2019FY 100700). Literature cited Castanieda-Ruiz RF. 1986. Deuteromycotina de Cuba. Hyphomycetes IV. Instituto de Investigaciones Fundamentales en Agricultura Tropical “Alejandro de Humboldt,’ Habana, Cuba. 17 p. 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Mycotaxon 136: 85-95. https://doi.org/10.5248/136.85 Zhang K, Guo WH, Heredia G, Delgado-Zuniga JP, Ma J, Castafieda-Ruiz RE 2020. Anasporidesmiella gen. nov. for an atypical Sporidesmiella species and for A. manifesta sp. nov. Mycotaxon 135: 719-727. https://doi.org/10.5248/135.719 MYCOTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2022 January-March 2022— Volume 137, pp. 73-87 https://doi.org/10.5248/137.73 Neotypification of Claviceps humidiphila and recognition of C. bavariensis sp. nov. M1Ao Liv", Eyt TANAKA?,, MIROSLAV KOLARIK? ‘Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, 960 Carling Ave. Ottawa, Ontario K1A0C6 Canada ? Ishikawa Prefectural University, 1-308 Suematsu, Nonoichi, Ishikawa 921-8836 Japan * Department of Botany Faculty of Science, Charles University, Benatska 2CZ-128 01, Praha 2 Czech Republic “CORRESPONDENCE TO: miaomindy.liu@agr.gc.ca; tanakae@ishikawa-pu.ac.jp ABSTRACT—Claviceps humidiphila |= C. purpurea var. phalaridis] was previously typified with the holotype collected by Tanda in Japan and an epitype from Bavaria, Germany. Phylogenetic analyses based on translation elongation factor 1-a (TEF1l-a) and RNA polymerase II second largest subunit (RPB2) indicated that the previously designated epitype from Germany was not conspecific with the Japanese species. The German specimen is proposed as a new species, C. bavariensis, and a specimen collected from the type location (Chiba, Japan) is designated as a neotype for C. humidiphila, replacing the lost holotype. Key worps—Ascomycota, Clavicipitaceae, ergot fungi, Hypocreales, taxonomy Introduction Claviceps was validly published by Tulasne (1853), as discussed by Donk (1963). Claviceps purpurea Tul. was selected as lectotype by Clements & Shear (1931) and accepted by Donk (1963). Tulasne (1853) noted the hosts of C. purpurea include cereal crops (e.g., Avena, Secale, Triticum) and grasses (e.g., Alopecurus, Dactylis, Lolium, Poa). Thereafter, the reported host range increased to more than 400 species in Poaceae. Molecular studies (Pazoutova & al. 2000, Douhan & al. 2008) revealed cryptic speciation within the pre- molecular concept of C. purpurea s.lat. Pazoutova & al. (2015) provided 7A ... Liu, Tanaka, Kolatik formal taxonomic descriptions for four phylogenetic species to replace C. purpurea s.lat.: C. purpurea s.str., C. arundinis, C. humidiphila, and C. spartinae. Claviceps humidiphila was characterized by its frequently larger conidia than C. purpurea, sclerotia capable of floating on water, and typical habit on Calamagrostis, Deschampsia caespitosa, and Phalaris arundinacea. Previously, Tanda recognized three varieties: C. purpurea var. alopecuri, C. purpurea var. dactylidis, and C. purpurea var. phalaridis, producing conspicuously larger conidia and on specific host plants. Claviceps purpurea var. alopecuri was found on Alopecurus, but also on Agrostis clavata, A. palustris, Calamagrostis epigejos, C. hakonensis, Holcus lanatus, Phleum pratense, Poa spp., Polypogon fugax, and Trisetum bifidum (Tanda 1978a,b,c, 1979a,c, 1981; Tanda & Kawatani 1980). Claviceps purpurea var. dactylidis was found on Dactylis glomerata and Calamagrostis epigejos (Tanda 1980). Claviceps purpurea var. phalaridis occurred on Phalaris, Calamagrostis epigejos, and C. pseudophragmites (Tanda 1979a). Pazoutova & al. (2015), who noted that the conidial dimension and host range of C. purpurea var. dactylidis and C. purpurea var. alopecuri overlapped with both C. purpurea s.str. and C. humidiphila, treated the varieties as taxa of uncertain identities. However, C. purpurea var. phalaridis was considered as representing the same taxon as the species later named C. humidiphila. The name C. purpurea var. phalaridis could not be elevated to species rank as a stat. nov. because this would create an illegitimate later homonym of C. phalaridis J. Walker [= Aciculosporium phalaridis (J. Walker) M. Kolarik & Pichova]. Instead, Claviceps humidiphila was proposed as a nom. nov. based on the replaced synonym Claviceps purpurea var. phalaridis. Tanda (1979b) designated a holotype from Chiba, Japan, now believed to have been lost (see TaxONOMY: COMMENTS below, p. 78). Pazoutova & al. (2015) designated an epitype from Bavaria, Germany, but phylogenetic analysis indicates that the epitytpe is not conspecific with Japanese collections. Here, we propose Claviceps bavariensis as a new species holotypified by the Bavarian collection and designate a recent specimen from Chiba, Japan, as the neotype of C. purpurea var. phalaridis [= C. humidiphila]. Material & methods DNA extraction, PCR, sequencing, analyses Axenic cultures were developed for two samples collected from Canada (Alberta) and gDNA extracted following Shoukouhi & al. (2019). For 29 samples from Japan Claviceps bavariensis sp. nov. & C. humidiphila neotypified ... 75 (Chiba, Gifu, Ishikawa, Kanagawa, Miyagi, Nagano, Niigata, Tochigi, Toyama), axenic cultures were developed from peeled ergots that were surface-disinfected in 2% sodium hypochlorite solution for 1 min; gDNA was extracted according to Izumitsu & al. (2012). Two genomic regions, RPB2 (RNA polymerase II second largest subunit) and TEF1-a (translation elongation factor 1-a), were amplified and sequenced using modified fRPB2-5F (5’-rTTCGTGGTATTGTTCGCAGA-3’) specific for ergot fungi (Pazoutova & al. 2015) and fRPB2-7cR (Liu & al. 1999), and the primer pair EF1-983F and EF1-2218R (Rehner & Buckley 2005). For 29 samples from Japan, the RPB2 regions were amplified and sequenced using Cla-RPB2 (5’-CAGTGAAACCAGAAAGGCCTTC-3’) and modified fRPB2-7cR (5’-CCCATGGCCTGCTTACCCAT-3’). Polymerase chain reactions (PCRs) were performed in 10 uL volumes containing final concentrations of 10x Titanium Taq buffer (with 3.5 mM MgCl), 0.1 mM dNTPs, 0.08 uM each of forward and reverse primer, 50x Titanium Taq polymerase (Takara Bio, California), 0.01 mg BSA, and 1 uwL of DNA template. A touchdown protocol was applied with an initial denaturation at 95 °C for 3 min, followed by 5 cycles of 95 °C for 1 min, annealing at 63 °C (decrease 1 °C per cycle) for 45 s, and extension at 72 °C for 1 min 30 s followed by 30 cycles of 95 °C for 1 min, annealing at 58 °C for 45 s, extension at 72 °C for 1 min 30 s, concluding with a final extension at 72 °C for 8 min. PCR products were sequenced using a ABI BigDye Terminator 3.1 cycling sequencing kit on Applied Biosciences Prism 3130xl Genetic Analyzer. Fifty-nine reference RPB2 and TEFl-a sequences from previous studies downloaded from GenBank were compiled and aligned with sequences generated in the present study (31 strains) using MAFFT online version 7 (Katoh & al. 2019, https://mafft.cbrc.jp/alignment/server/, accessed in Aug 2020). Alignments of concatenated sequences were generated in Geneious Prime 2020.1.2. (http://www.geneious.com). Maximum parsimony (MP) analyses were performed using PAUP* 4.0b10 (Swofford 2002). Heuristic searches with 200 replicates of random stepwise addition and tree bisection-reconnection branch swapping were conducted with a limit of 1,000,000 re-arrangements for each replicate. Bootstrapping analyses used 2000 replicates of a full heuristic search with random stepwise addition of 20 replicates with limit of 50,000 rearrangements per replicate. Bayesian inference analyses were conducted using MrBayes 3.2 (Ronquist & al. 2012) with a GTR model chosen by a previous study (Liu & al. 2021). Each run was set to four chains of 100,000,000 MCMC generations, sampling frequency was every 2000 generations, terminated when the standard deviation of the average split frequency was lower than 0.01. The BI consensus tree was directly generated after 25% burn-in. Morphological examination Specimens were conserved in the Herbarium, Department of Botany, National Museum of Nature and Science, Tsukuba, Japan (TNS) and the Canadian National Mycological Herbarium (DAOM); cultures were conserved in NARO Genebank, 76 ... Liu, Tanaka, Kolatik Microorganism Section, Genetic Resources Center, National Agriculture and Food Research Organization, Tsukuba, Japan (MAFF). Sclerotia were tested for floating ability using the protocol developed by Pazoutova & al. (2000). The shape, size, color, and surface of sclerotia were recorded from observations of available sclerotia. Specimens were photographed using a DFC425 camera and Leica Application Suite 4.12.0 software attached to Leica M165C or Motic SMZ-168-BL stereo microscopes. Colors were characterized using html color HEX codes at the maximum approximation (https://htmlcolorcodes.com/color-names/). Sclerotial tissues were sectioned by hand for microscopical examination. Conidia were washed off from sclerotial surfaces and mounted in water or lactic acid for examination using a Zeiss Axio Scope.Al microscope with differential interference contrast (DIC) illumination, or Zeiss Imager M2. Microphotographs were taken with either a Jenoptik ProgRes SpeedXTcore 5 digital camera using ProgRes image processing software for CCC samples or with (alternatively) an Axiocam 503 color camera using a Zeiss ZEN (blue edition) 2.6 pro imaging processing for Canadian samples, and for Japanese samples a MicroPublisher 5.0 or 3.3 RTV using QImaging QCapture Pro software or a WRAYCAM-NOA2000 using the Wraymer MicroStudio processing package. Conidiogenesis in sclerotia was observed under a Zeiss Imager M2 by mounting small fragments of internal sclerotial tissue obtained by slicing or picking. Germinated sclerotia were obtained by chill-treating sclerotia on moistened quartz sand in sealed cups at 4°C about 3 months followed by incubation under 12 h light/12 h dark condition at 20 °C for a few weeks. Ascostromal sections were prepared as described by Tanaka & al. (2020). Examination and microphotography of asci and ascospores followed the same procedures as used for conidia. Phylogenetic results DNA sequences generated in this study were submitted to GenBank. Including 59 reference sequences, the TEFl-a and RPB2 matrices comprised 90 taxa and 918 and 1027 characters. The phylogenetic tree based on the concatenated alignment of TEFl-a and RPB2 placed the specimen collected from the type location of C. purpurea var. phalaridis (TNS-F-60506 on Phalaris arundinacea Chiba) in a different clade, sister to CCC 434 (= DAOMC 251717), the epitype from Germany. Here, we retain the name C. humidiphila for the Japanese clade and propose C. bavariensis for the German clade. The two are sister species and closely related to C. arundinis and C. perihumidiphila (Fic. 1). Phylogenetic trees based on the individual genes showed localized low resolution as discussed by Shoukouhi & al. (2019 and Liu & al. (2020). C. citrina CCC 265 100/ 100/1 C. sect. Claviceps Epichloe bromicola AL0434 Epichloe glyceriae E277 Claviceps bavariensis sp. nov. & C. humidiphila neotypified ... C. sect. Pusillae C. paspali CCC 130 99/1 C. occidentalis DAOMC 250578 C. quebecensis DAOMC 2518985" 7510.98— ¢. cyperi CCC 1219 roel C. fimbristyliais CCC 14726" ‘ C. nigricans CCC 802 C. grohii CBS124x47 7110.95;— ©. capensis CCC 1504 oe C. macroura CCC 1482&T C. pazoutovae CCC 1485" p8/1 C. monticola CCC 1483 oot C. purpurea DAOMC 250822 C. purpurea DAOMC 251723°T C. purpurea DAOMC 250877 [__ C. zizaniae DAOMC 252145 93/1 C. ripicola DAOMC 251844€T C. ripicola DAOMC 252149 C. ripicola DAOMC 251923 C. spartinae DAOMC 251720 400/1 C. spartinae DAOMC 251721 C. spartinae RUTPP 3491HT p8/).977 C. perihumidiphila DAOMC 250581 C. perihumidiphila DAOMC 252161" C. arundinis CCC 1094 C. arundinis DAOMC 251724=CCC 933€t C. arundinis CCC 974 C. arundinis CCC 236 C. arundinis CCC 902 C. arundinis CCC 480 TNS-F-60476 Phalaris arundinacea Toyama JA TNS-F-60478 P. arundinacea Ishikawa JP TNS-F-60488 P. arundinacea Ishikawa JP TNS-F-60492 P. arundinacea Tochigi JP TNS-F-60499 P. arundinacea Gifu JP TNS-F-60498 Phieum pratense Gifu JP TNS-F-60500 P. arundinacea Tochigi JP TNS-F-60506 P. arundinacea Chiba JP"T TNS-F-60507 P. arundinacea Chiba JP TNS-F-60512 P. pratense Miyagi JP TNS-F-60514 P. arundinacea Nagano JP TNS-F-60516 P. pratense Miyagi JP DAOM 984879 P. arundinacea Alberta CA DAOMC 251717=CCC 434 Dactylis sp. Bavaria DEFT DAOMC 251722=CCC 588 Phieum pretense Panska Skala CZ TNS-F-60470 Poa annua Ishikawa JP TNS-F-60472 Poa trivialis Ishikawa JP TNS-F-60473 Alopecurus aequalis var. amurensis Ishikawa JP TNS-F-60477 Dactylis glomerata Ishikawa JP TNS-F-60481 D. glomerata Niigata JP TNS-F-60482 Poa palustris Niigata JP TNS-F-60489 Poa acroleuca Ishikawa JP TNS-F-60490 D. glomerata Chiba JP TNS-F-60491 D. glomerata Tochigi JP TNS-F-60493 Calamagrostis epigeios Ishikawa JP TNS-F-60495 Agrostis gigantea Ishikawa JP TNS-F-60497 Festuca rubra Ishikawa JP TNS-F-60503 Polypogon fugax Ishikawa JP TNS-F-60504 Poa pratensis Ishikawa JP TNS-F-60505 Poa annua Kanagawa JP TNS-F-60508 A. aequalis var. amurensis Niigata JP TNS-F-60518 D. glomerata Miyagi JP DAOM 984880 F. rubra Alberta CA » CCC 691 P. arundinacea Dzungar Alatau KZ CCC 1020 Molinia caerulea Bozi Dar CZ 94/1 40.0 C. bavariensis wl ¥ 77 Fic. 1. Phylogenetic tree based on the concatenated sequence alignments of TEFl-a and RPB2 showing the separation and close relationship of Claviceps humidiphila and C. bavariensis. Reference sequences are in gray font, showing species names and voucher numbers; 24 taxa in C. sect. Pussillae are abbreviated to a single branch. The labels of the sequences in clades C. humidiphila and C. bavariensis include voucher numbers, hosts, locations, and country abbreviations: CA Canada; CZ Czech Republican; DE Germany; JP Japan; KZ Kazakhstan. Superscripts ET = ex-type, NT = neotype. Values on branches are bootstrapping values for MP analyses/posterior probability of BI. 78 ... Liu, Tanaka, Kolafik Taxonomy Claviceps humidiphila Pazoutova & M. Kolatik, Fungal Biol. 119(1): 22 (2015) Fic. 2 = Claviceps purpurea var. phalaridis Tanda, J. Agric. Sci. (Tokyo) 24: 84 (1979). Ho .oryPE (lost)—Japan, Chiba, Chiba, Yukijirushi Farm on Phalaris arundinacea, Aug 1, 1969, Seinosuke Tanda (TUAMH-PA 921). NEOTYPE (here designated, MBT395372)—Japan, Chiba, Chiba, Inage [= the holotype locality], 35.6672°N 140.1353°E, on Phalaris arundinacea, June 23, 2017, Eiji Tanaka (Neotype, TNS-F-60506; ex-type culture MAFF 247310; GenBank LC598958, LC598987). SCLEROTIA dark purple (#100336) to black (#05000A), 4-15 x 0.8-1.4 mm, cylindrical, floating in fresh water. Conrp14 from sclerotial surface hyaline, aseptate, allantoid, oblong, ovoid, ellipsoid, lunate, 6.0-16.9 x 3.6-5.9 um, L/W ratio 2.3-2.6. ASCOSTROMATA light orange (#E2C86C) to purplish red (#E6BOAA). STIPE filiform, 6-13 mm long, 0.1-0.4 mm wide. CAPITELLUM 0.5-1.9 mm diam. PERITHECIA obovoid, slightly fusiform to pyriform, 154-314 x 100-157 um. Asci cylindrical 50-110 x 2.5-5.5 um. Ascosporss filiform, 85-110 um long. ADDITIONAL SPECIMENS EXAMINED—JAPAN, CuiBA, Sakura, KODAKE, 35.7388°N 140.1661°E, on Phalaris arundinacea, June 23, 2017, Eiji Tanaka (TNS-F-60507; GenBank LC598959, LC598988), (TNS-F-60525, fruiting bodies derived from TNS-F-60507); GiFu, Takayama, Hirayu, 36.1917°N 137.5510°E, on P. arundinacea, Aug 14, 2016, Kazuhito Tanada (TNS-F-60499; GenBank LC598963, LC598992); 36.1914°N 137.5503°E, on Phleum pratense, Aug 14, 2016, Kazuhito Tanada (TNS-F-60498; GenBank LC598967, LC598996); IsH1kAwa, Nomi, NABETANI, 36.4189°N 136.5575°E, on P. arundinacea, June 30, 2016, Eiji Tanaka (TNS-F-60488; GenBank LC598961, LC598990); Uchinada, Kose, 36.6936°N 136.6842°E, on P. arundinacea, June 18, 2016, Eiji Tanaka (TNS-F-60478, MAFF 247302; GenBank LC598960, LC598989), (TNS-F-60524, fruiting bodies derived from TNS-F-60478); Mryaa1, Ohira, 38.4683°N 140.8833°E, on P. pratense, Aug 10, 2017, Kazuhito Tanada (TNS-F-60512; GenBank LC598968, LC598997); NAGANO, Nagano, TOGAKUSHI, 36.7711°N 138.0900°E, on P arundinacea, Aug 1, 2018, Eiji Tanaka (TNS-F-60514; GenBank LC598966, LC598995); TocuiGl, Tochigi, Fujioka, 36.2711°N 139.6567°E, on P. arundinacea, July 6, 2016, Kazuhito Tanada (TNS-F-60492, MAFF 247305; GenBank LC598965, LC598994); Nikko, Okukinu, 36.8681°N 139.3947°E, on P. arundinacea, Aug 15, 2016, Kazuhito Tanada (TNS-F-60500, MAFF 247310; GenBank LC598964, LC598993); ToyAMa, Nanto, FUKUMITSU, 36.5636°N 136.8778°E, on P. arundinacea, June 16, 2016, Eiji Tanaka (TNS-F-60476, MAFF 247301; GenBank LC598962, LC598991). Hosts—Phalaris, Phleum ComMMENTS—In the original description of C. purpurea var. phalaridis (Tanda 1979b), the specimens TUAMH-PA 820 and TUAMH-PA 921 were listed as the first and second specimens examined followed by the word “Holotype.” It is Claviceps bavariensis sp. nov. & C. humidiphila neotypified ... 79 Fic. 2. Claviceps humidiphila: A. Sclerotia on Phalaris arundinacea (TNS-F-60478); B. Ascostromata (TNS-F-60524) developed from germinated sclerotia (TNS-F-60478); C. Conidia washed off sclerotia (TNS-F-60478); D. Perithecia semi-embedded in stromata; E. Asci with thick caps and filiform ascospores (TNS-F-60524). Scale bars: A = 10 mm; B = 2 mm; D = 50 um; C, E= 10 um. likely that TUAMH-PA 921 was designated as holotype, although PA 820 was recorded as holotype in Pazoutova & al. (2015), possibly because PA 921 was erroneously overlooked. Nevertheless, we searched all of Tanda’s research collections (1979b) using several approaches. Firstly, theabbreviation TUAMH is not cited in Index Herbariorum (http://sweetgum.nybg.org/science/ih/) where TUAT is cited as the correct code for Tokyo University of Agriculture Herbarium (Museum); however, TUAT is currently inactive, and _ its 100,000 specimens were transferred to the National Museum of Nature and Science, Tokyo (TNS) in 2007. We searched the TNS online database (http://db.kahaku.go.jp/webmuseum_en) for Claviceps purpurea collected in Japan; of the 18 specimens located, none had been collected from Chiba or 80 ... Liu, Tanaka, Kolatik related to TUAMH PA 820 or PA 921. Next, we contacted the herbarium director and curator (Dr. Masanobu Higuchi and Dr. Tsuyoshi Hosoya), who recommended contacting Dr. Keiichi Motohashi at Tokyo University directly. According to a personal communication with Dr. Seinosuke Tanda, Tanda left all his un-submitted specimens in the laboratory at Tokyo University of Agriculture, where Dr. Motohashi checked 6000 specimens that had been collected by Dr. Tanda; 90% represented powdery mildews and the rest were rust fungi; no Claviceps specimens were found, possibly because other specimens had been discarded. Based on the collective evidence, it is likely that the specimens linked to the name C. purpurea var. phalaridis were lost. Therefore, we hereby designate a new specimen from the type location as neotype. According to Tanda (1979b), the sclerotia were of different sizes. One specimen collected from Asahi-mura, Niigata (PA007), possessed much shorter sclerotia (2.3-7.7 x 0.6-1.3 mm; n = 215), while others had a wider size range (2.2-15.4 x 0.5-1.5 mm; n = 599). The number of stromata developed from sclerotia varied from 1 to 11, for which Tanda (1979b) cited the dimensions of 0.1-1.6 x 0.2-1.9 mm (capitellum), 2-13 x 0.1-0.9 mm (stipe), and 4.4-16.9 x 2.4-5.9 um [L/W 2.2-2.6] (conidia; n = 1350). Our sclerotial, stromatal, and conidial measurements match those cited by Tanda. In addition, Tanda (1979b) also recorded perithecia at 161-217 x 91-130 um (a slightly narrower range than our data), asci at 74-133 x 1.8-4.2 um (slightly longer than our data), and ascospores at 72-129 um (similar to our data). Claviceps bavariensis M. Kolatik, E. Tanaka & M. Liu, sp. nov. Fig. 3 MB 838352 [= “Claviceps humidiphila” sensu epitype of Pazoutova & al. 2015, non Tanda 1979.] TypE—Germany, Bavaria, Philippsreut, 48.8564°N 13.6763°E, on Dactylis sp., 1988, Pazoutova (Holotype, PRM922708 [dried culture on T2 media]; ex-type culture CCC434; GenBank JX083704, JX083635). = Claviceps purpurea var. alopecuri Tanda, J. Agric. Sci. (Tokyo) 22: 295 (1977). = Claviceps purpurea var. dactylidis Tanda, J. Agric. Sci. (Tokyo) 25: 266 (1980). Differs from C. huimidiphila in amplified TEF1-a region (918 nts) by at least seven sites (C. humidiphila/C. bavariensis = 271 A/G; 348 A/G; 642 T/C; 675 T/C; 688 C/T; 690 T/C; 860 T/C) that are identical within species. EryMoLoGcy—referring to the provenance of the type specimen SCLEROTIA purplish brown (#452D2E), dark brown (#2D1E1E) to black (#272424), 3-10 x 0.3-1.4(-1.7) mm, ovoid, obclavate, narrow cylindrical, Claviceps bavariensis sp. nov. & C. humidiphila neotypified ... 81 en wg ie ay} BN. ! ij vn Fic. 3. Claviceps bavariensis. A. Sclerotia on Dactylis glomerata (TNS-F-60477); B. Sclerotia on Poa annua (TNS-F-60470); C. Sclerotia on Alopecurus aequalis var. amurensis (TNS-F-60473); D. Sclerotia on Festuca rubra (DAOM 984880); E. Sclerotia on Phalaris arundinacea (DAOM984879); F. Sclerotia detached from PRM922708 (holotype); G. Sclerotia detached from DAOM 984880; H. Sclerotia detached from DAOM 984879; I. Longitudinal section near the rind showing textura prismatica (DAOM 984879); J, K. Ascostromata developed from germinated sclerotia from D. glomerata (TNS-F-60523), and A. aequalis var. amurensis (TNS-F-60521); L, M. Cross sections of sclerotia showing the color of inner tissue (DAOM 984879, 984880); N. Asci showing the thick caps with a central pore (TNS-F-60522); O. Conidiogenesis in sclerotia (DAOM 984879, 984880); P. Conidia washed off sclerotia (PRM922708, holotype); Q. Filiform ascospores (TNS-F-60520). Scale bars: A = 5 mm; B, C, F-H, J, K= 2 mm; D, E= 1 mm; L, M = 200 um; I = 20 um; N-Q = 10 um. 82 ... Liu, Tanaka, Kolatik curved, or subulate, floating in fresh water, interior white, greyish yellow (#DOCBAO) to olive brown (#4A423B) from center to margin, or vivid yellow (#FEE302) to deep yellow (#FFC40C) in center (3A8-4A8). CONIDIOGENOUS CELL cylindrical, or obovoid, 12-23(-27) x 5-7 um. Conrp1A ovoid, reniform, cylindrical, (5.5—-)6.1-13.1(-14.3) x (2.3-)2.4-3.9(-4.4) um, L/W ratio 2.1-2.2. ASCOSTROMATA pinkish (#FFCOCB) to light brown (#5D4037). STIPE filiform 5-8 mm long. CaPITELLUM 0.5-1 mm diam. Asci cylindrical, 50-155 x 2.8-5.0 um. Ascosporsgs filiform. ADDITIONAL SPECIMENS EXAMINED—CANADA, ALBERTA, Beaverlodge, on Festuca rubra, Aug. 2019, Henry Klein-Gebbinck (DAOM 984880; GenBank MW411019, MW411021), on Phalaris arundinacea, Aug. 2019, Henry Klein-Gebbinck (DAOM 984879; GenBank MW411018, MW411020). JAPAN, CuHrspa, Sakura, UsuIDEN, 35.7416°N 140.1850°E, on Dactylis glomerata, Jul 4, 2016, Kazuhito Tanada (TNS-F-60490; MAFF 247304; GenBank LC598969, LC598998); IsH1KAWA, Hakusan, SHIRAMINE, 36.1150°N 136.6994°E, on Poa acroleuca, Jul 1, 2016, Eiji Tanaka (TNS-F-60489; GenBank LC598981, LC599010); YORISHINBO, 36.4770°N 136.5647°E, on Poa annua, Apr 29, 2016, Eiji Tanaka (TNS-F-60470; GenBank LC598977, LC599006); Kanazawa, MINATO, 36.6450°N 136.6631°E, on Calamagrostis epigejos, Jul 18, 2016, Eiji Tanaka (TNS-F-60493; MAFF 247306; GenBank LC598985, LC599014); Komatsu, IMAEMACHI, 36.3772°N 136.4492°E, on Poa trivialis, May 9, 2016, Eiji Tanaka (TNS-F-60472; GenBank LC598979, LC599008); Nomi, SUEDERA, 36.4442°N 136.5081°E, on Poa pratensis, June 9, 2017, Eiji Tanaka (TNS-F-60504; GenBank LC598976, LC599005); Nonoichi, NAKABAYASHI, 36.5064°N 136.5994°E, on Polypogon fugax, June 5, 2017, Eiji Tanaka (TNS-F-60503; GenBank LC598986, LC599015); Suematsu, 36.5103°N 136.5967°E, on Alopecuru. aequalis var. amurensis, May 23, 2016, Eiji Tanaka (TNS-F-60473; culture MAFF 247299; GenBank LC598974, LC599003); (TNS-F-60521, fruiting bodies derived from TNS-F-60473); Tsubata, 36.6875°N 136.7589°E, on F. rubra, Jul 24, 2016, Eiji Tanaka (TNS-F-60497; GenBank LC598984, LC599013); Uchinada, KosE, 36.6758°N 136.6706°E, on D. glomerata, June 18, 2016, Eiji Tanaka (TNS-F-60477; GenBank LC598970, LC598999); (TNS-F-60523, fruiting bodies derived from TNS-F-60477); Koyopal, 36.6375°N 136.6300°E, on Agrostis gigantea, Jul 24, 2016, Eiji Tanaka (TNS-F-60495; GenBank LC598982, LC599011); IwaTE, Miyako, Kuzakal, 39.6503°N 141.3574°E, on D. glomerata, Aug 7, 2019, Eiji Tanaka (TNS-F-60518; GenBank LC598973, LC599002); 39.6512°N 141.3574°E, on Phleum pratense, Aug 7, 2019, Eiji Tanaka (TNS-F-60516; GenBank LC598983, LC599012); KANAGAWA, Atsugi, NURUMIZU, 35.4347°N 139.3503°E, on PB annua, June 16, 2017, Kazuhito Tanada (TNS-F-60505; GenBank LC598978, LC599007); NiiGATa, Sanjo, KamiourA, 37.5883°N 139.0124°E, on A. aequalis var. amurensis, July 3, 2017, Kazuhito Tanada (TNS-F-60508; GenBank LC598975, LC599004); Tokamachi, MATSUNOYAMA, 37.1189°N 138.5908°E, on D. glomerata, June 24, 2016, Eiji Tanaka (TNS-F-60481; GenBank LC598972, LC599001); 37.0885°N 138.6097°E, on Poa palustris, June 24, 2016, Eiji Tanaka (TNS-F-60482; GenBank LC598980, LC599009); TocuiGl, Tochigi, Fujioka, 36.2436°N 136.6614°E, on D. glomerata, Jul 6, 2016, Kazuhito Tanada (TNS-F-60491; GenBank LC598971, LC599000). Claviceps bavariensis sp. nov. & C. humidiphila neotypified ... 83 Hosts—Alopecurus, Agrostis, _Ammophila, Calamagrostis, Dactylis, Deschampsia, Festuca, Molinia, Phalaris, Phleum, Phragmites, Poa, Polypogon COMMENTS—In our phylogenetic tree (Fic. 1), two samples on Alopecurus, four on Dactylis, six on Poa, and one on Polypogon group in the Claviceps bavariensis clade, suggesting that Tanda’s two varieties—C. purpurea var. alopecuri (Tanda 1977), C. purpurea var. dactylidis (Tanda 1981)—and the samples from Poa and Polypogon (Tanda 1980; Tanda & Kawatani 1980) might represent this species. In that case, a species name, “C. alopecuri” stat. nov., could be coined. However we considered the following: 1) the type specimen of C. purpurea var. alopecuri designated by Tanda (1977) was lost due to the same reason as C. purpurea var. phalaridis, therefore it is not possible to verify molecularly that the lost type truly belonged to the clade of C. bavariensis, which is essential for species delimitation; 2) the two specimens on Alopecurus in this study were not from Suginami, Tokyo, Japan (the type location); 3) the name “C. alopecuri” might give the false impression that the species has a host range limited to Alopecurus, contrary to the wide host range of the species. Therefore, we avoided elevating the varietal name to species level, proposing instead a new species name, C. bavariensis. Normally an epitype can only be replaced via conservation, but in this case the holotype specimen to which the epitype specimen would be attached has been lost, and an epitype no longer has nomenclatural status (Turland & al. 2018, Art. 9.20 Note 8). This allows us to use PRM922708, the epitype for C. humidiphila Pazoutova & M. Kolarik, as a holotype for C. bavariensis. Tanda (1977, 1980, 1981) and Tanda & Kawatani (1980) recorded detailed sexual and asexual morphological features of samples from Alopecurus, Dactylis, Poa, and Polypogon, which could represent Claviceps bavariensis. In general, sclerotia are cylindrical with pointed or blunt ends; stromatal stipes are reddish brown, cylindrical or filiform, and curved or straight; capitella are light orange, oblate globose, and verrucose; perithecia are pyriform or ovoid; asci are narrowly cylindrical with thickened caps; ascospores are colorless and filiform; and the conidia are 1-celled, hyaline, and cylindrical or ovoid. The sizes of these characters sclerotia, stromata, perithecia, asci, ascospores and conidia varied significantly from different host plants (TaBLE 1). Pazoutova & al. (2015) also provided more detailed morphological description and comparison with other species for C. bavariensis (as C. humidiphila). The conidia of C. bavariensis tend to be larger than C. purpurea s.str. and 84 ... Liu, Tanaka, Kolatik TABLE 1. Claviceps bavariensis: observations cited in Tanda (1977; 1980; 1981) and Tanda & Kawatani (1980) A—Host, sclerotial & ascomal dimensions Host SCLEROTIA (mm) STIPE (mm) CAPITELLA* height x diameter (mm) Alopecurus 1.5-5.1 x 0.3-0.9 1.0-4.0 x 0.3-0.5 0.4-0.9 x 0.6-1.3 Dactylis 3.2-11.7 x 0.6-1.7 1.0-14 x 0.1-1.4 0.3-1.5 x 0.3-2.3 Poa 1.5-5.8 x 0.2-1.5 1.0-9.0 x 0.1-0.7 0.4-1.0 x 0.5-1.9 Polypogon 1.2-4.0 x 0.2-1.0 1.0-7.0 x 0.2—-0.6 0.4-1.1 x 0.5-1.4 B—Anatomical dimensions PERITHECIA ASCI ASCOSPORE CONIDIA CONIDIA (um) (um) LENGTH (tm) (um) L/W 151-221 x 77-144 75-138 x 2.5-3.7 74-126 5.0-11.3 x 2.6-5.0 2.1-2.3 154-270 x 88-168 81-151 x 1.8-4.2 74-119 4.0-14.8 x 1.8-4.7 1.9-2.8 147-256 x 60-154 77-158 x 1.8-5.3 63-151 3.2-12.6 x 1.6-5.8 2.0-3.0 119-210 x 56-126 91-133 x 2.5-3.9 84-123 5.0-12.8 x 2.4-5.4 — C. arundinis, although the size ranges can overlap. Although these three species shared several host genera, population genetic analyses suggested significant genetic differentiation and limited gene flow among them (Pazoutova & al. 2015). The three species also differ in their ergot alkaloid spectra (Pazoutova & al. 2000, Negard & al. 2015) and ergochrome pigments (Flieger & al. 2019). Nevertheless, separation between C. bavariensis and C. humidiphila relies essentially on molecular evidence (mainly TEF1-a, at least seven constant nucleotide variance). The host species cited by Pazoutova & al. (2015) included 10 genera mainly from Europe (Belgium, Czech Republic, Germany, France, Norway, Poland, Turkey), but also from Central Asia (Kazakhstan, one specimen on P. arundinacea) and North America (USA, one specimen on Ammophila breviligulata). The present study expanded sample locations and host range by adding Canada (on Festuca, Phalaris) and Japan (on Agrostis gigantea, Alopecurus aequalis var. amurensis, Calamagrostis epigejos, Dactylis glomerata, Festuca rubra, Poa acroleuca, P. annua, P. palustris, P. pratensis, P. trivialis, Polypogon fugax). Claviceps bavariensis sp. nov. & C. humidiphila neotypified ... 85 Acknowledgments We are grateful to MSc Kazuhito Tanada for providing many ergot samples from various regions in Japan and Dr. Henry Klein-Gebbinck for samples from Alberta (Canada). We also thank Drs. Masanobu Higuchi, Tsuyoshi Hosoya, Keiichi Motohashi and Seinosuke Tanda for their efforts in searching for type specimens; Dr. Scott Redhead (DAOM, Agriculture and Agri-Food Canada, Ottawa, ON Canada) for nomenclature advice and pre-submission manuscript review; and Dr. Joey Tanney (DAVP, Pacific Forestry Centre, Victoria, BC, Canada) for pre-submission review. We thank Dr. Shaun Pennycook for the nomenclature review and the meticulous edits that have significantly improved the clarity and efficiency of the presentation. This work was supported by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) (grant number 16K07238); and Agriculture and Agri-Food Canada STB fungal and bacterial biosystematics J-002272. Literature cited Clements FE, Shear CL. 1931. The genera of fungi. New York, The H.W. Wilson Company Donk MA. 1963. Claviceps L. Tul. (1853) not illegitimate. Taxon 12: 264-266. Douhan GW, Smith ME, Huyrn KL, Westbrook A, Beerli P, Fisher AJ. 2008. Multigene analysis suggests ecological speciation in the fungal pathogen Claviceps purpurea. Molecular Ecology 17(9): 2276-2286. https://doi.org/10.1111/j.1365-294X.2008.03753.x Flieger M, Stodtilkova E, Wyka SA, Cerny J, Grobarova V, Pichova K, Novak P, Man P, Kuzma M, Cvak L. 2019. Ergochromes: heretofore neglected side of ergot toxicity. 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Mycologia 112: 974-988. https://doi.org/10.1080/00275514.2020.1797372 Liu M, Shoukouhi P, Bisson KR, Wyka SA, Broders KD, Menzies JG. 2021. Sympatric divergence of the ergot fungus, Claviceps purpurea, populations infecting agricultural and nonagricultural grasses in North America. Ecology and Evolution 11(1): 273-293. https://doi.org/10.1002/ece3.7028 Negard M, Uhlig S, Kauserud H, Andersen T, Hgiland K, Vralstad T. 2015. Links between genetic groups, indole alkaloid profiles and ecology within the grass-parasitic Claviceps purpurea species complex. Toxins 7(5): 1431-1456. https://doi.org/10.3390/toxins7051431 86 ... Liu, Tanaka, Kolatik Pazoutova S, Olsovska J, Linka M, Kolinska R, Flieger M. 2000. Chemoraces and habitat specialization of Claviceps purpurea populations. Applied and Environmental Microbiology 66(12): 5419-5425. https://doi.org/10.1128/aem.66.12.5419-5425.2000 Pazoutova S, Pesicové K, Chudi¢kova M, Sritka P, Kolatik M. 2015. Delimitation of cryptic species inside Claviceps purpurea. Fungal Biology 119(1): 7-26. https://doi.org/10.1016/j.funbio.2014.10.003 Rehner SA, Buckley E. 2005. A Beauveria phylogeny inferred from nuclear ITS and EF1-a sequences: evidence for cryptic diversification and links to Cordyceps teleomorphs. Mycologia 97(1): 84-98. https://doi.org/10.1080/15572536.2006.1 1832842 Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Héhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP. 2012. MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61(3): 539-542. https://doi.org/10.1093/sysbio/sys029 Shoukouhi P, Hicks C, Menzies JG, Popovic Z, Chen W, Seifert KA, Assabgui R, Liu M. 2019. Phylogeny of Canadian ergot fungi and a detection assay by real-time polymerase chain reaction. Mycologia 111(3): 493-505. https://doi.org/10.1080/00275514.2019.1581018 Swofford DL. 2002. PAUP*. Phylogenetic analysis using parsimony (*and other methods). version 4.0 b10 (computer program). Sunderland, Massachusetts, Sinauer Associates. https://doi.org/10.1017/cbo9780511819049.010 Tanaka E, Shrestha B, Shivas RG. 2020. Commelinaceomyces gen. nov. for four clavicipitaceous species misplaced in Ustilago that infect Commelinaceae. Mycologia 112: 649-660. https://dx.doi.org/10.1080/00275514.2020.1745524. Tanda S. 1977. Mycological studies on ergot in Japan (Part 3): a new variety of Claviceps purpurea Tul. Parasitic on Alopecurus aequalis Sobol. var. amurensis Ohwi. Journal of Agricultural Science, Tokyo Nogyo Daigaku 22: 293-299. Tanda S. 1978a. Mycological studies on ergot in Japan (Part 4): two varieties of Claviceps purpurea Tul. parasitic on timothy. Phleum pratense L. Journal of Agricultural Science, Tokyo Nogyo Daigaku 23: 141-150. Tanda S. 1978b. Mycological studies on ergot in Japan (Part 6): a physiologic race of Claviceps purpurea Tul. var. alopecuri Tanda collected from Trisetum bifidum Ohwi. Journal of Agricultural Science, Tokyo Nogyo Daigaku 23: 207-214. Tanda S. 1978c. Mycological studies on ergot in Japan (Part 7): two varieties of Claviceps purpurea Tul. parasitic on Agrostis spp. Journal of Agricultural Science, Tokyo Nogyo Daigaku 23: 215-221. Tanda S. 1979a. Mycological studies on ergot in Japan (Part 8): ergots on Calamagrostis spp. Journal of Agricultural Science, Tokyo Nogyo Daigaku 23: 67-78. Tanda S. 1979b. Mycological studies on ergot in Japan (Part 9): distinct variety of Claviceps purpurea Tul. on Phalaris arundinacea L. and P. arundinacea var. picta L. Journal of Agricultural Science, Tokyo Nogyo Daigaku 24: 67-95. Tanda S. 1979c. Mycological studies on ergot in Japan (Part 10): ergots on velvet grass, Holcus lanatus L. Journal of Agricultural Science, Tokyo Nogyo Daigaku 24: 145-148. Tanda S, Kawatani T. 1980. Mycological studies on ergot in Japan (Part 11): ergots on Polypogon fugax. Transactions of the Mycological Society of Japan 21: 97-101. Tanda S. 1980. Mycological studies on ergot in Japan (Part 16): ergots on orchard grass, Dactylis glomerata L. Journal of Agricultural Science, Tokyo Nogyo Daigaku 25: 263-271. Claviceps bavariensis sp. nov. & C. humidiphila neotypified ... 87 Tanda S. 1981. Mycological studies on ergot in Japan (Part 19): ergots on bluegrasses (Poa spp.). Journal of Agricultural Science, Tokyo Nogyo Daigaku 26: 179-192. Tulasne LR. 1853. Mémoire sur lergot des Glumacées. Annales des Sciences Naturelles, Botanique 20: 5-56. Turland NJ, Wiersema JH, Barrie FR, Greuter W, Hawksworth DL & al. 2018. International Code of Nomenclature for algae, fungi, and plants (Shenzhen Code) adopted by the Nineteenth International Botanical Congress, Shenzhen, China, July 2017. Regnum Vegetabile 159. Glashiitten, Koeltz Botanical Books. https://doi.org/10.12705/code.2018 MY COTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2022 January-March 2022— Volume 137, pp. 89-94 https://doi.org/10.5248/137.89 Passalora golaghati comb. nov. from India GARGEE SINGH’, SANJAY YADAV’, RAGHVENDRA SINGH?, SHAMBHU KUMAR? ‘Department of Botany, DDU Gorakhpur University, Gorakhpur, U.P, India 273009 ? Centre of Advanced Study in Botany, Institute of Science, Banaras Hindu University, Varanasi, U.P, India 221005 ° Forest Pathology Department, KSCSTE-Kerala Forest Research Institute, Peechi, Thrissur, Kerala, India 680653 " CORRESPONDENCE TO: drsinghtaxon@gmail.com, singhr.bot@bhu.ac.in ABSTRACT—The hyphomycete Cercospora golaghati is recombined as Passalora golaghati based on critical re-examinations of original type material and a fresh topotypic collection and comparison with closely related species of cercosporoid taxa. Key worps—foliicolous, anamorph, Mycosphaerellaceae, nomenclature, taxonomy Introduction The significant characteristic that separates the two cercosporoid genera Cercospora Fresen. ex Fuckel and Passalora Fr. is colouration of conidia. When Crous & Braun (2003) emended the circumscription of Passalora based on ITS and 5.8S rDNA sequence analyses, they observed that the formation of single or catenate conidia is not a stable feature for diagnosis at the generic level in cercosporoid hyphomycetes (Crous & al. 2001). Their contributions have confirmed that presence or absence of thickened conidiogenous loci and pigmentation in conidiophores and conidia are important features of taxonomic relevance (Crous & al. 2009, 2013; Videira & al. 2017). During the last decade a large number of cercosporoid fungi have been recombined in the genus Passalora (Crous & Braun 2003; Braun 90 ... Singh & al. & al. 2013, 2014, 2015a,b, 2016), particularly from India (Kamal 2010). Recently, the addition of several novel taxa of foliicolous cercosporoid fungi described from India (Kumar & Singh 2015a,b, 2016; Awasthi & al. 2016; Singh & al. 2011, 2012, 2013) suggests that cercosporoid diversity is still insufficiently known in this region. A new collection from the type locality of Cercospora golaghati (Saikia & Sarbhoy 1980) confirmed that this species is characterized by thickened scars and coloured conidiophores and conidia, indicating that it should be transferred to Passalora (Crous & Braun 2003). Materials & methods The holotype [conserved in the Herbarium, Division of Mycology & Plant Pathology, Indian Agricultural Research Institute, New Delhi, India (HCIO)] and a fresh specimen collected from the same host species in the same locality [conserved in the Mycological Herbarium of the Gorakhpur University, Gorakhpur, UP, India (MH-GPU)] were critically analyzed, and slides from infection spots were mounted in distilled water, lactophenol and cotton-blue mixture. Observation of microscopic characters was recorded under an Olympus CH20i-TR compound microscope to understand the exact morphology of the fungus. Measurements of 30 conidia, hila, conidiophores, and conidiogenous cells were recorded with the help of stage and ocular micrometers, and Lucida drawings were also made. The morphology of the fungus was compared with closely related cercosporoid taxa with the help of current literature. Taxonomy Passalora golaghati (Saikia & A.K. Sarbhoy) Gargee Singh, Sanj. Yadav, Raghv. Singh & Sh. Kumar, comb. nov. Figs 1-4 MB 835579 = Cercospora golaghati Saikia & A.K. Sarbhoy, Curr. Sci. 49: 830. 1980, as “golaghatii”. INFECTION SPOTS amphigenous, brown to dark blackish brown, initially circulartosubcircular, later spreading over the leafsurface to become irregular, 3-15 mm in diam. CoLonigs hypophyllous and effuse. MycELrum internal. STROMATA present, epidermal to subepidermal, pseudoparenchymatous, brown to dark brown, 30-32 x 18-19 um in diam. CONIDIOPHORES fasciculate, macronematous, brown to dark brown, unbranched, cylindrical, erect to procumbent, straight to flexuous, geniculate, smooth, thick- walled, 2—7-septate, (62—)80-102(-152) x 3-5 um. CONIDIOGENOUS CELLS polyblastic, integrated, terminal, apex often slightly wider than at the base, 20-40 x 3-5 um, loci thickened and darkened, 2-2.5 um. Conip1a solitary, acropleurogenous, obclavate-cylindrical, simple, dry, straight to slightly Passalora golaghati comb. nov. (India) ... 91 Fics 1-4. Passalora golaghati (holotype, HCIO 32660). 1. Symptoms of infection; 2. Stroma; 3. Conidiophores; 4. Conidia. 92 ... Singh & al. curved, thin-walled, smooth, brown to mid brown, 3-6-septate, tip acute to sub-obtuse, base obconico-truncate, (35-)50-60(-75) x 2.5-6 um, hilum protuberant, thickened and darkened, 2-2.5 um. SPECIMENS EXAMINED: INDIA, Assam, Golaghat, Salikihat, on living leaves of Dioscorea alata L. (Dioscoreaceae), 21 November 1977, leg. U.N. Saikia (HCIO 32660, holotype); 26.5239°N 93.9623°E, on living leaves of Dioscorea alata, December 2019, leg. Gargee Singh (MH-GPU 1, topotype). Discussion A literature review revealed that seven Passalora taxa have been reported on Dioscoreaceae: P. dioscoreae (Ellis & G. Martin) U. Braun & Crous, P. dioscoreae-nipponicae Y.L. Guo, P. dioscoreae-subcalvae Y.L. Guo, P. dioscoreicola Y.L. Guo, P. dioscoreigena U. Braun & Crous, P. tranzschelii (Vassiljevsky) U. Braun & Crous var. tranzschelii, and P._ tranzschelii var. chinensis Y.L. Guo (Braun & al. 2014, Crous & Braun 2003; Guo 2001, 2011). Passalora dioscoreae has been subsequently recombined in Distocercosporaster by Videira & al. (2017). Passalora golaghati differs distinctly from all the remaining Passalora species in its conidiophore and conidial sizes. Morphological comparisons of all Passalora species reported on Dioscoreaceae are presented in TABLE 1. Key to Passalora spp. reported on Dioscoreaceae DesConidial leriot ie 75 phi’. se Pan. otteela-nongdanaotedasscrtlancs aden scar a da sarte tends, sels. dee 2 ESCO eial Pei ot ly A Ty hy aha ect obs oct tey Iafemattee Bhanchey inc epaorabay bhsuably beube weed ial ate 3 2. Conidiophores 35-175 x 4-6 um, conidia 10-55 x 7.5-10 um, Pe2eseptate or dihioes shines cGine Ling iat PGA whut eae 8 NGUoS P. dioscoreicola 2. Conidiophores 80-102 x 3-5 um, conidia 50-60 x 2.5-6 um, S=G-SEPlate: 2 Sis isos we Seb vG Seo eG s 6. Bye 8 we Ben vG ed So Rea P. golaghati 3-Conidiophorelenctins5 Oui, <4 sei «2 ven cam 8 cGy epee any eee eee es 4 8. Conidie plore: le tect i 50) quite 1k 5 hae ee Prt PS ee Md Re el 5 4. Conidiogenous loci 1-2 um diam., conidia 15-90 x 2-5 um, W=S=septate”, sh. eg gis ea gistn +a site tga pera 4s bier ds P. tranzschelii var. tranzschelii 4, Conidiogenous loci 1.5-3 um diam., conidia 30-125 x 4-7 um, SS VOAseptateny cin. egies gd ge eddie ya ceerds Herd he P. tranzschelii var. chinensis 5s Comidiaphorelene ty AT OOM: i..5-3 dreseu-g dros setog gras sekeg rhs pebog echt pebon xis feds (9 pete g tt pede 6 SeGonidiophore lemethy SOO I ses otatse-o + eatery 4 tarsal ata cy aaa awe ote P. dioscoreigena 6. Conidiophores 10-70 x 4-7 um, conidia 30-125 x 4-6.5 um, S—PO=SE PLA LES toh su sea tsa testes a cet bed cp sto Srsghiuova aeblade asses P. dioscoreae-nipponicae 6. Conidiophores 25-65 x 4.5-6.5 um, conidia 30-85 x 4.5-6.5 um, DA~GUSEPLALG Eh Ore stance ries veks Sere aseantes tame cic ldes Russab Veh gcc Taneg P. dioscoreae-subcalvae Passalora golaghati comb. nov. (India) ... 93 TABLE 1. Comparative morphology of Passalora taxa reported on Dioscoreaceae. CONIDIA TAXON CONIDIOPHORE SIZE (um) SEPTATION SIZE (um) P. dioscoreae-nipponicae 10-70 x 4-7 3-10 30-125 x 4-6.5 P. dioscoreae-subcalvae 25-65 x 4.5-6.5 2-6 30-85 x 4.5-6.5 P. dioscoreicola 35-175 x 4-6 1-2 10-55 x 7.5-10 P. dioscoreigena 40-120 x 5-8.5 1-6 25-110 x 4.5-6.5 P. golaghati 80-102 x 3-5 3-6 50-60 x 2.5-6 P. tranzschelii var. tranzschelii 5-50 x 3-7 0-3 15-90 x 2-5 P. tranzschelii var. chinensis 5-50 x 3-7 3-10 30-125 x 4-7 Acknowledgments Sincere thanks are due to Curator of the HCIO (New Delhi) for making it possible to examine the holotype collection of Cercospora golaghati. We express our deep gratitude to Dr. R.E Castafeda-Ruiz (INIFAT Alejandro de Humboldt, Habana, Cuba) and Dr. Flavia Rodrigues Barbosa (Universidade Federal de Mato Grosso, Brazil) for presubmission critical review. We express our deep appreciation to Prof. Dr. Kamal (Emeritus Scientist, DST) for his valuable suggestions and kind help. We are also thankful to the Head of the Department of Botany of D.D.U. Gorakhpur University, Gorakhpur, U.P., for providing necessary facilities. Literature cited Awasthi N, Singh R, Kumar S. 2016. A new species of Pseudocercosporella on Andrographis paniculata from Central India. Sydowia 68: 27-33. Braun U, Nakashima C, Crous PW 2013. Cercosporoid fungi (Mycosphaerellaceae) 1. Species on other fungi, Pteridophyta and Gymnospermae. IMA Fungus 4: 265-345. https://doi.org/10.5598/imafungus.2013.04.02.12 Braun U, Crous PW, Nakashima C. 2014. Cercosporoid fungi (Mycosphaerellaceae) 2. Species on monocots (Acoraceae to Xyridaceae, excluding Poaceae). IMA Fungus 5: 203-390. https://doi.org/10.5598/imafungus.2014.05.02.04 Braun U, Crous PW, Nakashima C. 2015a. Cercosporoid fungi (Mycosphaerellaceae) 3. Species on monocots (Poaceae, true grasses). IMA Fungus 6: 25-97. https://doi.org/10.5598/imafungus.2015.06.01.03 Braun U, Crous PW, Nakashima C. 2015b. Cercosporoid fungi (Mycosphaerellaceae) 4. Species on dicots (Acanthaceae to Amaranthaceae). IMA Fungus 6: 373-469. https://doi.org/10.5598/imafungus.2015.06.02.09 Braun U, Crous PW, Nakashima C. 2016. Cercosporoid fungi (Mycosphaerellaceae) 5. Species on dicots (Anacardiaceae to Annonaceae). IMA Fungus 7: 161-216. https://doi.org/10.5598/imafungus.2016.07.01.10 Crous PW, Braun U. 2003. Mycosphaerella and its anamorphs: names published in Cercospora and Passalora. CBS Biodiversity Series 1. CBS Utrecht, The Netherlands. 571 p. 94 ... Singh & al. Crous PW, Kang JC, Braun U. 2001. A phylogenetic redefinition of anamorph genera in Mycosphaerella based on ITS rDNA sequence and morphology. Mycologia 93: 1081-1101. https://doi.org/10.1080/00275514.2001.12063243 Crous PW, Summerell BA, Carnegie AJ, Wingfield MJ, Hunter GC, Burgess TI, Andjic V, Barber PA, Groenewald JZ. 2009. Unravelling Mycosphaerella: do you believe in genera? Persoonia 23: 99-118. https://doi.org/10.3767/003158509X479487 Crous PW, Braun U, Hunter GC, Wingfield MJ, Verkley GJM, Shin HD, Nakashima C, Groenewald JZ. 2013. Phylogenetic lineages in Pseudocercospora. Studies in Mycology 75: 37-114. https://doi.org/10.3114/sim0005 Guo YL. 2001. Studies on Cercospora and allied genera in China IX. Mycosystema 20(3): 301-303. Guo YL. 2011. Studies on Cercospora and allied genera of China XIV. Mycosystema 30(6): 865-869. Kamal. 2010. Cercosporoid fungi of India. Bishan Singh Mahendra Pal Singh Publication, Dehradun (UK), India. 351 p. Kumar S, Singh R. 2015a. Passalora musicola sp. nov. a new Indian hyphomycete. Sydowia 67: 2Z1>23; Kumar S, Singh R. 2015b. Pseudocercospora bischofigena, a new Cercosporoid fungus from northeastern Uttar Pradesh, India. Czech Mycology 67: 39-44. https://doi.org/10.33585/cmy.67 105 Kumar S, Singh R. 2016. Passalora caesalpiniicola sp. nov. from India on Caesalpinia bonduc. Mycotaxon 131: 25-30. https://doi.org/10.5248/131.25 Saikia UN, Sarbhoy AK. 1980. A new species of Cercospora from India. Current Science 49: 830-831. Singh R, Kumar S, Kamal. 2011. Two new species of Passalora and Pseudocercospora from northeastern Uttar Pradesh, India. Mycotaxon 117: 137-143. https://doi.org/10.5248/117.137 Singh R, Chaurasia B, Shukla K, Upadhyaya PP. 2012. Passalora aseptata, a new Cercosporoid fungus from northeastern Uttar Pradesh, India. Mycotaxon 120: 461-463. https://doi.org/10.5248/120.461 Singh R, Kumar S, Saini DC, Upadhyaya PP, Kamal, Braun U. 2013. Diversity of Passalora on Ficus. Mycological Progress 12: 637-643. https://doi.org/10.1007/s11557-012-0870-6 Videira SIR, Groenewald JZ, Nakashima C, Braun U, Barreto RW, de Wit, PJGM, Crous PW. 2017. Mycosphaerellaceae - chaos or clarity? Studies in Mycology 87: 257-421. https://doi.org/10.1016/j.simyco.2017.09.003 MY COTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2022 January-March 2022— Volume 137, pp. 95-107 https://doi.org/10.5248/137.95 Stigmatomyces aff. limnophorae on dipteran hosts in Peninsular Malaysia NATASHA AZMI NurR-ALIAH’, JINGYU LIU’, Nurut AZMIERA', CHONG CHIN HEO??” ' Department of Medical Microbiology and Parasitology, Faculty of Medicine, Universiti Teknologi MARA Selangor Branch, Sungai Buloh Campus, 47000 Sungai Buloh, Selangor, Malaysia ? Department of Botany and Plant Pathology, College of Agriculture, Purdue University, West Lafayette IN 47907 U.S.A. ° Institute for Pathology, Laboratory and Forensic Medicine (I-PPerForM), Universiti Teknologi MARA Selangor Branch, Sungai Buloh Campus, 47000 Sungai Buloh, Selangor, Malaysia * CORRESPONDENCE TO: chin@uitm.edu.my ABSTRACT—Flies parasitized by Laboulbeniales, captured in Selangor state, were examined and identified as Boettcherisca javanica, Boettcherisca sp., and Hypopygiopsis violacea. The fungus was identified as Stigmatomyces aff. limnophorae based on morphology and phylogenetic analysis of sequences of the partial nuclear small and large subunit ribosomal RNA genes. This fungus represents a new record for Malaysia; and these are the first reports of Boettcherisca and Hypopygiopsis as hosts for any laboulbenialean species. Key worps—Calliphoridae, Laboulbeniomycetes, Sarcophagidae, southeastern Asia. Introduction Fungi in Laboulbeniales (Ascomycota: Laboulbeniomycetes) are ectoparasitic fungi that are obligatorily associated with arthropods as ectoparasites (Haelewaters & al. 2012, Melo & Melo 2019, Blackwell & al. 2020). These fungi are characterized by the presence of a three-dimensional thallus (plural: thalli), instead of hyphae with mycelial growth like many other fungi (Blackwell & al. 2020). Representatives of three arthropod subphyla 96 ... Nur Aliah & al. (Chelicerata, Myriapoda, Hexapoda) and various insect orders including Coleoptera, Diptera, Hemiptera, and Hymenoptera are known as hosts for these fungi (reviewed in Haelewaters & al. 2021). In the past 40 years, only two species Laboulbeniales have been reported in Malaysia: Laboulbenia admirabilis, found on the body of an unidentified Spaniocelyphus (Diptera) in Pahang state (Lee & Majewski 1986), and Diphymyces sabahensis, on three Ptomaphaginus spp. (Coleoptera) in Sabah state (Haelewaters & al. 2014). Stigmatomyces sensu lato is a large, paraphyletic genus of 176 species on dipteran hosts (Haelewaters & al. 2020a; Species Fungorum 2020). Species within this heterogeneous assemblage (sensu lato) are parasites on hosts in many different families—including Anthomyiidae, Calliphoridae, Chamaemyiidae, Diopsidae, Drosophilidae, Ephydridae, Fanniidae, Muscidae, Nycteribiidae, Sarcophagidae, Sphaeroceridae, and Streblidae (Thaxter 1901, 1905, 1917; Rossi 1998; Hyde & al. 2019; Haelewaters & al. 2018, 2020a). Thus far, five species of Stigmatomyces sensu stricto have been reported in Malaysia: S. dacinus, S. limosinoides, S. tortimasculus, and S. venezuelae in Malaysian Borneo (Thaxter 1915, 1918); and S. neurochaetae in Peninsular Malaysia (Sugiyama & Majewski 1985; Rossi & Weir 2007). Note that Thaxter (1915) reported S. stilici from Malaysian Borneo and Sugiyama & Majewski (1985) reported S. orientalis from Peninsular Malaysia—both associated with staphylinid beetles (Coleoptera: Staphylinidae) and both species later recombined in Zeugandromyces. Here, we provide the first records of Stigmatomyces aff. limnophorae from Malaysia. Our material was studied based on morphological characters and sequence data. The parasitized fly genera are for the first time reported in the literature as hosts for Laboulbeniales. Material & methods Collection & identification of flies An entomological survey was conducted in September 2019 in the state of Selangor, Peninsular Malaysia, to investigate the biodiversity of carrion flies. Chicken liver (200 g, 2d old) was used as bait and flies were collected using sweep nets at two different forests in the town of Rawang. Collected adult flies were then placed in a cloth-lid jar and brought back to the Parasitology Laboratory, Institute for Medical Molecular Biotechnology, Universiti Teknologi MARA (UiTM) in Sungai Buloh. The flies were incubated at -4 °C for 15 minutes, after which they were pinned and dried at room temperature. During microscopic examination for species determination, we observed four fly specimens (out of >100 observed) with thalli of Stigmatomyces aff. limnophorae on dipteran hosts (Malaysia) ... 97 Laboulbeniales. These specimens were carefully examined and photographed using an Olympus SZ51 stereomicroscope equipped with a digital camera and CellD Imaging Software. The parasitized adult flies were identified using Kurahashi & al. (1997) and Kurahashi & Samerjai (2018). Microscopic study of Laboulbeniales Parasitized flies were shipped to Purdue University for microscopic study of the Laboulbeniales (by J.L.). Thalli were taken from the host fly using a BioQuip #1208SA entomological pin dipped in Hoyer’s medium (30 g arabic gum, 200 g chloral hydrate, 16 ml glycerol, 50 ml ddH20). Thalli were mounted in Amann’s medium applying a double coverslip technique using Solakryl BMX as outlined in Liu & al. (2020). Microscope mounts were viewed at 200-400x using an Olympus BH2 bright field compound microscope. Line and stipple drawings were made with PITT artist pens based on photomicrographs taken with an Olympus SC30 camera and cellSens 1.18 imaging software. Permanent slides are deposited at PUL (Kriebel Herbarium) under numbers PUL F25943-F25950. DNA extraction, PCR amplification, sequencing DNA was extracted from 2-4 thalli of Stigmatomyces using the REPLI-g Single Cell Kit with modifications by Haelewaters & al. (2019). The nuclear ribosomal RNA small (SSU) and (LSU) large subunits were amplified using primer pairs NSL1/NSL2 for SSU (Haelewaters & al. 2015), and LROR/LR5 and LIC24R/LR5 for LSU (Vilgalys & Hester 1990, Hopple 1994, Miadlikowska & Lutzoni 2000). The DNA was amplified using an Eppendorf pro S Mastercycler in 25 uL volumes containing 12.5 ul 2x MyTaq Mix (Bioline, Swedesboro, New Jersey), 9.5 uL ddH20, 1.0 uL forward and reverse primer, and 1.0 uL DNA. Cycling conditions—for SSU: initial denaturation at 95 °C for 5 min; 40 cycles of denaturation at 95 °C for 30 sec, annealing at 55 °C for 45 sec, extension at 72 °C for 45 sec; and final extension at 72 °C for 1 min and for LSU: initial denaturation at 94 °C for 5 min; 35 cycles of denaturation at 94 °C for 30 sec, annealing at 50 °C for 45 sec, extension at 72 °C for 1 min; and final extension at 72 °C for 7 min. The PCR amplicons were sent to Genewiz (South Plainfield, New Jersey) for purification and sequencing. Raw sequence reads were assembled and edited in Gene Codes Sequencher 5.2.3. Sequences were deposited at the National Center for Biotechnology Information (NCBI) GenBank database; accession numbers MT341792—-M1T341794 (SSU) and MT341789-MT341791 (LSU). These sequences were then BLAST searched against NCBI's nucleotide collection to establish a rough relationship with existing sequences. Sequence alignments & phylogenetic analysis SSU and LSU sequences of Stigmatomyces species representing the same clade (clade IV sensu Haelewaters & al. 2020a) were downloaded from GenBank (https://www.ncbi.nlm.nih.gov/genbank/). Gloeandromyces dickii was selected as outgroup. Details for all isolates are presented in TABLE 1. Sequences of both regions 98 ... Nur Aliah & al. TABLE 1. Isolates and sequences used in phylogenetic analysis. SPECIES ISOLATE Country SSU LSU REFERENCE Gloeandromyces dickii D.Haelew. Panama MG958011 MH040582 Haelewaters & al. 2018 1323b Stigmatomyces borealis AW-979 USA JN835186 — A. Weir (unpublished) S. chamaemyiae D.Haelew. Portugal MH040564 — Haelewaters & al. 2018 1137a D.Haelew. Portugal MH040565 — Haelewaters & al. 2018 1137c S. limnophorae AW-785 USA AF407576 — Weir & Blackwell 2001b S. aff. limnophorae D.Haelew. Malaysia MT341792 MT341789 This study 1802c D.Haelew. Malaysia MT341793 MT341790 This study 1802d D.Haelew. Malaysia MT341794 MT341791 This study 1820e S. protrudens AW-793 USA AF298232 AF298234 Weir & Blackwell 42001a S. rugosus — — AF431759 — Weir & Hughes 2002 D.Haelew. Portugal MH040563 — Haelewaters & al. 2018 1138a were aligned with MUSCLE (Edgar 2004) on the Cipres Science Gateway web portal (Miller & al. 2010). For both datasets, the appropriate nucleotide substitution model was selected by considering the corrected Akaike Information Criterion (AICc) using ModelFinder Plus (Kalyaanamoorthy & al. 2017). Models selected were HKY+F+I (SSU, -InL = 2420.547) and TIM2+F+I (LSU, -InL = 1837.675). SSU and LSU aligned datasets were combined using MEGA7 (Kumar & al. 2016). A Maximum likelihood analysis of the concatenated two-locus dataset was performed using IQ-TREE (Nguyen & al. 2015) with partitioned models (Chernomor & al. 2016) and ultrafast bootstrapping with 1000 replicates (Hoang & al. 2018). The best-scoring tree was visualized in FigTree 1.4.3 (http://tree.bio.ed.ac.uk/ software/figtree/) and edited in Adobe Illustrator 24.1.1. Host identification The infected insect hosts (n = 4) belonged to two families, Calliphoridae (n = 1) and Sarcophagidae (n = 3). The calliphorid fly was identified as Hypopygiopsis violacea (Fic. 1). Two sarcophagid flies were identified as Boettcherisca javanica and the third as Boettcherisca sp. (Fics 2, 3). All four specimens bore thalli at their abdominal segments (TABLE 2). Stigmatomyces aff. limnophorae on dipteran hosts (Malaysia) ... 99 Fic. 1. Adult fly of Hypopygiopsis violacea (Diptera: Calliphoridae): A. Habitus of H. violacea at 0.8x magnification. B. The silver white facial tomentum. C. Stigmatomyces aff. limnophorae thalli on 5th tergite (black arrow). D. Stigmatomyces aff. limnophorae thalli on sternites 3 and 4 (black arrows). Scale bars: A, B= 2mm; C, D=1 mm. tom ity Tea Fic. 2. Adult fly of Boettcherisca sp. (Diptera: Sarcophagidae): A. Heavy infection with Stigmatomyces aff. limnophorae at abdominal tergites 4 and 5 (black arrow); B. Close-up view of the parasitized tergites 4 and 5 at 2.5x magnification. Scale bars: A = 2 mm; B = 1 mm. Fig. 3. Adult fly of Boettcherisca javanica (Diptera: Sarcophagidae): A. Stigmatomyces aff. limnophorae thalli on abdominal sternites 4 and 5 (black arrow); B. Tuft of S. (aff.) limnophorae thalli on abdominal sternites 4 and 5 at 3.2x magnification. Scale bars” A = 2 mm; B= 1 mm. 100 ... Nur Aliah & al. TABLE 2. Host specimens examined and position of thalli on the host body. DIPTERAN HOST SPECIES FAMILY LOCATION OF THALLI Hypopygiopsis violacea Calliphoridae Tergite 5; sternites 3, 4 (Fic. Q 1C, D) Boettcherisca sp. Sarcophagidae Abdominal tergites 3, 4 Q (Fic. 2) Boettcherisca javanica Sarcophagidae Abdominal sternites 4, 5 rou (Fia. 3) Taxonomy Stigmatomyces aff. limnophorae Thaxt., Proc. Am. Acad. Arts Sci. 36: 400. 1901. Fia. 4 Thallus hyaline, dark amber brown that gradually attenuates as the cell wall thickens; 647 um long from foot to perithecial tip. Cell I] <2 times longer than cell I, striate on the cell surface. Cell III longer than wide, rounded externally, not protruding abruptly below basal cell of appendage. Appendage free, slender, elongated, about as long as the perithecial venter; distal portion distinctly curved; bearing a single or multiple antheridia. Antheridia, short, broad, slightly recurved. Perithecium amber brown, 220 x 64 um; venter relatively small, ellipsoid, subsymmetrical, with wall cells powdered by darker maculation, spirally twisted, separated by corresponding number of well-defined longitudinal ridges, somewhat oblique; neck 96 x 16 um, abruptly distinguished from venter. MATERIAL EXAMINED/SEQUENCED—PENINSULAR MALAYSIA: SELANGOR, Gombak District, RAWANG, forested area, 3.281°N 101.261°F, 4 m a.s.l., ex. chicken liver, 11 Sep. 2019, leg. N.A. Nur Aliah & N. Azmiera, on Q Boettcherisca sp. D. Haelew. 1796 [host label] (slides PUL F25943, PUL F25944, PUL F25945); 3.296°N 101.611°E, 69 ma.s.l., ex. chicken liver, 7 Sep. 2019, leg. N.A. Nur Aliah & N. Azmiera, on & Boettcherisca javanica Lopes, D. Haelew. 1801 [host label] (slides PUL F25947, PUL F25948); on CO B. javanica, D. Haelew. 1802 [host label] (slides PUL F25949, PUL F25950) isolate 1802c [4 mature thalli] GenBank MT341792, MT341789; isolate 1802d [3 mature thalli] GenBank MT341793, MT341790; isolate 1802e [2 perithecia] GenBank MT341794, MT341791); 3.296°N 101.611°E, 101 ma.s.l., ex. chicken liver, 7 Sep. 2019, leg. N.A. Nur Aliah & N. Azmiera, on Q Hypopygiopsis violacea (Macquart), D. Haelew. 1800 [host label] (slide PUL F25946). Phylogenetic results All three newly generated SSU sequences share highest similarity (99.62%) with Stigmatomyces chamaemyiae (GenBank MH040565), followed by Stigmatomyces aff. limnophorae on dipteran hosts (Malaysia) ... 10.1 Fig. 4. Stigmatomyces aff. limnophorae: A [PUL F25945], B [PUL F25948]. Details of primary appendage; C [PUL F25950]. Detail of spirally twisted perithecium; D [PUL F25950]. Receptacular cells I and II with longitudinally striped ornamentation; E [PUL F25950]. Habitus of mature thallus; F. Stipple drawing. Scale bars: A, B = 10 um; C, D = 20 um; E = 50 um; F= 100 um. S. rugosus (MH040563) with 98.39-98.54% similarity (TaBLE 1). Based on morphology, we thought the fungus might represent S. limnophorae, but that species was not among the BLAST results despite the availability of an SSU sequence in GenBank (AF407576; isolate AW-785). Comparison of our Malaysian SSU sequences with S. limnophorae AW-785, however, shows a 99.08-99.36% similarity. All newly generated LSU sequences are most closely related to Gloeandromyces nycteribiidarum (MH040566) with 85.78% similarity (TABLE 1). The highest percentage similarity (87.30%) with any Stigmatomyces species is with S. protrudens (AF298234), but with only over a 42% query cover. The three Malaysian isolates group together in a maximum-supported clade that also includes S. limnophorae AW-785 and two S. chamaemyiae isolates from Portugal. The branch length among the Malaysian isolates and between the USA isolate AW-785 is very short. As a result, we believe the Malaysian fungus may be identified as S. aff. limnophorae. Discussion Fungal thalli were removed from all four fly specimens and subsequently identified as Stigmatomyces aft. limnophorae based on morphological (Fic. 4) 102 ... Nur Aliah & al. Gloeandromyces dickii D. Haelew. 1323b Stigmatomyces protrudens AW-793 Stigmatomyces borealis AW-979 Stigmatomyces rugosus Stigmatomyces rugosus D. Haclew. 1138a Stigmatomyces cf. limnophorae D. Haclew. 1802¢. Stigmatomyces cf. limnophorae D: Haclew.1802d Stigmatomyces\limnophorae AW-185) Stigmatomyces chamaemyiae D. Haelew. 1137a 0.02 Stigmatomyces chamaemyiae D. Haelew. 1137¢ Fig. 5. Phylogeny of Stigmatomyces isolates reconstructed from a combined SSU-LSU rDNA dataset, with Gloeandromyces dickii as outgroup. Shown is the best-scoring tree (-InL = 4263.622) as a result of maximum likelihood inference performed with IQ-TREE. For each node, the ML bootstrap (if >60) is presented at the branch leading to that node. Stigmatomyces aff. limnophorae is highlighted in grayscale. and phylogenetic (Fic. 5) analyses. The global distribution of S. limnophorae is presented in TABLE 3. Stigmatomyces limnophorae was described from a Limnophora fly (Muscidae) in California, USA. Isolate AW-785 originated from a Muscidae gen. sp. indet. collected in Louisiana, USA (Weir & Blackwell 2001b). This isolate will be regarded as representing the species until fungal sequences are obtained from a muscid fly identified to genus-level collected in the type locality of Berkeley, California. Morphologically our material resembles typical S. limnophorae morphology (Thaxter 1901, 1908), but our phylogenetic placement of the Malaysian isolates sister to S. chamaemyiae + S. limnophorae AW-785, suggests that they might represent another species closely related to S. limnophorae. Since the vast majority of thalli were heavily damaged, we were unable to describe the material accurately based on all morphological features. Stigmatomyces aff. limnophorae on dipteran hosts (Malaysia) ... 103 TABLE 3. World distribution of Stigmatomyces limnophorae, with host species and reference of first report. CONTINENt COUNTRY Host (FAMILY) FIRST REPORT North & Central America Cuba Limnophora arcuata (Muscidae) Krejzova & Weiser 1968 Grenada Anthomyiidae gen. sp. indet. Thaxter 1917 Guatemala = Limnophora sp. (Muscidae) Thaxter 1917 Jamaica Leucomelina sp. (Muscidae) Thaxter 1917 Mexico Onesia sp. (Calliphoridae) Thaxter 1917 USA Limnophora sp. (Muscidae) [type] Thaxter 1901 Anthomyiidae gen. sp. indet. Thaxter 1917 South America Bolivia Limnophora sp. (Muscidae) Rossi 1998 Brazil Limnophora sp. (Muscidae) Bergonzo & al. 2004 Venezuela Sarcophaga sp. (Sarcophagidae) Thaxter 1905 Europe Portugal Limnophora obsignata (Muscidae) Rossi & al. 2013 Africa Cameroon Anthomyiidae gen. sp. indet. Thaxter 1917 Canary Limnophora obsignata (Muscidae) Rossi & al. 2013 Islands Kenya Rhyncomya forcipata (Rhiniidae) Rossi & al. 2013 Morocco Limnophora obsignata (Muscidae) Rossi & al. 2013 Sierra Leone _ Lispe desjardinsii (Muscidae) Rossi & Leonardi 2018 Uganda Fainia albitarsis (Rhiniidae) Rossi & al. 2013 Asia Indonesia Lucilia dux (Calliphoridae) Thaxter 1917 Philippines — Lucilia dux (Calliphoridae) Thaxter 1917 Thailand Heliographa ceylanica (Muscidae) Rossi & al. 2013 Israel Limnophora quaterna (Muscidae) Rossi & al. 2013 Saudi Isomyia terminata (Rhiniidae) Rossi & al. 2013 Arabia Limnophora quaterna (Muscidae) Rossi & al. 2013 Taiwan Sumatria flava (Rhiniidae) Rossi & al. 2013 Turkey Dasyphora albofasciata (Muscidae) Rossi & al. 2013 Australasia Australia Calliphora augur (Calliphoridae) Rossi & al. 2013 As a result, for the time being, we refer to the species as S. aff. limnophorae. Efforts will be ongoing to sample flies using chicken liver and other baits and to collect additional Stigmatomyces-infected fly specimens. 104 ... Nur Aliah & al. Stigmatomyces limnophorae was discovered by Thaxter (1901) as a parasite of Limnophora sp. [misspelt as “Limnophorus”] (Muscidae) in California, USA. The fungus appears to be a very widespread and plurivorous species and has been reported on all continents except Antarctica from hosts in different dipteran families including Anthomyiidae, Calliphoridae, Muscidae, Rhiniidae, and Sarcophagidae (TABLE 3). Despite its wide distribution, the fungus has not yet been reported in Malaysia. Stigmatomyces limnophorae has, however, been reported in neighbouring countries—Indonesia, the Philippines, Thailand— on different dipteran hosts such as Chrysomya megacephala [as “Lucilia dux”] (Calliphoridae) and Heliographa ceylanica (Muscidae) (Thaxter 1917, Rossi & al. 2013). Here we report the occurrence of S. aff. limnophorae in Malaysia on different hosts in Calliphoridae and Sarcophagidae. While it is not uncommon for S. limnophorae to parasitize flies in either of these families (TABLE 3), the Malaysian hosts are in genera that have not previously been observed with thalli of Laboulbeniales. Thalli of Stigmatomyces aff. limnophorae were always observed at the abdominal segments, which might indicate the original infection site; however, our observations are based on only three host specimens. Limited data are available on Laboulbeniales. For example, the impact of infection on their hosts is still poorly studied (Nalepa & Weir 2007, Riddick 2010, Bathori & al. 2017, Haelewaters & al. 2020b). Further exploration of the diversity of ectoparasitic fungi is required to gain a complete picture of their distribution and interactions with insect hosts. In summary, we report two new hosts for Stigmatomyces limnophorae, a complex species able to infect wide number of species within different genera. This could be caused by evolution and adaption of the fungi for survival in different genera on various continents. As our sample was identified morphologically as S. limnophorae but phylogenetically as sister to S. chamaemyiae + S. limnophorae AW-785 (suggesting a different species), we could postulate that hybridization is occurring between these two species, thereby explaining adaptation of these fungi to different host species and within wide range of genera. Additional research is required to support this hypothesis. Acknowledgments We thank the Institute of Medical Molecular Biotechnology (IMMB), Universiti Teknologi MARA, for providing laboratory facilities. Special thanks to Dr. Matthew T. 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The taxonomic status of Corethromyces bicolor from New Zealand, as inferred from morphological, developmental, and molecular studies. Mycologia 94(3): 483-493. https://doi.org/10.1080/15572536.2003.11833213 MY COTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2022 January-March 2022— Volume 137, pp. 109-121 https://doi.org/10.5248/137.109 Pluteus variabilicolor and Volvopluteus earlei, new records for Pakistan JUNAID KHAN’, HASSAN SHER’, AIMAN IZHAR?’, MUHAMMAD HAQNAWAZ?’, ABDUL NASIR KHALID? "Center for Plant Sciences and Biodiversity, University of Swat, Mingora, Pakistan ? Institute of Botany, University of the Punjab, Quaid-e-Azam campus, Lahore, Pakistan *CORRESPONDENCE TO: junaid.botany@gmail.com Axsstract—Pluteus variabilicolor and Volvopluteus earlei are reported as new records for the Pakistani funga, with the former representing a new record for South Asia and the latter, a second report from Asia. Morphological descriptions are accompanied by colored photographs of basidiomata and microscopic structures. Identity of both species is confirmed by ITS sequence analyses. Comparison with previous descriptions and data on their distributions are briefly discussed. Key worps—Agaricales, phylogeny, Pluteaceae, P. sect. Hispidoderma, taxonomy Introduction Pluteaceae Kotl. & Pouzar (Agaricales) is a family of saprophytic fungi widely distributed in major parts of the world (Kirk & al. 2008). Molecular reassessment of the family (Justo & al. 2011a) supports two genera: Pluteus Fr. and Volvopluteus Vizzini & al. Pluteus, the larger genus, comprises >300 species (Kirk & al. 2008, He & al. 2019), distributed worldwide especially in boreal to tropical forested regions (Menolli & al. 2014). Pluteus species are primarily lignicolous and grow on partially or well-rotted angiosperm and gymnosperm wood (Orton 1986, Singer 1986, Vellinga 1990). The presence of free lamellae, pinkish 110... Khan & al. spore-print, inverse hymenophoral trama, and inamyloid basidiospores and lack of universal veil are some key Pluteus characters (Vellinga & Schreurs 1985, Orton 1986, Singer 1986, Vellinga 1990, Heilmann-Clausen 2012). Only eight Pluteus species have been reported from Pakistan: P ephebeus (Fr.) Gillet (as P murinus Bres.), P escharites (Berk. & Broome) Sacc., P. fusconigricans (Berk. & Broome) Sacc., P. leoninus (Schaeff.) P. Kumm., P. palumbinus (Berk.) Sacc., P. pellitus (Pers.) P. Kumm., P. petasatus (Fr.) Gillet, and P pulverulentus Murrill (Ahmad 1980, Iqbal & Khalid 1996, Ahmad & al. 1997). Volvopluteus, typified by V. gloiocephalus (DC.) Vizzini & al., was erected to accommodate some species formerly placed in Volvariella (Justo & al. 2011a). The genus is distinguished from Pluteus and Volvariella by a gelatinous pileipellis that is an ixocutis with relatively narrow hyphae (average <15 um diam.) and average basidiospore length >11 um (Justo & al. 2011a). Only one Volvopluteus species, V. gloiocephalus, has been reported from Pakistan (Ahmad 1980 and Iqbal & Khalid 1996, as Volvariella speciosa (Fr.) Singer). This paper, part of a larger project dealing with the exploration of macrofungi in northern Khyber Pakhtunkhwa reports two pluteaceous species as new records for the funga of Pakistan: Pluteus variabilicolor and Volvopluteus earlei. Materials and methods Collection & morphological Studies Specimens were collected during macrofungal surveys conducted between 2014 and 2019 in different parts of Pakistan. The specimens of P. variabilicolor were collected in village Ingaro Dherai, district Swat, Khyber Pakhtunkhwa province. Those of V. earlei were collected in the same locality and in the Sheikupura and Muzaffargarh districts of Punjab province. Basidiomata were dug out to the extreme base (to obtain some wood with specimens of Pluteus and volva with those of Volvopluteus) with a sharp knife. The specimens were photographed in their natural habitat and important macro-morphological characters were noted when fresh. Colors were coded according to soil color chart of Munsell (1975). Terminology for macroscopic and microscopic features follows Vellinga (1988). The specimens were dried in sun and deposited in the herbarium of the University of Swat, Kanju Township, Pakistan (SWAT) and the herbarium of the University of the Punjab, Lahore, Pakistan (LAH). Microscopic descriptions are based on free-hand sections from dried specimens mounted both in distilled water and in aqueous potassium hydroxide solution (5% w/v), with aqueous Congo red added for contrast. Basidiospore measurements are based on 60 elements randomly selected from all the available basidiomata; Pluteus & Volvopluteus spp. new to Pakistan ... 111 x presents the mean length and width of the basidiospores. The abbreviation Q refers to the length : width (L/W) ratio, with Q = the average length : width ratio. DNA extraction, PCR amplification, sequencing Genomic DNA from dried herbarium specimens was extracted using modified CTAB method of Gardes & Bruns (1993). The internal transcribed spacer region (ITS = ITS1-5.8S-ITS2) was amplified using the primer pair ITSIF (Gardes & Bruns 1993) and ITS4 (White & al. 1990). Polymerase chain reactions (PCR) were performed in 25 uL volume aliquots. Amplification involved initial 4 minutes denaturation at 94 °C, 40 cycles of 1 minute at 94 °C, 1 min at 55 °C, 1 min at 72 °C, and a final extension of 10 minutes at 72 °C (Naseer & al. 2020). PCR products were visualized in a UV illuminator loaded in 1% agarose gel added with 3 uL ethidium bromide. Purification and sequencing of the PCR amplicons were carried out at Beijing Genomic Institute (BGI) Hong Kong. Sequence alignment & phylogenetic analysis The forward and reverse reads were assembled into a consensus sequence using BioEdit software v. 7.2.5 (Hall 1999). The consensus sequences were used for a GenBank search, using the NCBI Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Matching sequences, especially those published in Justo & al. (2011b) and Lezzi & al. (2014), were downloaded for further phylogenetic analysis. Separate datasets were used for Pluteus and Volvopluteus. Multiple sequences were aligned using online Multiple Alignment using Fast Fourier Transform (MAFFT) algorithm at European Bioinformatics Institute website (https://www.ebi.ac.uk/Tools/msa/mafft/). The phylogenies were inferred by maximum likelihood (ML) analysis using model selection for the best DNA analysis in MEGA6 software (Tamura & al. 2013). Jukes-Cantor model Models with the lowest BIC scores (Bayesian Information Criterion) was considered to describe the substitution pattern the best. Non-uniformity of evolutionary rates among sites were modeled using a discrete gamma distribution (+G) with 5 rate categories and assuming that a certain fraction of sites is evolutionarily invariable (+1). The phylogenetic analyses included 1000 bootstrap replicates. Phylogenetic results The final Pluteus ITS dataset comprised 37 sequences representing P sect. Hispidoderma Fayod including the two sequences from our new Pakistani specimens. Pluteus diettrichii Bres. (HM562143) of P sect. Celluloderma Fayod was used as outgroup following Lezzi & al. (2014). Maximum likelihood analysis convincingly clustered the Pakistani collections with other sequences of P. variabilicolor in the Leoninus clade of P. sect. Hispidoderma with a 100% bootstrap value (Fic. 1). The ITS consensus sequences from our three new Pakistani Volvopluteus collections matched 99-99.5% with multiple V. earlei sequences (HM562205, 112... Khan & al. HM562139 Pluteus aff. leoninus TNSF11908 MGS544913 Pluteus roseipes HM562215 Pluteus leoninus DrewH HM562071 Pluteus leoninus Halling6546 t- HM562187 Pluteus leoninus SF17 KC147679 Pluteus roseipes UC 1861249 KC147681 Pluteus roseipes UC 1861251 P. leoninus complex 99) HM562188 Pluteus aff. leoninus | SF19 HMS562190 Pluteus aff. leoninus | SF21 HM562077 Pluteus leoninus Josserand s.n. MG544910 Pluteus leoninus 98 79) HM562045 Pluteus leoninus AJ212 MG544911 Pluteus leoninus KP192913 Pluteus variabilicolor MT351154 Pluteus variabilicolor (SWAT001367) KP192914 Pluteus variabilicolor MG544909 Pluteus variabilicolor MG544908 Pluteus variabilicolor 1'FJ774077 Pluteus aurantiorugosus P. variabilicolor 86 HM562099 Pluteus castroae HMS562092 Pluteus castroae MT351155 Pluteus variabilicolor (LAH31413) bi KP192911 Pluteus variabilicolor KP192912 Pluteus variabilicolor JN603206 Pluteus chrysaegis K13587 94] )HM562142 Pluteus conizatus var. africanus | P. chrysaegis 88. MG968799 Pluteus chrysaegis KF692077 Pluteus pantherinus 100. HM562089 Pluteus pantherinus 100) HM562048 Pluteus granulatus HM562048 Pluteus granulatus = 100} MH059512 Pluteus umbrosoides Teapdhveatdey 69 52 | P. pantherinus P. granulatus KX216348 Pluteus umbrosoides 69 KX216343 Pluteus umbrosus to0| | KX216347 Pluteus umbrosus 63] }HM562069 Pluteus granularis 96'HM562189 Pluteus granularis HM562143 Pluteus diettrichii | outgroup P. umbrosus/granularis 0.02 Fic. 1: Maximum likelihood phylogram of Pluteus variabilicolor and related species inferred from ITS data. Bootstrap values 250% are presented above nodes. Newly generated sequences of P. variabilicolor are presented in bold. HM562205, HM246496, HM246497, HM246498, HM246499, MK204987, MK204989) in GenBank. The final Volvopluteus dataset comprises 19 sequences including the three sequences from the new Pakistani specimens and from Pluteus heteromarginatus Justo (HM562058) and P longistriatus (Peck) Peck. (HM562082), the outgroup cited in Justo & al. (2011b). Sequences of the Pakistani collections clustered with other sequences of V. earlei, confirming their identity (Fic. 2). Pluteus & Volvopluteus spp. new to Pakistan ... 113 6g | HM562204 Volvopluteus earlei MA22816 HM246499 Volvopluteus earlei TO HG1973 HMS562205 Volvopluteus earlei Mamet7 MK204987 Volvopluteus earlei MK204989 Volvopluteus earlei HM246496 Volvariella cookei TO AV133 HM246498 Volvariella media TO HG2001 93 || MT353644 Volvopluteus earlei SWAT001366 MW362280 Volvopluteus earlei LAH35715 OM761893 Volvopluteus earlei LAH37018 HM246497 Volvopluteus earlei TO AV 134 MK944280 Volvopluteus sp. JPL 2019 100' MK 944281 Volvopluteus sp. JPL 2019 (GDGM73195) MK616346 Volvopluteus gloiocephalus MK616345 Volvopluteus gloiocephalus NR 119878 Volvopluteus asiaticus NR 119876 Volvopluteus michiganensis HMS562058 Pluteus heteromarginatus 100 HM562082 Pluteus longistriatus 61 99 = 0.02 Fic. 2: Maximum likelihood phylogram of Volvopluteus earlei and related species inferred from ITS data. Bootstrap values 250% are presented above nodes. Newly generated sequences of V. earlei are presented in bold. Taxonomy Pluteus variabilicolor Babos, Ann. Hist.-Nat. Mus. Natl. Hung. 70: 93 (1978) Fics 3a-c, 4 PiLEus 50-70 mm diam., convex to plano-convex with a central umbo, light orange yellow (10YR 9/8) to dark yellow (2.5Y 9/12), disc darker (2.5Y 7/12); surface smooth, glabrous to lightly velvety, margin striate or not; context concolorous with the pileus just beneath the cuticle, otherwise cream colored, unchanging upon cutting, thin, 2-3 mm at the disc, <1 mm at the margin. LAMELLAE free, close, sub-ventricose (<6 mm at the center); 114... Khan & al. white to pale pink (7.5YR 9/4) when young, pink (7.5R 9/4) when mature; lamellar edge even to slightly undulate; lamellulae present, short to long extending beyond the middle of the lamellae. StrpE 50-80 x 5-7 mm, central, cylindrical to very slightly thickening downward; pinkish (7.5YR 8/4); longitudinally striate in mature specimens, fistulose; context concolorous with the exterior or light yellowish brown (10YR 6/4), unchanging upon cutting. BASIDIOSPORES (5.1-)5.5-6.3(-6.8) x (4.4-)4.8-5.8(-5.9) um, x = 5.9 x 5.1 um, Q = 1.1-1.19(-1.2), Q. = 1.1, mostly sub-globose, rarely ovoid to broadly ellipsoid; smooth, thick-walled (2-3 um thick), mostly with one central guttula, rarely multi-guttulate. Basidia 20-25 x 6.0-7.0 um; cylindrical to broadly oblong, with 4 sterigmata. CHEILOCysTIDIA 55-90 x 20-30 um; fusiform with a short appendix at the tip to lageniform. PLEUROCYSTIDIA 60-120 x 20-40 um, narrowly utriform to utriform, rare. PILEIPELLIS a hymeniderm composed of clavate to rounded terminal elements, 50-200 x 20-40 um, without any yellow intracellular pigment. STIPITIPELLIS a cutis composed of narrow branched hyphae, 3.0-5.0 um diam., with rare claviform caulocystidia, 25-50 x 15-30 um, usually in clusters extending along the stipe length. CLAMP CONNECTIONS absent. MATERIALS EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat district, Ingaro Dherai village, 1000 m. a.s.l, on decomposing stump of Populus nigra L., 7 August 2014, Junaid Khan ING-32 (SWAT001367; GenBank MT351154); Ingaro Dherai village, 1010 m. a.s.l., on decomposing stump of Populus nigra, 27 July 2015, Junaid Khan ING-1507 (LAH31413; GenBank MT351155). COMMENTARY—Pluteus variabilicolor, originally described from Hungary, is characterized by its pileus color varying from yellowish orange to chrome- yellow, pileipellis with morphologically variable elements (a mixture of spheropedunculate-vesiculose and elongated + cystidioid elements), the presence of caulocystidia, and growth on decaying sawdust (Babos 1978). Our description fits well with previous descriptions (Lohmeyer & al. 1994, Lanconelli & al. 1998, Migliozzi 2011, Lezzi & al. 2014, Kaygusuz & al. 2019). Lezzi & al. (2014) cite P variabilicolor as widespread but not common. Currently the species has been reported from numerous central and eastern European countries—Hungary (Babos 1978), Austria (Lohmeyer & al. 1994), Italy (Lanconelli & al. 1998, Migliozzi 2011), Germany (Ludwig 2007), central Russia (as P castri, Justo & al. 2011b), Romania (Béres 2012), Slovenia (Jogan & al. 2012), and Moldova (Lezzi & al. 2014). From Asia, the species has been reported from Japan (as P. castri, Justo & al. 2011b), Pluteus & Volvopluteus spp. new to Pakistan ... 115 Fic. 3: Field photographs of basidiomata. Pluteus variabilicolor: a—b. (SWAT001367); c. (LAH31413). Volvopluteus earlei: d & f (SWAT001366); e. (LAH37018); g & h. (LAH35715). Scale bars = 10 mm. and from Turkey and South Korea (Kaygusuz & al. 2019). Here we report P. variabilicolor from Pakistan and South Asia for the first time. Pluteus variabilicolor grows primarily on sawdust deposits and on branches and rotten wood of Fagaceae, mainly Quercus spp. (Migliozzi 2011, Lezzi & al. 2014). Both Pakistani collections, however, were collected from decaying stumps of Populus sp., which agrees with Russian reports of collections growing on Populus tremula stumps of (Justo & al. 2011b). 116... Khan & al. Fig. 4: Pluteus variabilicolor (SWAT001367): a. Basidiospores; b. Basidia; c-f. Cheilocystidia; g-i. Pleurocystidia; j, k. Pileipellis elements; |. Stipitipellis elements. Scale bars: a, b = 5 um; c-i = 20 um; j-l = 30 um. We suggest that P variabilicolor was introduced into Pakistan with the introduction of Populus nigra. Volvopluteus earlei (Murrill) Vizzini, Contu & Justo, Fungal Biology 115(1): 15 (2011) Fics 3d-h, 5 PiLeus 20-40 mm diam.; hemispherical to obtusely conical when young, applanate to plano-concave on maturity; white to greyish white with a greyish disc when young, in maturity white to moderate whitish pink Pluteus & Volvopluteus spp. new to Pakistan ... 117 Fic. 5: Volvopluteus earlei (LAH35715): a. Basidiospores; b. Basidia; c. Cheilocystidia; d. Pleurocystidia; e. Stipitipellis; f. Pileipellis elements. Scale bars = 10 um. (2.5YR 7/4) with reddish pink margins (10R 6/8); surface moist and viscid in young specimens, dry and dull when mature, glabrous; margin striate, striation short (<1/4 of pileus radius); context white, thin, 1-2 mm thick at the disc, <1 mm elsewhere. LAMELLAE free, broad (4-6 mm at the center), sub-crowded to close; white to cream-colored when young, pink (7.5YR 8/8) when mature; lamellulae present. Stipe 70-100 x 4-7 mm; central, tapering upward from slightly flattened base; surface smooth; white to cream colored, turning slightly pinkish (7.5 YR 9/4) upon maturity and handling; volva 118 ... Khan & al. present, small (<8 mm high), membranous, saccate, 2-3 lobed; white to pale gray. BasIDIOSPORES (11.0—)11.5-14.5(-15.0) x (7.0-)7.5-9.6(-10.0) um, x = 12.8 x 9.1 um, Q = (1.2-)1.3-1.6(-1.7), Q. = 1.4; ellipsoid to ovoid, smooth, thick-walled (<0.5 um), mostly with one large and many small guttulae. Basip1A 30-40 x 10.0-12.0 um, 4-spored, cylindrical to broadly clavate. CHEILOCYSTIDIA 35-60 x 8.0-15.0 um, fusiform to clavate mucronate. PLEUROCYSTIDIA 80-150 x 15-20 um, cylindrical to narrowly conical. PILEIPELLIS ixocutis, composed of cylindrical and scattered obovoid to broadly cylindrical and rarely branched terminal elements; 30-35 x 14.0-18.0 um. STIPITIPELLIS hyphae 7-10 um diam., + parallel, caulocystidia cylindrical to narrowly clavate, 40-55 x 8-11 um. CLAMP CONNECTIONS absent. MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat district, Ingaro Dherai village, 1000 m. a.s.l., among grasses in humus rich wet soil in the paddy fields, 27 June 2016, Junaid Khan ING-1501 (SWAT001366; GenBank MT353644); PUNJAB, Sheikhupura district, 236 m. a.s.l., solitary on muddy rich soil of a graveyard, 16 July 2017, Aiman Izhar SKP03 (LAH35715; GenBank MW362280); Muzaffargarh district, surrounding head Taunsa barrage, 65 m. a.s.l., in scattered groups on humus rich soil, 13 August 2019, Muhammad Haqnawaz TM04 (LAH37018; GenBank OM761893). COMMENTARY— Volvopluteus earlei, originally described from Cuba (Murrill 1911), is characterized by its dry to sub-viscid relatively small (25-45 mm) white pileus, narrowly cylindrical stipe with a small white basal volva, rare or absent broadly clavate pleurocystidia, rostrate cheilocystidia, and large (averaging >12.5 tm long) basidiospores (Shaffer 1957, Desjardin & Hemmes 2001, Contu 2007, Justo & al. 2011a). Murrill (1911) described only basidiospores in his description; other microscopical details were later provided by Coker (1947) and Shaffer (1957). Descriptions from different parts of the world (Pathak 1975, Heinemann 1975, Desjardin & Hemmes 2001, Contu 2007, Vizzini & Contu 2010, Justo & al. 201 1a) cite highly diverse cystidial forms for V. earlei. For instance, no cystidial forms were reported for the African (Pathak 1975) and Italian (Vizzini & Contu 2010) collections of V. earlei [= Volvariella acystidiata N.C. Pathak]. Elsewhere, Justo & Castro (2010) reported only cheilocystidia in the Spanish collection, while Shaffer (1957) report both cheilocystidia and pleurocystidia for the North American collections We observed both cheilocystidia and pleurocystidia in the Pakistani collection, which agrees with descriptions of the American collections (Shaffer 1957, Desjardin & Hemmes 2001). Pluteus & Volvopluteus spp. new to Pakistan ... 119 Volvopluteus earlei is known from the U.S.A (Murrill 1911; Coker 1947; Desjardin & Hemmes 2001), Mexico (Vazquez & al. 1989), Africa (Heinemann 1975, as Volvariella acystidiata), Sardinia (Contu 2007, as Volvariella earlei (Murrill) Shaffer), Spain (Justo & Castro 2010), and India (Amandeep & al. 2015; Atri & al. 1996, as Volvariella earlei). This is the first report of this species from Pakistan and the second record from Asia. 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Volvariella acystidiata (Agaricomycetes, Pluteaceae), an African species new to Europe, with two new combinations in Volvariella. Mycotaxon 112: 25-29. https://doi.org/10.5248/112.24 White T, Bruns TD, Lee S, Taylor J. 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. ©2022 January-March 2022— Volume 137, pp. 123-134 https://doi.org/10.5248/137.123 New Turkish records of Hebeloma excedens and H. geminatum, and confirmation of H. celatum AYTEN Dizxrricr’, AYSENUR KALMER’, ISMAIL ACAR” "Department of Molecular Biology and Genetics, Van Yiiziincti Yil University, 65080, Van, Turkey ? Department of Organic Agriculture, Baskale Vocational High School, Van Yiiztincti Yl University, 65080, Van, Turkey * CORRESPONDENCE TO: iacar2011@gmail.com ABSTRACT—Hebeloma excedens and H. geminatum are reported as new records and H. celatum is confirmed for the Turkish mycota, on the basis of morphological and phylogenetic data. Morphological descriptions, illustrations, and comparisons with closely related taxa are provided. Key worps—Agaricales, Basidiomycota, Hymenogastraceae, ITS, phylogeny Introduction Hebeloma (Fr.) P. Kumm. (Hymenogastraceae, Agaricales) is a genus of ectomycorrhizal and mostly poisonous basidiomycetes primarily distributed in temperate, boreal, arctic, and alpine habitats (Vesterholt 2005, Beker & al. 2016, Cripps & al. 2019). The genus is characterized by a mostly two coloured convex to plano-convex pileus, dull brown lamellae, presence of a veil, and morphologically distinctive cheilocystidia (Beker & al. 2016). Identification of Hebeloma at the species level is considerably challenged by morphological similarities and infrequent or transient characters (Eberhardt & al. 2012). Therefore, species determination and classification and their phylogenetic relationships within the genus have come to rely upon DNA genomic sequences such as the nuclear ribosomal DNA internal 124 ... Dizkirici, Kalmer, Acar transcribed spacer (nrDNA ITS) region. One advantage of selecting the ITS region for analysis is the many Hebeloma sequences available in GenBank. Moreover, the availability of universal primers, high rate of PCR success, and superior resolution at infrageneric levels are other important advantages of the ITS region (Aanen & al. 2000, 2001; Vesterholt & al. 2014; Eberhardt & al. 2015a,b). Twenty-eight Hebeloma species have been recorded from Turkey (Sesli & Denchev 2014; Giingor & al. 2015; Sesli & al. 2015, 2018; Solak & al. 2015; Dogan & Kurt 2016; Dizkirici & al. 2019). Beker & al. (2016) investigated seven Turkish specimens (representing six species, including H. celatum and H. subtortum) using molecular data but did not provide morphological details; subsequently Dizkirici & al. (2019) provided detailed descriptions of Turkish H. subtortum. Hebeloma celatum, which has been widely reported from Europe, known from Turkey only by a single 2008 collection from Adana province (Grilli & al. 2015). Here, we provide full descriptions and phylogenetic results for Hebeloma excedens, Hebeloma geminatum, and Hebeloma celatum. Hebeloma excedens is placed in H. sect. Hebeloma, characterized by the visible cortina seen in young basidiomes and cheilocystidia that are always lageniform or ventricose; H. geminatum belongs to H. sect. Denudata, characterized by cheilocystidia that are primarily clavate-stipitate or clavate- ventricose and basidiospores that are weakly dextrinoid; and H. celatum is placed in H. sect. Velutipes, characterized by a velutinate stipe, clavate cheilocystidia, strongly dextrinoid basidiospores, and absence of cortina (Vesterholt 2005, Beker & al. 2016, Grilli & al. 2015). In the present study, we used morphological and ITS sequence analyses to identify H. excedens and H. geminatum as new Turkish records and confirm the existing Turkish record of H. celatum. Materials & methods Taxon sampling & morphological studies Fresh basidiomata of H. excedens, H. geminatum, and H. celatum were collected during 2017-18 fieldwork in south-eastern Turkey. Collected specimens were photographed in situ using a Canon (EOS 60D) camera equipped with a Tokina 100 mm macro lens. Macromorphological characters were determined based on field notes and colour photographs of fresh fruiting bodies. Dried specimens were examined microscopically after sectioning and rehydration following procedures in Vesterholt (2005) and Beker & al. (2016). Structures were observed using a Leica DM500 research microscope and measured with the Leica Application Suite v.3.4.0. Hebeloma celatum, H. excedens, H. geminatum in Turkey... 125 The samples were dried and deposited as voucher specimens in the Fungarium of Van Yiiztincti Yil University, Van, Turkey (VANF). Abbreviations include: x = average length x width and Q = length/width ratio of basidiospores calculated based on the number (n) of spores measured. At least 100 spores, 30 basidia, and cheilocystidia from four samples were measured in distilled water and Melzer’s reagent for each specimen. Additional potentially diagnostic characters were abbreviated according to Vesterholt (2005) with “D” indicating the degree of basidiospore dextrinoidity in Melzer’s, “P” the degree of loosening of the basidiospore perisporium in Melzer'’s, and “O” the degree of ornamentation on the basidiospore surface. DNA isolation, amplification, sequencing Genomic DNA was extracted and purified directly from dried basidiomata using a modified CTAB protocol (Doyle & Doyle 1987). The purity and quantity of extracted DNA were determined by using a NanoDrop2000c UV-Vis Spectrophotometer and 0.8% agarose gel electrophoresis. Isolated stock DNA was stored at -20°C prior for further studies. Two different samples were amplified for each Hebeloma species to increase reliability of the sequences. DNA was amplified in 25 pl volume mixtures containing genomic DNA (10 ng/ul), 10X PCR Buffer, MgCl, (25 mM), dNTP mixture (10 mM), the primer pair (10 uM), Taq polymerase (5u/ul) and sterile water. The primer pair N-ncl8S10 5’AGGAGAAGTCGTAACAAG3’ / C26A 5’GTTTCTTTTCCTCCGCT3’ (Wen & Zimmer 1996) was used to amplify the ITS1-5.8S-ITS2 region. Amplicons were checked in 1% TAE agarose gel stained with Gelred dye. The PCR products were sequenced with forward and reverse primers in an ABI 3730XL automated sequencer. Sequences generated in the current study were submitted to GenBank. Sequence alignment and phylogenetic analysis All sequence chromatograms were opened using Finch TV (http://www.geospiza. com/finchtv/) and checked for reading errors, using the Q-Quality value to assess the accuracy of each base. BLAST analysis was performed using the UNITE (http://unite. ut.ee/) and NCBI (http://www.ncbi.nlm.nih.gov/) databases. Following a preliminary BLAST search that supported our newly amplified sequences in Hebeloma, we prepared a combined dataset containing sequences scoring the greatest number of BLAST hits. Sixty-three sequences representing different Hebeloma sections were downloaded from the database and aligned with the newly amplified sequences. The final alignment was trimmed, with the data retained for further phylogenetic analyses. Galerina pruinatipes (AJ585510) and G. pseudocamerina (AJ585508) were chosen as outgroup. The nucleotide evolutionary model for phylogenetic analyses was determined using MEGA 6.0 (Tamura & al. 2013), with the model with the lowest BIC (Bayesian Information Criterion) score selected. MrBayes v.3.2.6 (Ronquist & al. 2012) was employed for Bayesian phylogenetic analysis using the Markov chain Monte Carlo (MCMC) method (Yang & Rannala 1997) under K2P model. Two independent runs 126 ... Dizkirici, Kalmer, Acar with 4 chains each were run for 2 million generations, with trees sampled every 1000 generations. Analyses continued until the average standard deviation of split frequencies was <0.01. A Bayesian inference 50% majority rule tree and posterior probability values were estimated from the samples after discarding the first 25% of sampled trees and viewed with Figtree (Rambaut 2010). Taxonomy Fic 1. Hebeloma excedens [VANF1051]: a. Basidiomata; b. Basidiospores in distilled water; c. Basidiospores in Melzer’s reagent; d. Basidia; e. Cheilocystidia; f. Pileipellis. Scale bars: a-e = 20 um; f = 50 um. Hebeloma excedens (Peck) Sacc., Syll. Fung. 5: 806 (1887) FIG. 1 PiLeus 15-25 mm, convex then almost flat and slightly umbonate, darker in the center (cocoa brown, yellow brown, or cream brown when young; later darkening), pale brown on most of the pileus, lighter at margin and with/ without white velar remnants on the edges. CorTINA present. LAMELLAE sinuate, subdecurrent, whitish cream when young, then pale brown, wider and eroded in age. STIPE 30-60 x 3-5 mm, cylindrical, equal, slightly curved, pale cream to light brown, pruinose above cortina zone, lower parts dirty and pale brown, with zones of brown fibrils. Hebeloma celatum, H. excedens, H. geminatum in Turkey ... 127 BasiD1Iospores 7.8-11(-12) x 4.1-6.3 um, x = 9.8 x 5.2 um, (n = 40; Q = 1.8-1.9), light yellow-yellow brown, elliptical, slightly ovoid, slightly roughened (O1), non- to slightly dextrinoid (D0; D1), perispore not loosening (PO). Basip1a 19.7-31 x 7-9.8 um, clavate, 4-spored. PLEUROCYSTIDIA absent. CHEILOCYSTIDIA 35-58 x 5-7 x 4.6-6.8 x 5.6-11 tm, ventricose, cylindrical apex, swollen at the base, rarely fully cylindrical, sometimes septate. PILEIPELLIS an exocutis, <45 um thick, slightly brownish, encrusted. SPECIMEN EXAMINED—TURKEY, BiTLis, Van-Bitlis roadway, under Populus sp., 38.3625°N 42.7614°E, 1952 m asl, 15.05.2018, Acar 1051 (VANF1051; GenBank MW544166, MW544167). Hebeloma geminatum Beker, Vesterh. & U. Eberh., Persoonia 35: 122 (2015) FIG. 2 PiLeus 25-110 mm, usually convex, occasionally umbonate or flattened, edges generally smooth, slightly incurved or wrinkled when young, when young sticky or moist, usually uniformly coloured (occasionally bi-coloured), cream to beige or (sometimes) light yellow, whitish toward margin. CORTINA absent. LAMELLAE close, adnate to emarginate, notched, finely serrate at margin, droplets on margin when young or in humid conditions, color light pinkish-grey. Stipe 30-100 x 4-12 mm, flocculose, cylindrical to clavate (slightly swollen toward base), whitish. BASIDIOSPORES 10-11.8 x 5.5-6.5 um, x = 7.6 x 11.5 (n = 40; Q = 1.7-1.8), amygdaloid, non- to very slightly dextrinoid (D0,D1), ornamentation distinct Fic 2. Hebeloma geminatum [VANF1116]: a. Basidiomata; b. Basidiospores in distilled water; c. Basidiospores in Melzer’s reagent; d. Basidia; e. Cheilocystidia in Melzer’s reagent; f. Pileipellis. Scale bars: a—e = 20 um; f = 50 um. 128 ... Dizkirici, Kalmer, Acar verrucose (02,03), perispore loosening (P0,P2). BAsrp1a 31 x 8 um, 4-spored (rarely 2-spored). PLEUROCYSTIDIA absent. CHEILOCYSTIDIA 50-85 x 7.2-11 x 4-5.6 x 3.3-4.5 um, clavate-stipitate, spathulate, sometimes clavate-lageniform or sometimes capitate/bifurcate, septate, sinuate. PILEIPELLIS an ixocutis; with a medium thick epicutis 110-180 um thick, tramal hyphae beneath subcutis cylindrical, ellipsoid, sausage-shaped and inflating from septa <15 um diam. SPECIMEN EXAMINED—TURKEY, BINGOL, Elmali village, under Populus sp., 39.0256°N 40.7339°E, 1234 m, asl 20.05.2017, Acar 1116 (VANF1116; GenBank MW544168, MW544169). Fic 3. Hebeloma celatum [VANF1132]: a. Basidiomata; b. Basidiospores in distilled water; c. Basidiospores in Melzer’s reagent; d. Basidia; e. Cheilocystidia; f. Pileipellis. Scale bars: a-e = 20 um; f= 50 um. Hebeloma celatum Grilli, U. Eberh. & Beker, Mycol. Progr. 15(5): 23. 2015 [“2016”] FIG. 3 PiLeus 30-70 mm, convex, occasionally wrinkled, margin involute when young, sometimes smooth or wavy when mature, glutinous when damp, spotting variable, mono- or bi-coloured, cream, clay-buff, ochraceous or yellowish brown. Cortina absent. LAMELLAE adnexed to emarginate, white fimbriate margin usually present. STrPE 30-70 x 6-10(12) mm, white to light cream, surface with white powdery granules, fibrils, or floccules; cylindrical, clavate with bulbous base, rarely tapering, stuffed when young, later hollow. BASIDIOSPORES 9-15.2 x 5.5-8.5 um, x = 11.3 x 5.2 um, (n = 40; Q = 1.6-1.8), amygdaloid, limoniform, yellow, yellowish brown, light yellow, reddish yellow to brown, apiculus distinct, guttulate, ornamentation distinct Hebeloma celatum, H. excedens, H. geminatum in Turkey ... 129 verrucose (O2-O4), strongly dextrinoid (D2-D4), perispore loosening (PO-P2). Basip1a 25-38 x 7-9.8 um, cylindrical or clavate, 4-spored, rarely 2-spored. PLEUROCYSTIDIA absent. CHEILOCYSTIDIA 32-75 x 4-10 x 4-6.8 x 4-—11.2 um; cylindrical, clavate, clavate-lageniform, or ventricose, sometimes subcapitate, septate. PILEIPELLIS an ixocutis; epicutis 70-95 um thick, tramal hyphae beneath subcutis angular, ellipsoid, sausage-shaped, inflating from septa <18 um diam. CLAMP CONNECTIONS occasionally present on cheilocystidia. SPECIMEN EXAMINED— TURKEY, HAKKARI, Semdinli, DERYA VILLAGE, under Quercus sp., 37.3383°N 44.5322°E, 1616 m, 15.10.2018, Acar 1132 (VANF1132; GenBank MW544170, MW544171). Phylogenetic results Phylogenetic analyses were performed on the ITS dataset comprising 71 sequences, of which six were derived from our Turkish samples and two represented the outgroup. The 5’ and 3’ends were trimmed from the ~680 bp sequence alignment and of the 631 bp included in the final analysis, 130 were variable and 87 parsimony informative. Of the variable sites, the ITS1 possessed 72, the 5.8S gene only one, and the ITS2 57. BLAST and UNITE results of ITS sequences indicated 99% identity value for each sample. Our Bayesian phylogenetic tree (Fic. 4) outlines the phylogenetic relationships and taxonomic positions among the species selected. Our Hebeloma excedens samples (VANF1051A,B) clustered with H. excedens, H. mesophaeum, and H. subtortum in H. sect. Hebeloma with strong support (PP = 0.97). The two H. excedens sequences grouped together with a PP = 1 value, expected because no nucleotide variation was observed between them. Differences between our and the database sequences were noted at ITS1 base 150 (C to T) and ITS2 base 530 T to C). Our Hebeloma geminatum (VANF1116A, B) samples grouped closely with the database representatives in H. sect. Denudata (PP = 1), with H. geminatum closely related to H. alpinum (J. Favre) Bruchet, H. aanenii Beker & al., H. crustuliniforme (Bull.) Quél., H. eburneum Malencon, H. salicicola Beker & al., and H. minus Bruchet (Fic. 4). Hebeloma geminatum cannot be easily separated from its sister species based on ITS sequence data alone; here morphological characters are helpful. Eberhardt & al. (2015a) observed that a specimen almost certainly represents H. geminatum when the average width of the cheilocystidial apex exceeds 9 um. The average cheilocystidial width in our material was 8-9 um but its range extending to 11 um supports its identification as H. geminatum. 130 ... Dizkirici, Kalmer, Acar 0.05 Ii a hh , H_subtortum_KX76578S po) a rl a or | 0, F L H__excedens_VANFIOSIA H__excedens_VANF1051B H__excedens_MK2S0986 H__excedens_MK281081 H__excedens_MK2S1082 7 H__excedens_MK281124 B__secedate MIC) 121 “H__excedens_MK281122 H__exceders_MK281123 H__mezophaewm_KT071036 H__pubescens_KX765792 H__chunense_KX687200 H__margmatuhum_KT071029 H__fiseatum_KY271851 H__monticola_KX765772 H__oreophihum_KY271850 H__clavulipes_KX765771 H__hygophihun_KX765778 H__nigelhm_KX765786 H__gandispomm_KT071023 H__sordescerns_KX765787 H__cistophihun_EU570178 H__celatun_VANFI132B H__celatum_VANF1132A H__erebamm_KT218370 H__erebium_KT218321 H__quercetorum_KT218432 H__quercetoram_KT218433 H__celatum_KT218346 H__celatum_KT21$421 H__celatum_KT21845S H__celatum_KX905038 H__celatum_KT218301 H__celatum_KT218482 H__celatum_KT218490 H__leucozarx_KY081726 H__vehitipes_KT218456 H__bubiferum_KX266254 H__simapizans_KT218484 H__pumuhum_KX765808 H__laterimm_KX687214 H__binws_JF908029 H__cylindiospormm_KX687197 H_adicoaun_KX 765800 H__rsadicomum_FJ168582 H__namum_MK281018 H__nammm_MK280996 H__vesterholtu_FJ943240 H__theobromimun_JX275966 H__odoratissinmun_KX687216 H__naweosum_KX765763 H__sacchaniolens_KX449205 H__genunatom_VANF1116A H__gewunatum_VANF1116B H__geminatun_KM390606 H__geminatum_KM390636 H__geminatum_KM390770 H__geminatum_MF039233 H__geminatun_KM39055S H__alpirmm_KM390650 H__alpirwm_KF309411 H__envtuliniforme_KX657847 B H__salicicola_KM390543 H__salicicola_KM39075S H__aanenii_KX657845 H__ebumewn_KX657857 H__aanenii_KM390742 H__mins_KM390771 Galerina_pnuinatipes_AJ5S5510 Galenna_pseudocamenina_AJ5$550$ Fic 4. The Bayesian 50% majority rule consensus tree inferred from ITS region. PP> 0.5 are indicated above branches. Sequences from studied specimens are marked with red colour. Hebeloma sections are indicated with colours: yellow = Hebeloma, pale blue = Velutipes, dark blue = Sinapizantia, pink = Scabrispora & Myxocybe, green = Sacchariolentia & Theobromina, and orange = Denudata. Topology is rooted with Galerina pseudocamerina and G. pruinatipes. Hebeloma celatum, H. excedens, H. geminatum in Turkey... 131 Our Hebeloma celatum sequences (VANF1132A, B) clustered in H. sect. Velutipes (PP = 0.99) with H. celatum, H. erebium (Huijsman) Beker & U. Eberh., and H. quercetorum Quadr. (Fic. 4). This close phylogenetic relationship can be expected since these species are also morphologically very similar. Our H. celatum sequences were phylogenetically separated from representatives retrieved from the database due to nucleotide variations observed at base 497 (A to G). Discussion Hebeloma excedens and Hebeloma geminatum are recorded for the first time from Turkey, and Hebeloma celatum, previously reported for Turkey by Grilli & al. (2015), is confirmed for the country. Hebeloma excedens and H. mesophaeum are not easily separated from each other because of their close morphological and ecological similarities. Nonetheless, the differences in pileus morphology are useful for distinguishing the species. The pileus of H. excedens is less yellow brown overall and less brown at the disc, and its margin extends over the lamellae. Also, H. excedens has fibrils on the stipe surface that are not observed in H. mesophaeum (Cripps & al. 2019, Beker & al. 2016). Our H. excedens material produced evenly coloured basidiomes that are more slender than customary for H. mesophaeum and cheilocystidia that are generally swollen at the base. Molecular data also supported subtle differences between the two species. Nucleotide variations observed at bases 453 (A to G) and 512 (C to T) support the two species as independent. Hebeloma geminatum can be confused with H. alpinum and H. aanenii. Morphologically, the easiest way to separate H. geminatum from close relatives is through basidiospore and cheilocystidial comparison (Eberhardt & al. 2015a). The average basidiospore dimensions cited for H geminatum is <11 um long and 6 um diam. The basidiospores in our collections of H. geminatum measured 10-11.8 x 5.5-6.5 um, while the cheilocystidia were generally swollen in the lower half but appeared spathulate. Eberhardt & al. (2015a) noted that they regarded specimens with cheilocystidial apices with an average width >9 um to represent H. geminatum; our cheilocystidia measured <11 um at the apex, meeting their morphological criterion. Molecular data supported this morphological identification, with nucleotide variations at bases 140 (G to A), 220 (indel), 562 (A to G) and 565 (G to A) bases separating H. geminatum from two close relatives. 132 ... Dizkirici, Kalmer, Acar Grilli & al. (2015) selected a large number of collections from throughout Europe to infer species diversity and evolutionary history in the two main Hebeloma sections, Sinapizantia and Velutipes. They included one H. celatum sample (KT218421) collected from Turkey in 2008 in their study, but as there was no mention of H. celatum in the Turkish mycobiota since then, we reevaluated H. celatum through morphological comparisons and molecular analyses. Hebeloma celatum, which produces mature basidiomes that greatly resemble those of H. erebium and H. quercetorum, can be distinguished by its floccose stem and raphanoid odor. More robust basidiomes with larger stipes help separate H. celatum from H. erebium (Grilli & al. 2015, Cripps & al. 2019), and the Turkish H. celatum specimens were robust. Likewise, the clearly clavate and sometimes subcapitate cheilocystidia distinguish our H. celatum from H. quercetorum, which is characterized by more regularly ventricose to cylindrical cheilocystidia. Hebeloma celatum associates with a variety of trees, among which the most commonly recorded is Quercus (Cripps & al. 2019); our specimens from Turkey were associated with Quercus species. Sequence variations at bases 140 (A to G) and 475 (A to G) also support separation of H. celatum from its two close relatives. The above data well illustrate that macrofungal molecular analyses aid species identification and that the ITS region is particularly helpful in understanding the taxonomy and phylogeny of Hebeloma. 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Molecular Biology and Evolution 14: 717-724. https://doi.org/10.1093/oxfordjournals.molbev.a025811 MY COTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2022 January-March 2022— Volume 137, pp. 135-151 https://doi.org/10.5248/137.135 Ganoderma multipileum and Tomophagus cattienensis— new records from Pakistan AISHA UMAR", SHAKIL AHMED’, LAURA GUZMAN-DAVALOS?, MILAY CABARROI- HERNANDEZ”* ' Department of Botany, University of the Punjab, Quaid-e-Azam Campus, Lahore, Pakistan ? Department of Botany and Zoology, University of Guadalajara, Apdo. Postal 1-139, Zapopan, Jal., C.P. 45147, Mexico * CORRESPONDENCE TO: ash.dr88@gmail.com cabarroi80@gmail.com ABSTRACT—New records of Ganoderma multipileam and Tomophagus cattienensis collected from Changa Manga Forest and Lahore, Pakistan, are presented based on morphological and nuclear rDNA ITS sequence data from fresh basidiomata. Specimens previously treated as G. lucidum from Pakistan were reviewed and found to represent different species, among them G. multipileum. Specimens of T. cattienensis determined for the first time from Pakistan presented morphological features similar to T: colossus but corresponded molecularly to T. cattienensis; the morphological description for T. cattienensis is expanded accordingly. KEY WoRDS—Ganodermataceae, morphology, Punjab, taxonomy Introduction Ganodermataceae, a monophyletic polypore family with ganodermatoid basidiospores (Costa-Rezende & al. 2020) previously included in Polyporaceae (Justo & al. 2017, He & al. 2019), is characterized by its shelf-like basidiomes, poroid hymenophores, and complex-walled basidiospores. Amauroderma Murrill, Amaurodermellus Costa-Rezende & al., Cristataspora Robledo & Costa-Rezende, Foraminispora Robledo & al., Furtadoa Costa-Rezende & al., Ganoderma P. Karst., Haddowia Steyaert, Humphreya Steyaert, and Tomophagus Murrill are some genera currently included in Ganodermataceae (Murrill 1905; Moncalvo 1996; Moncalvo & 136 ... Umar & al. Ryvarden 1997; Ryvarden 2004; Kirk & al. 2008; Costa-Rezende & al. 2017, 2020). Several species of fungi with ganodermatoid basidiospores (specifically in Ganoderma) have been used as medicine in Asia for more than 2000 years where they have been cultivated on an industrial scale (Wasser & Weis 1997). Despite of their significance, this group of fungi remains poorly studied in many regions of the world such as in Pakistan, where Ganoderma ahmadii Steyaert, G. applanatum (Pers.) Pat., G. australe (Fr.) Pat., G. boninense Pat., G. chalceum (Cooke) Steyaert, G. curtisii (Berk.) Murrill, G. flexipes Pat., G. lipsiense (Batsch) G.F. Atk., G. lucidum (Curtis) P. Karst., G. perzonatum Murrill, G. philippii (Sacc.) Bres., G. praelongum Murrill, G. multicornum Ryvarden, G. multiplicatum (Mont.) Pat., G. resinaceum Boud., G. tornatum (Pers.) Bres., G. tsugae Murrill, and Tomophagus colossus (Fr.) Murrill [= G. colossus (Fr.) C.F. Baker] have been recorded (Ahmad 1956, 1972, Steyaert 1972, Irshad & al. 2012, Fakhar-ud-Din & Mukhtar 2019). These species were determined primarily based on morphological concepts; however, there is much uncertainty regarding which morphological criteria should be used to assign species to Ganodermataceae and specifically to Ganoderma (Moncalvo & Ryvarden 1997, Welti & Courtecuisse 2010). Clarification on the placement of species in Ganoderma or related genera is essential, not only for taxonomic reasons, but also to investigate the economical and pharmacological importance of the species that actually grow in the tropics. The aim of this study was to use morphological and molecular analyses to show that some specimens previously identified as G. lucidum in Pakistan actually represent G. multipileum, reported here as a first record for the country. We also report Tomophagus cattienensis from Pakistan for the first time. Materials & methods Collections & ecology Fungal specimens were collected in Punjab, Pakistan, during 2018 by the first author and deposited in the Herbarium of the Institute of Botany, University of the Punjab, Lahore, Pakistan (LAH). The material was collected in [1] Changa Manga Forest in the Kasur District, dominated by Dalbergia sissoo DC. and Vachellia nilotica (L.) PJ.H. Hurter & Mabb. (Fabaceae) and with an average annual rainfall of 1232 mm and average 24 °C temperature (Ahmad & al. 2014) and [2] the New Campus, University of the Punjab in Lahore, covered by D. sissoo with many rotting trunks and with an average annual rainfall of 607 mm and average 24 °C temperature (Shirazi & al. 2019). Additionally, a specimen from XAL was re-examined. Ganoderma & Tomophagus spp. new for Pakistan ... TABLE 1. Sequences of Ganoderma, Tomophagus, and Perenniporiella outgroup included in the analyses. [T] ex-type sequences. SPECIES G. ahmadii G. curtisii G. leucocontextum G, lingzhi G. lucidum G. martinicense G. mizoramense G, multipileum G. multiplicatum G. parvulum VOUCHER/STRAIN FWP 14329 [T] CBS 100132 UMNGAI AY2B GDGM 40400 [T] Cui 9166(67) Dai 12574 K 175217 MUCL 35119 CWN01740 BCRC36123 LIP SW-Mart08-44/ MUCL:GSP44 LIP SW-Mart08-55/ MUCL:GSP55 [T] UMN-MZ5 UMN-MZ4 CM10 CM110 CWN 04670 Dai 9447 HMAS242384 MN14091107 Dai 13710 Dai 12320 JFL 10004081328 SPC9 SPC5 MUCL 47096 MUCL 53123 URM80765 URM2948 LOCALITY Pakistan USA USA Pakistan China China China UK France Taiwan India Martinique Martinique India India Pakistan Pakistan China China China Myanmar China China China Brazil Brazil Cuba French Guiana Brazil Brazil GENBANK ITS1/ITS2 Z37047/ Z37098 JQ781848 MG654117 MN134012 KFO11548 MH109560 KJ143908 KJ14391 MK554779 EU021461 EU021459 KF963257 KF963256 KY643751 KY643750 MW349830 MW349829 KJ143913 KJ143914 JF915409 MK345439 KU572489 K00U572490 MH106879 KU569553 KU569549 MK554783 MK531814 JX310822 JX310821 REFERENCE Moncalvo & al. 1995 Cao & al. 2012 Loyd & al. 2018 Unpublished Li & al. 2015 Unpublished Zhou & al. 2015 Zhou & al. 2015 Cabarroi- Hernandez &al. 2019 Wang & al. 2009 Wang & al. 2009 Welti & al. 2015 Welti & al. 2015 Crous & al. 2017 Crous & al. 2017 This study This study Zhou & al. 2015 Zhou & al. 2015 Wang & al. 2012 Hapuarachchi & al. 2019 Unpublished Unpublished Hapuarachchi & al. 2018 Bolanos & al. 2016 Bolanos & al. 2016 Cabarroi- Hernandez &al. 2019 Cabarro0i- Hernandez &al. 2019 De Lima Jr & al. 2014 De Lima Jr & al. 2014 137 138 ... Umar & al. GENBANK SPECIES VOUCHER/STRAIN LOCALITY ITS1/ITS2 REFERENCE G. resinaceum CBS 194.76 Netherlan0ds KJ143916 Zhou & al. 2015 MUCL 52253 France MK554786 Cabarroi-Hernandez & al. 2019 G. sichuanense HMAS251146 China JF915401 Wang & al. 2012 G. valesiacum CBS 282.33 UK Z37081/ Z37056 Moncalvo & al. 1995 Ganoderma sp. AU-2019a/HP12 Pakistan MN006955 Unpublished P. chaquenia MUCL 47648 Argentina FJ411084 Robledo & al. 2009 P. pendula MUCL 47129 Cuba FJ411082 Robledo & al. 2009 T. cattienensis CATPU120 Pakistan MW737424 This study CATPU121 Pakistan MW737425 This study CT119 Vietnam JN184398 Le & al. 2012 CT99 [T] Vietnam JN184397 Le & al. 2012 T. colossus 255FL USA MG654427 Loyd & al. 2018 CGMCC5.763 Philippines JQ081068 Wang & al. 2012 UMNFPFL151 USA MG654431 Loyd & al. 2018 URM80450 Brazil JX310825 De Lima Jr & al. 2014 URM83330 Brazil JQ618247 De Lima Jr & al. 2014 TC-02 Vietnam KJ 143923 Zhou & al. 2015 Tomophagus sp. BAB-4989 India KR155077 Unpublished AUMC 14536 Egypt MW186858 Unpublished Morphology Size, shape, and color of basidiomata were noted from fresh material. Color descriptions follow Munsell (1975). For microscopical analyses, basidiome cross sections were soaked in 3% KOH, stained with 1% Congo red, and examined under 100x magnification using a Meiji MX4300H compound light microscope. At least 30 basidiospore measurements (face and side view, excluding the apical umbo) were recorded and rounded to the nearest 0.5 um; dimensions are presented as length x width (Nagy & al. 2010). Microscopical terms follow, in part, Torres-Torres & Guzman-Davalos (2012). Color descriptions follow Munsell (1975). DNA extraction, amplification, and sequencing Total genomic DNA was extracted from dried specimens following a modified CTAB procedure (Doyle & Doyle 1987). The ITS1+5.8S+ITS2 rDNA region (ITS) was amplified using primers ITS1 & ITS2 (White & al. 1990). Reaction mixtures (20 ul) containing 0.5 ul template DNA, 8.5 ml distilled water, 0.5 ul of each primer, and 10 ml DreamTaqGreen PCR Master Mix (2 X) ran 35 cycles of 95 °C for 30 s, 52 °C for 30 s, and 72 °C for 1 min, followed by a final extension at 72 °C for 10 min. The PCR amplicons were purified and sequenced by Tsingke Co. Ltd. (Tianjin, China) Ganoderma & Tomophagus spp. new for Pakistan ... 139 1/99 P. chaquenia MUCL 47548, Argentina Perenniporiella pendula MUCL 4712, Cuba Tomophagus sp. BAB-4989, India T. colossus UMNFL151, USA Tomophagus sp. AUMC 14536, Egypt 1/98 W T. colossus URM83330, Brazil T. colossus 2 1/81 N « COIOSSUS F ZI . 4 T. colossus URM80450, Brazil = cays, | 7: Colossus 255FL, USA ) 1/100 T. colossus CGMCCS.763,Philippines E T. colossus TC-02, Vietnam T. cattienensis CT99, Vietnam, TYPE 1am T. cattienensis CT119, Vietnam T. cattienlensis 1/89 | _T. cattienensis CATPU120, Pakistan T. cattienensis CATPU121, Pakistan Ganoderma lucidum MUCL 35119, France -/39 | G. lucidum K175217, UK ie 1/95) G, leucocontextum Code AY2B, Pakistan G. leucocontektum G. leucocontextum GDGM 40400, China, TYPE 1/100 G. ahmadii FWP 14329,Pakistan G. valesiacum CBS 282.33, UK 1/100 - G. parvulum MUCL 53123, French Guiana G. parvulum SS. 1/83 G. parvulum MUCL 47096, Cuba 1/100 G. resinaceum MUCL 52253, France G. resinaceum CBS 194.76, Netherlands G. lingzhi Dai 12574, China 1/93 |G, sichuanense HMAS251146, China G. sichuanense 1/94 G. lingzhi Cui9166(67), China 0.84/90. G. curtisii UMNGA1, USA “L. G. curtisii CBS 100132, USA G. multipileum HMAS242384, China G. multipileum CWN 04670, China 0.95/75 | | G. lucidum CWNO01740, Taiwan G. muttipieum CM110, Pakistan G. multipileum G, multipileum Dai9447, China G. multipileum CM10, Pakistan °°" 1 LG. lucidum BCRC36123, India pe { Ganoderma sp. Code HP12, Pakistan 0.90/77 G. martinicense LIP SW-Mart08-55, Martinique, TYPE 1/7 G. martinicense LIP SW-Mart08-44, Martinique 1/95} G. parvulum URM2948, Brazil G. parvulum URM80765, Brazil 1/100 G, mizoramense UMN-MZ5, India G. mizoramense UMN-MZ4, India 99 -— G. multiplicatum SPC9, Brazil G. multiplicatum G. multiplicatum SPCS, Brazil Brazil G. multiplicatum MN14091107, Myanmar he G. multiplicatum Dai 12320, China G, multipli tum G. multiplicatum Dai 13710, China Asia G. multiplicatum JFL 14081328, China G. lucidum s.s. 1/99 G. valesiacum-G. ahmadii G. resinaceum 1/- <_< G. curtisii Ganoderma 1/- G. martinidense G. parvulum 0.89/- G. mizoramens 0.02 Fic. 1. Phylogenetic tree of Ganoderma and Tomophagus based on ITS rDNA sequences generated by maximum likelihood. Perenniporiella pendula and P. chaquenia were chosen as outgroup. Posterior probabilities (>0.85) and bootstrap values (>75%) are shown at the branches. 140 ... Umar & al. Phylogenetic analysis The data set comprised four DNA sequences from Pakistani material and 45 ITS sequences downloaded from GenBank (www.ncbi.nlm.nih.gov/genbank/) (TaBLE 1). The ITS data set was subdivided into three parts: ITS1, 5.88, and ITS2. Perenniporiella chaquenia Robledo & Decock and P. pendula Decock & Ryvarden were selected as outgroup (Costa-Rezende & al. 2017). All sequences were automatically aligned with MUSCLE (Edgar 2004) and manually adjusted using PhyDe® (Miller & al. 2010). JModelTest (Posada & Crandall 1998) was used to determine the best evolutionary model using the corrected Akaike information criterion (AICc). Maximum Likelihood (ML) analyses were conducted using RAxML 7.0.4 (Stamatakis 2006) and Bayesian Inference (BI) analyses were conducted using MrBayes v.3.2.2 (Ronquist & Huelsenbeck 2003). In the ML analysis, the default priors were used, performing 1000 replicates under the GTRGAMMA model. BI analyses were run on CIPRES Science Gateway (Miller & al. 2010). Two independent runs, with 2,000,000 generations each, were carried out with a sampling frequency every 1000 generations and a burn-in of 25%. A 50% majority rule consensus tree with posterior probabilities (PP) was obtained. Convergence of the Markov chains to a stationary distribution was assessed by visual examination of the log likelihood values in the program Tracer v1.7.1 (Rambaut & al. 2018). Nodes were considered supported when bootstrap values (BS) were 275% and the PP was 20.85. The final alignments were deposited in TreeBASE (www.treebase.org). Phylogenetic results The evolutionary models that best fit the individual dataset according to the AICc were ITS] = GTR+G, 5.88 = K80, ITS2 = GTR+G. In BI analyses, the average standard deviation of split frequencies was 0.007460. Phylogenetic analysis of the ITS region indicated that our specimens represent Ganoderma multipileum and Tomophagus cattienensis (Fic. 1). All sequences of G. multipileum, including the Pakistani collections CM10 and CM110, are supported in one clade (PP = 0.95; BS = 75%). This clade, which included sequences of specimens from China, India, and Taiwan, also clustered with sequences from “G. lucidum” specimens and formed a sister group with G. martinicense Welti & Courtec. and G. parvulum Murrill (0.94 PP; 68% BS). The other two Pakistani sequences (from CATPU120 and CATPU121) clustered in the strongly supported Tomophagus group (PP = 1; BS = 100). The Pakistani sequences and the holotype of T! cattienensis (CT99) formed a monophyletic group with another T. cattienensis sequence from Vietnam and sequence TC-02 (as T. colossus, surely a misdetermination), also from Vietnam. Ganoderma & Tomophagus spp. new for Pakistan ... 141 Taxonomy A Fic. 2. Ganoderma multipileum (LAH36825). A. Pileus; B. Pore surface and stipe; C. Section of context and tubes; D. Basidiospores; E. Cells of crustohymeniderm; F. Basidium. Scale bars: A-C = 2. cm; D-F = 10 um. Ganoderma multipileum Ding Hou [as ‘multipilea’], Quart. J. Taiwan Mus. 3: 101 (1950) FIG: 2 BASIDIOMATA annual; stipitate, solitary, single or with a group of 3-4 pilei growing from the same stipe, light in weight, corky. PILEUs projecting <12 cm, 12.2 cm wide, and <2.9 cm thick at the base; reniform, dimidiate to flabelliform, laccate, sulcate, with concentric growing zones; crust thin, yellowish brown (2.5YR4/8) to brown (10R4/6); MARGIN 0.2-0.3 cm thick, entire, obtuse, orange-brown (10YR6/8) to brown (5YR 5/8). STIPE 9-9.6 x 142 ... Umar &al. 3-4.1 cm, sub-cylindrical, eccentric to lateral, strongly laccate, woody, maroon-brown (2.5YR3/6). Pores 6-7 per mm, 110-140 um diam., sub-round to round, straw cream (7.5YR8/4) to brown when bruised (2.5YR4/6). TUBES <1.3 cm long, brown (10YR4/6) to pale brown (2.5YR 8/4). CONTEXT $1.7 cm thick, with concentric growth zones, brown (7.5YR6/8) to yellowish-brown (5YR 5/8), with melanoid incrustations. HyYPHAL SYSTEM DIMITIC: 1) generative hyphae 3.5-5 um diam., thin-walled, hyaline, clamped; 2) arboriform skeleto-binding hyphae 2.5-7 um diam., thick-walled, red-brown to yellow brown. PILEIPELLIS a crustohymeniderm; cells 15-60 x 4.5-12.5 um, clavate, thick-walled. Basrp1a 20.5-29 x 5.5-8.5 um, claviform, hyaline. Bastp1osporgs 8.0-13.2 x 5.5-7.4 um, ellipsoid to ovoid, apex truncate, brown, double walled with endosporic ornamentation as solid, thin, free pillars or column-like projections. SPECIMENS EXAMINED—PAKISTAN, Punjab, Kasur district, Changa Manga Forest, 31.05°N 73.4072°E, 200 m a.s.L, gregarious on decayed hardwood, Dalbergia sissoo and Vachellia nilotica, 10 July 2018, Aisha Umar CM10 (LAH36826, GenBank MW349830). Lahore district, Lahore, New Campus, University of the Punjab, 31.4981°N 73.3044°E, 217 ma.s.l, gregarious on hardwood, on living tree trunk of V. nilotica, 10 April 2018, Aisha Umar CM110 (LAH36825, GenBank MW349829). DISTRIBUTION: China, India, Nepal, Pakistan, Philippines, Taiwan, Thailand. Tomophagus cattienensis X.T. Le & Moncalvo, Mycol. Prog. 11: 777 (2012) FIG. 3A—-E BASIDIOMATA annual, sessile, bulky, light in weight, spongy. PILEus projecting <9.2 cm, 10.2 cm wide, and <7.8 cm thick at the base, dimidiate to flabelliform, laccate, surface friable, rugose, golden (LOYR7/8), yellow-brown (10YR8/8), orange-brown (10YR6/8), colors patchy and not on a gradient; MARGIN 3.4-4 cm, very thick, entire to lobulated, obtuse, rugose, whitish, white creamy (10YR8/3) to pale yellow brown (10YR7/6). Pores 2-3 (-4) per mm, angular to round, creamy (7.7Y R8/4) to brown (10YR3/4) when bruised. TuBESs 0.9-1.2 cm long., golden yellow (10YR7/8). CONTEXT 3.8-7.6 cm thick, thick at the base, homogeneous, light, soft, creamy white (2.5YR8/4) when fresh to pale brown (10YR4/6) when dry, powdery. HYPHAL SYSTEM DIMITIC: 1) generative hyphae 2.5-3 um diam., thin- walled, clamped, hyaline; 2) skeletal hyphae 2-3 um diam., thick-walled, hyaline. PILEIPELLIs a crustohymeniderm; cells 40-80 x 7-15 um, narrowly clavate, thick-walled, apically ornamented. CHLAMYDOSPORES 22-24.5 um diam., globose, double walled, endosporic ornamentation with thick cylindrical projections, yellowish brown. Basrp1A not seen. BASIDIOSPORES Ganoderma & Tomophagus spp. new for Pakistan ... 143 Fic. 3. Tomophagus cattienensis (LAH36830). A. Pileus; B. Pore surface and context; C. Basidiospores; D. Chlamydospores; E. Crustohymeniderm cells. Tomophagus colossus (XAL-Guzman 35708). F Crustohymeniderm cell; G, H. Basidiospores. Scale bars: A = 2 cm; B=1 cm; C, D = 20 um. 144 ... Umar & al. 17.4-21.3 x 11.3-14.6 um, ellipsoid to ovoid, apex acute or subacute, or when collapsed then shortly truncated or even concave, reddish brown, double- walled with endosporic ornamentation as thick, partially reticulate pillars, apex hyaline. SPECIMENS EXAMINED— PAKISTAN, PunyjaB, Lahore district, Lahore, New Campus, University of the Punjab, 31.4981°N 73.3044°E, 217 m a.s.L, gregarious on hardwood, on living tree trunk of Dalbergia sissoo, 10 April 2018, Aisha Umar CATPU120 (LAH36830, GenBank MW737424). Kasur district, Changa Manga Forest, 31.05°N 73.4072°E, 200 m a.s.l., gregarious on decayed hardwood, Vachellia nilotica, 10 July 2018, Aisha Umar CATPU121 (LAH36839, GenBank MW737425). DISTRIBUTION: Pakistan, Vietnam. ADDITIONAL SPECIMEN EXAMINED (FIG. 3F-H)—Tomophagus colossus: MEXICO, VERACRUZ, Guzman 35708 (XAL) [as Ganoderma “colossum’]. Discussion Our morphological and molecular analyses confirm the presence of two polypores of Ganodermataceae from Pakistan: Ganoderma multipileum and Tomophagus cattienensis. For many years, the name Ganoderma lucidum has been misapplied to Ganoderma specimens from tropical Asia that represent other species (Wang & al. 2009, Hennicke & al. 2016, Raja & al. 2017). Collections labeled as ‘G. lucidum’ from world regions outside Europe (where G. lucidum was described) have appeared in several different lineages in numerous phylogenetic analyses (Moncalvo & al. 1995, Gottlieb & al. 2000, Smith & Sivasithamparam 2000, Hong & Jung 2004). Ganoderma multipileum is one species confused with G. lucidum that is now recognized as representing a different species (Wang & al. 2009) first described over a half century ago by Hou (1950). Loyd & al. (2018) showed that the “multipileum clade” is sister to the “curtisii clade, with the “multipileum clade” split into two subclades, one clustering specimens of G. multipileum from China and the other clustering specimens of G. martinicense from Martinique (type specimen) and from North America. Ganoderma multipileum, originally described from Taiwan (Wang & al. 2009), has been recorded from India, China, Nepal, and Philippines (Wang & al. 2012, Fryssouli & al. 2020). Welti & Courtecuisse (2010) suggested that G. martinicense might represent a “vicariant from the Caribbean area’ of G. multipileum. Our phylogeny, although based on a single DNA marker, agrees with that of Loyd & al. (2018) based on four markers and supports a relationship between G. multipileum and G. martinicense. Ganoderma & Tomophagus spp. new for Pakistan ... 145 However, our phylogeny also includes two sequences of G. parvulum (De Lima Jr & al. 2014) as sister taxon of G. martinicense. Cabarroi- Hernandez & al. (2019) suggested that the sequences of G. parvulum sensu De Lima Jr. & al. (2014) might represent G. bibadiostriatum Steyaert, a species described from Nicaragua (Steyaert 1962) and phylogenetically unrelated to G. parvulum s.str. (Cabarroi- Hernandez & al. 2019). Our “multipileum clade” comprises sequences of specimens from China, India, and Taiwan and CM10 and CM110 (previously labeled G. lucidum) from Pakistan. The clade also includes the sequences BCRC36123 from India and CWNO01740 from Taiwan, both referenced at GenBank as G. lucidum but identified as G. multipileum by Wang & al. (2009), as well as the sequence Code HP12 referenced at GenBank as Ganoderma sp. from Pakistan (very probably representing G. multipileum). The ITS tree corroborates our morphology-based identification and supports extending the distribution of G. multipileum to Pakistan. Furthermore, we suggest that G. lucidum s.stv. is not present in that country. Wang & al. (2009) concluded that, based on morphological characters and their ITS phylogeny, “G. lucidum” from tropical Asia is divided into two clades, both of separated from the European G. lucidum s.str. One clade contained tropical collections (labeled G. multipileum) while a second “unknown” clade clustered specimens from China and Japan (Wang & al. 2009). Wang & al. (2012) later recognized the “unknown” clade as G. sichuanense J.D. Zhao & X.Q. Zhang, while Cao & al. (2012) noted that G. multipileum also occurred in India and the Philippines. Ganoderma multipileum is recognized morphologically based on three features: basidiomes with pilei or with some stipes and pilei growing together, mostly regular clavate crustohymeniderm cells, and truncated ellipsoid basidiospores with free fine pillars (Wang & al. 2009, Zhou & al. 2015). According to Cao & al. (2012) G. multipileum inhabits fabaceous hosts and is distinguished from G. sichuanense (as G. lingzhi Sheng H. Wu & al.) by its “distinct concentric growth zones in context at maturity, and finely echinulate basidiospores.” These primary diagnostic features are also seen in the Pakistani specimens of G. multipileum (Fic. 2). Furthermore, our specimens were collected growing gregariously on decayed hardwood of Dalbergia and Vachellia (Fabaceae). The other Pakistani specimens studied here nested in the Tomophagus clade. Murrill (1905), who proposed the genus, characterized Tomophagus by its very lightweight basidiome with a pale soft spongy context and a 146 ... Umar & al. “labyrinthine” basidiospore surface (Le & al. 2012), as noted in both species covered here (FIG. 3c,G,H). Tomophagus was typified by Polyporus colossus Fr. (Murrill 1905). Furtado (1962, 1965), who described the neotype specimen as having “Ganoderma-type basidiospores,” accepted the species as G. colossus. Moncalvos (2000) phylogeny revealed an unclassified basal group composed of G. colossus, G. tsunodae (Lloyd) Sacc. & Trotter, and other taxa. Subsequent molecular phylogenetic studies confirmed the genus as a well- established group independent of Ganoderma (Hong & Jung 2004, Le & al. 2012, Xing & al. 2018, He & al. 2019, Costa-Rezende et al 2017, 2020). Tomophagus cattienensis and T: colossus have been distinguished primarily by color of the pileal surface and ITS sequence analyses (Le & al. 2012). Tomophagus cattienensis was distinguished from T!: colossus by its “red-brown or red-coffeate” (instead of yellow) pileal surface and by its harder crust and context that “turns pale brown upon drying (instead of remaining creamy white)” (Le & al. 2012). However, Pakistani specimens have yellow, yellow-brown, orange-brown pilei and lighter crusts (somewhat similar to T. colossus or intermediate between the two species) whereas the color change of the context agrees with that described for T! cattienensis (Fic. 24). Another character distinguishing T. cattienensis and T. colossus are the thick-walled crustohymeniderm cells, present in the Pakistani specimens of T: cattienensis but not previously described for this species. Tomophagus colossus characteristically has thin-walled cuticular cells (Ryvarden 2000) observed in the specimen of T: colossus from Mexico (Guzman 35708, XAL) (Torres-Torres & al. 2015). Our observations of the Pakistani specimens therefore expand the morphological description of T’ cattienensis. In our phylogeny, the ITS sequences of the Pakistani specimens showed only one nucleotide difference from ITS sequences of the T: cattienensis type specimen in contrast to the six-nucleotide difference with T. colossus. Our phylogeny included sequences labelled G. multiplicatum in GenBank, which separated into two different clades. One clade (PP 1/BS 100) clustered specimens from China and Myanmar, and the other (PP 1/BS 99) clustered specimens from Brazil. Morphologically, G. multiplicatum is distinguished from G. multipileum primarily by its cuticular cells with up to 14 lateral or apical protuberances (Torres-Torres & Guzman-Davalos 2012, Bolafos & al. 2016). Originally described from French Guyana, G. multiplicatum was subsequently found in other parts of South America, Africa, and Asia (Steyaert 1980, Zhao 1989), including India (Bhosle & al. 2010), Pakistan Ganoderma & Tomophagus spp. new for Pakistan ... 147 (Fakhar-ud-Din & Mukhtar 2019), and Taiwan (Wang & Wu 2008). The Asian specimens identified as G. multiplicatum should be re-evaluated and more DNA regions must be included, to define their taxonomical status. In this study, we combined macro and microscopic morphological characters, ecological aspects, as well as molecular data to determine two species of Ganodermataceae from Pakistan. This is the first report of the occurrence of G. multipileum and T: cattienensis in this country. Our results suggest that some species with ganodermatoid basidiospores previously registered from Pakistan (e.g., G. ahmadii, G. applanatum, G. australe, G. boninense, G. chalceum, G. curtisii, G. flexipes, G. lipsiense, G. lucidum, G. praelongum, G. multicornum, G. multiplicatum, G. resinaceum, G. tornatum, G. tsugae, and T. colossus) need thorough re-evaluation of both morphology and molecular data before their presence in the country or taxonomic status can be confirmed. Acknowledgments The authors thank Mario Rajchenberg (Centro de Investigacién y Extension Forestal Andino Patagénico, Chubut, Argentina) and Gerardo Robledo (Universidad Nacional de Cordoba, Ciudad Universitaria, Argentina) for helpful comments and their pre-submission expert reviews. MCH gratefully acknowledges financial support received from CONACYT, Mexico (Postdoc Scholarship). MCH and LGD thank the University of Guadalajara, Mexico for supporting their research. Literature cited Ahmad S. 1956. 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Via Cappuccini 78/8, I-33170 Pordenone, Italy > National Botanical Garden of Santo Domingo, Santo Domingo, Dominican Republic “CORRESPONDENCE TO: niconiveiro@gmail.com ABSTRACT—Two purple Marasmius species from the Dominican Republic are described and illustrated. Marasmius tageticolor is characterized by its radially striped pileus, distant lamellae, and elongated spores, and M. tucumanus is characterized by its purple to dark red pileus, fragile basidioma, and small spores. Both species were sequenced for the first time and phylogenetically resolved to Marasmius sect. Globulares, closely related to species of M. ser. Haematocephali + M. ser. Leonini. Key worps—Agaricales, ITS, Marasmiaceae, mushroom, diversity Introduction Marasmius tageticolor and M. tucumanus are members of M. sect. Globulares (Antonin & Noordeloos 2010, Oliveira & al. 2020), characterized by the purple coloration of their pileus surface (Singer 1976). Marasmius tageticolor is a relatively well-known species from the Caribbean and northern South America, but there are no complete modern descriptions or publicly available sequences. On the other hand, M. tucumanus has been found only once, as the type material from northwestern Argentina that has not been illustrated or sequenced. The aim of this work is to describe and 154 ... Niveiro, Ramirez, Angelini illustrate both species, evaluate their phylogenetic relationship, and expand their range of distribution to the Dominican Republic. Materials & methods Morphological studies The specimens, which were collected on litter from man-made lowland deciduous woods in the Dominican Republic, were photographed and described macroscopically in situ. The specimens were analyzed macro- and microscopically following the criteria and terminology proposed by Vellinga (1988) and Lodge & al. (2004). Color terminology follows Kornerup & Wanscher (1978). For microscopic analysis, tissue sections were made freehand and mounted in a solution of 5% KOH (v/w) with 1% phloxine aqueous solution. Melzer’s reagent (Wright & Alberté 2002) was used to verify amyloidity. The microscopic structures were measured directly through a 1000x oil-immersion objective or photographs taken with a Leica EC3 built-in camera using ImageJ software (Schneider & al. 2012). The notation “L’ refers to the number of true lamellae (extending from the stipe insertion to the pileus margin) counted at the margin of the pileus. The minimum-maximum interval was provided for the different microscopic structures. For basidiospores, n = number of spores measured, x = mean spore length x width, Q= length / width ratio, and Qx = mean Q value. The authors of scientific names agree with Index Fungorum (2020), and herbarium acronyms follow Thiers (2020). The collected material was dried at 40 °C and stored in a freezer for a week before being deposited in the Jardin Botanico Nacional Dr. Rafael M. Moscoso (JBSD) and in the Instituto de Botanica del Nordeste herbaria (CTES). DNA extraction, amplification, sequencing Genomic DNA was extracted from dried specimens implementing a modified CTAB protocol based on Murray & Thompson (1980). For PCR reactions, we followed the recommended cycling condition by Mullis & Faloona (1987). Primers ITSIF and ITS4 (White & al. 1990, Gardes & Bruns 1993) were employed to amplify the ITS rDNA region. PCR products were checked in 1% agarose gels and positive reactions were sequenced with one or both PCR primers. DNA extraction, amplification, and sequencing were performed by Alvalab (Spain). Sequences were edited using BioEdit 7.2.5 (Hall 1999). Phylogenetic analysis The nrITS dataset compiled included our three new sequences and 44 Marasmius sequences selected from GenBank based on BLAST results and previous studies (TABLE 1). Crinipellis zonata (Peck) Sacc. was used as outgroup (Aime & Phillips- Mora 2005). Sequences were aligned with MAFFT 7 (Katoh & Standley 2013) under the Q-INS-i criteria. The alignment was manually adjusted with MEGA 5 (Tamura & al. 2011). Potential ambiguously aligned ITS1-ITS2 segments were detected and deleted using Gblocks 0.91b (Castresana 2000). Two Marasmius spp. new to the Dominican Republic ... TABLE 1: Marasmius and outgroup sequences used in the phylogenetic analyses. Sequences obtained in this study are in bold. SPECIES Crinipellis zonata SERIES VOUCHER VPI3355 GENBANK # AY916692 REFERENCE Aime & Phillips-Mora 2005 15S M. sect. Marasmius M. rotalis M. cf. subruforotula M. tubulatus Marasmius Sicciformes Marasmius JES145 JES141 JES186 JES192 TYS502 TYS518 KX149000 KX148999 KX149017 KX149018 FJ431280 FJ431279 Shay & al. 2017 Shay & al. 2017 Shay & al. 2017 Shay & al. 2017 Tan & al. 2009 Tan & al. 2009 M. sect. Globulares M. maximus M. nivicola M. pseudo- purpureostriatus M. purpureostriatus M. corrugatiformis M. cystidiatus M. ochroleucus M. strobiluriformis M. bondoi M. confertus var. tenuicystidiatus M. haematocephalus M. magnus M. siccus M. sullivantii Globulares Globulares Globulares Globulares Atrorubentes Atrorubentes Atrorubentes Atrorubentes Haematocephali Haematocephali Haematocephali Haematocephali Haematocephali Haematocephali KG224 BRNM714571 BRNM714575 BRNM714572 NW286 BRNM7 14566 DED8233 DED8326 1672 CAL1669 LE 295978 BRNM714914 BRNM714915 NW320 BRNM718808 NW434 NW430 ICN179252 FLOR55963 BRNM552709 LE295980 MO218479 FJ904974 FJ904977 FJ904972 FJ904970 EU643513 FJ904978 KX953757 KX953756 MH2160421 MH216191 KF912952 GU266263 GU266264 EU935474 HQ607374 EU935529 EU935535 KX228848 KX228846 HQ607384 KF774130 MK607492 Antonin & al. 2010 Antonin & al. 2010 Antonin & al. 2012 Antonin & al. 2010 Wannathes & al. 2009 Antonin & al. 2010 Shay & al. 2017 Shay & al. 2017 Sharafudheen & Manimohan 2018 Sharafudheen & Manimohan 2018 Kiyashko & al. 2014 Antonin & al. 2012 Antonin & al. 2012 Wannathes & al. 2009 Antonin & al. 2012 Wannathes & al. 2009 Wannathes & al. 2009 Magnano & al. 2016 Magnano & al. 2016 Wannathes & al. 2009 Kiyashko & al. 2014 Russell & Grootmyers (unpub.) 156 ... Niveiro, Ramirez, Angelini SPECIES SERIES VOUCHER GENBANK # REFERENCE M. acerosus Leonini TYS427 FJ431214 Tan & al. 2009 TYS458 FJ431213 Tan & al. 2009 M. adhaesus Leonini TYS467 FJ431216 Tan & al. 2009 TYS464 FJ431217 Tan & al. 2009 M. olivascens Leonini TYS424 FJ431266 Tan & al. 2009 TYS426 FJ431265 Tan & al. 2009 M. plicatulus Leonini NW439 EU935480 Wannathes & al. 2009 M. tageticolor Leonini JBSD 130776 MT260146 _‘This paper JBSD130775 MT260147 This paper M. tucumanus Leonini JBSD 130778 MT260145_ _— This paper M. dendrosetosus Spinulosi JES205 KX148995 Shay & al. 2017 JES211 KX148996 Shay & al. 2017 M. longisetosus Spinulosi JO248 JX424040 Oliveira & al. 2014 M. neotrichotus Spinulosi CTES0568167 MF683958 Niveiro & al. 2018 M. nummularius Spinulosi NW266 EU935492 Wannathes & al. 2009 NW396 EU935493 Wannathes & al. 2009 M. paratrichotus Spinulosi DED8248 KX953749 Grace & al. 2019 M. trichotus Spinulosi NW262 EU935490 Wannathes & al. 2009 NW263 EU935491 Wannathes & al. 2009 Phylogenetic reconstruction was inferred using Maximum Likelihood (ML) and Bayesian Inference (BI). Maximum likelihood was carried out in RAxML-HPC v.8 (Stamatakis 2014), employing the GT[RGAMMA model for the entire dataset. The analyses first implemented 1000 ML independent searches, each one starting from one randomized stepwise addition parsimony tree. Only the best scored ML tree was kept, and node reliability was accessed through nonparametric Bootstrap (BS) pseudoreplicates under the same model, allowing the program to halt bootstrapping automatically using the autoMRE option. Bayesian Inference was performed in MrBayes 3.2.6 (Ronquist & al. 2012). The evolutionary model for BI was estimated using Akaike Information Criterion (AIC) as implemented in jModelTest2 v.1.6. (Guindon & Gascuel 2003, Darriba & al. 2012). We implemented two independent runs, each one beginning from random trees, with four simultaneous independent chains. A total of 2x10’ generations was carried out, sampling one tree every 1x10° generation. All phylogenetic analyses were conducted via CIPRES Science Gateway (Miller & al. 2010). Both analyses (ML and BI) produced similar topology trees. Only the BI 50% majority-rule consensus tree is shown, indicating support values Bayesian posterior probabilities (BPP) / rapid bootstrapping (BS) of each node. A node is considered strongly supported if it showed a BPP 20.98 and/or BS =90%; moderate support is indicated by BPP 20.95 and/or BS =70%. Two Marasmius spp. new to the Dominican Republic ... 157 0,98/81 1/97 1/75 1/99 1/99 0,95/87 1/- 1/99 1/100 0,99/71 1/97 1/78 M. bondoi EU935474 - SH M. confertus var. tenuicystidiatus HQ607374 - SH 100 7 ™. strobiluriformis GU266263 M. strobiluriformis GU266264 1/96 M. ochroleucus KF912952 400 [ ™. cystidiatus MH216191 M. cystidiatus MH2160421 ser. Atrorubentes 4/100 M. corrugatiformis KX953756 M. corrugatiformis KX953757 0.99/76 7 M. adhaesus FJ431217 M. adhaesus FJ431216 4/100 M. olivascens FJ431266 1/95 M. olivascens FJ431265 ser. Leonini M. acerosus FJ431214 1/96 M. acerosus FJ431213 M. purpureostriatus FJ904978 SG M. pseudopurpureostriatus EU643513 M. siccus.HQ607384 SH M. siccus KF774130 1/100 sect. Globulares M. nummularius EU935492 ser. Spinulossi 4/100 M. dendrosetosus KX148995 M. dendrosetosus KX148996 M. longisetosus JX424040 M. haematocephalus EU935535, SH M. haematocephalus EU935529 M. tageticolor MT260146 SL H) M. tageticolor MT260147 SL M. tucumanus MT260145 SL + ser. Leonini (SL) M. sullivantii MK607492 - SH ser. Haematocephalii (S M. plicatulus EU935480 - SL 1/97 1/99 1/100 1/95 1/97 0,99/100 M. nivicola FJ904970 M. nivicola FJ904972 400 [ M. maximus FJ904974 M. maximus FJ904977 M. magnus KX228846 ser. Globulares (SG) 77 M. neotrichotus MF683958 1/88 M. paratrichotus KX953749 M. trichotus EU935491 4100 M. trichotus EU935490 4/100 M. nummularius EU935493 M. magnus KX228848 4/100 M. rotalis KX149000 M. rotalis KX148999 1/100 M. tubulatus FJ431279 M. tubulatus FJ431280 4/100 M. cf. subruforotula KX149018 sect. Marasmius M. cf. subruforotula KX149017 Crinipellis zonata AY916692 0.04 Fic. 1: 50% majority-rule consensus tree from Bayesian Inference based on a dataset of nrITS sequences of Marasmius taxa. Bayesian posterior probability BPP 20.9, and Bootstrap value, BS =70% are shown. 158 ... Niveiro, Ramirez, Angelini Phylogenetic results The nrITS dataset included 48 sequences from 29 Marasmius taxa plus the outgroup, resulting in an alignment with 728 characters, of which 296 are parsimony informative. Our phylogenetic inference (Fic. 1) recovered two major, well-defined clades: M. sect. Marasmius (BPP = 1; BS = 97%) and M. sect. Globulares (BPP = 0.93). Within M. sect. Globulares, five moderately to highly supported subclades are recognized: M. ser. Globulares (BPP = 1; BS = 99%), M. ser. Atrorubentes (BPP = 0.98; BS = 81%), M. ser. Leonini (BPP = 1; BS = 95%), M. ser. Spinulosi (BPP = 1; BS =99%) and M. ser. Haematocephali+ M. ser. Leonini (BPP = 1; BS = 78%). Our target species, M. tageticolor and M. tucumanus, are sisters (BPP = 0.99; BS = 71%) and closely related to M. bondoi Wannathes & al. and M. confertus var. tenuicystidiatus Antonin within the subclade of M. ser. Haematocephali + M. ser. Leonini. Taxonomy Marasmius tageticolor Berk., Hooker’s J. Bot. Kew Gard. Misc. 8: 136. 1856. Fics 2, 3 BASIDIOMATA gregarious, in small groups. PiLEus <20 mm broad, campanulate to convex when young, then convex to broadly convex, umbonate or with depressed center, sulcate up to the center in all stages of development, with slightly crenate edge; surface striped in pigmentation (the colored stripes correspond to the lamellae and lamellulae lines), center and radial stripes purple (13E8 “deep magenta’ to 13F8 “dark magenta”) usually with lighter marginal zones (13C6-7 “greyish magenta’), and white (1A1), pinkish-white (13A2) to pinkish (13A3) between stripes, brown (7E5) to dark brown (7F5) with light yellow (4A4) to light orange (5A4) stripes in dehydrated specimens; dry, subvelutinous, dull. ConTEext thin, whitish (13A1), odor and taste not distinctive. LAMELLAE free to narrowly adnexed, broadly ventricose, <5 mm, distant, L = 8-9, white (13A1) at the marginal edge, pink (13A3) to purplish red (13A6) towards the pileus; usually with one series of lamellulae between lamellae. Stipe 15-45 x 1-1.5 mm, central, cylindrical thin, flared upwards, hollow, surface reddish purple brown (9D7-9E7 “reddish-brown’), lilac (13C7, “purplish red”) towards the apex, blackish (8F4 “greyish brown to 14F8 “dark purple”) towards the base, glabrous, dry, with a whitish ochre (5B3-4 “greyish orange”) basal mycelial patch. ANNULUs absent. SPORE-PRINT not observed. Two Marasmius spp. new to the Dominican Republic... 159 Fic. 2: Marasmius tageticolor, macroscopic views: a. JBSD 130776; b. JBSD 130775; c. Detail of basal mycelial patch. Scale bars: a, b = 10 mm. (Photo by C. Angelini). 160 ... Niveiro, Ramirez, Angelini BASIDIOSPORES 14-21.8 x 3-4(-4.5) um, x = 18.8 x 3.4 um, Q = 3.5-7.1, Qx = 5.5, n = 25; oblong, acicular to narrowly clavate, with suprahilar depression, inamyloid, hyaline, smooth, thin-walled. Bastp1a 22-25 x 5-6.5 um, clavate, 4-spored, hyaline, thin-walled. BastproLes 15-25 x 5-6.5 um, narrowly clavate to fusoid, hyaline, thin-walled. PLEUROCysTIDIA absent. CHEILOCYSTIDIA in form of Siccus-type broom cells, hyaline, with main body claviform, 9-15 x 4.5-6 um, setulae <6 um long, and base 1 um diam. HYMENOPHORAL TRAMA Subregular, hyphae 3-5(-9) um diam., hyaline, thin-walled, dextrinoid. PILEIPELLIs hymeniform, comprising Siccus-type broom cells with main body claviform, 9.5-11.5 x 5-7 um, setulae <7 um long, and bases 1.5 um diam.; hyaline in lighter or discolored interlamellar region, stramineous in the pigmented region. PILEOCysTrIDIA absent. STIPITIPELLIS a cutis of smooth hyphae, stramineous, appressed, 3-4 um diam. CAULOCYSTIDIA absent. CLAMP CONNECTIONS present. EcoLocy— Forming small groups on dried dicotyledonous twigs and leaves in the litter. SPECIMENS EXAMINED—DOMINICAN REPUBLIC: PUERTO PLATA PROVINCE, Sosua, PUERTO CHIQUITO, on the litter of an anthropized plain forest with broad-leaved trees, 8/8/2011, leg. C. Angelini 714 (JBSD130775!, GenBank MT260147; CTES!); 5/12/2013, leg. C. Angelini 291 (JBSD130776!, GenBank MT260146; CTES!). DIsTRIBUTION—Originally described from northwestern Amazonas State, Brazil (Berkeley 1856); also known from Venezuela, Mexico (Singer 1976), Panama (Desjardin & Ovrebo 2006), Colombia (Vasco-Palacios & al. 2005), and USA, Trinidad and Tobago, St. Vincent and the Grenadines (GBIF 2020). Newly recorded from the Dominican Republic. COMMENTS—Marasmius tageticolor is characterized by its pileus surface, which is conspicuously radially striped with a purple-red to purple center and lamellar stripes, interspersed with lighter interlamellar regions, distant lamellae, and elongated spores (Singer 1976). The Siccus-type broom cells of the pileipellis and the absence of pleurocystidia place this species in M. sect. Globulares (Antonin & Noordeloos 2010) and M. ser. Leonini (Singer 1976). A striped pileus is not a frequent feature in M. ser. Leonini (Antonin 2007). Marasmius poecilus Berk., found in Bolivia, Brazil, and Venezuela, is the closest species and occasionally considered a synonym (Singer 1965). However, Singer (1976) highlighted fine differences to separate these two species, such as the more darkly colored pileus (purplish brown to fulvous with white to yellow stripes), and longer (35-80 mm) stipe in M. poecilus (Singer 1976). Two Marasmius spp. new to the Dominican Republic... 161 b C Bey Fic. 3: Marasmius tageticolor (JBSD130775). a. Basidiospores; b. Basidia; c. Basidioles; d. Cheilocystidia. Scale bar = 10 um. Another striped Neotropical species is M. phaeus Berk. & M.A. Curtis, but it clearly differs by its dark reddish-brown coloration, lacking a purple hue (Singer 1976). Some African striped taxa such as M. lilacinoalbus Beeli var. 162 ... Niveiro, Ramirez, Angelini lilacinoalbus and M. lilacinoalbus var. lilacinocarmineus Singer are similar to M. tageticolor but differ in their yellowish pileus center and broader spores (4-5 um; Antonin 2007). Marasmius striipileus Antonin has orange brown (6C8) to brown (6D8) tints with paler stripes (Antonin 2004), but differs in the absence of marked streaks with white, yellowish or with pink tints (Antonin 2007). Berkeley (1856) described M. tageticolor (based on dehydrated material and black and white drawings submitted by Spruce from Brazil) as “convex, membranaceus, umbonate, varying from reddish-brown to deep crimson, adorned with from eight to ten yellow rays, very minutely wrinkled... Gills narrow, ventricose, attenuated behind and free, yellow like the rays.” Over a century later, Singer (1965) reported specimens of M. tageticolor having brown pileus with deep cream radial stripes and lamellae based on the type material (Spruce 37 K) and additional material from Bolivia (Singer B 1981 LIL) that was later identified as M. poecilus (Singer 1976). In that same work and with additional material from Mexico and Venezuela, Singer (1976) described M. tageticolor with red pileus and white to ochraceous- whitish stripes and white lamellae. More recently, Desjardin & Ovrebo (2006) described M. tageticolor based on specimens from Panama having a light buff pileus surface with beet red (11-12E-F-8) stripes. The specimens analyzed here agree with those described by Desjardin & Ovrebo (2006), but they present some minor differences compared with the protologue (Berkeley 1856) and descriptions made by Singer (1965, 1976), primarily regarding the color of the pileus and lamellae. Pigment variations may be due to different developmental stages, humidity, or observations based on dehydrated materials in which the lamellae and pileus stripes have yellowed. Despite these variables, the remaining characteristics agree with the concept of M. tageticolor. Likewise, the pigmentation of the specimens studied here and those described by Desjardin & Ovrebo (2006) are similar to M. haematocephalus var. pseudotageticolor Singer, with which they could easily be confused, although the M. haematocephalus variety differs by the presence of pleurocystidia and the insititious or subinsititious stipe base (Singer & Digilio 1952 as “M. tageticolor”; Singer 1959, 1965, 1976). Marasmius tucumanus Singer, Lilloa 25: 206. 1952. Fics 4, 5 BASIDIOMATA solitary or in small groups. PILEUs <15 mm broad, campanulate to convex with papillate center, striate-sulcate up to the center, Two Marasmius spp. new to the Dominican Republic ... 163 Fic. 4: Marasmius tucumanus, macroscopic views: a, b. JBSD 130777; c. JBSD130778; d. Detail of basal mycelial patch. Scale bars: a~c = 10 mm. (Photo by C. Angelini). 164 ... Niveiro, Ramirez, Angelini with entire to crenate edge, dark purple (11E7, 11F7 “violet brown”) at the center, lighter towards the margin, from vivid purple red (11A8, “vivid red”) to dark red (11C8); dry, subvelutinous, sometimes pruinose, mostly dull or semi-translucent at the margin. CONTEXT thin, whitish (13A1), odor and taste not distinctive. LAMELLAE free to narrowly adnexed, ventricose, <2 mm, distant, L = 11-15, white (11A1) at the marginal edge, pink (12A3-4) towards the pileus; with 3-4 series of lamellulae. StrpE 20-40 x 1.5 mm, central, cylindrical, equal, hollow, surface purple red (10A8 “vivid red”) when young, to violet brown (10F8) when mature, glabrous, dry, with a tomentose, whitish ochre (5B3-4, “greyish orange”) basal mycelial patch. ANNULUS absent. SPORE-PRINT not observed. BASIDIOSPORES 10-15 x 3-4.5 um, x = 13.2 x 3.7 um, Q =3.0-4.0, Qx = 3.5, n= 15; oblong, subclavate, with attenuated suprahilar depression, inamyloid, hyaline, smooth, thin-walled. Basrp1a 22-27 x 6-7.5 um, clavate, 4-spored, hyaline, thin-walled. BAsrpIoLeEs 15.5-34.5 x 4-6 um, narrowly clavate to fusoid, hyaline, thin-walled. PLEUROCysTIDIA absent. CHEILOCYSTIDIA in form of Siccus-type broom cells, stramineous colored with main body claviform, 11-19 x 5-9.5 um, setulae <7 um long, and base 1.5 um diam. HYMENOPHORAL TRAMA Subregular, hyphae 1.5-3.5 um diam., thin-walled, dextrinoid. PILEIPELLIS hymeniform, comprising Siccus-type broom cells with main body claviform, 15-22 x 6-7.5 um, hyaline with <7 um long setulae and base 1.5 um diam. with stramineous coloration. PILEOCYSTIDIA absent. STIPITIPELLIS an appressed cutis of smooth stramineous hyphae, 3-6 um diam. CAULOCYSTIDIA absent. CLAMP CONNECTIONS present. EcoLocy—Solitary or forming small groups on dried leaves or small twigs of dicotyledonous litter. SPECIMENS EXAMINED—DOMINICAN REPUBLIC, PUERTO PLATA PROVINCE, Sousa, VERTEDERO, on the litter of an anthropized hill forest with broad-leaved trees, 6/12/2013, leg. C. Angelini 136 (JBSD130777!; CTES!); PUERTO CHIQUITO, on the litter of an anthropized plane forest with broad-leaved trees, 13/12/2014, leg. C. Angelini 503 (JBSD130778!, GenBank MT260145; CTES!). ARGENTINA, TUuCUMAN PROVINCE, Tafi, PARQUE ACoNQquyA, 13/03/1951, leg. R. Singer T1460 (holotype, LIL!). DISTRIBUTION—Previously known only from the type locality, in Argentina (Singer & Digilio 1952). Newly recorded from the Dominican Republic. COMMENTS—Marasmius tucumanus is characterized by its relatively fragile basidiome, purple to dark red coloration, and spores smaller than those of its most closely related taxa: M. tageticolor or M. haematocephalus complex (Singer & Digilio 1952). The type specimen (Singer T1460 LIL!) consists of Two Marasmius spp. new to the Dominican Republic ... 165 a ZNG W d Fic. 5: Marasmius tucumanus (JBSD130777). a. Basidiospores; b. Basidia; c. Basidioles; d. Cheilocystidia. Scale bar = 10 um. a single, well-preserved specimen with macroscopic features that agree with the characters cited in its protologue. Unfortunately, it was not possible to 166 ... Niveiro, Ramirez, Angelini determine whether the microscopic structures are the same, as destructive sampling of the type specimen is not allowed. The presence of purple pigment in the pileus is considered an important feature by different authors in delimiting groups of species (Singer 1976, Desjardin & al. 2000, Tan & al. 2009, Wannathes & al. 2009, Shay & al. 2017). Species with similar pigmentation in M. ser. Leonini are: 1) M. tageticolor, which differs by its peculiar striped pileus and elongated basidiospores (Singer 1976, Desjardin & al. 2000); 2) M. amazonicus Henn., known from Bolivia and Brazil, which is distinguished by its larger (16-58 mm) pileus with oval to irregularly rounded buff-colored dots (Oliveira & al. 2008); 3) M. purpureotinctus Antonin & P. Roberts, an African species, which differs by its larger (17.5-20 x 4.5-5.7 um) basidiospores and its pileipellis with thick-walled broom-cells (Antonin 2013); and 4) M. aratus Massee [= M. masseei Tkaléec & MeSi¢] from southeastern Asia, which differs by larger spores (22-32 x 3.5-5 um; Tan & al. 2009). The M. haematocephalus complex also resembles M. tucumanus in pileal shape and coloration. However, all its taxa have well differentiated pleurocystidia and larger spores (14-25 x 3.5-6 um; Singer 1976, Tan & al. 2009, Wannathes & al. 2009, Shay & al. 2017). Discussion Marasmius tageticolor and M. tucumanus are sister species in the phylogenetic tree (Fic. 1) and closely related to some species belonging to the traditional M. ser. Haematocephali + M. ser. Leonini. This clade has been consistently recovered in different papers (Wannathes & al. 2009, Magnano & al. 2016, Shay & al. 2017, Grace & al. 2019). Among the other species in this clade included in our analyses, M. bondoi, M. confertus var. tenuicystidiatus, and M. sullivantii Mont. lacka purple pileus and have pleurocystidia (Antonin 2004, Wannathes & al. 2009), while M. plicatulus Peck has more robust basidiomata, a dark reddish brown, minutely velutinous pileus surface, and wider basidiospores (4.8-6.3 um; Desjardin 1987). Acknowledgments The authors wish to thank J.J.S. Oliveira (Instituto Nacional de Pesquisas da Amazonia, Rio de Janeiro) and R.A. Koch (University of Nebraska at Lincoln, USA) for their critical revision of the manuscript, C. Salvador Montoya for his advice on phylogenetic analysis, A. Michlig for a preliminary manuscript revision, and A. Hladky and P. Medina from LIL for photographs of the M. tucumanus type specimens. CA wishes to thank Ricardo G. Garcia, Francisco Jiménez, Brigido Two Marasmius spp. new to the Dominican Republic ... 167 Peguero (Jardin Botanico Nacional) and Dr. Rafael M. Moscoso (Santo Domingo, Dominican Republic) for their interest and encouragement in studying fungi of the Dominican Republic and for their active cooperation in providing herbarium material preserved in their institution. NN acknowledges the support of the Consejo Nacional de Investigaciones Cientificas y Técnicas (CONICET) from Argentina (PIP 2014-0714), the Secretaria General de Ciencia y Técnica, Universidad Nacional del Nordeste (SGCyT-UNNE - PI19-P001), and the Agencia Nacional de Promocién Cientifica y Tecnoldgica (PICT 2016-2529) that made this work possible with their funding. 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MYCOTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. © 2021 January-March 2022— Volume 137, p. 171 https://doi.org/10.5248/137.171 REGIONAL ANNOTATED MYCOBIOTA NEW TO THE MYCOTAXON WEBSITE ABSTRACT— The 14-page mycobiota reporting on new records of Chaetomium and Chaetomium-like species on Syagrus coronata from the Caatinga in Bahia, Brazil by Fortes & Vitéria may now be downloaded from Mycotaxon’s mycobiota webpage. This well-illustrated range extension, which includes a key to 8 species, brings to 153 the number of free-access fungae uploaded or linked to http://www.mycotaxon.com/mycobiota/index.html SOUTH AMERICA Brazil NILo GABRIEL SOARES FORTES & NADJA SANTOS VITORIA. New records of Chaetomium and Chaetomium-like species (Ascomycota, Chaetomiaceae) on Syagrus coronata from the Raso da Catarina Ecological Station (ESEC), Caatinga, Bahia, Brazil. 14 p. ABSTRACT—Studies on the mycobiota associated with the plants of family Arecaceae are scarce in Brazil, especially in semiarid ecosystems within the Caatinga domain, which comprises a unique biodiversity. During field expeditions to the Raso da Catarina Ecological Station, we found six new records of Chaetomium and Chaetomium-like species for the Caatinga domain in the State of Bahia. These fungi were colonizing vegetative and reproductive structures of Syagrus coronata (Mart.) Becc., a palm tree endemic to the Caatinga and particularly important for animals and people from this region. We present morphological descriptions, illustrations, comments, and distribution maps for the fungal species associated with S. coronata. KEY worps—Arecaceae, biodiversity, fungi, licuri, semiarid, taxonomy Beltrania shenzhenica sp. nov. (Zhang & al.— Fie. 2, p. 37)