MYCOTAXON THE INTERNATIONAL JOURNAL OF FUNGAL TAXONOMY & NOMENCLATURE VOLUME 135 (3) JULY-SEPTEMBER 2020 Guayaquilia cubensis sp. nov. (Magdama & al. — Fia. 3, p. 509) ISSN (PRINT) 0093-4666 https://doi.org/10.5248/135-3 ISSN (ONLINE) 2154-8889 MYXNAE 135(3): 471-718 (2020) EDITORIAL ADVISORY BOARD KAREN HANSEN (2014-2021), Chair Stockholm, Sweden P. BRANDON MATHENY (2013-2020), Past Chair Knoxville, Tennessee, U.S.A. ELsE C. VELLINGA (2019-2022) Berkeley, California, U.S.A. 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 JULY-SEPTEMBER 2020 VOLUME 135 (3) http://dx.doi.org/10.5248/135-3 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 MyYcoTAxONn, LTD. © 2020 www.mycotaxon.com & www.ingentaconnect.com/content/mtax/mt P.O. BOX 264, ITHACA, NY 14581-0264, USA Iv ... MYCOTAXON 135(3) MYCOTAXON VOLUME ONE HUNDRED THIRTY-FIVE (3) — TABLE OF CONTENTS Nomenclatural novelties & typifications .......... 0000 c cece cece ee ees vii ECVIEW CNS Wag ad te. Maat tA nes of Poise Sood ry rere NSS sdk yet By EN as got Bde area ix COLigeHda cnn oan moat abet, Hie AW RR ee Ne eras id dee Ai ad x BE POU Pde TE ILO ER Stok Path Pes OM alate te Plas ett ol Ain les alan ddahy LA xi ZOZOSUDTTISSIOM PNOCCH UTE S.n:. weal Rasier a Rime N,. crete okie cee. ea xiii TAXONOMY Lepiota punaensis sp. nov. from Hawai‘i Island, and a discussion of L. elaiophylla JEFFERY K. STALLMAN, DON E. HEMMES, NICOLE A. Hynson, MICHAEL H. SHINTAKU 471 Notes on rust fungi in China 8. Pucciniastrum tiliae life cycle and new host plants inferred from phylogenetic evidence JING-XIN JI, ZHUANG Li, Yu L1, MAKOTO KAKISHIMA 490 Guayaquilia gen. nov., typified by Idriella cubensis FREDDY MAGDAMA, DAYNET SOSA, FERNANDO ESPINOZA, LIZETTE SERRANO, SIMON PEREZ-MARTINEZ, ELAINE MALOosso, MARGARITA HERNANDEZ-RESTREPO, RAFAEL F, CASTANEDA-Ru1z 501 Distoseptispora longispora sp. nov. from freshwater habitats in China Hal-YAN SONG, ALY FaraG EL SHEIKHA, ZHI-JUN ZHAI, JIAN-PING ZHOU, Mrinc-Hutr CHEN, GuANG-Hua Huo, X1-GEN Huane, D1An-MinG Hu 513 Notes on rust fungi in China 9. Puccinia miscanthi life cycle and morphology confirmed by inoculation JING-XIN JI, ZHUANG LI, Yu L1, MAKOTO KAKISHIMA 525 Biodiversity of heat-resistant ascomycetes from semi-arid soils in Argentina STELLA MARIS ROMERO, ANDREA IRENE ROMERO, ALBERTO MIGUEL STCHIGEL, ERNESTO RODRIGUEZ ANDRADE, VIVIANA ANDREA BARRERA, JOSE FRANCISCO CANO, RICARDO COMERIO 535 Cortinarius rapaceoides, a new record for Turkey MERYEM SENAY SENGUL DEMIRAK & HAKAN IsIK 559 New records of Toninia from China Concconc M1Ao, MEUIE SUN, XIAO ZHANG, ZHAOJIE REN, LING Hu 569 JULY-SEPTEMBER 2020 ... V Serendipita sacchari sp. nov. from a sugarcane rhizosphere in southern China LinG X1g£, YAN- YAN LONG, YAN ZHANG, YAN-LU CHEN, WEN-LONG ZHANG 579 Five Nolanea spp. nov. from Brazil FERNANDA KARSTEDT, SARAH E. BERGEMANN, MARINA CAPELARI 589 First record of Trappea darkeri from Turkey YASIN UzuN, OSMAN BERBER, ABDULLAH Kaya 613 Mesocorynespora sinensis gen. & sp. nov. from southern China ZHAO-HUuAN Xu, KAI ZHANG, YOU-QIANG Luo, XIU-GUO ZHANG, RAFAEL FE. CASTANEDA-RUiZ, JIAN Ma 617 Corynesporopsis hainanensis sp. nov., a bambusicolous fungus from southern China ZHAO-HuaNn Xu, XuU-GEN SHI, WEI-GANG KUANG, XIU-GUO ZHANG, RAFAEL FE. CASTANEDA-RUiZ, JIAN Ma 623 Hymenochaete longisterigmata sp. nov. from India NAVPREET Kaur, AVNEET PAL SINGH, GURPAUL SINGH DHINGRA 631 Entoloma conferendum, Hygrocybe coccineocrenata, and Hypholoma ericaeum new to Montenegro ILINKA CETKOVIG, ZDENKO TKALCEC, SNEZANA DRAGICEVIC, ANTUN ALEGRO, VEDRAN SEGOTA, MARGITA JADAN, NEVEN MATOCEC, IvANA KuSAN, ZELJKO ZGRABLIC, ARMIN ME81¢ 637 First Pakistani report of Erysiphe betae on the invasive weed Chenopodium ambrosioides AYESHA ANWAR, NAJAM UL SEHAR AFSHAN, AAMNA ISHAQ, MARIA RIAZ, ABDUL NASIR KHALID, SIRAJ UDDIN 649 Crustose lichens new to India Rupyyoti GoGo, SILJo JOSEPH, Manas PRATIM CHOUDHURY, SANJEEVA NAYAKA, FARISHTA YASMIN 657 Chromelosporium re-evaluated, with Chromelosporiopsis gen. nov. and Geohypha stat. nov. GREGOIRE L. HENNEBERT 665 VI ... MYCOTAXON 135(3) PUBLICATION DATE FOR VOLUME ONE HUNDRED THIRTY-FIVE (2) MYCOTAXON for APRIL-JUNE 2020 (I-XIV + 235-471) was issued on July 13, 2020 JULY-SEPTEMBER 2020 ... NOMENCLATURAL NOVELTIES AND TYPIFICATIONS PROPOSED IN MYCOTAXON 135(3) Botrytis isabellina Preuss (lectotypified) [MBT 392406], p. 671 Chromelosporium herbicola (Ellis & Dearn.) Hennebert [MB 833990], p.673 Chromelosporiopsis Hennebert [MB 835622], p. 688 Chromelosporiopsis carnea (Schumach.) Hennebert [MB 835623], p. 689 Chromelosporiopsis coerulescens (Bonord.) Hennebert [MB 835625], p. 695 Corynesporopsis hainanensis Z.H. Xu, Jian Ma, X.G. Zhang & R.F. Castaneda [MB 836568], p. 624 Distoseptispora longispora H.Y. Song & D.M. Hu [MB 826916], p. 516 Geohypha (Fr.) Hennebert [MB 835628], p. 708 Geohypha terrestris (Fr.) Hennebert [MB 835629], p. 709 Guayaquilia R.F. Castafieda, Magdama, D. Sosa & Hern.-Restr. [MB 831849], p. 506 Guayaquilia cubensis (R.F. Castaheda & G.R.W. Arnold) R.F. Castaneda, Magdama, D. Sosa & Hern.Restr. [MB 831851], p. 507 Hymenochaete longisterigmata Nav. Kaur, Avn.P. Singh & Dhingra [MB 832678], p. 632 Lepiota punaensis Stallman [MB 832104], p. 477 Mesocorynespora Jian Ma, X.G. Zhang & R.E. Castaneda [MB 836511], p. 618 Mesocorynespora sinensis Jian Ma, X.G. Zhang & R.E Castaneda [MB 836512], p. 618 Nolanea albertinae Karstedt & Capelari [MB 826784], p. 596 Nolanea atropapillata Karstedt & Capelari [MB 805603], p. 600 Nolanea pallidosalmonea Karstedt & Capelari [MB 805604], p. 602 VII Vill ... MYCOTAXON 135(3) Nolanea parvispora Karstedt & Capelari [MB 805605], p. 603 Nolanea tricholomatoidea Karstedt & Capelari [MB 805607], p. 606 Polyactis carnea Ehrenb. (lectotypified) [MBT 392408], p. 689 Puccinia miscanthi Miura (neotypified) [MBT 388544], p. 531 Serendipita sacchari L. Xie, Y.Y. Long & Y.L. Chen [MB 836761], p. 582 Trichoderma laeve Pers. (lectotypified) [MBT 392413], p. 709 JULY-SEPTEMBER 2020 ... REVIEWERS — VOLUME ONE HUNDRED THIRTY-FIVE (3) 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. Vladimir Antonin Flavia Rodrigues Barbosa Timothy J. Baroni Maria Virginia Bianchinotti Uwe Braun Rafael E Castafieda-Ruiz Cvetomir M. Denchev Edit Farkas Patricia Oliveira Fiuza André Fraiture Shouyu Guo Nils Hallenberg David L. Hawksworth R.G.U. Jayalal Abdullah Kaya Ali Keles De-Wei Li Xiao- Yong Liu Jian Ma Eric H.C. McKenzie Andrew N. Miller A.R. Niazi Lorelei L. Norvell Shaun R. Pennycook Brian A Perry Keith Seifert B.M. Sharma Silvana Santos da Silva G.P. Sinha Ibrahim Tirkekul Yusuf Uzun Alfredo Vizzini Felipe Wartchow Jiwen Xia IX X ... MYCOTAXON 135(3) CORRIGENDA FOR MYCOTAXON 135-(3) Cited below are mistakes present in files submitted for PDF conversion in the current issue but not detected by the authors until after the PDF had been processed. p. 495, line 10 FOR: A-D READ: 5A-D p. 496, line 13 FOR: Helongjiang READ: Heilongjiang p. 531, line 8 FOR: Helongjiang READ: Heilongjiang p#533,-lines3 FoR: Wuhan READ: Wuchang JULY-SEPTEMBER 2020 ... XI FROM THE EDITOR-IN-CHIEF WHO ‘AUTHORS’ SCIENCE?—Fungal research is now so specialized that the single- authored paper has become a rare beast indeed. Modern mycological systematics now rely on contributions from masters from several different disciplines: alpha taxonomists and collectors well versed in morphological identification, ecologists able to link environmental requirements and taxa, physiologists and “beta” taxonomists who craft diagnostic matrices from colonies in Petri dishes, “gene jockeys” who reliably deliver pristine sequences from targeted DNA regions, and evolutionary biologists wielding Bayesian analysis and Markov chain Monte Carlo methods with morphological analytics to generate elegant and reliable phylogenies. To these may be added those who summarize, analyze, and wrap everything up in a research paper—the authors. In multi-authored papers, there is usually one individual who “drives” the paper, although sometimes two authors may be said to “contribute equally” The first-named (also called “senior”) authors should have participated in the research since its inception so that they understand all aspects well enough to create a clear and comprehensible presentation of their research. Unfortunately lists begin with one name, and often the author with the best English writing abilities ascends to first position by default, irrespective of the number of scientific contributions made during research. Alternately, the most fluent English speakers who are designated corresponding authors may appear anywhere in the lists. There is also the trend toward ‘anchoring’ authorship by placing the Lab supervisor in last place; here we devoutly hope that the lab head has read the paper in its entirety and corrected mistakes well before journal submission. Your editors recognize the problems encountered by contributing scientists in deciding which contributors to include as authors and which to recognize in the Acknowledgments. We definitely expect all authors to gather frequently to discuss the research while in progress, when being written, and when being prepared for submission. These discussions are true teaching moments, and authors who have eliminated mistakes during these stringent vetting processes will have much better success in publishing their science. MycoTaxon ART—Your editors are unusually fortunate to be able to present on our cover such beautiful art created by authors to illustrate their own research. The illustrations featured on our covers are selected from papers proposing taxa new to science. However, there are some truly spectacular plates in this issue accompanying previously named taxa that “escaped” cover selection. Part of the pleasure of opening a new Mycotaxon is leafing through the beautiful fungal portraits lurking inside. MYCOTAXON 135(3) contains 18 papers by 92 authors (representing 17 countries) as revised by 32 expert reviewers and the editors. The 2020 July-September MycoTAxon proposes FOUR new genera (Chromelo- sporiopsis, Geohypha, Guayaquilia, and Mesocorynespora) and TEN species new XII ... MYCOTAXON 135(3) to science representing Corynesporopsis, Distoseptispora, Mesocorynespora, and Serendipita from Cuina; Hymenochaete from INp1a; Lepiota from Hawai‘i (U.S.A.); and Nolanea from BraAziL. We also offer FIVE new combinations in Chromelosporium, Chromelosporiopsis, Geohypha, and Guayaquilia and FouR newly registered typifications for Botrytis, Polyactis, Puccinia, and Trichoderma. New species range extensions are reported for [ascomycetes] five heat resistant species in an elegant culture study from ARGENTINA; [basidiomycetes] Cortinarius & Trappea in TuRKEY and Entoloma, Hygrocybe, and Hypholoma in MONTENEGRO; and [lichens] Bacidia, Malmidea, Porina, and Pyrenula in Inv1a and Toninia in CHINA. New hosts are cited for Erysiphe in PAKISTAN and Pucciniastrum in China. We also offer keys to species of Lepiota in Hawati‘l and Toninia s.l. in CuH1Na along with worldwide keys to Corynesporopsis, Distoseptispora, and Mesocorynespora. The July-September issue now offers more molecularly supported taxonomic studies: nine new species representing Distoseptispora, Guayaquilia, Lepiota, Nolanea, and Serendipita), one Cortinarius range extension, an Erysiphe host association, and a full life cycle of the rust Pucciniastrum tiliae are all supported by phylogenetic analyses, while another life cycle is established for Puccinia miscanthi via culture studies. Alpha taxonomy is not dead, particularly when supported by a lifetime of close microscopical scrutiny and systematic research. The issue ends with “my final paper” by our co-founding editor, GREGOIRE HENNEBERT, who presents a masterful synopsis of a difficult (and often overlooked) group of fungi and concluding with a warm memorial of Agriculture Canada’s irreplaceable Stan Hughes, who died last year at the age of 102. Warm regards, Lorelei L. Norvell (Editor-in-Chief) 9 October 2020 JULY-SEPTEMBER 2020... XIII 2020 MyCOTAXON SUBMISSION PROCEDURE Prospective MycotTaxon authors should download the Mycotaxon 2020 guide, review & submission forms, and MycoTaxon sample manuscript by clicking the ‘file download page’ link on our INSTRUCTIONS TO AUTHORS page before preparing their manuscript. 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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 MycorTaxon 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. MYCOTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 July-September 2020—Volume 135, pp. 471-489 https://doi.org/10.5248/135.471 Lepiota punaensis sp. nov. from Hawai'i Island, and a discussion of L. elaiophylla JEFFERY K. STALLMAN’, DON E. HEMMEs?’, NICOLE A. HyNSON?, MICHAEL H. SHINTAKU* ™ Department of Tropical Conservation Biology & Environmental Science Graduate Program, University of Hawai‘i Hilo, Hilo, Hawai‘i 96720 USA ? Department of Biology, University of Hawai‘i Hilo, Hilo, Hawai‘i 96720 USA ° Pacific Biosciences Research Center, University of Hawai‘i Manoa, Honolulu, HI 96822 USA * College of Agriculture, Forestry, and Natural Resource Management and Department of Tropical Conservation Biology & Environmental Science Graduate Program, University of Hawai‘i Hilo, Hilo, Hawai‘i 96720 USA *CORRESPONDENCE TO: jstall@hawaii.edu ABSTRACT—A new species, Lepiota punaensis, in L. subsect. Helveolinae is described from the Puna District of Hawai‘i Island. A species closely resembling L. elaiophylla in morphology but genetically distant from the species paratype is also discussed. Both taxa and additional Hawaiian collections are analyzed in an nrITS phylogeny, and a key to Lepiota on Hawai‘i Island is given. KEY worps—Agaricaceae, Casuarina equisetifolia, cryptic species Introduction Lepiota (Pers.) Gray is a large genus of more than 400 species (Kirk & al. 2008) with widespread distribution that continues to see new species described regularly from temperate (Caballero & al. 2015), subtropical (Qasim & al. 2015), and tropical (Sysouphanthong & al. 2016) environments. Exceptions occur, but in general the genus is characterized macroscopically by white to yellow-spored agarics with free gills, evidence of partial and universal veils, but lacking a volva. Microscopically the lamellar trama are regular, and 472 ... Stallman & al. basidiospores are thin-walled, without a germ pore, generally dextrinoid, and not metachromatic in Cresyl Blue. Lepiota has historically been split into sections based on morphology (e.g., Bon 1993), and more recently, into clades based on molecular data (Vellinga 2003). Lepiota subsect. Helveolinae Bon & Boiffard [in L. sect. Ovisporae (J.E. Lange) Kuhner], which contains the two species closely examined in this manuscript, is characterized morphologically by ellipsoid to oblong spores with a pileus covering composed of a trichoderm of predominantly long cylindrical elements and has been consistently recovered as monophyletic in molecular analyses of Lepiota and its infrageneric sections (Vellinga 2003; Qasim & al. 2015, 2016; Sysouphanthong & al. 2012, 2016; Liang & al. 2018; but see Liang & al. 2011). This clade contains species such as Lepiota subincarnata J.E. Lange and L. brunneoincarnata Chodat & C. Martin, which are known to contain amatoxins (Benjamin 1995). Our project documenting the lepiotaceous fungi from Hawai‘i Island reports a new taxon from L. subsect. Helveolinae, Lepiota punaensis, and a new record for Hawai‘i, Lepiota cf. elaiophylla. In addition, Lepiota aspera (Pers.) Quél., Lepiota besseyi H.V. Sm. & N.S. Weber, Lepiota pseudorubella Gubitz, Lepiota rubrobrunnea Gubitz, and a poorly known taxon with an epithelial pileus covering (MK412575) are confirmed as occurring on Hawai‘i Island. All species are found in wet, lowland environments associated with alien vegetation and are therefore presumed to be introduced. Materials & methods Collection and morphologic description Basidiome collecting occurred from September 2016 to January 2019 on Hawai‘i Island. Photographs were taken in the field with a Canon Rebel T4i camera and have not been altered (PLATES 2a; 3a). Morphological details were recorded at the time of collection. Descriptions of Hawaiian habitats and vegetation zones are from Gagné & Cuddihy (1999). Macroscopic descriptions are based on terminology from Vellinga (2001) and color descriptions are from Kornerup & Wanscher (1978). After collection, specimens were desiccated before microscopic and molecular analysis, and are deposited at the Joseph F. Rock Herbarium, University of Hawaii at Manoa, Honolulu, HI, USA (HAW). Measurement of microscopic structures was performed on material rehydrated in 10% ammonia (pileus and stipe covering, spores), or 10% ammonia with 1% Congo Red (all other structures). Melzer’s reagent, distilled water, and 1% Cresyl Blue in distilled water were also used to observe natural colors and color- changing reactions. Mature spores were measured in side view (excluding the hilar appendage) using the program Piximétre version 5.9 (http://ach.log.free. fr/Piximetre/). Measurements represent values falling within a 95% confidence Lepiota punaensis sp. nov. (Hawai'i USA) ... 473 interval; the annotation [30, 4, 3] indicates that thirty spores from four specimens in three collections were measured. Micrographs for import to Piximétre were captured using an Olympus XM10 microscope camera attachment on an Olympus BX53 microscope with the software cell-sens Dimension version 1.5. Molecular analysis The primers ITSIF and ITS4 were used to amplify the nuclear ribosomal internal transcribed spacer region (nrITS; Schoch & al. 2012) via the polymerase chain reaction from dried fungal tissue of eleven specimens from Hawai‘i Island. Amplicons were Sanger sequenced in both forward and reverse directions using the primers ITSIF and ITS4, and sequences were trimmed, edited, and forward and reverse reads aligned into consensus sequences in Geneious v. 9.1.8 (Kearse & al. 2012). Sequences generated were further investigated using the BLAST tool in GenBank (NCBI 2015), and the top sequences by similarity to Hawaiian taxa, as well TABLE 1. Sequences newly generated and downloaded from GenBank for phylogenetic analysis. TAXON (COLLECTION #) GENBANK # TAXON (COLLECTION #) GENBANK # C. hetieri AY176459 L. luteophylla AY176475 C. pulverulenta AF391035 L. magnispora AF391005 C. seminuda JF907983 L. pilodes AY 176476 C. sp. (Hawai‘i) (JKS 143) MK412600 L. punaensis (JKS 64) MK412572 C. sp. (Hawai‘i) (JKS 140) MK412604 L. punaensis (JKS 86) MK412577 Ch. molybdites (JKS 96) MK412591 L. punaensis (JKS 87) MK412578 L. andegavensis KP004931 L. rubrobrunnea (JKS 118) MK412583 L. apatelia AY176462 L. rhodophylla AY176480 L. aspera KP843884 L. sp. (O‘ahu) (SS 1) MK412617 L. aspera MK412598 L. sp. (Australia) KP012693 L. brunneoincarnata FJ998395 L. sp. (Australia) KP012854 L. castanea AY176463 L. sp. (Hawai‘i) (JKS 74) MK412575 L. castaneidisca AF391061 L. sp. (Kaua‘i) AY176402 L. cristata U85327 L. sp. (Thailand) HQ647293 L. cf. elaiophylla MK412581 L. sp. (Thailand) JN224825 L. cf. elaiophylla (JKS 71) MK412589 L. sp. (Thailand) JN224826 L. cf. elaiophylla (JKS 77) MK412588 L. sp. (Thailand) JN224828 L. elaiophylla MH979467 L. subincarnata AY176491 L. elaiophylla AF391024 L. thiersii AY176492 L. erminea AY176470 L. tomentella EF080868 L. farinolens AY176368 L. vellingana HE974764 L. felina U85330 L. xanthophylla AY176405 L. helveola MH979466 M. haematospermum KF953545 L. himalayensis HE614898 Uncultured fungus (Australia) FJ528742 Sequences with collection numbers were newly generated; Hawaiian sequences are in bold. C. = Cystolepiota, Ch. = Chlorophyllum, L. = Lepiota, and M. = Melanophyllum. 474. ... Stallman & al. as representatives from each of the clades of Lepiota identified in Vellinga (2003) were included in our phylogenetic analyses (TaBLE 1). Pairwise genetic distances (TABLE 2) were calculated in Geneious by comparing aligned, overlapping regions, excluding gaps and ambiguous positions. Lepiota besseyi was excluded from our phylogenetic analysis and key to Lepiota because it falls outside of Lepiota in the Leucocoprinus Pat. and Leucoagaricus Locq. ex Singer clade in Agaricaceae Chevall., although a formal transfer has not been made (Vellinga & al. 2011). TABLE 2. Pairwise genetic distance comparison between Lepiota cf. elaiophylla and closest Lepiota relatives in GenBank. elaiophylla elaiophylla cf. elaiophylla cf. elaiophylla | cf. elaiophylla (Canada) (Netherlands) (Hawai‘i) (Hawai‘i) (Hawai‘i) MH979467 AF391024 MK412581 MK412581 MK412588 elaiophylla (Netherlands) 92.9% AF391024 cf. elaiophylla (Hawai‘i) 99.9% 92.9% MK412581 cf. elaiophylla (Hawai ‘i) 99.9% 92.9% 99.9% MK412589 cf. elaiophylla (Hawai ‘i) 99.9% 92.9% 99.9% 99.9% MK412581 L. sp. (Australia) 98.3% 93.2% 98.3% 98.3% 98.3% KP012854 Sequences were aligned in Geneious with the default settings of the MAFFT (Katoh & Standley 2013) plugin, then imported to Molecular Evolutionary Genetics Analysis (MEGA) version X (Kumar & al. 2018). Based on the “Find Best DNA Model” tool in MEGA, Maximum Likelihood (ML) phylogenetic trees were created with all sites with gaps and/or missing data eliminated resulting in 465 total positions in the final dataset using the following settings: General Time Reversible (GTR) model (Nei & Kumar 2000) including gamma distributed (G) and invariant sites (I), moderate branch swap filter, and Subtree-Pruning-Regrafting (Extensive, level 5) for the ML heuristic method. The initial tree was built using the default neighbor-joining/bioneighbor-joining method, and the phylogeny was tested with 1000 bootstrap replicates. The alignment was further analyzed using the Geneious plugin MrBayes (Huelsenbeck & Ronquist 2001). The tree was constructed with a GTR model including G and I sites with default settings (four chains for 1,100,000 generations, sampled every 200 generations with the first 100,000 generations discarded as burn- in). Tree images were edited in Inkscape version 3 (https://inkscape.org/). Lepiota punaensis sp. nov. (Hawai'i USA) ... 475 Results & discussion Our Bayesian and ML phylogenetic analyses generated identical tree topologies when considering our ML tree with the highest log likelihood (-4599.22) shown in Fic. 1. The topology of our Lepiota phylogeny is similar to prior analyses from the last 16 years and does not differ greatly from Vellinga’s (2003) work using nrITS and nrLSU sequences, Qasim & al’s (2015; 2016) works using nrITS sequences, Sysouphanthong & al’s (2012; 2016) works using nrITS sequences, and Liang & al’s (2018) work using nrITS, nrLSU, IGS, and mtSSU sequences (but see Liang & al. 2011 using nrITS, nrLSU, IGS, and mtSSU sequences where species with a trichoderm of long elements in clade 2 were dispersed). Bootstrap support values for clades 2 and 4 (as defined by Vellinga 2003) are too low to draw general conclusions about the deeper relationships of our Hawaiian taxa within the genus, but our species clusters do reveal interesting information about close relatives of Hawaiian Lepiota s.l. species. Lepiota punaensis is always on a long branch by itself with the closest nrITS match in GenBank an 87% similar Australian sequence (KP012854). Lepiota punaensis is sister to the cluster of species from Hawai‘i, Canada, Australia, and Europe, all of which (except the Australian collection) are morphologically identified as L. elaiophylla. The Hawaiian L. cf. elaiophylla is genetically identical (99.9% similar, TABLE 2) to a collection identified as L. elaiophylla from a greenhouse in Canada, close to (98.3% similar, TABLE 2) a collection from a native Eucalyptus and Acacia swamp in Australia (M. Barrett, pers. comm.); these three geographically separated collections form a clade sister to the paratype of L. elaiophylla from the Netherlands, where the species was originally described. The collection from the Netherlands is 92.9% similar to the Hawaiian collections based on our alignment (TABLE 2). Regarding other Hawaiian collections with a trichoderm of predominantly long elements, L. rubrobrunnea is on a long branch in a poorly supported group and its closest relatives are unclear. A Lepiota sp. collected on the UH Manoa Campus on O‘ahu clusters in a well-supported clade with L. vellingana Nawaz & Khalid, currently known from Pakistan (Nawaz & al. 2013), and L. farinolens Bon & G. Riousset, from France (Bon 1992). A species collected on the Island of Kaua‘i clusters in a well-supported clade with three taxa from Thailand and L. subincarnata, a species known from Europe, North America, and South Asia (Razaq & al. 2014). It may be conspecific with sequence JN224826, a species collected in Chiang Mai Province in 2008 that, according to notes in GenBank, is “close to L. subincarnata? 476 ... Stallman & al. . Cf. mg a A MK412589 . cf. elaiophylla MK412588 . cf. elalophy lla MK412581 ees a {Canada MH979467 oat (Australlia) KP012854 ‘elaiopnytia (Netherlands) AF391024 L. punaensis MK412577 99/10] [ punaensis MK412578 L. punaensis MK412572 99/1.0 rae sp. (Kaua‘i) AY176402 L. sp. AM ) JN224826 subincarnata AE. 76491 sp. (Thailand) JN224825 73/1.0 © sp. (Thailand) JN224828 L. brunneoincarnata FJ998395 1.0 L. farinolens AY 176368 oa vellingana HE974764 6/0.97 98/1.0 (0 op O‘ahu) MK412617 L. helveola mriosba L. himalayensis HE614898 rubrobrunnea MK412583 86/0.99-— | . [reaopril. AY 176480 © Q oO oO oO clade 2 trichoderm of long elements o6it10 Thailand) HQ647293 Bo L. andegavensis KP004931 + 2 89/1.0 L. castanea AY176463 Sie, eu L. pilodes AY 176476 oa L. tomentella EF080868 ods L. castaneidisca AF391061 L. cristata U85327 95/1.0 L. apatelia AY176462 L. thiersti AY176492 99/1.0 L. erminea AY176470 aanof— L. magnispora AF391005 *4 L. felina U85330 L. xanthophylla AY176405 L. sp. (Hawai‘i) MK412575 99/1.0; C. sp. (Hawai‘i) MK412604 ano C. sp. (Hawai‘i) MK412600 99/10 Uncultured Fungus (Australia) FJ F 1528742 sp. (Australia) KP012693 C. pulverulenta AF391035 M. haematospermum KF953545 zo C. seminuda JF907983 C. hetieri AY176459 99/1.0, L. aspera MK412598 L. aspera KP843884 L. cf. luteophylla AY176475 Ch. molybdites MK412591 | outgroup 0.03 s & e) clade 1 clade 4 Fic. 1. Phylogenetic analysis of selected sequences and species of Lepiota based on nrITS sequences. The phylogeny was inferred using the Maximum Likelihood method and is drawn to scale, with branch lengths measured in the number of substitutions per site. Maximum Likelihood bootstrap support values >70% are shown in bold, and Bayesian posterior probabilities 20.95 are shown in normal type. Hawaiian taxa are in bold, and islands are indicated for taxa not identified to species. C. = Cystolepiota, Ch. = Chlorophyllum, L. = Lepiota, and M. = Melanophyllum. The Hawaiian Cystolepiota species clusters in a well-supported clade with two unknown taxa from Australia, and these are sister to Cystolepiota pulverulenta (Huijsman) Vellinga [= C. petasiformis (Murrill) Vellinga], a purportedly widespread species found in Europe (Vellinga 2001), western North America (Vellinga 2006), and India (Kumar & Manimohan 2009). The closest Australian sequence (FJ528742) is 96% similar to the Hawaiian Lepiota punaensis sp. nov. (Hawai'i USA) ... 477 Cystolepiota species, and its lack of any closer matches in GenBank and association with native vegetation supports the inference that it is a Hawaiian endemic (Hemmes & Desjardin 2002). A poorly known Lepiota (MK412575) that was only collected once in this study is on its own long branch in clade 1 (with only 74% support), and its closest relatives are unclear. There are few data at this time to support broader conclusions about the origins and full ranges of Hawaiian Lepiota s.l. taxa. Lepiota pseudorubella lacks molecular data, L. rubrobrunnea and L. sp. (MK412575) have uncertain placement in our tree due to low support values, and L. sp. (MK412617) is on a relatively long branch in a clade with dispersed taxa. Lepiota aspera and Melanophyllum haematospermum (Bull.) Kreisel (for which no Hawaiian sequences are available) have widespread distributions, and L. pseudorubella, L. rubrobrunnea, and L. cf. elaiophylla have all been collected in artificially warm environments in temperate areas such as Canada and Germany (Gubitz 2008). The only putatively native Hawaiian taxon, Cystolepiota sp., is likely of Australian origin, while a collection from Kaua‘i (AY176402) clusters with several species from Thailand. Lepiota punaensis and L. cf. elaiophylla have a close relative found in native Australian vegetation, but this is far from conclusive in establishing a location of origin or theory of dispersal. Taxonomy Lepiota punaensis Stallman, sp. nov. PLATE 2 MB 832104 Differs from Lepiota elaiophylla by its white lamellae and from L. subincarnata by its pileus covering containing short and narrowly clavate cells. Type: USA, Hawai‘i, Hawai‘i Island, Puna District, MacKenzie State Recreation Area, 19°26’11”N 154°51’57”W, in duff under Casuarina equisetifolia L. and Cocos nucifera L., 9 Dec 2016, coll. JKS 86 (Holotype, HAW-F-00256, GenBank: MK412577). ETYMOLOGY: from the Puna District of Hawai‘i Island, the only known locality of this species. PrLEus 8-24 mm broad, at first hemispherical, becoming obtusely conical to obtusely broadly conical, then convex to plano-convex in age; pileus covered in a dense mat of warm brown (6E6 to 7E4-5; occasionally 7F6) fibrils when young, quickly breaking into minute to small scales, composed of generally appressed aggregated fibrils, over a white to off-white (6B2) background as pileus expands, fibrils denser towards the cracked, or remaining intact disc surface, lessening towards margin, margin aspect straight, shape entire to eroded in age, often with remnants of the pileus covering adhering when young, dry, non-hygrophanous; CONTEXT thin (<3 mm), white, unchanging; 478 ... Stallman & al. LAMELLAE, L = 36,1 = 1-3, free, close, subventricose to ventricose, <3 mm broad, white to off-white, edge even to eroded in age; stiPE 14-35 x 1-3 (-4 at base) mm, cylindrical, equal to tapering upwards, off-white to light brown (6B2, 8C2), covered with aggregated fibrils of similar appearance to pileus covering on lower 1/2 to 1/3 of stipe that are easily removed and become less conspicuous in age, appearing glabrous above this zone, but usually with thin covering of white hyphal strands oriented parallel to the stipe (lens), also easy to remove; stipe darkening over time to brown (6F5) where handled; ConTExT thin, concolorous with exterior of stipe, stuffed with white hyphae when young, hollow in age, base equal to slightly enlarged; BASAL TOMENTUM absent to inconspicuous; RHIZOMORPHS absent or present, white; ANNULUS present only as annular zone; SPORE DEPOSIT white; EDIBILITY toxic, likely contains amatoxins; ODOR none to fungal; TASTE none to mildly fungal. BasIDIOSPORES [94, 6, 4] (5.4-)6.3-6.6(-7.5) x (3.2-)3.7-3.8(-4.4) um [x= 6.4 x 3.8 um], Q=(1.4—)1.6-1.8(-2.0) [Qm = 1.7], ellipsoid to subcylindrical (generally oblong) in side view, usually with a straight adaxial (suprahilar depression absent) and convex abaxial side; oblong to rarely obovoid in frontal view, hyaline in H,O and NH,OH, dextrinoid, congophilous, weakly cyanophilous, non-metachromatic in Cresyl Blue, germ pore absent, mono to less commonly biguttulate; BAsIp1A [63, 6, 4] (21.2-)25.6-33.3(-34.7) x (5.9-) 6.5-8(-8.3) um [x = 28.9 x 7.2 um], hyaline, thin-walled, narrowly clavate to subclavate, sometimes with flexuous wall, with 4 sterigmata; LAMELLAR-EDGE sterile; CHEILOCYSTIDIA [44, 6, 4] (15.6—)22.3-24.6(-31.3) x (5.1-)8.9-10.2 (-14.0) um [x = 23.5 x 9.5 um], narrowly clavate to more commonly clavate or broadly clavate, hyaline, thin-walled; PILEUS COVERING an intricate trichoderm of long and shorter inflated cylindrical, sometimes flexuous, golden-colored hyphae with parietal pigment, thin to slightly thick-walled, shorter elements are present and common, but not in a distinctive layer, terminal elements narrowly clavate, cylindrical, inflated cylindrical, or less commonly irregular/flexuose narrowly clavate, or with a mucronate tip, <160 um long and 12 um diam, clamp connections common; STIPITIPELLIS a cutis of tightly packed hyaline, narrowly cylindrical hyphae <5 um diam, scales below annular zone similar to those on pileus but generally composed of shorter elements, above annular zone irregularly present and mostly composed of long, narrowly cylindrical, hyaline, clamped hyphae; ANNULUS as zone only; CLAMP CONNECTIONS present in all tissues. ADDITIONAL SPECIMENS EXAMINED—USA, Hawai‘i, Hawai‘i Island, Puna District, MacKenzie State Recreation Area, 19°26’11”N 154°51’57”W, 17 Sep 2016, coll. JKS Lepiota punaensis sp. nov. (Hawai'i USA) ... 479 20 um PLATE. 2. Lepiota punaensis (holotype, HAW-F-00256). a. Basidiomata; b. Basidiospores; c. Basidia (with four sterigmata; only two shown in this basidial view); d. Cheilocystidia; e. Stipe covering; f. Pileus covering. 64 (HAW-F-00257; GenBank MK412572); 5 Jan 2017, coll. JKS 87 (HAW-F-00258; GenBank: MK412578); 1 February 2018, coll. JKS 186 (HAW-F-00255); 2 January 2019, coll. JKS 204 (HAW-F-00259). 480 ... Stallman & al. ECOLOGY & DISTRIBUTION — Solitary to scattered on needles and seed pods of Casuarina equisetifolia in wet, lowland, alien environments. January, February, September, and December. Currently known only from the Puna District of Hawai'i Island. CoMMENTS—Lepiota punaensis is known only from Casuarina equisetifolia duff in the Puna District of Hawai‘i Island. The species is recognized by its small size, lack of an annulus, and small, appressed brown fibrils over an off-white cap context. Its oblong spores and pileus covering of long elements without a layer of shorter clavate elements (although shorter elements are present) place this species in L. subsect. Helveolinae with other species that contain amatoxins. Although L. punaensis is a small, relatively nondescript species, few described species provide similar morphological matches, and its nrITS sequence is unique. Recent studies from around the world such as Thailand (Sysouphanthong & al. 2012), China (e.g., Liang & al. 2018), Pakistan (Qasim & al. 2016), and the Dominican Republic (Justo & al. 2015) have described new Lepiota species from the (neo)tropics, and have included DNA sequence data that excludes any species from these studies, or other studies incorporating nrITS sequence data, from providing a match to L. punaensis. Older literature can be more difficult to interpret, but a review of species sharing similarities, and newly described species without molecular data also do not reveal a close match. Lepiota zalkavritha T.K.A. Kumar & Manim. is a species of small size in L. sect. Ovisporae described from India with no nrITS sequence (Kumar & Manimohan 2009). Lepiota zalkavritha differs from L. punaensis in having occasional apical outgrowths on its cheilocystidia, being more robust, having darker brown coloration on pileus and stipe, and growth in soil (compared to Casuarina equisetifolia duff). Lepiota brevipes Murrill is a small species described from Florida in L. sect. Ovisporae with similar coloration to L. punaensis, but the stipe is often much wider, and the spores are smaller at 4.1-5 x 2.5-3.1 um (Akers 1997). Lepiota cinnamomea Cleland is a small species from Australia in L. sect. Ovisporae (Aberdeen 1992), but the pinkish-brown and dark-brown color descriptions of the scales and disc, and shorter elements in the pileus covering (only <68 um long) rule it out. Other small species described from Australia and stated to be in L. sect. Ovisporae (Grgurinovic 1998) have annuli (e.g., L. rimosa Murrill) or color-changing reactions not present in L. punaensis. Lepiota punaensis sp. nov. (Hawai'i USA) ... 481 Pegler’s work from Ceylon (1972) reveals species in L. sect. Ovisporae that are much larger, have unique red or yellow colors, or have annuli that do not match L. punaensis, and his works from Africa (1977) and the Caribbean (1983) do not reveal close matches. Rick’s (1905, 1907, 1920, 1926, 1930, 1937, 1938, 1961) works from South America present few good matches for L. punaensis and Pereira’ (1998, 2000, 2001) present none. Rick does describe a variety of Lepiota species with arachnoid veils (e.g., L. flavipes Rick and L. hypholoma Rick), but other features such as the coloration or spore sizes are different. The closest morphological match appears to be Lepiota subincarnata, a well-known species described from Europe that is also found in North America and Asia (Razaq & al. 2014). Lepiota punaensis differs in having a typically smaller pileus and containing pileus covering elements that can have flexuose walls, be narrowly clavate, and contain shorter elements (although not in a distinctive layer), all of which L. subincarnata lacks. Lepiota cf. elaiophylla Vellinga & Huijser PLATE 3 Piteus (15-)25-40(-50) mm broad, at first paraboloid to narrowly hemispherical, then convex, expanding to plano-convex or applanate in age, completely covered in warm brown to dark brown (7F5, 8F4, 8F6, 8F8, 9F5) fibrils when young, quickly breaking up as the pileus expands into small to medium, generally appressed scales of aggregated fibrils over a dull yellow (3A4, 3B5, 4A3, 4A2) background; fibrils concentrated at disc (sometimes remaining unbroken) and gradually lessening towards margin to reveal more of surface; margin aspect straight, shape entire to eroded in age, sometimes with medium to large remnants of pileus covering adhering to margin, non-hygrophanous, dry; CONTEXT <5 mm thick, yellow (3A3, 3A4, 4B4, 4A3) fading to brown or reddish brown over time; LAMELLAE L = 32,1 = 3 (usually), free, ventricose, up to 9 mm broad, dull yellow to sulphur yellow (3B6, 3B4), sometimes darkening in age (3A4, 4B4, 4C5); stripe 20-40 x 3-7 mm, cylindrical (although sometimes flattening near the pileus) and equal (rarely tapering downwards), yellow (3A3, 3B4, 3A4, 4B3, 4A2, 3B4), with a clear annular zone beginning *% to % from the bottom of the stipe, above which the stipe appears glabrous, but remnants of the white, cortina-like partial veil generally remain, uncommonly aggregating into larger visible structures, below covered in medium to large, horizontally- oriented brown scales, generally appressed, but more uplifted than those on the pileus, concolorous with those on the pileus; CONTEXT fleshy-fibrous, color as in pileus or slightly lighter; when young solid to stuffed with translucent to 482 ... Stallman & al. white hyphae, hollow in age; RHIZOMORPHS present, white; BASAL TOMENTUM generally present; SPORE DEPOSIT white; EDIBILITY toxic, likely contains amatoxins; ODOR none to mildly fungal; TAsTE none to mild. BASIDIOSPORES [150, 7, 5] (5.8-)7.0-7.2(-8.4) x (3.3-)3.7-3.8(-4.2) um [x = 7.1-3.8 um], Q = (1.5-)1.9(-2.3) [Qm = 1.9], narrowly ellipsoid to subcylindrical (usually oblong) in side view with a convex abaxial side and straighter adaxial side (mild suprahilar depression rarely present), oblong to subcylindrical in face view, hyaline in H,O and NH,OH, dextrinoid, congophilous, weakly cyanophilic to cyanophilic and non-metachromatic in Cresyl Blue, germ pore absent, mono- or (less commonly) bi-guttulate; BAsIDIA [57,7,5] (20.6-)23.1-28.7(-34.6) x (5.7-)7.2-8.8(-9.2) um [x = 26 x 8 um], narrowly clavate to clavate, hyaline, thin-walled, 4-spored, rarely 2-spored; LAMELLAR-EDGE Sterile; CHEILOCYSTIDIA [39, 5, 4] (15.5-)20.1-30.1(-39.3) x (5.2-)6.1-9.8(-10.3) um [x = 24.5 x 7.9 um], generally narrowly clavate to clavate, but also cylindrical and somewhat flexuous, sometimes with an apical extension, narrowly utriform, or less commonly subconical, often septate with one to two short, cylindrical basal cells, with or without clamp connections, hyaline, thin-walled; PLEUROCYSTIDIA absent; PILEUS COVERING an intricate trichoderm of mostly long erect and repent, generally cylindrical hyphae with brown intracellular and parietal pigments, lacking a layer of short clavate elements, terminal cells 25-240 x 4-15 um, most commonly 60-100 um in length, inflated cylindrical to narrowly clavate, often flexuous; STIPITIPELLIS a cutis of narrowly cylindrical hyaline hyphae 3-7 um diam with stipe scales below annular zone similar to scales on pileus, above this zone a covering of thin, hyaline, cylindrical, often flexuous hyphae up to 4 um diam and 100 um long; ANNULUS as a cortinoid zone only; rarely with stipe covering elements aggregating to create a medial to superior raised region resembling an adhering “ring”; CLAMP CONNECTIONS present in all tissues; may be absent in cheilocystidia. SPECIMENS EXAMINED—USA, Hawai‘i, Hawai‘i Island, Hilo District, University of Hawai‘i Hilo Botanical Garden, 19°42’10”N 155°04’56”W, 28 September 2016, coll. JKS 69 (HAW-F-00244); Hilo, ‘Imiloa Astronomy Center, 19°42’07”N 155°05’16’W, 5 October 2016, coll. JkKS 71 (HAW-F-00245; GenBank MK412589); 6 November 2016, coll. JKS 77 (HAW-F-00246; GenBank MK412588); Puna District, MacKenzie State Recreation Area, 19°26’22”N 154°51’4”W, 15 November 2017, coll. JKS 174 (HAW-F-00247). ECOLOGY & DISTRIBUTION—Scattered to gregarious, sometimes cespitose, in wood chips, on nutrient-rich soil in disturbed settings, or on Casuarina equisetifolia duff in wet, lowland, alien environments. January, May, and July Lepiota punaensis sp. nov. (Hawai'i USA) ... 483 Pirate. 3. Lepiota cf. elaiophylla (HAW-F-00246). a. Basidiomata; b. Basidiospores; c. Basidia (generally with four sterigmata; only two shown in these basidial views); d. Cheilocystidia; e. Pileus covering. to December. Currently only known from the Puna and South Hilo Districts on Hawai‘i Island; L. elaiophylla in the broad sense (see discussion) is reported 484 ... Stallman & al. from Australia, Europe, North America, South America, India, and possibly Africa (as L. xanthophylla P.D. Orton). ComMMENTS—Lepiota cf. elaiophylla occurs on wood chips, nutrient-rich soil in disturbed environments such as gardens, and under coastal Casuarina equisetifolia where it can fruit singly, or in large groups and cespitose clusters. Although many yellow lepiotaceous fungi occur in Hawai‘i, L. cf. elaiophylla is easily distinguishable by its overall sulphur yellow coloration (including the gills) with brown scales on the pileus, robust stature (differentiating it from delicate yellow species in Leucocoprinus), and clear annular zone on the stipe with cortina-like partial veil remnants above a base covered in brown scales. Its oblong spores and pileus covering of primarily long elements place this species in L. subsect. Helveolinae with other species containing amatoxins. A BLAST search reveals the top match by identity in GenBank to represent a collection identified as Lepiota elaiophylla by Else Vellinga, the co-describer of this species, from a greenhouse in Canada at 99.9% sequence similarity (KP012854). The next closest match (at 98.3% similarity) is MH979467 from a specimen collected near Darwin Australia growing in a native Eucalyptus and Acacia swamp habitat (M. Barrett, pers. comm.). Micrographs of the Australian collection (T. Lebel, pers. comm.) reveal spores that are more ellipsoid than in most collections of L. cf. elaiophylla, so it is unclear without additional Australian specimens whether these two taxa are morphologically identical. Our phylogeny of Lepiota (Fic. 1) shows the Hawaiian L. cf. elaiophylla and the Canadian collection identified as L. elaiophylla as identical, with the Australian collection close on its own short branch. The Dutch paratype of L. elaiophylla is sister to the clade containing the Hawaiian, Canadian, and Australian collections, and a well-supported (84% bootstrap support, 1.0 Bayesian posterior probability) clade is formed when considering all taxa, including the paratype collection. See TABLE 2 for a comparison of pairwise genetic distances between the species discussed. Lepiota elaiophylla was described from the Netherlands and is reported throughout Europe (e.g., Holec & Halek 2008), Brazil (Wartchow & al. 2008, Ferreira & Cortez 2012), India (Kumar & Manimohan 2009), and possibly Africa (as L. xanthophylla; Pegler 1977). The Hawaiian L. cf. elaiophylla is clearly closely related to L. elaiophylla, from which it is macroscopically indistinguishable. The only differences are (morphologically) a more variable cheilocystidia shape in L. cf. elaiophylla and (molecularly) a different nrITS sequence, although some European collections outside the Netherlands and Brazilian collections show variation in cheilocystidia shape. Lepiota punaensis sp. nov. (Hawai'i USA) ... 485 For a discussion of other Lepiota spp. with yellow lamellae (including the macroscopic lookalike L. xanthophylla, which is differentiated by containing a layer of short, clavate elements in the pileus covering and which has not been recorded from Hawai‘i), see Vellinga & Huijser (1997). Collections of the European L. xanthophylla and L. elaiophylla are described with differently colored dried lamellae (yellow in xanthophylla and dirty olive-brown for elaiophylla). Collections of L. cf. elaiophylla generally have dark yellow lamellae when dried, although older, poorly dried, or damaged specimens may exhibit brown colors; so this does not appear to be a useful taxonomic feature at this time. More information is needed to distinguish species concepts among L. cf. elaiophylla, the Australian collection, and L. elaiophylla from the Netherlands and throughout its reported range. One option is to argue that L. cf. elaiophylla falls within normal morphological variation (minor cystidia differences) of L. elaiophylla, a widespread species with high molecular variation and some morphological variation as evidenced by its presence in India (Kumar & Manimohan 2009), Brazil (Wartchow & al. 2008, Ferreira & Cortez 2012), and Australia. This option cannot be proven but falls on the conservative side of not describing a new taxon without first examining molecular data from other regions that might reveal whether there is a range of variation in the nrITS or two distinct taxa. The other option is to argue that L. cf. elaiophylla is clearly molecularly different from the Dutch L. elaiophylla (over 7% pairwise distance) and supported by a morphological difference (cheilocystidia shape). This argument is supported by pointing out that the related species L. brunneoincarnata and L. subincarnata initially showed little to no molecular variation across their respective geographic ranges (Europe and Central Asia; Europe, South Asia, and North America) when examined by Razaq & al. (2014). At this time, more molecular data from Europe, South America, India (and possibly Africa) and morphological data from India and Africa are needed to clarify the species concept. The Indian collection was not given a full species description, but collections from South America reportedly have smaller spores as well as lageniform (Ferreira & Cortez 2012) or subfusoid (Wartchow & al. 2008) cheilocystidia, while collections from greenhouses in Europe primarily have narrowly clavate to clavate cheilocystidia (with the exception of the subglobose, and oval to pyriform cheilocystidia noted by Breitenbach & Kranzlin 1995). At this time, habitat does not seem to be a useful taxonomic feature as both Hawaiian and Dutch collections have been found in greenhouse 486 ... Stallman & al. environments, although only the Hawaiian L. cf. elaiophylla is currently confirmed from non-artificial habitats in (sub)tropical environments. Key to the Lepiota of Hawai'i Island if — Oo OW on a Pileus brownish with covering of acute pyramidal scales composed of elobase on inflated, Clements t..:, 3.) ft os 2h 2 Se Se, sgce th eget Ser arp sca 2 . Pileus color variable (including brownish) with covering lacking acute pyramidal scales, at least some long (2100 ttm) cylindrical ClEMIENtS: PRESEHIS. fat. oo eee vate ten Un Mee cet MhrY cag stains Ovid ip ey « 3 . Basidiome with large membranous annulus, spores oblong to cylindrical, averaging 6.4 x 2.7 um ................ L. aspera . Basidiome annulus and other features poorly known, spores ellipsoid to oblong, averaging 4.4 x 2.7m .............. 0.0. eee L. sp. (MK412575; poorly known; see text) . Basidiome with ellipsoid spores averaging 5X 3UM..... eee eee eee 4 . Basidiome with ellipsoid to oblong spores averaging 7 x 3-4um _............... 5 . Basidiome with prominent basal stipe ornamentation concolorous with pileus scales; farinaceous odor when crushed; pileus and stipe covering of long flexuose hyphae lacking abundant shorter clavate Clemence s.r hen ween. Mer ety atten Sea eee ent ete eee, L. rubrobrunnea . Basidiome generally lacking prominent basal stipe ornamentation, stipe scales present usually lighter than pileus scales; lacking a farinaceous odor when crushed; pileus and particularly stipe covering with abundant short clavate elements ............... L. pseudorubella . Basidiome with brown scales over a dull yellow pileus background, lamellae and stipe also yellow; arachnoid partial veil usually leaving only zone on stipe; pileus covering a trichoderm of long (<240 um) elements lacking shorter cells..................0..000- Lepiota cf. elaiophylla . Basidiome with warm brown scales over an off-white pileus background lacking yellow coloration in all tissues; annulus a zone only; pileus covering a trichoderm of long (<160 um) elements with shorter, sometimes clavate hyphae interspersed but not in a distinct layer; shorter hyphae more common in stipe covering, currently known only from Casuarina equisetifolia duff in the Puna District of Hawai‘i 1S ha (6 hee pes A De nt ye prea, UM Rec Slay. pe Saas Ch ear L. punaensis Acknowledgments We would like to acknowledge the National Geographic Society, Puget Sound Mycological Society, and Sonoma County Mycological Society for funding. Thank you to Dr. Teresa Lebel and Dr. Matthew Barrett for sharing information on the Australian collection (GenBank KP012854), to Sean Swift for a collection made on O‘ahu (GenBank MK412617), and to Dr. Else Vellinga on a variety of topics related Lepiota punaensis sp. nov. (Hawai'i USA) ... 487 to Lepiota taxonomy in general and L. elaiophylla in particular. We also thank Dr. Brian A. Perry (Department of Biological Sciences, California State University East Bay, USA) and Dr. Alfredo Vizzini (Department of Life Sciences & Systems Biology, University of Torino, Italy) for presubmission review. Literature cited Aberdeen JEC. 1992. Lepiotoid genera (Agaricales) in south-eastern Queensland. Gailes, Queensland: Aberdeen Publications. Akers B. 1997. The family Lepiotaceae (Agaricales, Basidiomycetes) in Florida. PhD dissertation. Southern Illinois University, Carbondale, IL. Benjamin DR. 1995. Amatoxin syndrome poisoning by the amanitins. 198-241, in: Mushrooms poisons and panaceas. New York: W.H. Freeman and Company. Bon M. 1992. Clé analytique des cortinaires alpins (avec quelques incursions dans la rhodoraie et laulnaie verte). Documents Mycologiques 22: 43-67. Bon M. 1993 Flore mycologique d’Europe, 3. Les Lépiotes. Lepiotaceae Roze. 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Acta Botanica Brasilica 22: 287-299. https://doi.org/10.1590/S0102-33062008000100026 MYCOTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 July-September 2020—Volume 135, pp. 491-500 https://doi.org/10.5248/135.491 Notes on rust fungi in China 8. Pucciniastrum tiliae life cycle and new host plants inferred from phylogenetic evidence JING-XIN Jr, ZHUANG L?’, Yu Lr’, MAKOTO KAKISHIMA®?® ‘Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi, Jilin Agricultural University, Changchun, Jilin 130118, China College of Plant Protection, Shandong Agricultural University, Taian 271000, China >University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan * CORRESPONDENCE TO: * 1096314395@qq.com * kakishima.makoto.ga@u.tsukuba.ac.jp ABSTRACT—The life cycle connection between spermogonial and aecial stages of a rust fungus found on Abies holophylla and uredinial and telial stages on Tilia mongolica and T. mandshurica collected in northeast China were confirmed by phylogenetic analyses. The rust, identified as Pucciniastrum tiliae, was confirmed by morphological observations. The life cycle of this rust fungus is reported for the first time in China, and A. holophylla and T: mongolica represent new host plants for the species. Key worps—phylogeny, Pinaceae, Pucciniastreae, Tiliaceae, tree disease Introduction Pucciniastrum tiliae was first described with uredinial and telial stages on Tilia cordata var. japonica [= T: japonica; Tiliaceae| collected in Hokkaido, Japan (Hiratsuka 1897). This species has been since reported on T: amurensis in Korea, China, and Russian Far East; T: americana and T. kiusiana in Japan; T. mandshurica in China and Russian Far East; T. maximowicziana [= T. miyabei] in China and Japan; T: platyphyllos in Japan; and T! tuan in China (Hiratsuka 1936, 1958; Ito 1938; Cummins 1950; Cummins & Ling 1950; Wang 1951; Kim 1963; Tai 1979; Hiratsuka & al. 1992; Azbukina 2005, 492 ... Ji &al. 2015). The life cycle of this fungus was revealed by Kamei in Japan in 1934 through inoculations of basidiospores from teliospores on T: japonica and T. maximowicziana into needles of Abies sachalinensis var. mayriana (Pinaceae) (Hiratsuka & al. 1992). Although P tiliae is distributed in northeast China (Tai 1979), there are no reports of a spermogonial and aecial host in the country. During field investigation of rust fungi in northeast China, we collected uredinial and telial stages of Pucciniastrum on T. mandshurica and T: mongolica in the mountains of Jilin and Heilongjiang Provinces. We also observed occurrences of aecia producing long peridia on Abies holophylla near infected trees of Tilia. Therefore, we suspected life cycle connections between the rust on Abies and Tilia. Inoculation of plants by spores are an appropriate method to clarify rust life cycles (Ji & al. 2017a, b). However, collection and propagation of plants from the mountains of Maple Valley (Jilin) and Fenix National Park (Heilongjiang) where we collected specimens are strictly prohibited as they are nature conservation areas. Therefore, we applied molecular analyses to resolve these problems and to clarify the life cycle (Liu & Hambleton 2013, Ji & al. 2016, Padamsee & McKenzie 2017, Scholler & al. 2019). We report here the results of phylogenetic analyses and morphological observations of the specimens. Materials & methods Molecular analyses Uredinial and telial stages on Tilia mandshurica and T: mongolica collected in mountains in Jilin and Heilongjiang Provinces, China, were used for molecular analyses. The spermogonial and aecial stages on Abies holophylla collected in Jiaohe, Jilin Province, China, were also used for analyses. The total genomic DNA was directly extracted from c. 200 spores obtained from single sori on the leaf of each specimen using similar methods reported by Ji & al. (2016, 2019). Specimens used in the experiments were deposited in the Herbarium of Mycology, Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi, Jilin Agricultural University, China (HMJAU); all data sequenced in this experiment were deposited in GenBank as shown in specimens examined. The sequences obtained from specimens were aligned following Ji & al. (2019). ITS and 28S sequences related to P tiliae were retrieved from GenBank and added to phylogenetic analyses (Ji & al. 2019). The accession numbers of these data are shown in the phylogenetic trees (Fics 1, 2). Phylogenetic trees were constructed using Melampsora laricis-populina Kleb. as outgroup according to Ji & al. (2019). The alignment and trees were deposited in TreeBASE under http://purl.org/phylo/ treebase/phylows/study/TB2:S24909 (Fics 1, 2). Pucciniastrum tiliae in China ... 493 Pucciniastrum tiliae HMJAU8581 [Tilia mandshurica|UT Pucciniastrum tiliae HMSJAU8582 [Tilia mongolica|UT satepii.nd Pucciniastrum tiliae HMJAU8583 [Tilia mongolica|UT =< Pucciniastrum tiliaeg HMJAU8584 [Tilia mongolica|UT 65\68\0,94| [Pucciniastrum tiliae (AB 221412) [Tilia mandshurica] Pucciniastrum tiliae (AB 221415) [Tilia japonica] Pucciniastrum tiliae HMJAU8641 [Abies holophylla|SA Thekopsora triangula (KF551204) [Cornus macrophylla] Thekopsora ostryae (KC416004) [Ostrya japonica] Thekopsora ostryae (KC415993) [Ostrya japonica] Pucciniastrum agrimoniae (MG787142) [Agrimonia pilosa] Pucciniastrum agrimoniae (MG787143) [Agrimonia pilosa] Pucciniastrum agrimoniae (MG787145) [Agrimonia pilosa] Pucciniastrum agrimoniae (KJ725375) Thekopsora lanpingensis (KF551208) [Cornus schindleri] 100\100\1;7ekopsora lanpingensis (KF551211) [Cornus walteri] Thekopsora lanpingensis (KF551213) [Cornus schindleri] Thekopsora lanpingensis (KF551214) [Cornus officinalis | Pucciniastrum coronispora (MH780998) [Galium trifidum] Pucciniastrum coronispora (MG787127) [Galium davuricum] Pucciniastrum coronispora (MG787128) [Galium boreale] Pucciniastrum coronispora (MG787132) [Galium bungei| Thekopsora nipponica (KC416003) [Galium aparine] L00\100\I-7hekopsora nipponica (KC415995) [Galium aparine] Pucciniastrum rubiae (MG787136) [Rubia cordifolia] #1*\0.56| [Pucciniastrum rubiae (MG787139) [Rubia chinensis] Pucciniastrum rubiae (MG787140) [Rubia chinensis] Pucciniastrum guttata (KC415999) [Galium odoratum] Pucciniastrum rubiae (KC616007) [Rubia cordifolia] Pucciniastrum annulospora (MH781004) [Kalimeris integrifolia] 97 | Pucciniastrum annulospora (MH781003) [Aster tataricus] Pucciniastrum annulospora (MG787133) [Kalimeris lautureana] ‘Melampsora laricis-populina (MG787151) [Populus sp.] 70\*\0.75 98\97\0,89 79\82\0.86 99\L00\0. 0.04 Fic. 1. Phylogenetic tree constructed by Bayesian method based 28S rDNA sequences. MP/ML bootstrap values precede Bayesian posterior probabilities (Bpp) on the nodes. Asterisks (*) indicate bootstrap values <50% or Bpp <0.5. Sample data include species name, (voucher specimen or GenBank accession number), and [host plant]. Sequences generated in this study are shown in bold face. SA= Spermogonial and aecial stages; UT = Uredinial and telial stages. Morphological observations Light (LM) and scanning electron (SEM) microscopy were used to examine morphological characters of rust specimens, including the size and shape of sori and spores following Ji & al. (2019). Phylogenetic results The 28S dataset comprised 33 sequences with 496 total characters, of which 394 were constant, 30 parsimony-uninformative variable, and 72 parsimony-informative. Parsimony analysis yielded one parsimonious tree with TL = 156, CI = 0.7692, RI = 0.9231, and RC = 0.7101. Bayesian analysis resulted in average standard deviation of split frequencies of 0.008983. The ITS dataset comprised 39 sequences of 745 total characters, of which 228 were parsimony-informative. Parsimony A494 ... Ji & al. Pucciniastrum coronispora (MH780990) [Galium trifidum] 98\97\1] Pucciniastrum coronispora (MG787102) [Galium davuricum] Pucciniastrum coronispora (MG787103) [Galium boreale] 1OO\OO Pr cciniastrum coronispora (MG787107) [Galium bungei| Pucciniastrum nipponica (KC415792) [Galium davuricum] 16\9 1O0\1 00} Mcciniastrum nipponica (KC415794) [Galium aparine] Pucciniastrum nipponica (KC415793) [Galium aparine] Pucciniastrum annulospora (MG787108) [Kalimeris lautureana] 00\TOO q Pucciniastrum annulospora (MH780995) [Aster tataricus] Pucciniastrum annulospora (MH780996) [Kalimeris integrifolia] Pucciniastrum guttatum (KC415789) [Galium odoratum] Pucciniastrum guttatum (KC415790) [Galium odoratum] Pucciniastrum guttatum (KC415791) [Galium odoratum] Pucciniastrum rubiae (MG787114) [Rubia chinensis] Pucciniastrum rubiae (MG787115) [Rubia chinensis] Pucciniastrum rubiae (KC415799) [Rubia cordifolia] 100\9 Pucciniastrum rubiae (KC415802) [Rubia cordifolia] Pucciniastrum rubiae (KC415801) [Rubia cordifolia] 83\59\0.67 Pucciniastrum rubiae (MG787111) [Rubia cordifolia] Pucciniastrum tiliae HMJAU8581 [Tilia mandshurica}UT Pucciniastrum tiliae HMJAU8583 [Tilia mongolica}UT Pucciniastrum tiliae HMJAU8641 [Abies holophylla]SA 65\*\0.8] Pucciniastrum tiliae HMJAU8582 [Tilia mongolica|UT Pucciniastrum tiliae HMJAU8584 [Tilia mongolica}UT 9ON\9G\1I| Pucciniastrum tiliae (AB 221453) [Tilia japonica] SOM Pucciniastrum tiliae (AB 221455) [Tilia mandshurica] INIG\I Pucciniastrum kusanoi (AB221426) [Clethra barbinervis] 9NIN\I" Pucciniastrum boehmeriae (AB221449) [Boehmeria tricuspis] Thekopsora triangula (KF551219) [Cornus macrophylla] Pucciniastrum agrimoniae (MG787117) [Agrimonia pilosa] 100\100\1|Pucciniastrum agrimoniae (MG787118) [Agrimonia pilosa] 99\100 Pucciniastrum agrimoniae (MG787120) [Agrimonia pilosa] ‘Pucciniastrum agrimoniae (KJ486537) Thekopsora ostryae (KC415796) [Ostrya japonica] 71\85\0.81 Thekopsora lanpingensis (KF551220) [Cornus walteri] 100\100\1| 7hekopsora lanpingensis (KF551222) [Cornus schindleri] Thekopsora lanpingensis (KF551225) [Cornus schindleri] Thekopsora lanpingensis (KF551224) [Cornus officinalis | Melampsora laricis-populina (MG787126) [Populus sp.] 9ON\I\I Fic. 2. Phylogenetic tree constructed by Bayesian method based on ITS rDNA sequences. MP/ ML bootstrap values precede Bayesian posterior probabilities (Bpp) on the nodes. Asterisks (*) indicate bootstrap values <50% or Bpp <0.5. Sample data include species name, (voucher specimen or GenBank accession number), and [host plant]. Sequences generated in this study are shown in bold face. SA= Spermogonial and aecial stages; UT = Uredinial and telial stages. analysis yielded one parsimonious tree with TL = 543, CI = 0.8066, RI = 0.9417, and RC = 0.7596. Bayesian analysis resulted in average standard deviation of split frequencies of 0.003112. Tree topologies formed by MP, ML, and MCMC methods were identical among trees. The Bayesian phylogenetic trees are shown in Fic. 1 (28S) and Fie. 2 (ITS). Both 28S and ITS phylogenetic trees included the uredinial and telial stages on T. mandshurica (HMJAU 8581) and T: mongolica (HMJAU 8582, 8583, 8584) and the spermogonial and aecial stages on A. holophylla (HMJAU 8641) in a monophyletic clade (Fics 1, 2). Genetically identical to one another, they can be considered the same species. The life connection between spermogonial and aecial stages on A. holophylla and uredinial and telial stages on T: mandshurica and T: mongolica is therefore strongly supported by the phylogenetic analyses. Pucciniastrum tiliae in China ... 495 Taxonomy The phylogenetic analyses reveal that this rust fungus has a heteromacrocyclic life cycle, producing spermogonia and aecia on Abies holophylla (Fic. 3p) and uredinia and telia on Tilia mandshurica and T. mongolica (Figs 3a-c). LM and SEM observations showed that the morphological characteristics of spermogonial and aecial specimens on A. holophylla collected from the field were identical with the descriptions of Pucciniastrum tiliae reported by Hiratsuka (1936) and Hiratsuka & al. (1992), except for outer wall surface structure of the peridial cells (Fics 3p, 4A-C, A-D). Under SEM, these walls are minutely verrucose, in contrast to the smooth surface described by Hiratsuka & al. (1992). Previously, P. tiliae spermogonial and aecial stages have been reported on Abies species only from Japan, so this represents the first report of the P. tiliae life cycle from China. Abies holophylla is a new spermogonial and aecial host plant and occurs in northeastern China, Korea, and Russian Far East. After clarifying the life cycle connection of P tiliae with phylogenetic analyses, we described its morphology based on specimens collected in the field. The morphological characters of uredinial and telial specimens on T. mandshurica and T: mongolica were similar to one another (Fics 34-c, 4D-F, 5E,F) and identical to the descriptions of P. tiliae by Hiratsuka (1936), Hiratsuka & al. (1992) and Azbukina (2005, 2015). No researcher has previously reported P. tiliae on T. mongolica (Hiratsuka 1936, 1958; Kim 1963; Tai 1979; Hiratsuka & al. 1992; Azbukina 2005, 2015), so we add T: mongolica (known from Mongolia, northern China, and eastern Russia) as a new host for the rust. Pucciniastrum tiliae Miyabe, Bot. Mag. Tokyo 11: 47, 1897. Figs 3-5 Spermogonia amphigenous, pale yellow to yellowish, subcuticular, type 3 (Cummins & Hiratsuka 2003). Aecia with long peridia, Peridermium-type, hypophyllous, cylindrical, orange-yellow. Peridia colorless, rupturing at the sides and apex; peridial cells ovate to ellipsoid, slightly overlapping, inner and outer walls thin, minutely verrucose. Aeciospores globose to ellipsoid, 15.0-22.0 x 13.0-16.5 um (av. = 18.5 x 15.0 um), walls hyaline, 1.0-2.0 um (av. = 1.5 um) thick, densely verrucose with annulate verrucae. Uredinia hypophyllous, scattered to gregarious, subepidermal, small, round, covered by the epidermis, rupturing with pores, pulverulent. Peridia hemispherical, delicate, firm, rupturing at the apex, peridial cells small, 496 ... Ji & al. Fic. 3. Pucciniastrum tiliae. A. Uredinia and telia on lower leaf surface of Tilia mongolica. B. Telia produced on lower leaf surface of T: mongolica. C. Uredinia produced on lower leaf surface of T. mongolica. D. Aecia with long peridia produced on needles of Abies holophylla. irregularly polygonal, walls smooth, thin, ostiolar cells rounded, walls smooth. Urediniospores subglobose, ellipsoid, obovate, or oblong, 16.5-25.5 x 8.0-15.5 um (av. = 20.5 x 12.5 um), walls echinulate, 0.5-2.0 um (av. = 1 um) thick. Telia hypophyllous, subepidermal, scattered or in small groups, at first orange-yellow, then brown to reddish in color. Teliospores forming under the epidermis, attached laterally, subglobose, oblong, or somewhat angular, divided into 2-6 cells by vertical or oblique septa, 21.0-37.0 x 5.5-14.0 um (av. = 30.5 x 9.5 um), walls thin, 0.5-1.0 um (av. = 0.7 um) thick, smooth. SPECIMENS EXAMINED—CHINA, Jilin Province, Jiaohe, stages 0 & I, on Abies holophylla Maxim., 16 June 2019 (HMJAU8641, GenBank MN294706, MG787116); stages II & IH, on Tilia mongolica Maxim., 11 September 2017 (HMJAU8583, GenBank MG787149, MG787124; HMJAU8584, GenBank MG787150, MG787125); Helongjiang Pucciniastrum tiliae in China ... 497 Fic. 4. Pucciniastrum tiliae observed with LM. A. Vertical section of spermogonium (type 3). B. Aeciospores with verrucose surface. C. Vertical section of aecium surround by peridium (P). D. Urediniospores with echinulate surface. E. Vertical section of uredinium with peridium (P), opening by central pore. F. Vertical section of telia with laterally attached teliospores, covered by host epidermis. Scale bars: A, C = 20 um; B = 30 um; D, E= 10 um; F = 15 um. Province, Wuchang, stages II & III, on T: mongolica, 9 September 2017 (HMJAU8582, GenBank MG787148, MG787123); stages Il & II, on T: mandshurica Rupr. & Maxim., 9 September 2017 (HMJAU8581, GenBank MG787147, MG787122). 498 ... Ji & al. Fic. 5. Pucciniastrum tiliae observed with SEM. A. Aeciospores with densely verrucose surface. B. Aecium with long peridium. C. Outer walls of peridial cells. D. Inner walls of peridial cells. E. Urediniospores with echinulate surface. F. Uredinium with peridium (P) and ostiolar cells (O), opening by central pore. Scale bars: A, E = 5 um; B = 100 um; C, D = 20 um; F = 10 um. Pucciniastrum tiliae in China ... 499 Hosts & DISTRIBUTION WITHIN CHINA—Abies holophylla, Jilin (new host; this paper). Tilia amurensis Rupr., Jilin (Ito 1938, Tai 1979). Tilia mandshurica, Heilongjiang (Ito 1938, Hiratsuka 1958, Tai 1979). Tilia mongolica, Heilongjiang, Jilin (new host; this paper). Tilia tuan Szyszyt., Guizhou (Hiratsuka 1958, Tai 1979). Acknowledgments This work was financed by the Fungal Flora in Jilin Province (20130206073NY). We thank Dr. E.H.C. McKenzie (Manaaki Whenua Landcare Research, Auckland, New Zealand) and Prof. C.M. Denchev (Bulgarian Academy of Sciences, Sofia, Bulgaria) for critical reading of the manuscript and suggestions. Literature cited Azbukina ZM. 2005. Rust fungi. Cryptogamic plants, fungi and mosses of the Russian Far East, vol. 5. Dalnauka, Vladivostok. (In Russian) Azbukina ZM. 2015. Definitorium fungorum Rossiae, Ordo Pucciniales 1. Dalnauka, Vladivostok. Cummins GB. 1950. Urediniales of continental China collected by S.Y. Cheo. I. Mycologia 42: 779-797. https://doi.org/10.1080/00275514.1950.12017881 Cummins GB, Hiratsuka Y. 2003. Illustrated genera of rust fungi, 3% ed. American Phytopathological Society, St. Paul, Minnesota. Cummins GB, Ling L. 1950. An index of the plant rusts recorded for continental China and Manchuria. Plant Disease Reporter (Supplement) 196: 520-556. Hiratsuka N. 1897. Notes on some Melampsorae of Japan. Botanical Magazine Tokyo 11: 45-49. Hiratsuka N. 1936. A monograph of the Pucciniastreae. Memoirs of Tottori Agricultural College 4. 374 p. Hiratsuka N. 1958. Revision of taxonomy of the Pucciniastreae. Memoirs of the Faculty of Agriculture, Tokyo University of Education 5. 167 p. Hiratsuka N, Sato S, Katsuya K, Kakishima M, Hiratsuka Y, Kaneko S, Ono Y, Sato T, Harada Y, Hiratsuka T, Nakayama K. 1992. The rust flora of Japan. Tsukuba-shuppankai, Tsukuba. Ito S. 1938. Mycological flora of Japan, vol. 2, no. 2, Urediniales-Melampsoraceae. Yokendo, Tokyo. Ji JX, Li Z, Wan Q, Li Y, Kakishima M. 2016. Notes on rust fungi in China 1. Autoecious life cycle of Puccinia tatarinovii on Prenanthes. Mycotaxon 131: 653-661. http://dx.doi.org/10.5248/131.653 Ji JX, Li Z, Wan Q, Li Y, Kakishima M. 2017a. Life cycle of Aecidium klugkistianum on Ligstrum and its new combination, Puccinia klugkistiana. Mycoscience 58: 307-311. https://doi.org/10.1016/j.myc.2017.01.004 Ji JX, Li Z, Wan Q, Li Y, Kakishima M. 2017b. Notes on rust fungi in China 3. Puccinia adenocauli comb. nov. and its life cycle and new host. Mycotaxon 132: 141-148. http://dx.doi.org/10.5248/132.141 Ji JX, Li Z, Li Y, Kakishima M. 2019. Two new species of Pucciniastrum producing dimorphic sori and spores from northeast of China. Mycological Progress 18: 529-540. https://doi.org/10.1007/s11557-018-1460-z Kim CJ. 1963. A provisional list of Uredinales of Korea. Korean Journal of Microbiology 1: 51-64. 500 ... Ji & al. Liu M, Hambleton S. 2013 Laying the formation for a taxonomic review of Puccinia coronata s.l. in a phylogenetic context. Mycological Progress 12: 63-89. https://doi.org/10.1007/s11557-012-0814-1 Padamsee M, McKenzie EHC. 2017. The intriguing and convoluted life of a heteroecious rust fungus in New Zealand. Plant Pathology 66: 1248-1257. https://doi.org/10.1111/ppa.12672 Scholler M, Lutz M, Aime MC. 2019. Repeated formation of correlated species in Tranzschelia (Pucciniales). Mycological Progress 18: 295-303. https://doi.org/10.1007/s11557-018-1417-2 Tai FL. 1979. Sylloge fungorum sinicorum. Science Press, Beijing. Wang YZ. 1951. Index Uredinearum sinensium. Academia Sinica, Beijing. MYCOTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 July-September 2020—Volume 135, pp. 501-512 https://doi.org/10.5248/135.501 Guayaquilia gen. nov., typified by /driella cubensis FREDDY MAGDAMA’, DAYNET SOSA’, FERNANDO ESPINOZA’, LIZETTE SERRANO’, SIMON PEREZ-MARTINEZ’, ELAINE MALOSSO}, MARGARITA HERNANDEZ-RESTREPO*, RAFAEL F. CASTANEDA-RUIZ> ' Escuela Superior Politécnica del Litoral, ESPOL, Centro de Investigaciones Biotecnologicas del Ecuador, Campus Gustavo Galindo, Km. 30.5 Via Perimetral, RO. Box 09-01-5863, Guayaquil, Ecuador ? Universidad Estatal de Milagro (UNEM1I), Facultad de Ingenieria, Cdla. Universitaria Km. 1.5 via Milagro-Km26. Milagro 091706, Guayas, Ecuador > Centro de Biociencias, Departamento de Micologia, Universidade Federal de Pernambuco, Avenida da Engenharia, s/n Cidade Universitaria, Recife, PE, 50.740-600, Brazil * Westerdijk Fungal Biodiversity Institute, 3508 AD Utrecht, The Netherlands ° Instituto de Investigaciones Fundamentales en Agricultura (INIFAT), Tropical Alejandro de Humboldt, OSDE, Grupo Agricola, Calle 1 Esq. 2, Santiago de Las Vegas, C. Habana, Cuba, C.P. 17200 * CORRESPONDENCE TO: dasosa@espol.edu.ec ABSTRACT—A new genus Guayaquilia is established to accommodate Idriella cubensis based on morphology and phylogenetic analysis. DNA sequence data place these specimens as incertae sedis separate from Microdochiaceae (Xylariales), forming a monophyletic lineage separated from Neoidriella desertorum and phylogenetically distant from Idriella. The novel genus is characterized by macronematous, tree-like, fasciculate, profuse dichotomously, alternately, or irregularly branched, brown conidiophores with polyblastic, denticulate, sympodial extended, intercalary and terminal conidiogenous cells that produce solitary, sublunate, subnavicular, lunate, inequilateral, (0-)1-septate, hyaline conidia. KEY worps—asexual fungi, hyphomycetes, Microdochiaceae, taxonomy, tropics Introduction Idriella, introduced by Nelson & Wilhelm (1956) with I. lunata as type species, is distinguished by macronematous, mononematous, unbranched, and basally 502 ... Magdama & al. inflated and apically slightly geniculate brown conidiophores, mostly reduced to polyblastic, denticulate, sympodial extended conidiogenous cells. Conidia are typically lunate or falcate, unicellular, hyaline, and acuminate towards the apex (Nelson & Wilhelm 1956). Morgan-Jones (1979) updated and provided brief nomenclatural commentaries on Idriella australiensis, I. couratarii, I. desertorum, I. lunata, I. mycogonoidea, I. ramosa, I. vandalurensis, and I. variabilis and proposed two new species, I. angustispora and I. bambusae. Von Arx (1981), who presented a combined key for Idriella and Microdochium species, proposed the conidial shape and the habitat as the main characters to separate the genera; he included the Idriella species treated by Morgan-Jones (1979), excluded I. tropicalis as similar to I. variabilis, and added five new combinations (J. bolleyi, I. caespitosa, I. falcata, I. grisea, and I. tainanensis). Subsequently, 16 new species have been described I. acerosa, I. amazonica, I. angamosensis, I. asaicola, I. cagnizarii, I. campnospermae, I. cubensis, I. euterpes, I. jambosae, I. licualae, I. mycophila, I. rara, I. rhododendri, I. setiformis, I. stilboidea, and I. uncinospora (Castaheda-Ruiz 1985, 1986; Castaneda-Ruiz & Arnold 1985; Castafteda-Ruiz & Kendrick 1991; Castafieda- Ruiz & al. 1997; Matsushima 1985, 1995; Rodrigues & Samuels 1990, 1992; Subramanian & Bhat 1987; Wu & al. 1997). When the generic concept of Idriella was expanded, it becamea heterogeneous genus (Seifert & al 2011). In the generic type, I. lunata, conidiophores are unbranched and mostly reduced to conidiogenous cells producing unicellular conidia on tiny denticles arranged along the apical and subapical surface of each conidiogenous cell after several sympodial holoblastic extensions (Nelson & Wilhelm 1956), but many other Idriella species produce conidiophores branched in various patterns, sympodial extensions of the conidiogenous cells confined to a small, nodose area at the apex, and conidiogenous loci as inconspicuous denticles that produce unicellular or septate conidia. DNA sequences of several Idriella species revealed the polyphyletic nature of this genus, showing phylogenetic separation between I. lunata (type species) and four other Idriella species, which consequently were transferred to four new genera: Castanediella Hern.-Restr. & al., Idriellopsis Hern.-Restr. & Crous, Neoidriella Hern.-Restr. & Crous, and Paraidriella Hern.-Restr. & Crous (Crous & al. 2015, Hernandez-Restrepo & al. 2016). During a workshop on fungal diversity conservation in Cacao forest areas, two surveys of microfungi were conducted in Theobroma cacao plantations in Guayaquil, Guayas Province, Ecuador, and several Idriella-like specimens were found. LSU and ITS sequence analyses place these specimens as incertae sedis Guayaquilia cubensis gen. & sp. nov. (Cuba, Ecuador) ... 503 and separate from Microdochiaceae (Xylariales). They are not congeneric with Idriella lunata (the generic type), and we propose a new genus Guayaquilia to accommodate them. Materials & methods Sampling and fungal strains studied Samples of decaying plants were placed in plastic bags, transported to the laboratory, placed in moist chambers, and treated according to Castafeda-Ruiz & al. (2016). Individual conidia were separated from the plant material under a stereoscope using an entomological needle and cultured in two different media: V8 (V8: 125 ml V8 juice, 18 g agar, 1000 ml distilled water, pH 6.3) and Cornmeal agar (CMA: 20 g cornmeal, 18 g agar, 1000 ml distilled water, pH 6.3). Morphological observations were made from cultures grown on CMA after five days, incubated at 25 + 1°C under 12 h alternating near-UV light and darkness (using Vica, FLB-20W T10 near-UV lamp in an irradiation box). Colony colors were coded according to Rayner (1970). Mounts were prepared in lactic acid (90%) or in polyvinyl alcohol-glycerol (8 g PVA in 100 mL of water + 5 mL of glycerol) and lactofuchsin (0.1 g acid fuchsin, 100 mL 85% lactic acid) (Carmichael 1955). Microscopic characters were measured at x1000 using a Nikon Eclipse Ni-U microscope with DIC optics and a Nikon DS-Fi2 camera. Pure cultures were deposited at the Microbial Culture Collection of the Biotechnology Research Center, Guayaquil, Ecuador (CCM-CIBE), and the voucher specimens and slide preparations were deposited in the Herbarium, Departamento de Micologia, Universidade Federal de Pernambuco, Recife, Brazil (URM). Also examined were the holotype and another specimen deposited as Idriella cubensis in the INIFAT Fungus Collection. DNA extraction, sequencing, and phylogenetic analysis Genomic DNA was extracted from fresh mycelia grown on V8 Agar at 25°C using a modified rapid extraction method for filamentous fungi according to Cenis (1992). The primer pairs ITS1/ITS4 (White & al. 1990) and LROR/LRS (Vilgalys & Hester 1990) were used for the amplification of the internal transcribed spacers (ITS), and part of the large subunit of the nuclear ribosomal RNA gene (LSU). PCR cycling conditions followed Korabecna (2007; ITS) and Yang & al. (2017; LSU). PCR products were sent to Macrogen Korea for purification and sequencing with the same primers. Sequences were aligned and edited using MEGA v.6.0. Phylogenetic relationships among taxa were addressed through Maximum Likelihood (ML) analysis using MEGA v.6.0 and Bayesian analysis using Beast v1.10.4 (Drummond & al. 2012). Data for each gene or region was analyzed both individually and together as a combined data set. jModeltest v.2.1.10 (Darriba & al. 2012) was used to determine the best nucleotide substitution model for both studies. Congruence between individual gene data sets was tested using the partition homogeneity test (Farris & al. 1995) implemented in PAUP v4.0b10 (Swofford, 2001), using a heuristic search option with random taxon addition and TBR branch swapping with 1000 replicates. Tree topologies from ML 504 ... Magdama & al. analysis of individual genes were also compared visually for congruence. For the combined ML study, GTR was specified as the evolutionary model with estimated proportion of invariables sites and gamma distribution as default parameters. Number of substitution rates was set to four and topology search changed to SPR for tree improvement. Nodal support was assessed by bootstrap analysis from 1000 replicates. Bootstrap values equal or higher than 70% were considered significant. Bayesian inference was run with four Monte Carlo Markov (MCM) chains over 1 million generations with a sampling frequency of 1000 trees using the GTR+I+G as the best model (Drummond & al. 2012). To detect if the sample distribution has reached stationarity, convergence and the effective sample size (ESS) were checked for each run. Posterior probabilities (PP) for the Bayesian analysis were determined by calculating 50% majority rule consensus tree and added onto congruent nodes of the ML tree topology. PP equal or above 0.95 were considered significant. Relevant sequences of ITS and LSU from Hernandez-Restrepo & al. (2016) obtained from GenBank, were included in the phylogenetic inference for comparison purposes (TABLE 1). TABLE I. Sequences of Guayaquilia cubensis and related species used in the phylogenetic analysis. New sequences are indicated in bold. SPECIES STRAIN # ITS LSU REFERENCE Astrocystis IT1612 KP297404 KP340545 Daranagama & al. 2015 concavispora Castanediella acaciae CBS 139896 KR476728 KR476763 Crous & al. 2015 C. cagnizarii CBS 542.96 KP859054 KP858991 Hernandez-Restrepo & al. 2016 CBS 101043 KP859051 KP858988 Hernandez-Restrepo & al. 2016 C. couratarii CBS 579.71 KP859050 KP858987 Hernandez-Restrepo & al. 2016 Guayaquilia cubensis MUCL 39017 KC775733 KC775708 Becerra-Hernandez & al. 2016 CCMCIBE-H312 MH777025 MH777024 This Study CCMCIBE-H320 MH777026 MH777023 This Study Idriella lunata CBS 177.57 KP859043 KP858980 Hernandez-Restrepo & al. 2016 CBS 204.56 KP859044 KP858981 Hernandez-Restrepo & al. 2016 CBS 209.60 KP859045 KP858982 Hernandez-Restrepo & al. 2016 CBS 736.74 KP859046 KP858983 Hernandez-Restrepo & al. 2016 Idriellopsis CBS 575.92 KP859052 KP858989 Hernandez-Restrepo & uncinospora al. 2016 Kretzschmaria deusta CBS 163.93 KC477237 KY610458 Wendt & al. 2018 Guayaquilia cubensis gen. & sp. nov. (Cuba, Ecuador) ... 505 Microdochium CBS 243.83 KP858994 KP858930 Hernandez-Restrepo & albescens al. 2016 M. citrinidiscum CBS 109067 KP859003 KP858939 Hernandez-Restrepo & al. 2016 M. lycopodinum CBS 109398 KP859005 KP858941 Hernandez-Restrepo & al. 2016 M. majus CBS 741.79 KP859001 KP858937 Hernandez-Restrepo & al. 2016 M. CBS 445.95 KP858997 KP858933 Hernandez-Restrepo & neoqueenslandicum al. 2016 M. nivale CBS 116205 KP859008 KP858944 Hernandez-Restrepo & al. 2016 M. seminicola CBS 122707 KP859007 KP858943 Hernandez-Restrepo & al. 2016 Microdochium sorghi CBS 691.96 KP859000 KP858936 Hernandez-Restrepo & al. 2016 M. tainanense CBS 269.76 KP859009 KP858945 Hernandez-Restrepo & al. 2016 M. trichocladiopsis CBS 623 77 KP858998 KP858934 Hernandez-Restrepo & al. 2016 Neoidriella CBS 985.72 KP859048 KP858985 Hernandez-Restrepo & desertorum al. 2016 Paraidriella jambosae CBS 374.90 KP859049 KP858986 Hernandez-Restrepo & al. 2016 Poronia punctata CBS:656.78 KT281904 KY610496 Wendt & al. 2018 Rosellinia aquila MUCL 51703 KY610392 KY610460 Wendt & al. 2018 R. corticium MUCL 51693 KY610393 KY610461 Wendt & al. 2018 R. necatrix CBS 349.36 AY909001 KF7 19204 Pelaez & al. 2008 Sarcoxylon CBS 359.61 KT281903 KY610462 Wendt & al. 2018 compunctum Selenodriella cubensis CBS 683.96 KP859053 KP858990 Hernandez-Restrepo & al. 2016 S. fertilis CBS 772.83 KP859055 KP858992 Hernandez-Restrepo & al. 2016 Xylaria arbuscula CBS:126415 KY610394 KY610463 Fournier & al. 2011; Wendt & al. 2018 X. hypoxylon CBS 122620 KY610407 KY610495 Wendt & al. 2018 X.polymorpha MUCL 49884 KY610408 KY610464 Wendt & al. 2018 Phylogeny The LSU-ITS dataset comprised 36 aligned sequences with 1482 positions. Fic. 1 presents the ML tree including BS and PP values. The partition homogeneity test showed no significant incongruence between the combined data sets (p >0.05). Trees obtained from ML and Bayesian analysis of the individual loci and the combined analysis produced congruent topologies. Phylogenetic inferences grouped the three strains identified as Idriella cubensis (MUCL 39017, CCMCIBE-H312, CCMCIBE-H320) together in a 506 ... Magdama & al. 92/1 - Microdochium citrinidiscum CBS 109067 Microdochium sorghi CBS 691 96 100/17 Microdochium seminicola CBS 122707 Microdochium albescens CBS 243 83 76/1 Wl Microdochium tainanense CBS 269 76 Microdochium trichocladiopsis CBS 623 77 89/1] |400/17 Microdochium majus CBS 741 79 Microdochium nivale CBS 116205 Microdochium neoqueenslandicum CBS 445 95 Microdochium lycopodinum CBS 109398 100/1 | Selenodriella fertilis CBS 772 83 Selenodriella cubensis CBS 683 96 -/0.96 Idriella lunata CBS 736 74 100/1 |- !driella lunata CBS 209 60 Idriella lunata CBS 204 56 100/1! Idriella lunata CBS 177 57 Neoidriella desertorum CBS 985 72 “j0.96| Uayaquilia cubensis sp. nov. CCMCIBE-H312_ _ | Guayaquilia cubensis sp. nov. CCMCIBE-H320 Paraidriella jambosae CBS 374 90 Idriellopsis uncinospora CBS 575 92 Castanediella acaciae CBS 139896 Castanediella couratarii CBS 579 71 Castanediella cagnizarii CBS 542 96 99/1 Castanediella cagnizarii CBS 101043 “ft Sarcoxylon compunctum CBS 359 61 Poronia punctata CBS 656 78 86/1 -/t -/t 100/1 Rosellinia corticium MUCL 51693 fi J 96/0.98 Rosellinia aquila MUCL 51703 oe il Astrocystis concavispora IT1612 78/1 Rosellinia necatrix CBS 349 36 |i Xylaria polymorpha MUCL 49884 86/0.99 Xylaria hypoxylon CBS 122620 -/0.99 Kretzschmaria deusta CBS 163 93 Xylaria arbuscula KY610463 0.050 Fic. 1. Maximum Likelihood (ML) non-rooted tree inferred from the combined LSU and ITS sequences of Guayaquilia cubensis and related species. Numbers on the branches are support values >75% from BS and >0.95 from PP. The scale bar shows the expected changes per site. supported clade and sister to Neoidriella desertorum, both clades separate from, and basal to, the Microdochiaceae Hern.-Restr. & al. clade containing Idriella lunata, Microdochium spp., and Selenodriella spp. We therefore propose a new genus Guayaquilia typified by Idriella cubensis. Taxonomy Guayaquilia R.F. Castafieda, Magdama, D. Sosa & Hern.-Restr., gen. nov. MB 831849 Differs from Neodriella and Paraidriella by its macronematous, tree-like, irregularly multibranched conidiophores and its intercalary, discrete conidiogenous cells. Guayaquilia cubensis gen. & sp. nov. (Cuba, Ecuador) ... 507 Type Species: Idriella cubensis R.F. Castahteda & G.R.W. Arnold [= Guayaquilia cubensis (R.E Castafieda & G.R.W. Arnold) R.F. Castafeda & al.]. EryMo.Loey: Guayaquilia (Latin), referring to Guayaquil city, Ecuador. ASEXUAL. COLONIES on natural substrate effuse, pulvinate-velutinous, golden brown to brown. CONIDIOPHORES macronematous, mononematous, fasciculate, erect, cylindrical, irregularly branched, septate, smooth, brown. CONIDIOGENOUS CELLS polyblastic, denticulate, sympodial extended, terminal or intercalary, cylindrical or slightly inflated, pale brown to brown. Conidial secession schizolytic. Conip1a solitary, acropleurogenous, sublunate, subnavicular, inequilateral, unicellular or septate, hyaline, smooth. CHLAMYDOSPORES solitary, bicellular, brown and the apical cell globose, thick walled, smooth. Guayaquilia cubensis (R.F. Castafieda & G.R.W. Arnold) R.F. Castafieda, Magdama, D. Sosa & Hern.-Restr., comb. nov. FIGS 2, 3 MB 831851 = Idriella cubensis R. F. Castaheda & G.R.W. Arnold, Rev. Jard. Bot. Nac., Univ. Habana 6(1): 50 (1985). COLONIES on V8 agar at 25°C attaining 60 mm diam after five days, floccose, pale lavender grey to buff, sporulation poor, sparse. On CMA reaching 50 mm diam, felted, dark brick at the center, whitish toward the periphery. CONIDIOPHORES macronematous, mononematous, tree-like, fasciculate, profuse, dichotomously, alternately or irregularly branched toward the apex, slightly inflated or bulbous at the base, smooth, brown below, pale brown to subhyaline at the tip of branches, <500 um long, 4-7.5 um wide at the base, branches 2-3.5 um wide. CONIDIOGENOUS CELLS polyblastic, denticulate, intercalary and terminal, in branches integrated, cylindrical, or slightly curved, brown to pale brown or subhyaline, 8-25 x 2-3 um. CONIDIA solitary, acropleurogenous, sublunate, subnavicular, lunate, inequilateral, (0-) l-septate, 14-17 x 2.5-3 um. CHLAMyDospoRESs bicellular, the basal cells subcampanulate, subcuneiform to hemispherical, 5-8 um wide, and the apical cells globose, 18-22 um diam, brown, thick walled; both cells with lumina granulose, brown; terminal, smooth, arising from assimilative hyphae. SPECIMENS EXAMINED: ECUADOR, Guayas PROVINCE, Naranjal, 2°48’S 79°40’W, on decaying leaves of Theobroma cacao L. (Malvaceae), 8 July 2017, F. Espinoza & S. Pérez-Martinez (URM 91815 = CCMCIBE-H312; GenBank MH777025, MH777024); 2°41’S 79°36’W, on decaying leaves of Theobroma cacao, 8 July 2017, F. Espinoza & S. Pérez-Martinez (URM 91815a = CCMCIBE-H320; GenBank MH777026, MH777023). CUBA, La HABANA PROVINCE, Santiago de Las Vegas, 22°58’N 82°22’W, on decaying leaves of Calophyllum calaba L. (Clusiaceae), 29 Jun 1983, RF Castaneda Ruiz (holotype, 508 ... Magdama & al. 50pm 50 um Fic. 2. Guayaquilia cubensis (holotype, INIFAT C83/57-1). Conidiophores, conidiogenous cells, and conidia. Guayaquilia cubensis gen. & sp. nov. (Cuba, Ecuador) ... 509 C, D. Conidiophores; E-G. Conidiogenous cells; H-L. Conidia; M, N, Chlamydospores. Scale bars = 10 um. INIFAT C83/57-1); PINAR DEL R10 PROVINCE, Los Portales, 22°40’N 83°28’W, on decaying leaves of Cupania sp. (Sapindaceae), 5 Feb. 1994, RF Castafieda Ruiz, (INIFAT C94/27 = MUCL 39017). 510 ... Magdama & al. Note: Guayaquilia shares some morphological characters with the genera Castanediella, Idriella, Idriellopsis, Neoidriella, and Paraidriella, such as blastic conidial ontogeny and denticulate conidiogenous loci, but the tree-like, dichotomously, alternately or irregularly profuse branched conidiophores with intercalary and terminal conidiogenous cells are present only in Guayaquilia. Also, the bicellular, globose, brown chlamydospores that arise from assimilative hyphae in Guayaquilia are another distinctive character. The conidiophore morphology in Guayaquilia resembles that of some Phaeodactylium species (P. biseptatum, P. curvularioides, and P. stadleri) described by Castafeda- Ruiz & al. (2009, 2013) and Matsushima (1980), but these taxa have clavate, ellipsoidal, to obovoid, brown to subhyaline conidia. Acknowledgments We are indebted to Dr. Flavia Rodrigues Barbosa (Instituto de Ciéncias Naturais, Humanas e Sociais, Universidade Federal de Mato Grosso, Brazil) and Dr. De-Wei Li (The Connecticut Agricultural Experiment Station Valley Laboratory, USA) for their critical reviews. The authors are grateful to Escuela Superior del Litoral (ESPOL), CIBE for financial support and the International Society for Fungal Conservation for facilities. RFCR is grateful to the Cuban Ministry of Agriculture. We acknowledge the websites provided by Dr. P.M. Kirk (Index Fungorum) and Dr. K. Bensch (MycoBank). Dr. Lorelei Norvell’s editorial review and Dr. Shaun Pennycook’s nomenclature review are greatly appreciated. Literature cited Arx JA von. 1981. Notes on Microdochium and Idriella. Sydowia 34: 30-38. Becerra-Hernandez CI, Gonzalez D, Luna ED, Mena-Portales J. 2016. First report of pleoanamorphy in Gyrothrix verticiclada with an Idriella like synanamorph. 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MYCOTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 July-September 2020—Volume 135, pp. 513-523 https://doi.org/10.5248/135.513 Distoseptispora longispora sp. nov. from freshwater habitats in China Hal-YAN SONG’, ALY FARAG EL SHEIKHA”?*>*, ZHI-JUN ZHADL, JIAN-PING ZHOU’, MING-HuI CHEN’, GUANG-Hua Huo}, X1-GEN HuANG?®, Dian-MInG Hu”? ' Key Laboratory of Crop Physiology, Ecology and Genetic Breeding, Ministry of Education of the P. R. China, ? Bioengineering and Technological Research Centre for Edible and Medicinal Fungi, ’ Jiangxi Key Laboratory for Conservation and Utilization of Fungal Resources, and * College of Bioscience and Bioengineering, ™4 Jiangxi Agricultural University, 1101 Zhimin Road, Nanchang, Jiangxi 330045, China ° Department of Food Science and Technology, Minufiya University, Shibin El Kom, Minufiya Government, Egypt “ CORRESPONDENCE TO: * elsheikha_aly@yahoo.com ®=hxg208@163.com “hudianming1@163.com ABSTRACT—A novel species, Distoseptispora longispora, from submerged wood in Yunnan Province, China, is described and illustrated. The fungus is characterized by macronematous, mononematous, septate conidiophores with monoblastic, integrated, determinate, terminal conidiogenous cells that produce acrogenous, solitary, obclavate, elongated, distoseptate conidia. SSU-ITS-LSU sequence analyses were used to infer the phylogenetic relationship between D. longispora and related species. Both molecular analyses and morphological data well support D. longispora as an independent taxon. A key to Distoseptispora species is provided. KEY worDs—Ascomycota, asexual fungi, Distoseptisporaceae, hyphomycetes Introduction Distoseptispora K.D. Hyde & al. was introduced by Su & al. (2016) to accommodate two species, D. fluminicola McKenzie & al., the type species, 514 ... Song & al. and D. aquatica Z.L. Luo & al. The genus is characterized by hairy colonies, macronematous conidiophores, monoblastic conidiogenous cells, and distoseptate acrogenous cylindrical conidia., Distoseptispora represents a phylogenetically distinct lineage in Sordariomycetes for which Su & al. (2016) proposed a new family, Distoseptisporaceae K.D. Hyde & McKenzie. Subsequently, 13 more species have been added: D. cangshanensis Z.L. Luo & al., D. dehongensis W. Dong & al., D. guttulata Jing Yang & K.D. Hyde, D. martinii (J.L. Crane & Dumont) J.W. Xia & X.G. Zhang, D. obpyriformis Z.L. Luo & H.Y. Su, D. palmarum S.N. Zhang & al., D. phangngaensis Jing Yang & al., D. rostrata Z.L. Luo & al., D. submersa Z.L. Luo & al., D. suoluoensis Jing Yang & al., D. thailandica Tibpromma & K.D. Hyde, D. thysanolaenae Goonas. & al., and D. xishuangbannaensis Tibpromma & K.D. Hyde (Hyde & al. 2019, Luo & al. 2018, Phookamsak & al. 2019, Tibpromma & al 2018, Xia & al. 2017, Yang & al. 2018). Many aquatic fungi have been discovered in China’s freshwater habitats, which are rich in lignicolous fungi (Hu & al. 2007, 2012a,b; Huang & al. 2016; Song & al. 2018). Our ongoing survey of aquatic fungi in Yunnan Province revealed an interesting Distoseptispora species that we describe as new based on morphological characters and molecular analyses (SSU-ITS-LSU). Materials & methods Sample collection & morphological studies Debarked wood samples from different freshwater habitats in Yunnan Province, China, were taken to the lab for detection of fungi using dissecting and compound microscopes. The dried specimens were deposited in the Herbarium of Fungi, Jiangxi Agricultural University, Nanchang, China (HFJAU). DNA extraction, sequencing, and phylogenetic analyses Genomic DNA was extracted from fresh fungal materials found on the natural substrate following Hu & al. (2012c). We amplified three rDNA regions: internal TABLE 1. Isolates included in the phylogenetic analyses. GENBANK ACCESSION NUMBERS SPECIES STRAIN ITS LSU SSU Distoseptispora “adscendens” HKUCC 10820 — DQ408561 — D. aquatica MFLUCC 15-0374" NR_ 154040 KU376268 — SNJ14 MH555360 — a D. fluminicola MFLUCC 15-0417 =NR_154041 KU376270 — D. guttulata MFLUCC 16-0183 MF077543 MF077554 MF077532 D. “leonensis” D. longispora D. martinii D. multiseptata D. phangngaensis D. suoluoensis D. tectonae D. tectonigena Distoseptispora sp. Dothidea sambuci Ellisembia bambusicola E. calyptrata E. minigelatinosa Ellisembia sp. Sporidesmium aquaticivaginatum S. aquaticum S. australiense S. fluminicola S. macrurum S. obclavatulum S. olivaceoconidium S. pachyanthicola S. parvum S. pyriformatum S. submersum S. tengii S. thailandense S. tropicale Sporidesmium sp. Sporidesmium sp. Sporidesmium sp. Distoseptispora longispora sp. nov. (China) ... 515 HKUCC 10822 HFJAU 0705" HSAUP myr4280 MFLUCC 15-0609" MFLUCC 16-1044 MFLUCC 16-0857 MFLUCC 17-1305 MFLUCC 17-0224 MFLUCC 12-0291 GZ25 MFLUCC 12-0292 HLXM 15-1 AFTOL-ID 274 HKUCC 3578 HKUCC 10821 NN47497 HKUCC 10558 MFLUCC 15-0624 MFLUCC 15-0420 HKUCC 10833 MFLUCC 15-0346 HKUCC 2740 HKUCC 10834 MFLUCC 15-0380 HKUCC 10835 HKUCC 10836 MFLUCC 15-06207 MFLUCC 15-0627 MFLUCC 15-0421 HKUCC 10837 MFLUCC 15-0964" MFLUCC 16-0185 HKUCC 10838 KUFA 0043 MFLUCC 15-0617 MFLUCC 16-0186 MH555359 KU999975 NR_154017 MF077544 MF077545 MF077547 MF077546 KX751711 MH555361 NR_154018 DQ491505 KX710147 KX710144 KX710146 KX710148 MF374361 MF077551 KT824799 MF077550 MF077549 DQ408566 MH555431 KX033566 KX710140 MF077555 MF077556 MF077558 MF077557 KX751713 KX751714 KU376269 AY544681 DQ408562 DQ408564 DQ408567 DQ408565 KX710142 KU376273 DQ408554 KU376271 DQ408555 DQ408556 KX710139 DQ408557 DQ408558 KX710141 KU376272 DQ408559 MF374370 MF077562 DQ408560 MF077561 MF077560 MH555357 KX033537 MF077540 MF077533 MF077534 MF077536 MF077535 MH555358 AY544722 MF077541 MF077542 KX710143 MF077539 MF077538 516 ... Song & al. transcribed spacer (ITS) with the primer pair ITS1 & ITS4 (White & al. 1990), partial small subunit (SSU) with NS1 & NS4 (White & al. 1990), and large subunit (LSU) with LROR & LR6 (Vilgalys & Hester 1990, Rehner & Samuels 1995). PCR protocols followed the conditions set by Hu & al. (2012b). The PCR products were purified and sequenced by the same primers used for PCR at Sangon Biotech (Shanghai) Co. Ltd. In this study, we generated six novel sequences (MH555360, MH555359, MH555431, MH555357, MH555361 & MH555358) and retrieved 36 reference sequences from GenBank (TABLE 1) that were aligned using MAFFT v7. The ML analyses were produced with RAxML v7.2.6 (Stamatakis & Alachiotis 2010) using a GTRGAMMA substitution model with 1000 bootstrap replicates and evaluated by bootstrap support (MLBS). The best-fit evolutionary models were estimated using MrModeltest V2.2. Posterior probabilities (PP) (Zhaxybayeva & Gogarten 2002) were determined by Markov chain Monte Carlo sampling (BMCMC) in MrBayes 3.0b4 (Huelsenbeck & Ronquist 2001). Six simultaneous Markov chains were run 1,500,000 generations, with trees sampled every 100 generations and the first 25% deleted as burn- in. Posterior probabilities were calculated based on the remaining trees. The novel taxonomic descriptions and nomenclature were deposited in MycoBank (http://www.mycobank.org/). Phylogenetic results The six new sequences generated in our study included one LSU, two SSU, and three ITS sequences. The phylogenetic tree based on the three-locus analysis (Fic. 1) shows the relationship between the new taxon and other related species. The concatenated aligned dataset comprised 17 isolates from 13 Distoseptispora spp., 23 isolates from 21 Sporidesmium (or “Ellisembia”) spp., and one isolate of Dothidea sambuci (Pers.) Fr. (Dothideomycetes) as an outgroup. The dataset including alignment gaps comprised 2817 characters: 1348 for SSU, 629 for ITS, and 840 for LSU. The combined dataset ML tree (Fic. 1) with bootstrap support values (MLBS) and Bayesian posterior probabilities (BPP) indicates several well-supported clades, with the Distoseptispora longispora strain forming a well-supported clade (MLBS = 94%, BPP = 0.68) with other Distoseptispora species. Taxonomy Distoseptispora longispora H.Y. Song & D.M. Hu sp. nov. Fic. 2 MB 826916 Differs from Distoseptispora aquatica by its larger and more septate conidia; and from D. tectonae and D. tectonigena by its smooth and brown to yellowish brown conidia. Distoseptispora longispora sp. nov. (China) ... 517 66/0.99 Distoseptispora tectonae MFLUCC 12-0291 60/0.98|! nistoseptispora tectonae GZ25 Distoseptispora tectonigena MFLUCC 12-0292? Distoseptispora “adscendens” HKUCC 10820 79/0.96: Distoseptispora fluminicola MFLUCC 15-0417? Distoseptispora aquatica SNJ14 abet Li Dinseviees basis MFLUCC 15-0374? 73/0.76| | Distoseptispora longispora HFJAU 0705t v1 Distoseptispora sp. HLXM 15-1 65/0.68 Distoseptispora multiseptata MFLUCC 15-0609T Distoseptispora multiseptata MFLUCC 16-1044 98/0.67 Distoseptispora phangngaensis MFLUCC 16-0857 Distoseptispora martinii HSAUP myr4280 Distoseptispora guttulataMFLUCC 16-0183 94/0.68] | 100/1)Distoseptispora suoluoensis MFLUCC 17-0224 Distoseptispora suoluoensis MFLUCC 17-1305 63/- PONS Distoseptispora “leonensis” HKUCC 10822 100/1 >- Sporidesmium olivaceoconidium MFLU! oC Sporidesmium aquaticivaginatum MI FLUCC 99/0.94 Sporidesmium pyrif 100/1 iE 1! 'Sporidesmium pyriformat, 100/1 93/0.73 ‘Sporidesmiine a c 100/0.85 96/1 = Sl Sporidesmium sp. MFLUCC 15-0 100/1 Sporidesmium thailandense MFLU 87/0.94, Sporidesmium submersum MFLU u /0. Ellisembia minigelatinosa NN47497 7 100/0.93 99/0. Br ON zitisembia bambusicola HKUCC 3578 Ellisembia sp. HKUCC 10558 S Sporidesmium aquaticum MFLUCC 15-0420 100/1/ Sporidesmium tropicale HKUCC 10838 100/0.99 Sporidesmium tropicale MFLUCC 16-0185 64/0.8. Sporidesmium macrurum HKUCC 2740 100/1 Sporidesmium sp. MFLUCC 16-0186 -/67 Ellisembia calyptrata HKUCC 10821 Sporidesmium sp. KUFA 0043 99/1 Sporidesmium tengii HKUCC 10837 100/1 Sporidesmium australiense HKUCC 10833 Sporidesmium obclavatulum HKUCC 10834 Sporidesmium pachyanthicola HKUCC 10835 Dothidea sambuci AFTOL-ID 274 0.1 Fic. 1. Maximum Likelihood (ML) phylogenetic tree of Distoseptispora longispora and allied species based on concatenated SSU-ITS-LSU data. The RAxML bootstrap support values (MLBS) and Bayesian posterior probabilities (BPP) are given at the nodes (MLBS/BPP). Type: China, Yunnan Province: Mengla, a small stream, on submerged wood, 13 Apr 2017, H.Y. Song (Holotype, HFJAU 0705). EtyMo.oey: longispora, referring to the long conidia of the fungus. COLONIES on submerged wood superficial, effuse, scattered, hairy, brown to dark brown. Mycelium mostly immersed, composed of branched, septate, smooth, subhyaline to pale brown hyphae. CONIDIOPHORES macronematous, mononematous, 2-4-septate, unbranched, single, straight or slightly flexuous, erect, cylindrical, smooth, brown to dark brown, 17-37 x 6-10 um. CONIDIOGENOUS CELLS monoblastic, integrated, determinate, terminal, light brown to dark brown, cylindrical. Conidial secession schizolytic. CoNIDIA acrogenous, solitary, dry, obclavate, elongated, straight or slightly curved, rounded at the apex, truncate at the base, 31—56-distoseptate, smooth, brown 518 ... Song & al. to yellowish brown, slightly paler towards the apex, 189-297 x 16-23 um (mean = 204 x 21 um, n= 50). ADDITIONAL SPECIMEN EXAMINED: CHINA, YUNNAN PROVINCE, Mengla, a small stream, on submerged wood, 13 Apr 2017, H.Y. Song (HFJAU 0706). Notes: Distoseptispora longispora is characterized by its macronematous, mononematous, septate conidiophores, monoblastic, integrated, determinate, terminal conidiogenous cells, and acrogenous, solitary, dry, obclavate, elongated, distoseptate conidia, which fit well with the generic concepts of Distoseptispora (Su & al. 2016). The molecular phylogeny (Fic. 1) clusters the D. longispora strain well within the Distoseptispora clade and closely related to D. aquatica but with an ITS six base pair (bp) difference separating it from D. aquatica (MFLUCC 15-0374). Distoseptispora longispora resembles D. aquatica, D. tectonae, and D. tectonigena in conidial shape. However, its larger and more septate conidia separate D. longispora from D. aquatica (189-297 x 16-23 um; 15-28 septa). In addition, D. tectonae has verruculose, dark reddish conidia, and D. tectonigena has dark reddish-brown conidia (Hyde & al. 2016). The multilocus molecular phylogeny and morphological data fully support separation of our new species, D. longispora. Morphologically, Distoseptispora resembles Ellisembia Subram., first established to accommodate the species with distoseptate conidia in Sporidesmium Link (Subramanian 1992). However, a molecular phylogeny by Shenoy & al. (2006) showed that Ellisembia and Sporidesmium were polyphyletic. Su & al. (2016) later clarified differences between Ellisembia and Sporidesmium, noting that euseptate/distoseptate characters were not supported by molecular evidence and treating Ellisembia as a synonym of Sporidesmium. Distoseptispora species differ from Ellisembia (Sporidesmium) species in having relatively short conidiophores and darker conidia with slightly paler, but not hyaline, rounded apices, basal cells cut off by cross walls, and of indeterminate length (Su & al. 2016). There are various hyphomycetes in freshwater habitats, and several studies have reported Distoseptispora species, e.g., D. aquatica, D. fluminicola, D. guttulata, D. multiseptata, D. phangngaensis, and D. suoluoensis (Hu & al. 2007, 2010a,b; Huang & al. 2018; Hyde & al. 2016; Su & al. 2016; Yang & al. 2018). Most of these species were identified based on morphology, which proved to be insufficient for accurate identification (Hu & al. 2017). Numerous other studies (Réblova & al. 2016, Su & al. 2016, Zhang & al. 2017) have revealed many different freshwater lineages using molecular analyses. Distoseptispora longispora sp. nov. (China) ... 519 d b Fic. 2. Distoseptispora longispora (holotype, HFJAU 0705). a. Colonies on submerged wood; b, c. Conidia; d. Conidiophore. Scale bars: a = 100 um, b-d = 5 um. 520 ... Song & al. Key to Distoseptispora species 1. Conidia muriform, having both longitudinal and transverse septa ...... D. martinii b-@oniciawathy Only transverse septa: 4) ef aia n he Seed ert bs ae natok Sue hes 2 PAC OMIA CUSe Plate .cA Ars Hs AMS sR tee ode gt AE Tee ppc i oan: ood aPag ET NR OVS CP 3 2 COniatacdistosep tate. 7. Reed oe AR ER Ne He see hy wasn aeale pS Ree ea 4 3. Conidia 75-130 x 7-11 um, 11-14(-20)-euseptate, smooth ........... D. guttulata 3. Conidia 80-125 x 8-13 um, 8-10-euseptate, verrucose............. D. suoluoensis 4, Conidiogenous cells polyblastic, conidia 35-180 x 7-ll um ......... D. palmarum 4. Conidiogenous:cellsimonoblastic «5, ose ages axis sean nied omens Panaee ken 5 5. Conidiophores having percurrently proliferating conidiogenous cellsatthe apex Or comidiophoress:,. <. eactcmtics aha sate Ke sent teen sede oP Seek 6 5 Conidiophores Ot AG ADOVE Bi sts remiaginw Hits te aca hbo aaitess ertticen peta Sabghomem ae 7 6; Conidial PaaS 5 8 Orr pas An. Se, Scene ae tl Rd, Bre mle AN al D. rostrata Gs ConidianAs 22 51 az ties sea tetiaeale Sat wants. eee D. tectonigena 7 @onidiaverruculoses7..5=9:5-X SiS SYM 25s eens eke aod ete alae os D. tectonae Fe LOT Cia NOt eTTUCULOSE Pic etn)... Aas 5) ARN oe Sng. cats stata tar Ate so IRB. 8 8. Conidia 3-5-distoseptate, 17-30 x 7.5-10 um................004. D. dehongensis 8. Gonidia with more-than-6. distosepta: «oe. .5 da.04 wedeae vee ee ae cea 9 9. Saprobic on submerged wood in freshwater habitats ...................0000-% 12 9. Not saprobic on submerged wood in freshwater habitats...................-. 10 10. Saprobic on dead culms of Thysanolaena maxima, conidia 8-14-distoseptate, 21.5-80 x 6.5-12.8 um ........... D. thysanolaenae 10Saprobic-on-deadsleaves*ot Pandanussp. bchie aiuto ag ths to ethuseen nore hoh wht ie 11 11. Conidia reddish brown to brown, 130-230 x 13.5-17 um ......... D. thailandica 11. Conidia green-brown to brown, 160-305 x 8-15 um...... D. xishuangbannaensis 12. Conidia 9-11-distoseptate, 53-71 x 12-16um .................. D. obpyriformis 12. Comidia with-morediant 12 aistos ep ta gets ssi cists sis ae todays aloe fel tte tess 13 13. Conidiogenous cells yellow, conidia 125-250 x 13-15 um ......... D. fluminicola 13¥Gonidiogenous cells: brownish: 22.0202 42-02-50u nessa ee Qo ueeee ee ou eeeeeees 14 14. Conidia brown to yellowish brown, 31-56-distoseptate, 1B oe Pde eo Ad CePA on Ae Roa ee a ai D. longispora 14. Conidia greenish brown or dark olivaceous green ............. 0.0 c cece eee 15 15. Conidiogenous cells subhyaline to pale brown, conidia 58=1665¢ 10-14 yiml e255 toe tsa pee, Pee ed ee D. cangshanensis 15. Conidiogenous cells brown to dark brown ............ 0.00. eee eee eee eee 16 16. Conidiogenous cells dark brown to olivaceous, conidia 110-157" 13:5.51:625 im" * 4 254 ak. 7 5 eis ef eal D. aquatica 16.-Gonidiogenious cells brown oo... 4h oi. gee eee ete a ede te deen ce dae 17 Distoseptispora longispora sp. nov. (China) ... 521 17. Conidia dark olivaceous green, multi-distoseptate, 99 = 29096 TP ONE em ae ERS Sin Ss ike ced eS ad D. multiseptata 17. Conidia dark or mid olivaceous to mid or dark brown ...................04- 18 18. Conidia dark olivaceous to mid or dark brown, 16553507% 14 S19 spirrn Vaan ae VR Brn tae eNe Bote Ee, ese ae D. phangngaensis 18. Conidia mid-olivaceous to brown, 95-123 x15-19 um .............. D. submersa Acknowledgments The authors express their sincere gratitude to Dr. Rafael EF Castafieda-Ruiz (Alejandro de Humboldt—INIFAT, Havana, Cuba) and Dr. Jian Ma (College of Agronomy, Jiangxi Agricultural University, Nanchang, China) for their critical review of the manuscript, to Dr. Shaun Pennycook for nomenclatural review, and to Dr. Lorelei L. Norvell for editorial review. 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BMC Genomics 3: 4. https://doi.org/10.1186/1471-2164-3-4 MYCOTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 July-September 2020—Volume 135, pp. 525-534 https://doi.org/10.5248/135.525 Notes on rust fungi in China 9. Puccinia miscanthi life cycle and morphology confirmed by inoculation JING-XIN J’*, ZHUANG LY’, Yu Lt, MAKOTO KAKISHIMA??® ‘Engineering Research Center of Chinese Ministry of Education for Edible & Medicinal Fungi, Jilin Agricultural University, Changchun, Jilin 130118, China College of Plant Protection, Shandong Agricultural University, Taian 271000, China >University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan * CORRESPONDENCE TO: * 1096314395@qq.com * kakishima.makoto.ga@u.tsukuba.ac.jp ABSTRACT— The Heteroecious and macrocyclic life cycle of Puccinia miscanthi is confirmed for the first time with inoculation experiments in China. The rust produces spermogonial and aecial stages on Plantago asiatica and uredinial and telial stages on Miscanthus sacchariflorus. Morphological characters of these stages are described based on field collections and specimens that confirmed their life cycle connections. The neotype of P miscanthi is designated. Key worps—Plantaginaceae, plant disease, Poaceae, Pucciniales, Uredinales Introduction Puccinia miscanthi was first described with uredinial and telial stages on Miscanthus sacchariflorus (Poaceae) collected in northeastern China (Miura 1928). The life cycle of P. miscanthi was reported in Japan by Hiratsuka (1933), Ito (1934), and Asuyama (1936) under the name of P. eulaliae Barclay. These authors demonstrated life cycle connection between spermogonial and aecial stages on Plantago asiatica (Plantaginaceae) and uredinial and telial stages on M. sinensis Andersson through inoculations. Ito (1950) considered the rust fungus as producing spermogonial and aecial stages on Plantago and uredinial and telial stages on Miscanthus and Imperata (as P. eulaliae) and treated P. miscanthi as a synonym of this species. Later, however, this rust fungus 526 ... Ji & al. was considered to represent a species different from P. eulaliae because of morphology and host relations, and it was treated as P. miscanthi (Cummins 1971, Hiratsuka & al. 1992). Sato & Kakishima (1982) reported Lysimachia clethroides Duby (Primulaceae) as an additional host of the rust’s spermogonial and aecial stages. This rust fungus has been reported on wide range of host plants from northeastern Asia (TABLE 1, p. 532). During investigation of rust fungi in northeastern China, a Puccinia species producing uredinial and telial stages on M. sacchariflorus was collected in Heilongjiang Province. Spermogonial and aecial stages of a rust fungus on Plantago asiatica were also found near infected M. sacchariflorus plants. The spermogonial and aecial stages were suspected to represent different stages of Puccinia miscanthi. However, life cycle connection of P miscanthi between different host plants has not been clarified in China, although PI. asiatica, Pl. depressa Willd., and Pl. major L. have all been listed as spermogonial and aecial host plants, and M. floridulus (Labill.) K. Schum. & Lauterb., M. sacchariflorus, M. sinensis, Saccharum spontaneum L., and Thysanolaena maxima (Roxb.) Kuntze as uredinial and telial hosts (Wang & Zhuang 1998, Cao & Li 1999). Furthermore, Puccinia cynodontis Lacroix ex Desm. also has been reported to produce spermogonia and aecia on Plantago species including Pl. asiatica (Cummins 1971, Wang & Zhuang 1998). Worldwide, uredinial and telial stages of six morphologically similar Puccinia species have been recorded on Miscanthus (TABLE 1; Cummins 1971, Hiratsuka & al. 1992, Wang & Zhuang 1998). Among them, P. miscanthi, P. miscanthicola EL. Tai & C.C. Cheo, P. melanocephala Syd. & P. Syd., and P. erythropus Dietel have been recorded in China (Tai 1979, Wang & Zhuang 1998). Therefore, we carried out inoculation experiments to clarify the life cycle connection between spermogonia and aecia on PI. asiatica and uredinia and telia on M. sacchariflorus collected in Heilongjiang Province on 2 July 2018. We also observed morphology of these specimens for identification of species. Materials & methods Inoculation experiments On 2 July 2018 uredinia and telia on Miscanthus sacchariflorus were found at Wuchang, Heilongjiang Province, China (44°12’22”N 128°03’33”E). Infected plants were transplanted in plastic pots and placed on the ground at the campus of Jilin Agricultural University, Changchun, Jilin Province. In late summer, abundant telia appeared on the plants, and these telia were overwintered on dead leaves. Teliospores obtained from the telia were observed to germinate and basidiospores produced in the spring of 2019 were used for inoculations. On 17 June 2019 small pieces of leaves Puccinia miscanthi life cycle (China) ... 527 - P XG) Le = te! Fic. 1. Puccinia miscanthi on Plantago asiatica (A, C, E) and Miscanthus sacchariflorus (B, D, F). A. The plants in plastic pots, producing spermogonia and aecia by basidiospore inoculation. B. Plant transplanted from Heilongjiang Province and cultured at campus of Jilin Agricultural University. C. Spermogonia produced on leaf surface by basidiospore inoculation. D. Brown uredinia produced on leaf surface by aeciospore inoculation. E. Aecia on lower leaf surface produced by basidiospore inoculation. F. Dark brown telia on leaf. with teliospores were attached to the surfaces of young healthy leaves of Pl. asiatica for inoculation, using a similar method previously reported by Ji & al. (2017a,b). The inoculated plants were collected at campus of Jilin Agricultural University and had been maintained in plastic pots. No natural infection of the field plants had 528 ... Ji&al. been confirmed prior to inoculation. The inoculated plants were kept in a humid environment in a plastic box in darkness at 20-23 °C for 3 days and then transferred to a place near windows at 20-23 °C for observation. Aeciospores obtained from aecia on the basidiospore-inoculated Plantago asiatica leaves were dusted onto small pieces of wet filter paper (about 3 mm?’) in a Petri dish. These papers were then placed on young healthy leaves of M. sacchariflorus, which had been transplanted from Wuchang, Heilongjiang Province in 2018 and maintained in plastic pots at campus of Jilin Agricultural University. Plants with new leaves were used for the inoculation. The inoculated plants were kept in a humid environment in a plastic box under the same conditions as the basidiospore inoculations, and later transferred to the place near windows. Morphological observations Specimens collected at Wuchang, and specimens obtained by inoculations were morphologically examined. The spores and size and shape of sori were microscopically examined using Light (LM) and scanning electron (SEM) microscopes according to Ji & al. (2019). The specimens are deposited in the Herbarium of Mycology, Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi, Jilin Agricultural University, Changchun, China (HMJAU). Results & discussion Life cycle About 10 days after inoculation of Plantago asiatica with basidiospores from germinating teliospores, small yellow spots of spermogonia appeared on both surfaces of the inoculated leaves (Fics 1A, C). These spots were frequently observed on the leaf, and the leaf tissue around infected veins was sometimes malformed when many spots were produced. About 10 to 12 days later, orange- yellow aecia were produced around the spots on lower surfaces of the leaves (Fic. 1E). About 5 days after aeciospore inoculation, pale yellow spots appeared on the leaves of Miscanthus. sacchariflorus, after which then brown uredinia were produced (Fic. 1D). One to two months after that, blackish telia appeared on M. sacchariflorus (Fic. 1F). The inoculation results confirmed that the rust fungus producing spermogonial and aecial stages on PI. asiatica was conspecific with the rust fungus producing uredinial and telial stages on M. sacchariflorus. Morphology & identification Theinoculations show that this rust fungus has an heteromacrocycliclife cycle, producing spermogonia and aecia on Plantago asiatica and uredinia and telia on Micanthus sacchariflorus. The uredinial and telial stages of our Heilongjiang Province rust on M. sacchariflorus are morphologically similar to those of Puccinia miscanthi life cycle (China) ... 529 ore seed fl —- Fic. 2. Puccinia miscanthi on Plantago asiatica (HMJAU8642: A, B, D) and Miscanthus sacchariflorus (neotype, HMJAU8643: C, E, FE, G) observed by LM. A. Vertical section of spermogonium (type 4). B. Aeciospores with verrucose surface. C. Urediniospores with equatorial germ pores. D. Vertical section of aecium with catenulate aeciospores surrounded by peridium. E. Vertical section of uredinium mixed with paraphyses. F. Teliospores. G. Vertical section of telia, surrounded by host epidermis. Scale bars: A, C = 30 um; B, E = 20 um; D = 100 um; F G = 25 um. Puccinia erythropus, P. melanocephala, and P. miscanthi in urediniospore and teliospore shapes. However, teliospores of P. erythropus and P. melanocephala 530 ... Ji&al. are shorter, and their apical walls thinner, than those of our M. sacchariflorus rust fungus (TABLE 1). Furthermore, although Ito (1950) described paraphyses (TABLE 1), P. erythropus has been reported to have no paraphysis in uredinia, whereas, our M. sacchariflorus rust fungus produces abundant paraphyses (Fics 2E, 3D). The morphological characters of our M. sacchariflorus rust fungus are very close to those previously reported for P miscanthi, but as its teliospores are only slightly shorter than these descriptions (TABLE 1), we identified our rust fungus as P. miscanthi. Host range and morphological features of P. miscanthi reported by different authors as shown in TABLE 1; the differences may be considered variations within species caused by host plants and geographical distribution. Puccinia miscanthi has been reported to be genetically very close to P melanocephala although they are morphologically different (Virtudazo & al. 2001, Dixon & al. 2010). Phylogenetic analyses with specimens from different regions and host plants may clarify their relationships. Spermogonial and aecial stages of Puccinia miscanthi have been reported to be produced on Plantago and Lysimachia species (Cummins 1971, Hiratsuka & al. 1992, Wang & Zhuang 1998). However, no confirmation of these stages of P. miscanthi has been reported in China, although its life cycle connection was reported in Japan with inoculations. Therefore, Pl. asiatica is the first host plant confirmed by inoculations of its spermogonial and aecial stages in China. The morphology of these stages is almost identical with the descriptions reported from Japan by Ito (1950), Sato & Kakishima (1982), and Hiratsuka & al. (1992), except for the slightly smaller aeciospores. Wang & Zhuang (1998) described the rust’s spermogonial and aecial stages on Pl. depressa in China; however, a connection to its uredinial and telial stages was not confirmed. Two Plantago species, Pl. asiatica and Pl. lanceolata L., were also reported as spermogonial and aecial host plants of P. cynodontis without confirmation of their life cycle connections in China (Wang & Zhuang 1998). Therefore, rust species identifications of spermogonial and aecial stages on species of Plantago should be reconsidered. Ono & Azbukina (1997) reported that P. erythropus, morphologically similar to P miscanthi, produced spermogonia and aecia on Cynanchum sublanceolatum var. obtusum (Franch. & Sav.) Matsum. (Apocynaceae), suggesting that this species differs from P miscanthi in its spermogonial and aecial hosts. Miura (1928) described Puccinia miscanthi based on a specimen on Miscanthus sacchariflorus collected in Teikaton, Manchuria (northeast China) on 22 September in 1919. However, this specimen has not been found in any herbarium, we therefore designate a neotype. We collected specimens on the Puccinia miscanthi life cycle (China) ... 531 Fic. 3. Puccinia miscanthi on Plantago asiatica (HMJAU8642: A, B) and Miscanthus sacchariflorus (neotype, HMJAU8643: C, D) observed by SEM. A. Aeciospores with densely verrucose surface. B. Aecium with peridium. C. Urediniospore with echinulate surface. D. Uredinium mixed with paraphyses. Scale bars: A, C = 5 um; B = 40 um; D = 30 um. same host plant from the same area of China, as reported by Miura (1928) and used for inoculations. We believe that specimens collected and obtained from inoculations are morphologically close to the original specimen and describe its morphology based on these specimens. Puccinia miscanthi Miura, Fl. Manch. E. Mong., III Cryptogams, Fungi: 302, 1928. Figs 1-3 Type: Uredinia and telia on Miscanthus sacchariflorus (Maxim.) Franch. (Poaceae), China, Helongjiang Province, Wuchang, 9 September 2017, leg. M. Kakishima & J-X. Ji (neotype designated here, HMJAU8643; MBT388544). Spermogonia amphigenous, surrounded by greenish yellow lesions, densely grouped, yellow, subepidermal, flask-shaped, type 4 of Cummins & Hiratsuka (2003). Aecia hypophyllous, densely grouped, yellow, cupulate with peridia, Aecidium-type. Aeciospores catenulate, globose to subglobose, Ji & al. 2h. uoT}dIIOsap [RUIBTIO :, (€ ‘UdAT3 JON : 8661 SueNYZ 2» SUM C66T ‘TR 28 eNS EIT TZ6T suture) OS6I OI xSO6T [PIC Z66T ‘Te 29 eYNsyeITH IZ61 suru OS6T OY 8661 SueNYZ 2» Sue Z66T ‘Te 29 eYNsIeITY LZ6T sururuns «L061 Mopds 29 MopdS LZ6T sururuns 8661 SueNYZ 2» Sue LZ6T suru so0c eURIngzZy 8661 SueNYyZ 2» Sue Z66T ‘Te 29 eyNsIeITY TZ6T surtuuine) OS6I OI x8C6I PANIY] jaded yuasoid ayy, TONAU AAA Y S-€ SC-ST I@-LI X SP-CE S-€ SC-ST OC-9I X SP-EE c-€ SC-ST OC-9I X SP-EE E56 CC-VL X CS-CE v 2 I@-9L X SP-8T €I-Z c-ST CC-ST X 9S-SE €I-Z c-S'T CC-ST X 9S-GE ScI-6 é I@-ST X 09-CP v-€ C-S'T CC-LI XEV—-OE v-€ c-S'T I@-LI X €h-O€ v-€ c-ST I@-LI X €P-0€ 9-9 é OC-VI X SP-SE 6-S £-¢ LC-OC X 9P-€E v-T f° VC-SI X SS-CE €-¢ E-G VC-SI X SS-CE 9-9 c-ST VC-9L X 09-CE 8-T C-S'T VC-9L X OL-TE 9-9 C-SI €C-9L X 09-O0F 9-9 CSA. €C-9L X 09-0F LE. é VC-9T X OL-TE S8-Z é SC-ITC X 19-€P 9-9°C I-S'0 CC-VLI X G'9S-S'6E xody apts (ur) STAOASOITAY, b-€ p-€ b-€ v S-) OI V ‘ou 210g €T-8I X TE-HT €T-8I X €€-F7 €T-8I X €€-F7 ST-O7 X PE-8Z €T-8I X 8Z-€Z €T-6I X €€-97 €T-6I X €£-97 87-17 X PE-87 PT-8I X PE-LZ €T-8I X €£-8Z €T-8I X €£-8Z SZ-8I X OF-77 €Z-61 X O€-F7 uUMOUYUN eIUTpar—, CE-OC X BE-LT LE-OC X 8E-ST 9C-61 X SE-6T 9T-61 X SE-6T T¢e-07 X 8e-ST GC X €€ SECT-G'6T X GSE-LT (um) 9215 SHUOdSOINIGHY) + + + + + + + + + + + + -uInTuUIpein ul SaSAHAVUVg PISsSny ‘sautddrryg ‘uede( ‘WeMIey, “euryD) vissny ‘uede( 2PIMPHOM BLY YINOS euryo BISsny ‘soutddrryg ‘uede( ‘ueMIey, “eury) NOLLOGISLSIG, W'S = = ai a Sess n Bown ;VWuUANAD LSOP 3 ‘Sgouasqy :— ‘souasarg :+ (7 ‘snAnjaovYyg :q ‘vuavjouvsdy], :], SWinsvyrIvG :S ‘snyguvosIPy 7 ‘vJosadwy :] ssnyguvLag vy (1 sndosyjasa g sisuauaswp J vjvydasouvjau upiguvssiu g DIOINYJUDISIM J [Aepieg uou ‘oy *S avyyjna q =| iyjuvostur J SaIOadS ‘dds snyjuvosip uo payiodas saisads viuioong Jo ela} pue erurpein jo A8ojoydiow saneseduroy “| ATAVI, Puccinia miscanthi life cycle (China) ... 533 angular, 17.5-23.5 x 14.5-19.0 um (av. = 20.5 x 17. 0 um); walls hyaline, 0.6-1.2 um (av.= 0.9 um) thick, densely verrucose. Uredinia mostly on abaxial surface, scattered or gregarious, yellow- brown, subepidermal, erumpent, with abundant paraphyses. Paraphyses capitate; walls hyaline, thicker at the apical parts. Urediniospores pedicellate, obovoid to ellipsoid, 27.0-35.5 x 19.5-23.5 um (av. = 30.0 x 21.5 um); walls brown, echinulate, 0.7-1.5 um (av. = 1.0 um) thick; germ pores mostly 4, equatorial. Telia mostly on abaxial surface, blackish brown, erumpent, pulvinate. Teliospores pedicellate, oblong-clavate, 39.5-56.5 x 14.0-22.0 um (av. = 47.0 x 19.0 um); walls chestnut-brown, 0.5-1.5 um (av. = 1.0 um) thick at sides, 2.5-6.0 um (av. 4.0 um) thick at apices, smooth; pedicels persistent, brown, thick-walled, short. ADDITIONAL SPECIMENS EXAMINED — CHINA, HEILONGJIANG PROVINCE, Wuhan: Spermogonia and aecia on Plantago asiatica, 2 July 2018, (HMJAU8644); cultured at Jilin Agricultural University, Changchun, 5 July 2019 (HMJAU8642). Uredinia and telia on Miscanthus sacchariflorus, 2 July 2018 (HMJAU8645); cultured at Jilin Agricultural University, Changchun, 5 July 2019 (HMJAU8646). Acknowledgments This work was financed by the Fungal Flora in Jilin Province (20130206073NY). We wish to thank Dr E.H.C. McKenzie (Manaaki Whenua Landcare Research, Auckland, New Zealand) and Prof. C.M. Denchev (Bulgarian Academy of Sciences, Sofia, Bulgaria) for critical reading of the manuscript and suggestions. Literature cited Asuyama H. 1936. A telial host of an Aecidium on Plantago asiatica L. Journal of Plant Protection (Japan) 23: 206-207. (In Japanese) Azbukina ZM. 2005. Rust fungi. Cryptogamic plants, fungi and mosses of the Russian Far East, vol. 5. Dalnauka, Vladivostok. (In Russian) Cao ZM, Li ZQ. 1999. Rust fungi of Qinling Mountains. China Forest Publishing House. Beijing. Cummins GB. 1971. The rust fungi of cereals, grasses and bamboos. Springer-Verlag, New York. https://doi.org/10.1007/978-3-642-88451-1 Cummins GB, Hiratsuka Y. 2003. Illustrated genera of rust fungi, 3" ed. American Phytopathological Society, St. Paul, Minnesota. Dietel P. 1905. Uredineae Japonicae. VI. Botanische Jahrbiicher fiir Systematik Pflanzengeschichte und Pflanzengeographie 37: 97-109. Dixon LJ, Castlebury LA, Aime MC, Glynn NC, Comstock JC. 2010. Phylogenetic relationships of sugarcane rust fungi. Mycological Progress 9: 459-468. https://doi.org/10.1007/s11557-009-0649-6 Hiratsuka, N. 1933. Inoculation experiments with heteroecious species of Japanese rust fungi. Botanical Magazine Tokyo 47: 710-714. https://doi-org/10.15281/jplantres1887.47.710 Hiratsuka N, Sato S, Katsuya K, Kakishima M, Hiratsuka Y, Kaneko S, Ono Y, Sato T, Harada Y, Hiratsuka T, Nakayama K. 1992. The rust flora of Japan. Tsukuba-shuppankai, Tsukuba. 534 ... Ji &al. Ito, S. 1934. Cultures of Japanese Uredinales I. Botanical Magazine Tokyo 48: 531-539. https://doi.org/10.15281/jplantres 1887.48.531 Ito S. 1950. Mycological flora of Japan, vol. 2, no. 3, Urediniales-Pucciniaceae, Uredinales Imperfecti. Yokendo, Tokyo. Ji JX, Li Z, Wan Q, Li Y, Kakishima M. 2017a. Life cycle of Aecidium klugkistianum on Ligstrum and its new combination, Puccinia klugkistiana. Mycoscience 58: 307-311. https://doi.org/10.1016/j.myc.2017.01.004 Ji JX, Li Z, Wan Q, Li Y, Kakishima M. 2017b. Notes on rust fungi in China 3. Puccinia adenocauli comb. nov. and its life cycle and new host. Mycotaxon 132: 141-148. https://dx.doi.org/10.5248/132.141 Ji JX, Li Z, Li Y, Kakishima M. 2019. Two new species of Pucciniastrum producing dimorphic sori and spores from northeast of China. Mycological Progress 18: 529-540. https://doi.org/10.1007/s11557-018-1460-z Miura M. 1928. Flora of Manchuria and east Mongolia III. Cryptogams, Fungi. Minamimanshutetsudo, Dalian. Ono Y, Azbukina ZM. 1997. Heteroecious life cycle of two graminicolous Puccinia (Urediniales). Mycoscience 38: 281-286. https://doi.org/10.1007/BF02464085 Sato T, Kakishima M. 1982. Life cycles and morphology of two Puccinia species that produce aecidioid sori on Lysimachia clethroides. Transactions of Mycological Society of Japan 23: 293-300. Sydow H, Sydow P. 1907. Fungi Indiae orientalis pars II. Annales Mycologici 5: 485-515. Tai FL. 1979. Sylloge fungorum Sinicorum. Science Press, Beijing. Virtudazo EV, Nakamura H, Kakishima M. 2001. Phylogenetic analysis of sugarcane rusts based on sequences of ITS, 5.88 rDNA and D1/D2 regions of LSU rDNA. Journal of General Plant Pathology 67: 28-36. https://doi.org/10.1007/PL00012983 Wang YZ, Zhuang JY. 1998. Flora Fungorum Sinicorum vol. 10, Uredinales (I). Science Press, Beijing. MYCOTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 July-September 2020—Volume 135, pp. 535-558 https://doi.org/10.5248/135.535 Biodiversity of heat-resistant ascomycetes from semi-arid soils in Argentina STELLA MARIS ROMERO??*, ANDREA IRENE ROMERO’”, ALBERTO MIGUEL STCHIGEL?, ERNESTO RODRIGUEZ ANDRADE?, VIVIANA ANDREA BARRERA‘, JOSE FRANCISCO CANO}, RICARDO COMERIO® ' Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales, DBBE, Intendente Giiiraldes 2160, Pab. II Ciudad Universitaria, C1428EGA, Buenos Aires, Argentina ? CONICET-Universidad de Buenos Aires, Instituto de Micologia y Botanica (InMiBo), Intendente Giiiraldes 2160, Pab. II Ciudad Universitaria, C1428EGA, Buenos Aires, Argentina * Mycology Unit, Medical School and IISPV, Universitat Rovira i Virgili (URV), Sant Lloreng 21, 43201 Reus, Tarragona, Spain * Instituto Nacional de Tecnologia Agropecuaria (INTA), IMyZA-CICVyA, N. Repetto y De los Reseros, CC25 1712, Castelar, Pcia. Buenos Aires, Argentina ° Instituto Nacional de Tecnologia Agropecuaria (INTA), EEA Anguil Ing. Agr. G. Covas, Ruta Nacional N° 5, km 580, CC 11 6326 Anguil, Pcia. La Pampa, Argentina * CORRESPONDENCE TO— smromero@conicet.gov.ar ABSTRACT—Artificial thermal shock conducted on 50 soil samples from a semi-arid geographic region (Catamarca and La Rioja provinces) in northern Argentina yielded 34 heat-resistant fungal strains. These strains were assigned to seventeen taxa in ten ascomycete genera: Arthrinium (1), Aspergillus (3), Epicoccum (1), Gilmaniella (1), Hamigera (2), Leiothecium (1), Penicillium (2), Talaromyces (4), Trichocladium (1), and Trichoderma (1). All strains were identified by phenotypic features, with molecular data additionally obtained for eleven strains. Five species are reported for the first time in Argentina. Key worps —Ascomycota, Eurotiales, South America, Monte, Chaco. Introduction Temperature is an environmental parameter that plays a key role in the survival, growth, distribution, and speciation of microorganisms on earth (Mouchacca 1993). Fungi growing at high temperatures are generally classified 536 ... Romero &al. in two groups: thermophilic and thermotolerant. Thermophilic fungi are those able to grow in a temperature range of 20-50 °C or above; while the maximum temperature for growth of thermotolerant fungi is near 50 °C with a minimum of below 20 °C (Cooney & Emerson 1964, Mouchacca 2007). Alternatively, heat-resistant fungi are defined as those capable of surviving to a thermal shock, e.g., temperatures at or above 75 °C for 30 minutes or more (Samson & al. 2000). The fungal structures able to survive these extreme temperatures are mainly ascospores, but other propagules such as chlamydospores, thick-walled hyphae, and sclerotia, also may survive such temperature extreme (Scholte & al. 2004). Dijksterhuis & Samson (2006) expanded the concept of heat-resistant fungi to include most of fungal structures that survive after a thermal shock between 55-95 °C. Heat resistance is associated with the ability to maintain the viability of certain fungal structures in order to overcome natural thermal shock. Thus, heat- resistant fungi can be mesophilic, thermotolerant, or thermophilic. Soil, the primary reservoir of fungi, is also the main repository of dormant propagules of true soil-borne fungi as well as of fungi that grow elsewhere. Most fungi achieve survival through the production of both dormant and dispersal spores (Carlile & Watkinson 1994). Latent spores normally germinate due to slow dormancy decay or in response to a specific (physical or chemical) stimulus that breaks the dormancy (Carlile & Watkinson 1994), such as a thermal shock. Constitutive dormancy is a condition in which the development is delayed by an innate property, such as a barrier to the penetration of nutrients (including water), a metabolic blockage, or the action of a self-inhibitory molecule (Dijksterhuis & Samson 2006, Dijksterhuis 2007). Ascospores, the sexual propagules of the Ascomycota, often have a long survival capability (Dijksterhuis 2007). Ascospores of heat-resistant fungi exhibit constitutive dormancy and require a robust signal (heat, high pressure, or exposure to organic compounds, such as dimethylketone) to break the dormancy (Dijksterhuis 2007, Stchigel 2000). Warcup & Baker (1963) noted an increase in the number of ascomycete colonies when soil samples were exposed to temperatures between 50-75 °C for 30 minutes before culturing, compared with those not exposed to this thermal shock. The fungal soil communities from arid regions are mainly characterized by a low population coupled with a high biodiversity (Mouchacca 1993). During broad research on heat resistant fungi from the semi-arid region of Northern Argentina 194 isolates were obtained. Most isolates were related to Aspergillus sect. Fumigati, but 34 isolates represented different Heat resistant ascomycetes in Argentina ... 537 interesting taxa. This study documents those 34 miscellaneous heat-resistant ascomycetes isolated from soil. Materials & methods Description of the sampling area Fifty 200-g soil samples were collected in the summer of 2009 and winter of 2011 from several sites in Catamarca and La Rioja provinces, Northern Argentina. Of the six phytogeographical regions of Catamarca (Morlans 1995), most samples were collected from the “monte” region and only a few from the “chaquefia’” region; additionally three collections were sampled from the “monte” region of La Rioja (with five phytogeographical regions). The monte region in Catamarca, which comprises c. 14,600 km? (Manghi & al. 2005) and extends through the center of La Rioja province, is characterized by steppe vegetation dominated by xerophilous, psammophilous, or halophilous shrubs, and characterized by the almost constant presence of Larrea (Zygophyllaceae) and Prosopis (Leguminosae). The climax community is Larrea divaricata Cav. (“jarillal”), which develops in the sandy pockets and plains or stony- sandy soils (Cabrera 1971). Climate in the monte is subtropical, arid, with an annual 150-200 mm precipitation occurring mostly in the summer (60-70% of total rainfall; Morlans 1995). Temperature shows a relatively broad seasonal and daily variation, with autumn/winter lows (-30 °C in west mountain region) and spring/summer highs (45 °C in the east and central regions) (Gobierno de Catamarca 2018, Morlans 1995). The strong winds throughout the year favor environmental dryness. The monte elevation ranges from 600-3000 m a.s.l. (Morlans 1995). Fungal isolation From each sample, c. 5 g of soil was placed into a conical flask, mixed with 100 ml of sterile melted malt extract agar (Oxoid MEA: malt extract 30.0 g, mycological peptone 5.0 g, agar 15.0 g) plus 50 ppm of chloramphenicol, and heated at 75 °C for 30 min. The suspension was plated into sterile Petri dishes and, once gelled, incubated at 30 °C <30 d (Samson & al. 2000). All colonies were transferred to individual Petri dishes containing MEA to obtain pure cultures. The fungal strains were deposited in the culture collection of the Herbarium, Universidad de Buenos Aires, Argentina (BAFC) and the culture collection of Faculty of Medicine, Universitat Rovira i Virgili, Reus, Catalonia, Spain (FMR). Author abbreviations include SMR (S.M. Romero), AIR (A.I. Romero), AMS (Stchigel), ERA (Rodriguez Andrade), VB (Barrera), and RC (Comerio). Abbreviations under CULTURE(S) EXAMINED are RN (Ruta Nacional) and RP (Ruta Provincial). Morphological study Preliminary characterization at the genus level was conducted on MEA (Domsch & al. 2007, Guarro & al. 2012). Species were identified by growing the fungal strains on different culture media and temperatures (following the bibliography for each genus mentioned in each description). Vegetative and reproductive structures were 538 ... Romero &al. microscopically examined by mounting directly into lactic acid from cultures on MEA and then observed under a Zeiss Axioskop bright field microscope. Mature ascomata were crushed, coated with gold, and observed and photographed with a Zeiss Supra 40 (extra high tension = 3 Kv; working distance = 3.7-6 mm) scanning electron microscope. Molecular study EXTRACTION: DNA was extracted directly from colonies on potato dextrose agar (PDA) after 7-10 d at 25 °C in darkness, through the modified protocol of Miiller & al. (1998). The following genetic markers were sequenced: fragments of -tubulin (BenA), calmodulin (CaM), RNA polymerase II second largest subunit (rpb2) (Peterson & al. 2010, Samson & al. 2014), and the DNA replication licensing factor (Mcm7) (Schmitt & al. 2009, Raja & al. 2011). The primers used were Bt2a and Bt2b for BenA, CMD5 and CMD6 for CaM (Glass & Donaldson 1995), RPB2-5F and RPB2-7R for rpb2 (Liu & al. 1999), and Mcm7-709For and Mcm7-1348Rev for Mcm7 (Schmitt & al. 2009). PCR products were sequenced in both directions using the same primers at Macrogen Europe (Macrogen Inc., Madrid, Spain). Sequences were assembled and edited using Sequencher v.4.1.4. The sequences generated were deposited in the GenBank database (https:// www.ncbi.nlm.nih.gov/genbank) using the Webin platform of the European Bioinformatics Institute (EMBL-EBI) (http://www.ebi.ac.uk/ena). PHYLOGENETIC ANALYSIS: Multiple sequence alignment was conducted using ClustalW (Thompson & al. 1994) and MUSCLE (Edgar 2004) within MEGA v.6 software (Tamura & al. 2013), with manual adjustments for refinement. The Maximum Likelihood (ML) phylogenetic method was also run with MEGA v.6, as well as the estimation of the best nucleotide substitution model. Support of the internal branches was assessed by the bootstrap method with 1000 replications, where values 270 were considered significant. The phylogenetic analyses were carried out first individually for each gene followed by a concatenated study. To support our analyses, sequences were obtained from GenBank and added to the analyses. Taxonomy In our study, 34 heat-resistant fungal strains from the semiarid region in Argentina were all morphologically characterized and assigned to 17 taxa in 10 genera—Arthrinium (1), Aspergillus (3), Epicoccum (1), Gilmaniella (1), Hamigera (2), Leiothecium (1), Penicillium (2), Talaromyces (4), Trichocladium (1), and Trichoderma (1). For 11 strains, the morphological identification was confirmed by molecular analyses. Five taxa are reported for the first time in Argentina. Arthrinium phaeospermum (Corda) M.B. Ellis, Mycol. Pap. 103: 8. 1965. Cotontes on MEA, 25 °C, 7 d, floccose, covering the whole culture plate, greyish, with dark specks and areas due to the anamorph presence. Heat resistant ascomycetes in Argentina ... 539 Conrpi1a lenticular, dark brown, 9-11 x 5-7 um, smooth with a conspicuous pale equatorial stripe. CULTURES EXAMINED—ARGENTINA. CaTaMARCA: RP 46, km 70, 28°05’35”S 66°12’55”W, 859 m a.s.l., 24.VHI.2011, leg. SMR, identified by SMR & RC, isolation 22.X.2011 (BAFCcult 4624); 27°35’13”S 66°22’11’W, 996 m a.s.l, 24.VIII.2011, leg. SMR, identified by SMR & RC, isolation 15.X1.2011 (BAFCcult 4625). DISTRIBUTION— Worldwide, including Argentina (Broggi & al. 2007, Crous & Groenewald 2013, Domsch & al. 2007). Hasitat—Cereals and vegetable materials, dung, fresh water, and soil (Domsch & al. 2007). CoMMENTS—Otur isolates agreed morphologically with descriptions by Ellis (1971), Domsch & al. (2007), and Crous & Groenewald (2013). In Argentina, this species was isolated from soil in Buenos Aires (Cabello 1986) and cereals collected in Entre Rios (Broggi & al. 2007, Sacchi & al. 2009). Aspergillus flavus Link, Mag. Ges. Naturf. Freunde, Berlin 3: 16. 1809. Cotoniges on MEA, 25 °C, 7 d, plane, granulose, floccose at the centre, 50-54 mm diam., yellowish green, exudate scarce uncolored; asexual sporulation abundant. On Czapek yeast agar (CYA), 25 °C, 7 d, sulcate, granulose, floccose at the centre, 60-68 mm diam., yellowish green, exudate scarce uncolored; asexual sporulation abundant. CONIDIAL HEADS commonly columnar, radiate, mainly uniseriate. STIPES roughened, vesicles spherical. Conip1a globose, 3.5-4.5 um, with finely roughened walls. CULTURE EXAMINED—ARGENTINA. La Rioja: RN 60, km 1172, 28°40'30’S 66°30'23”W, 653 m a.s.l., 12.1.2009, leg. SMR, identified by SMR, isolation 1.III.2011 (BAFCcult 4615). DIsTRIBUTION— Worldwide and cosmopolitan (Domsch & al. 2007; Ramirez- Camejo & al. 2012). HasitatT—Soil, cultivated or not (Domsch & al. 2007). This is the most widely cited fungal species as a food contaminant (Klich 2002, Pitt & Hocking 2009). ComMMENTS— The micromorphology of our isolate, and even its cultural features, agreed with the descriptions of Klich (2002) and Pitt & Hocking (2009). Horn & al. (2009) described the sexual stage for this heterothallic species as Petromyces flavus B.W. Horn &al., but according to the International Code of Nomenclature (Turland & al. 2018) and following Samson & al. (2014), the correct name for this species is Aspergillus flavus. In Argentina, 540 ... Romero & al. it has been isolated several times from soil (Allegrucci & al. 2009, Cabello 1986, Godeas & al. 1977, Mangiaterra & al. 2006, Winitzky 1948), foods (Bresler & al. 1991, Castellari & al. 2015, Dalcero & al. 1998, Etcheverry & al. 1999, Romero & al. 2005, Sepulveda & Piontelli 2005) and medicinal herbs (Sanchez & al. 2006). Aspergillus montevidensis Talice & J.A. Mackinnon, C.r. Seanc. Soc. Biol. 108: 1007. 1931. BIGiwTASB CoLonies on MEA, 25 °C, 7 d, plane and dense, 14 mm diam., yellowish; after 14 incubation d 26-28 mm diam., yellowish, with or without brown center. On Czapek yeast agar with 20% sucrose, 25 °C, 7 d, velutinous, 35-47 mm diam., yellow, with green center due to the anamorph presence; after 14 incubation d covering the whole culture plate, velutinous to granulose due to the abundant cleistothecia production, yellow with green center; asexual sporulation copious; reverse yellowish under cleistothecia and greenish under anamorph structures. At 37 °C, 7 d, 18-26 mm diam., yellow. CLEISTOTHECIA superficial, mainly globose, discrete, 90-150 um diam., yellow, covered by a hyaline hyphal net. Asci globose, 8-spored, 10-12 x 8-11 um, evanescent. Ascosporss lenticular, 5-4 x 3-4 um, with rough convex surfaces, furrowed and double-crested along the equator; SEM images revealed pores distributed in the whole ascospore surface, but mainly on crests. CONIDIAL HEADS radiate, uniseriate. STIPES 150-210 x 6-8 um, smooth, vesicles globose, 15-27 um diam. PHIALIDEs ampulliform, 5-8 x 4 um. Conrp1A globose to subglobose, 5-4 x 4-3 um, spinulose. CULTURES EXAMINED—ARGENTINA. CATAMARCA: RN 46, km 185, 27°45712”S 66°47°59”W, 1069 m a.s.l.. 9.1.2009, leg. SMR, identified by SMR, isolation 21.111.2011 (BAFCcult 4613; GenBank LT964777, LT964778); Papachacra, 2564 m a.s.l., 10.1.2009, leg. SMR, identified by SMR, isolation 1.[1I.2011 (BAFCcult 4614; GenBank LT964779, LT964780). DIsTRIBUTION—Argentina (Winitzky 1952), Australia, Brazil, India, Israel, Japan, Pakistan, Peru, South Africa, Syria, Tuamotu Archipelago, Turkey (Domsch & al. 2007). Hasitat—Soil, stored and/or decaying food products among other substrates (Domsch & al. 2007, Pitt & Hocking 1997). ComMMENTS—The morphological features of our strains agreed with those described by Thom & Raper (1941), Raper & Fennell (1965), Klich (2002) and Hubka & al. (2013). This species was also known as Eurotium amstelodami because Thom & Raper (1941) changed the original species Heat resistant ascomycetes in Argentina ... 541 concept of Mangin (1909), from a species with smooth-walled ascospores to a species with ornamented ones. Their 1941 published name, E. amstelodami was, in fact, a later illegitimate homonym. Pitt (1985) noted this confusion and speculated that Thom & Church (1926) and Thom & Raper (1941) did not base their description on the Mangin species concept. Hubka & al. (2013) concluded that the first available and validly published name for the species matching the concept of Thom & Raper (1941) is A. montevidensis, which includes both the anamorph and the teleomorph states. Aspergillus sydowii (Bainier & Sartory) Thom & Church, Aspergilli: 147. 1926. CoLoniEs on MEA, 25 °C, 7 d, velutinous to slightly floccose, 14-15 mm diam., blue green, asexual sporulation copious. On CYA, 25 °C, 7 d, sulcate, velutinous, 20 mm diam., blue green, exudate brown copious; asexual sporulation abundant; reverse orange brown. On 25% glycerol nitrate agar (G25N), 25 °C, 7 d, plane and dense, 11-13 mm diam., bluish grey. CONIDIAL HEADS radiate, biseriate; small and short penicillate heads produced. Stipes 250-350 um, pigmented. Conip1A globose, 3.5-4 um, very rough to spinose. HULLE CELLS abundantly produced. CULTURES EXAMINED—ARGENTINA. Catamarca: Rincon, 28°22’03”S 66°13’39”W, 8.1.2009, leg. SMR, identified by SMR, isolation 4.VI.2009, (BAFCcult 4589); RP 25, 28°22’41”S 66°11'50”W, 1304 m a.s.l., 23.VIII.2011, leg. SMR, identified by SMR, isolation 10.11.2012 (BAFCcult 4612). DisTRIBUTION—Argentina, Australia, Austria, Brazil, British Isles, Canada, Czechoslovakia, Egypt, India, Italy, Japan, Kuwait, Malaysia, Namibia, Pakistan, Peru, Singapore, South Africa, Spain, Syria, ex USSR, U.S.A. (Domsch & al. 2007). Hasitat—Mainly isolated from soil, but also from air (indoor and outdoor) (Klich 2002). This species has been reported from numerous foods, especially dried ones (Pitt & Hocking 2009). It has been recovered also from marine sponges (Ein-Gil & al. 2009). CoMMENTS— The features of our isolates matched those described by Raper & Fennell (1965) and Klich (2002). In Argentina, A. sydowii has been previously isolated from air (Winitzky 1948), amaranth seeds (Bresler & al. 1995), dried vine fruits (Romero & al. 2005), corn and soybean seeds (Sepulveda & Piontelli 2005), soil (Allegrucci & al. 2007, Eliades & al. 2006a, Mangiaterra & al. 2006) and medicinal herbs (Sanchez & al. 2006). This is the first report for Catamarca province. 542 ... Romero &al. Epicoccum nigrum Link, Mag. Gesell. Naturf. Freunde Berlin 7: 32. 1816. Co.ontes on Blakeslee’s MEA, 25 °C, 7 d, floccose, 53-58 mm diam., orange brown, exudate and soluble pigment absent; reverse orange. On oatmeal agar (OA), 25 °C, 7 d, lanose to floccose, 60 mm diam., orange with yellowish centre, exudate absent, soluble pigment yellow; reverse yellowish orange; after 12 d of incubation brownish green centre due to sporulation. On PDA, 25 °C, 7 d, 23-30 mm diam., orange brown, exudate red caramel, soluble pigment amber; reverse red brown. CONIDIOPHORES crowded, even forming pustules. Conrp1A globose to pyriform 22-30 x 18-29 um, orange brown to dark brown, verrucose, several septa which divide the conidia in different directions, pale extending cell at the base. CULTURE EXAMINED— ARGENTINA. CATAMARCA: Papachacra, 2564 m as.l., 10.1.2009, leg. SMR, identified by SMR, isolation 1.[II.2011 (BAFCcult 4622). DISTRIBUTION— Worldwide (Domsch & al. 2007). Hasitat—Cereals and grains, fruits, soil, among others (Domsch & al. 2007). CoMMENTS—The morphologic characteristics of our isolate agreed with the descriptions of Epicoccum nigrum by Ellis (1971) and Domsch & al. (2007). In Argentina there are numerous records of this species, which were isolated from soil samples of Jujuy province (Giusiano & al. 2002), wheat and soybean of Entre Rios province (Broggi & al. 2007), soil and litter of Buenos Aires province (Allegrucci & al. 2005, 2007), and alpataco fruits from La Pampa province (Canafoglia & al. 2007), among others. Gilmaniella humicola G.L. Barron, Mycologia 56: 514. 1964. FIG. 1P,Q Cotontes on MEA, 25 °C, 7 d, velutinous to slightly floccose, 59-75 mm diam., grayish, darkening in age; reverse very dark to black. At 30 °C, 7 d, velutinous to slightly floccose, 79-83 mm diam., grayish, darkening in age; reverse very dark to black. Hypuae light brown in age, smooth. Conip14 globose, terminal or lateral, usually in clusters and provided with a distinct germ pore, smooth, brown 7-9 um. CULTURES EXAMINED—ARGENTINA. CaTAMaARca: RP 25, 28°15’31”S 66°08’47’W, 1424 m a.s.l., 23.VIH.2011, leg. SMR, identified by SMR & RC, isolation 11.1.2012 (BAFCcult 4610, 4588). DIsTRIBUTION—Canada, Egypt, England, France, India, Japan, Namibia, Solomon Islands, The Netherlands, United States of America (Domsch & al. 2007). Heat resistant ascomycetes in Argentina ... 543 Fic. 1. Aspergillus montevidensis (BAFCcult 4613): A. Colonies on Czapek yeast extract agar (CYA) with 20% of sucrose, 14 d, 25 °C; B. Ascospore (SEM). Hamigera paravellanea (BAFCcult 4605): C. Colonies on CYA, 7 d, 25 °C; D. Ascospores (SEM); E. Conidiophore and conidia. Hamigera terricola (BAFCcult 4580): E Colonies on CYA, 7 d, 25 °C; G-H. Conidiophores and conidia; I. Ascospores (SEM). Leiothecium ellipsoideum (BAFCcult 4598): J. Colony on MEA, 7 d, 25°C; K, L. Ascospores (SEM); M. Chlamydospore. Penicillium capsulatum (BAFCcult 1627): N. Colonies on CYA, 7 d, 25 °C; O. Conidiophore and conidia. Gilmaniella humicola (BAFCcult 4588): P. Conidia with germination pores (arrows); Q. Colony on MEA 7 d, 30 °C. Trichocladium pyriforme (BAFCcult 4623): R. Colony on MEA 14 d, 25 °C; S, T. Chlamydospores. 544 ... Romero &al. Hasitat—Dung, groundnuts, plant debris, soil, among others (Domsch & al. 2007). COMMENTS—Otur isolates were phenotypically in accordance with Barron (1964) and Ellis (1971). Gilmaniella humicola is a good representative example of a heat-resistant fungus (Domsch & al. 2007), and it was the most thermal- resistant fungus found by Bollen (1969), tolerating a temperature of 90 °C for 30 minutes. All our isolates were recovered from samples collected in winter. In Argentina, the fungus was previously reported from soil in a Nothofagus dombeyi forest in Rio Negro province (Godeas 1977). Hamigera paravellanea S.W. Peterson, Jurjevi¢, Bills, Stchigel, Guarro & EE. Veja, Mycologia 102: 852. 2010. FIG. 1C-E Cotoniges on MEA, 25 °C, 7 d, plane somewhat floccose, 47-67 mm diam., pale orange to tan; reverse dark, mainly at colony centre. On CYA, 25 °C, 7 d, low, slightly sulcate, 50-70 mm diam., pale orange to tan; reverse dark reddish-brown. GYMNOTHECIA globose, discrete, 120-280 um diam., cream coloured, surrounded by a profuse hyphal net. Asci subglobose to claviform, 8-spored, 15-20 x 11-12 um, evanescent. Ascospores broadly ellipsoidal, 7—-7.5 x 5-6 um, hyaline, thick-walled, echinulate. ConrpIOPHORES penicillate, 120-480 x 3-5 um. Stipes slightly spathulate, smooth or roughened towards their bases, widest at their apices. METULAE with wide apices, 10-15 x 4-6 um. PHIALIDES ampulliform, 8-6 x 2-3 um. Conrp1a ellipsoidal, 3-4 x 2-3 um., smooth. CULTURES EXAMINED—ARGENTINA. CATAMARCA: RP 25, 28°15’31”S 66°08’47’W, 1424 ma.s.L., 6.1.2009, leg. SMR, identified by SMR, AMS & ERA, isolation 20.1X.2011 (BAFCcult 4605, FMR 16758; GenBank LT991984, LT991994); 23. VII.2011, leg. SMR, identified by SMR, AMS & ERA, isolation 11.1.2012 (BAFCcult 4606, FMR 16762; GenBank LT992033, LT992032). DIsTRIBUTION— Poland, Spain (Peterson & al. 2010). Hasitat—Soil, dung (Peterson & al. 2010). CoMMENTS—Morphological characters observed agreed with the description of the protologue (Peterson & al. 2010). This is the first report for Argentina. Hamigera terricola S.W. Peterson, Jurjevi¢, Bills, Stchigel, Guarro & EE. Veja Mycologia 102: 855. 2010. FIG. 1F-I COLONIES on MEA, 25 °C, 7 d, low, sulcate, velutinous, 58-70 mm diam., ochraceous buff to tan; reverse dark reddish-brown. On CYA, 25 °C, 7 d, low, Heat resistant ascomycetes in Argentina ... 545 plane, velutinous, 40-43 mm diam., ochraceous buff with yellowish margins; reverse dark reddish-brown. GYMNOTHECIA globose, discrete, 80-180 um diam., cream coloured, surrounded by a profuse hyphal net. Asci subglobose, 8-spored, 15-17.5 x 11-13 um, evanescent. AscosPorEs broadly ellipsoidal, 7-8 x 5-6 um, hyaline, thick-walled, echinulate. CoNIDIOPHORES penicillate, 170-400 x 3-6 um. STIPES sometimes spathulate, with apical vesicles 7-9 um wide, smooth or slightly roughened. METULAE with wide apices, 7-11 x 3-6 um. PHIALIDES ampulliform, 7-8 x 3 um. Conrp1a ellipsoidal, 3-4 x 2-3 um., smooth. CULTURE EXAMINED—ARGENTINA. CATAMARCA: RN 60, km 1102, 28°55’15"S 66°08'46’W, 338 ma.s.l., 5.1.2009, leg. SMR, identified by SMR, AMS & ERA, isolation 4.V1.2011 (BAFCcult 4580, FMR 16756; GenBank LT991985, LT991995). DIsTRIBUTION—Costa Rica, Equatorial Guinea, French Guiana, Guatemala, Panama (Peterson & al. 2010). HasitaT—Textile sample in contact with soil (Peterson & al. 2010). CoMMENTS—In a broad sense, the features of this isolate matched with those described by Peterson & al. (2010), but ascospores are slightly larger and the asci smaller than in the original description. This is the first report of Hamigera terricola for Argentina. Leiothecium ellipsoideum Samson & Mouch., Can. J. Bot. 53: 1634. 1975. FIG. 1J-M Cotonigs on MEA, 25 °C, 7 d, forming concentric rings, 82-83 mm diam., dark, superficial white mycelium present; reverse dark. At 35 °C, covering the whole culture plate before 7 d; reverse dark due to ascomata abundance. On OA, 25 °C, 7 d, colonies presenting a continuous layer of ascomata, dark, almost lacking aerial mycelium. At 35 °C, covering the whole culture plate before 7 d; subtle annularly developed, less aerial mycelium than on MEA. VEGETATIVE HYPHAE smooth, 3-11 um diam., hyaline. CLEISTOTHECIA globose, discrete, 40-90 um diam., dark brown, superficial or semi-submerged in the substratum, covered by white mycelium supporting ascomata as well; wall persistent, textura angularis, 5-8 um wide, composed by 15-25 um diam. dark brown cells. Asci1 globose to subglobose, 14-18 x 13-15 um, evanescent. Ascosporss ellipsoidal, 7-9 x 5.5-7 um, hyaline, spinulose under low magnifications, broadly reticulate under high magnifications; young ascospores presented an internal hyaline sheath which fades with maturity; SEM observations revealed alveolate plaques on ascospore wall, alveoles presented conspicuous projections at their edges which look as low frills. 546 ... Romero &al. CHLAMYDOSPORES isolated, subglobose to ellipsoidal, umbonate, 4.5-11 um diam., smooth, endogenous, and also terminal in maturity. CULTURES EXAMINED— ARGENTINA. CATAMARCA: RN 60, km 1146, 28°13’05"S 66°22’41”W, 1051 m a.s.L., 5.1.2009, leg. SMR, identified by SMR, RC & AMS, isolation 13.IX.2011 (BAFCcult 4598, FMR 15064; GenBank LT992254, LT992257); km 1016, 29°30'04”S 65°37'57”W, 237 m a.s.L, 22.VIH.2011, leg. SMR, identified by SMR, RC & AMS, isolation 12.XI.2011 (BAFCcult 4604, FMR 16765; GenBank LT992253, LT992256); RP 47, km 35.5, 27°26'50”S 66°24’26”W, 2700 m a.s.l., 24.VHI.2011, leg. SMR, identified by SMR, RC & AMS, isolation 18.1.2012 (BAFCcult 4609, FMR 16768; GenBank LT992255, LT992258). DISTRIBUTION—Canada (Samson & Mouchacca 1975), Greece, Hungary, Portugal, Spain, United States of America, and Venezuela (CBS-KNAW Culture Collection 2018). HaBitTat—Soil, seeds of Capsicum annuum (Samson & Mouchacca 1975), nest material of Nomia sp., a ground-nesting solitary bee (CBS-KNAW Culture Collection 2018). CoMMENTS—Argentinian isolates largely agreed with the description given by Samson & Mouchacca (1975) and Guarro & al. (2012). However, these strains grew faster on MEA at 35 °C than the ex-type strain. Regarding micromorphology, no chlamydospores in chains were observed (solitary chlamydospores were seen on small hyphal protrusions) and ascomata were smaller (40-90 um vs. 125 um diam) than those described in the protologue. This is the first report of Leiothecium ellipsoideum for Argentina. Penicillium capsulatum Raper & Fennell, Mycologia 40: 528. 1948. Fic. 1N,o Cotonigs on MEA, 25 °C, 7 d, low, plane, velutinous, 14-18 mm diam., white mycelium at the edges, grey green to dull green; conidiogenesis moderate; exudate and soluble pigment absent; reverse pale to yellowish. On CYA, 25 °C, 7 d, moderately deep, sulcate, slightly fasciculate, 12-15 mm diam.; conidiogenesis dense, grey green to dull green, white mycelium at the edges; exudate and soluble pigment absent; reverse pale to yellowish. At 37 °C, 7 d, funiculose, 18-21 mm diam., with white mycelium at the edges; exudate and soluble pigment absent; reverse pale to buff. At 5 °C germination absent. On G25N, 25 °C, 7 d, sulcate, velutinous to floccose, 6 mm diam., white mycelium at the edges, conidia in mass grey green to dull green; exudate and soluble pigment absent; reverse pale to yellowish. TELEOMORPH absent. CONIDIOPHORE monoverticillate, arising from substrate surface or from superficial mycelium. STIPES somewhat sinuose, 10-40 um long., smooth, mainly non-vesiculate, but sometimes vesicles up to Heat resistant ascomycetes in Argentina ... 547 4 um diam. PHIALIDEs 5-8, ampulliform to acerose, 6 x 2 um, occasionally up to 8 um in length, with short collula. Conrp1a ellipsoidal to cylindrical, 3-3.5 x 2 um, smooth-walled. CULTURE EXAMINED—ARGENTINA. CaTAMARCA: RP 33, 28°42’03”S 65°46'08”W 418 m as.l., 22.VIH.2011, leg. SMR, identified by SMR & RC, isolation 23.X1.2011 (BAFCcult 1627). DISTRIBUTION—China (Chen & al. 2013), Kiribati (Gilbert Islands), Panama (Raper & Fennell 1948). Hasitat—Optical instrument, environmentally exposed canvas and deteriorated military equipment (Raper & Fennell 1948), and contaminated beet pulp (Puls & Coughlan 1996). CoMMENTS—Otr soil-borne isolate matched the Pitt (1979) description. We also observed vesiculate conidiophores, but phialides were a bit shorter than in the holotype. Pitt (1979) described the colonies on CYA at 25 °C as depressed at the center, whereas the Argentinian strains presented a high and fasciculate center. This is the first report of Penicillium capsulatum for Argentina. Penicillium citrinum Thom, U.S.D.A. Bur. Animal Industr. Bull. 118: 61. 1910. CoLonigs on MEA, 25 °C, 7 d, low, plane, centrally umbonate, velutinous, 18-22 mm diam., grey green to dull green; conidiogenesis dense; exudate pale, soluble pigment absent; reverse pale. On CYA, 25 °C, 7 d, moderately deep, sulcate, velutinous, 24-30 mm diam., grey green to dull green; conidiogenesis dense, white mycelium at the edges; exudate pale, soluble pigment yellowish; reverse yellow to orange. At 37 °C, 7 d, 2-11 mm diam. At 5 °C no germination. TELEOMORPH absent. CONIDIOPHORES biverticillate, arising from substrate surface or from superficial mycelium. STIPEs with smooth walls and a conspicuous divaricate metulae whorl. METULAE of uniform length, 3-5 on each stipe. PH1ALIDES ampulliform, short collula. Conip1a globose to subglobose, 2.5-3 um diam., smooth-walled. CULTURE EXAMINED—ARGENTINA. CATAMARCA: Corral Quemado to Papachacra, 27°07'33"S 66°56'36"W, 2152 m a.s.l.: 10.1.2009, leg. SMR, identified by SMR & RC, isolation 5.III.2011 (BAFCcult 4616). DisTRIBUTION—Penicillium citrinum is considered a cosmopolitan and frequently isolated fungus (Domsch & al. 2007). Hasitat—The fungus has been reported from many substrates, including food (Domsch & al. 2007). CoMMENTS—Otmr isolate matched with the description of Penicillium citrinum provided by Pitt (1979). In Argentina, it has been isolated many times, generally 548 ... Romero & al. from food, e.g., soybeans (Bonera & al. 1982), amaranth seeds (Bresler & al. 1991, 1995), balanced feed for birds (Magnoli & al. 1998), and dried vine fruits (Romero & al. 2005), among others. Talaromyces macrosporus (Stolk & Samson) Frisvad, Samson & Stolk, Antonie van Leeuwenhoek 57: 186, 1990. Cotonigs on MEA, 25 °C, 14 d, almost covering the whole culture plate, moderate deep, plane, granulose, covering the whole culture plate, yellow, becoming orange at the centre. CLEISTOTHECIA superficial, mainly globose, commonly confluent but at the margin discrete, 200-400 um diam., yellow. Asci globose, 8-spored, evanescent; initials showing thin antheridia coiled around slender clavated ascogonia. ASCOSPORES ovoidal to broadly ellipsoidal, 5.5-6.5 x 3.5-4 um, pale yellow, with thick walls and spines. ANAMORPH absent. CULTURE EXAMINED—ARGENTINA. CaTAMARCA: RP 33, 28°42’03”S 65°46’08”W, 418 m asl, 22.VIL2011, leg. SMR, identified by SMR & AIR, isolation 23.X1.2011 (BAFCcult 4617). DISTRIBUTION—Ghana, Japan, Korea, Malaysia, New Guinea, Panama, Poland, South Africa, The Netherlands, United States of America (CBS-KNAW Culture Collection 2018, Stolk & Samson 1972). HasitatT—Soil, canned apples (Stolk & Samson 1972), freshly harvested strawberries (Frison & al. 2012). CoMMENTS—Our isolate agreed with the description of Talaromyces macrosporus presented by Stolk & Samson (1972), who recognize two varieties: T. flavus var. flavus and T. flavus var. macrosporus. Regarding these varieties, Beuchat (1988) pointed out that the strains with small ascospores (T. flavus var. flavus) are less thermal resistant than those with larger ones (T. flavus var. macrosporus). Frisvad & al. (1990) raised them to species rank based on ascospore size, heat-resistance, and secondary metabolite production. In Argentina, the species has previously been recorded in soils either as T: flavus s.l. or as T: flavus var. flavus (Bertoni 1973, Eliades & al. 2006 a,b, Magnoli & al. 1998). Frison & al. (2012) reported T: macrosporus on fresh strawberries. Talaromyces pinophilus (Hedgc.) Samson, N. Yilmaz, Frisvad & Seifert, Stud. Mycol. 70: 176. 2011. CoLoniges on MEA, 25 °C, 7 d, moderately deep, plane, floccose, 34-36 mm diam., green; conidiogenesis sparse, yellow mycelium present, almost dominating colony colour; exudate pale, soluble pigment absent; reverse Heat resistant ascomycetes in Argentina ... 549 yellowish. On CYA, 25 °C, 7 d, moderately deep, plane, floccose, 19-20 mm diam., greenish yellow; conidiogenesis sparse, yellow mycelium present predominating in colony colour; exudate pale, soluble pigment absent; reverse brown. At 37 °C, 7 d, 24-26 mm diam., with white mycelium at the edges; exudate and soluble pigment absent; reverse buff. At 5 °C no germination. On G25N, 25 °C, 7 d, plane, floccose, 3-5 mm diam., white mycelium; sporulation absent; exudate pale, soluble pigment absent; reverse buff. TELEOMORPH absent. CONIDIOPHORES biverticillate, arising from substrate surface and also from aerial hyphae. Stipes 100-150 um long., smooth. MetuLae in whorls mainly of 5 elements, 10-12 um long. PHIALIDEs 5-8, acerose, 8-10 um long., tapered. Conip1A globose to subglobose, 3-4 um diam., smooth-walled. CULTURE EXAMINED—ARGENTINA. CATAMARCA: Corral Quemado to Papachacra, 27°07'33"S 66°56”36"W, 2152 m a.s.l., 10.1.2009, leg. SMR, identified by RC, isolation 5.I11.2011 (BAFCcult 4618). DIsTRIBUTION—Argentina (Magnoli & al. 1998); Australia, France, India, Papua New Guinea, United States of America (Pitt 1979); Uruguay (Galvalisi & al. 2012). Hasitat—Soil, decaying plants, deteriorated material, barley grains, fermented dry sausages, balanced feed for birds (references as above). ComMMENTS—Excluding conidia, which in this case were a little bit bigger (in contrast to 2.5-2.8 x 2.2-2.5 um), we did not find morphological differences compared to those of Talaromyces pinophilus given by Pitt (1979). In Argentina, it has been previously reported as Penicillium pinophilum from corn, balanced feed for birds, and soil (Etcheverry & al. 1999, Magnoli & al. 1998, Nesci & al. 2006, Mangiaterra & al. 2006). Talaromyces trachyspermus var. macrocarpus J.E. Wright & Loewenb., Bol. Soc. Argent. Bot. 15(1): 100. 1973. CoLonigs on MEA, 25 °C, 14 d, moderately deep, plane, granular, 37-44 mm diam., white to cream-colored; exudate absent; reverse orange. CLEISTOTHECIA superficial, mainly globose, usually confluent, 400-930 uum diam., at first white becoming cream-colored in age, covered by a network of hyphal net. Ascr globose to ovoid, 8-spored, 7-8 x 6-7 tm, evanescent. AscosPpores ellipsoidal, 3-4 x 2-3 um, spinulose. ConrDIOPHORES slender monoverticillate and biverticillate, short. METULAE 8-11 x 2 um. PHIALIDES 2-5 in verticils, lanceolate, tapering to neck, 9-10 x 2 um. Conrp1a ellipsoidal, 2-2.5 x 2 um, smooth-walled. 550 ... Romero & al. CULTURE EXAMINED—ARGENTINA. CATAMARCA: RP 46, km 185, 27°45’12”S 66°47'59”’W, 1069 m a.s.l., 9.1.2009, leg. SMR, identified by SMR & AIR, isolation 21.111.2011 (BAFCcult 4621; GenBank LT968851). DISTRIBUTION—Argentina (Bertoni & al. 1973). HasitatT—Soil (Bertoni & al. 1973). COMMENTS—The characteristics of isolate BAFCcult 4621 agreed with the description of Talaromyces trachyspermus var. macrocarpus by Bertoni & al. (1973). This variety was not accepted by Pitt (1979); however, we prefer to recognize it until examination of the holotype, and study of more isolates and their molecular data. T’ trachyspermus var. macrocarpus is distinguished from T. trachyspermus by presenting bigger cleistothecia (500-1500 vs 50-350 um) and ascospores (4-4.5 x 2.8-3.2 vs 3-3.5 x 2-2.5 um). Talaromyces trachyspermus (Shear) Stolk & Samson, Stud. Mycol. 2: 32 (1973) var. trachyspermus CoLonigs on MEA, 25 °C, 14 d, moderately deep, plane, granular, 42-50 mm diam., white to cream-colored; exudate absent; reverse light orange and orange to brown in the centre. CLEISTOTHECIA superficial, mainly globose, usually confluent, 150-350 um diam., at first white becoming cream-colored in age, covered by a hyphal network. Ascr globose to ovoid, 8-spored, 7-9 x 6-8 um, evanescent. AscosPorss ellipsoidal, 3.5-4 x 2-3 um, spinulose. CONIDIOPHORES slender monoverticillate and biverticillate, short. METULAE 8-11 x 2 um. PHIALIDES 2-5 in verticils, lanceolate, tapering to neck, 9-10 x 2 um. Conrp1a ellipsoidal, 2-2.5 x 2 um, smooth-walled. CULTURES EXAMINED—ARGENTINA. CaTAMARCA: RP 46, 27°35'13”S 66°22’11”W, 996 m a.s.l., 9.1.2009, leg. SMR, identified by SMR & AIR, isolation 21.11.2011 (BAFCcult 4619; GenBank LT968849); (BAFCcult 4620; GenBank LT968850); 28°46'22”S 66°09'56”W, 1317 m a.s.l., 24.VIII.2011, leg. SMR, identified by SMR & AIR, isolation 4.1.2012 (BAFCcult 4629); Belén, 27°07’33”S 66°56'36’W, 2152 m a.s.L., 25.VIII.2011, leg. SMR, identified by SMR, isolation 27.1.2012 (BAFCcult 4630); RP 47, km 35.5, 27°26'50”S 66°24’26”W, 2700 m a.s.l., 25.VII.2011, leg. SMR, identified by SMR, isolation 18.1.2012 (BAFCcult 4631). DIsTRIBUTION—Argentina, Bangladesh, France, Germany, India, Japan, Nepal, Pakistan, South Africa, Tahiti, Uganda, United States of America (Domsch & al. 2007). Hasitat—Soil, cereals and grains, dung (Domsch & al. 2007). CoMMENTS—The morphological features of our isolates agreed with the original description of Talaromyces trachyspermus (Stolk & Samson 1972). In Heat resistant ascomycetes in Argentina ... 551 Argentina Bertoni & al. (1973) and Godeas (1975) isolated this fungus from soil. Trichocladium pyriforme M. Dixon, Trans. Br. Mycol. Soc. 51: 160. 1968. FIG. 1R-T Co.tontges on MEA, a 25 °C, 7 d, plane, slightly floccose, 36-40 mm diam., grey green to olive, darkening in age; reverse very dark; covering the whole culture plate in 21 d. Hypuae hyaline when young, yellow brown in age, 2-5 um wide, smooth. CHLAMYDOSPORES (holothallic conidia) terminal, pyriform, 3-4 celled, 16-19 x 6-7.5 um, smooth-walled; distal cell dark brown, with an acute apex and germinative pore. CULTURES EXAMINED—ARGENTINA. CaTAMaARCA: RN 60, km 934, 29°33’35’S 64°52’56’W, 186 ma.s.l., 5.1.2009, leg. SMR, identified by SMR & AIR, isolation 5.1.2011 (BAFCcult 4623); RN 40, km 4243.5, 26°51’23”S 66°05’52”W, 2031 ma.s.l., 25.VHIL2011, leg. SMR, identified by SMR & AIR, isolation 12.X1.2011 (BAFCcult 4611). DISTRIBUTION—Czech Republic (Mantle & al. 2006), Ireland (Dixon 1968). Hasitat—Soil (Dixon 1968, Goh & Hyde 1999). CoMMENTS—The morphological features of the Argentinian strains of Trichocladium pyriforme agreed with those described by Dixon (1968) and by Goh & Hyde (1999). Domsch & al. (2007) pointed out the remarkable thermal resistance of the spores of this species, and Dixon (1968) stated that it tolerated up to 95 °C for 10 min. Bollen (1969) isolated T. pyriforme from greenhouse soil using a thermal shock at temperatures greater than 55 °C. This is the first report of this fungus in Argentina. Trichoderma saturnisporum Hammill, Mycologia 62: 112. 1970. COLONIES on special nutrient agar (SNA), 72 h, 30 °C, in darkness, somewhat annellated, 38-44 mm diam., green. At 35 °C, 72 h, 38 mm diam. On PDA, 72 h, 30 °C, slightly annellated, 60 mm, dull green; at 35 °C, 69 mm diam. in darkness. CONIDIOPHORES associated in tufts up to 4 mm diam., branched, short sterile projections present. PHIALIDES mainly in verticils, cylindrical, 8.5-11 x 3.3-4 um. Conrp1A subglobose to broadly ellipsoidal, 4-4.8 x 2.9-3.7 um, greenish, walls with few notorious warts. CHLAMYDOSPORES not observed. CULTURES EXAMINED—ARGENTINA. CATAMARCA: RN 60, km 1016, 29°30'04’S 65°37'57”W, 237 m a.s.l., 5.1.2009, leg. SMR, identified by SMR & RC, isolation 3.VHI.2010 (BAFCcult 4584, 4586, 4587); RP 25, 28°14’36”S 66°08’51”W, 1507 m a.s.l., 6.12009, leg. SMR, identified by SMR & VB, isolation 1.X1.2010 (BAFCcult 4626); 552 ... Romero &al. Rincon, 28°22’03"S 66°13’39’W, 8.1.2009, leg. SMR, identified by SMR & VB, isolation 19. VIII.2010 (BAFCcult 4627, 4628. DIsTRIBUTION—Argentina (Godeas & al. 1977), Australia, Italy, South Africa, Turkey, United States (Samuels & al. 1998). HaBitaT—Soil. CoMMENTS—The morphological features of the examined culture agreed with those given by Samuels & al. (1998). Trichoderma saturnisporum is easily recognized by conidia with large warts mimicking wings, which give a saturnian appearance to the conidia. Discussion In previous papers we described two new species of Eurotiales, Talaromyces systylus S.M. Romero & al. (Romero & al. 2016) and Aspergillus fuscicans S.M. Romero & al. (Romero & al. 2018) from the same locations and substrate (soil) as in the present work. ‘The isolates in Aspergillus sect. Fumigati, which were prevalent in the analyzed samples, are currently under study. Some of the fungal species found have been previously cited as heat-resistant, such as Arthrinium phaeospermum (Kashiwagi & al. 2009, Pitt & Hocking 2009), Gilmaniella humicola (Bollen 1969, Jesenska & al. 1992), Leiothecium ellipsoideum (Samson & Mouchacca 1975), Talaromyces macrosporus (Beuchat 1988, Frison & al. 2012) and Trichocladium pyriforme (Bollen 1969, Dixon 1968). It was not possible to obtain information about the heat-resistance of Aspergillus montevidensis; however, Aspergillus glaucus (L.) Link, another species in A. sect. Aspergillus, was isolated from contaminated jellies and grape marmalades, and its thermal resistance was analyzed (Splittstoesser & al. 1989). No information about the heat resistance was found for Trichoderma saturnisporum; however, chlamydospore production (Samuels & al. 1998) would explain its survival after a thermal shock performed in the lab. Byssochlamys nivea, perhaps the most well-known heat-resistant fungal species, was not isolated during this study. This could be attributed to our thermal treatment (75 °C, 30 min), since other authors have determined that its ascospores can survive at 70 °C for 60-75 min but not at 80 °C for 1 min (Pieckova & al. 1994). The same authors also mentioned that at 80 °C the ascospores of Hamigera avellanea Stolk & Samson survive 120 min, whereas the conidia of Gilmaniella humicola survive between 8-14 min. Although no data were found on the thermal resistance of H. paravellanea and H. terricola ascospores, given the heat resistance described for H. avellanea (Pieckova & al. Heat resistant ascomycetes in Argentina ... 553 1994, Scaramuzza & Berni 2014), it can be assumed that other species of the genera would possess a similar capacity. Acknowledgements S.M. Romero and A.I. Romero thank the Consejo Nacional de Investigaciones Cientificas y Técnicas (CONICET-Argentina), PIP1086 and PICT-2018-03781 for the financial support to perform the present study. In addition, the Instituto de Microbiologia y Zoologia Agricola (Instituto Nacional de Tecnologia Agropecuaria) is particularly recognized for the provision of supplies and facilities to carry out this work. The authors acknowledge Dr. Maria Virginia Bianchinotti (CERZOS- CONICET, Universidad Nacional del Sur, Argentina) and Dr. Andrew Miller (Illinois Natural History Survey, University of Illinois, U.S.A.) for reading and improving the manuscript as pre-submission reviewers. The insightful and careful review of the Nomenclature Editor, Dr. Shaun R. Pennycook, is especially appreciated. The design and preparation of the plate by the technician Mariana Valente (InMiBo-CONICET) is held in high esteem. Literature cited Allegrucci N, Cazau MC, Cabello MN, Arambarri AM. 2005. Analisis de las comunidades de microhongos de la hojarasca de Scutia buxifolia (Rhamnaceae) en el este de la provincia de Buenos Aires, Argentina. Darwiniana 43: 1-9. Allegrucci N, Eliades L, Bucsinszky AM, Cabello M, Arambarri A. 2007. Diversidad de Anamorfos de Ascomycota en bosques nativos de Celtis tala (Ulmaceae) en la Provincia de Buenos Aires, Argentina. Bol. Soc. Argent. Bot. 42: 79-86. 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MYCOTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 July-September 2020—Volume 135, pp. 559-568 https://doi.org/10.5248/135.559 Cortinarius rapaceoides, a new record for Turkey MERYEM SENAY SENGUL DEMIRAK * & HAKAN IsIk? 'Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Tokat Gaziosmanpasa University, Tasliciftlik Campus, 60250, Tokat, Turkey ?Tokat M. Emin Sarac Anatolian Religious High School, 60030, Tokat, Turkey *CORRESPONDENCE TO: senay.sengul@gop.edu.tr ABSTRACT—A macrofungus collected from Akbelen village in Tokat has been identified based on molecular evidence and morphology as Cortinarius rapaceoides, a new record from Turkey. Sequence data generated from the nuclear ribosomal internal transcribed spacer and ribosomal large subunit regions were deposited in GenBank and used to determine phylogenetic relationships within Cortinarius subg. Phlegmacium. A morphological description, illustrations, and phylogeny of the Turkish C. rapaceoides are provided. Key worps—Cortinariaceae, ITS, LSU, taxonomy Introduction The ectomycorrhizal basidiomycete genus Cortinarius (Agaricales) is the world’s most speciose mushroom genus. Ectomycorrhizal associations formed with trees (e.g., Nothofagus, Quercus, Pinus) and herbaceous plants are important for forest ecosystem (Moser & Horak 1975, Breitenbach & Kranzlin 2000, Stensrud & al. 2014, Itoo & al. 2015). Morphological features such as cortina covering young gills, rusty brown mature gills, ochraceous- rusty to deep rusty spore print, and bulbous stipe distinguish this genus. Species separation within Cortinarius is greatly complicated by a high degree of inter- and intra-specific morphological variation. Molecular markers have become important in helping resolve and refine Cortinarius taxonomic classification. Previous studies have used molecular markers derived from ribosomal gene loci containing the internal 560 ... Sengiil Demirak & Isik transcribed spacer regions (ITS1 and ITS2), ribosomal large subunit (LSU), and the two largest subunits of RNA polymerase II (RPB1, RPB2) (Garnica & al. 2003, 2009; Froslev & al. 2005, 2007; Niskanen & al. 2009; Liimatainen & Niskanen 2013; Stensrud & al. 2014; Liimatainen & al. 2017; Sesli & Liimatainen 2018). Among those, ITS data strongly supports phylogenetic delimitation of species among Cortinarius sections and our understanding of the evolution of the genus (see Garnica & al. 2009). Most studies stress the necessity of combining sequence data with morphological studies to resolve the relationships among Cortinarius species. Cortinarius species have been previously recorded in Turkey (Sesli & Denchev 2014; Akata & al. 2015; Giingor & al. 2015; Sesli & al. 2015, 2016; Sesli 2018; Sesli & Liimatainen 2018; Kalmer & al. 2019). Our research provides the first identification of C. rapaceoides in Turkey with detailed description of morphological characteristics and evidence at the molecular level. Materials & methods Morphological studies Fresh Cortinarius basidiomes were collected from Akbelen village (Tokat) in the autumn of 2018, photographed in the field, and important morphological and ecological features noted. Basidiomes were stored in paper bags and kept in a box. The fresh collection was transported to the laboratory in paper bags, and collection numbers were assigned. The dried basidiomes were placed into polyethylene bags for storage. Tissues were mounted in Melzer’s reagent, distilled water, Congo red, and KOH prior to examination under a light microscope. At least 25 measurements from each microscopic structure were obtained from a mature basidiome, which was initially identified based on its morphological features with the help of Boisselet (2012a) and Tanchaud (2016). The identified collection was deposited in the Fungarium of the Department of Biology, Tokat Gaziosmanpasa University, Tokat, Turkey (GOPUF). Molecular studies Genomic DNA was extracted from lamella using the ZR Fungal/Bacterial DNA MiniPrep kit as described by the manufacturer's protocol. The primer pair ITS4- ITS5 (White & al. 1990) was used to amplify ITS1-5.8S-ITS2 region and the primer pair LROR-LRS (Vilgalys & Hester 1990) was used to amplify 28S LSU rRNA gene region. The DNA was amplified in a 30 ul volume mixture containing 3 ul 10X buffer, 3 ul dNTP mix, 3 ul degenerate primer pair (final concentration of 1 uM each), 0.3 ul Dream Taq DNA polymerase (Thermo), 10 ul gDNA and 7.7 ul sterile ddH,O. PCR amplification for ITS region was set as: A negative control reaction using only sterile ddH,O was also run in parallel. 5 min initial denaturation at 95°C + 40 denaturation Cortinarius rapaceoides new for Turkey ... 561 cycles (95°C for 30 sec) + annealing (53°C for 30 sec) + extension (72°C for 1 min) and a final extension for 10 min. The LSU PCR protocol comprised: a 3 min initial denaturation at 95°C + 40 denaturation cycles (95°C for 30 sec) + annealing (48°C for 30 sec) + extension (72°C for 1 min) and a final extension for 10 min. PCR products were run in a 1% agarose gel electrophoresis and positive PCR products were gel purified by using Wizard SV Gel and PCR Clean-up System. Purified PCR products were sequenced in both directions using the same primer pairs by BM Labosis Inc. (Ankara). Sequence and phylogenetic analysis Chromatograms were checked for any nucleotide errors for each genomic sequence generated from both ends and errors were corrected manually. Assembled rDNA sequences were examined using Basic Local Alignment Search Tool (BLAST) programme to select the most closely related ITS and LSU sequences in the database. Representative ITS and LSU sequences of Cortinarius species were retrieved from GenBank for phylogenetic analysis. The sequence alignments and phylogenetic trees for each genomic region were analysed using MEGA 6.0 (Tamura & al. 2013). In the CLUSTALW alignment, sequences were trimmed at both ends. Phylogenetic trees were constructed using the maximum likelihood (ML) and maximum parsimony (MP) methods. Tamura-Nei model (Tamura & Nei, 1993) was used to construct the ML tree with bootstrap support of 1000 replicates and default settings. The bootstrap support values >50% were marked on the branches of the tree. Taxonomy Cortinarius rapaceoides Bidaud, G. Riousset & Riousset, Micologia 2000: 68 (2000) Fic. 1 PitEus 50-100(-120) mm diam., at first convex then plane-convex to plane; surface dull when dry, lubricous when moist; colour overall yellowish to ochre-yellow, in the centre orange-yellow with darker brown squamules. LAMELLAE pale lilac, soon rust-brown, broad, crowded, edges smooth to slightly crenate. Stipe whitish, sometimes gray-purplish at the apex, fragile, solid, cylindrical, base with a marginate bulb, surface longitudinally whitish- fibrillose when young, brownish when old. FLEsH whitish sometimes bluish in the upper and lower stipe. KOH negative on the flesh, amber yellow colour on the pileal surface. ODorR of chocolate. BASIDIOSPORES (9.6—)10-13(-13.8) x (6.0-)6.5-8(-8.5) um, elliptical to amygdaliform, strongly verrucose, yellow-brown. BasIpIA, (30—)35-40(-44.6) x (9-)10.5-12(-12.4) um, with 2-4 sterigmata, clavate. SPECIMENS EXAMINED: TURKEY, TOKAT, Akbelen village, 40°27’0”N 36°39'16’E, 1050 m, among dead leaves of Quercus sp, 8 November 2018, leg. Isik 798 (GOPUF; GenBank MN094878, MN099705). 562 ... Sengiil Demirak & Isik Cortinarius rapaceoides new for Turkey ... 563 HABITAT & DISTRIBUTION—Among fallen leaves on calcareous soils in deciduous woods. Previously recorded from France and Italy (Liimatainen & al. 2014). Molecular phylogeny The ITS1-5.8S-ITS2 (666 bp) and LSU rRNA (996 bp) sequences were deposited in GenBank. ITS sequence MN094878 BLAST results matched closely with the sequences representing C. caroviolaceus (EU057049), C. rapaceoides (KF732407, ex-holotype), and C. saporatus (DQ663413), which are included in the /Caroviolacei clade (Garnica & al. 2009). The remaining BLAST hits were also matched with species within the same clade. Accordingly, 67 sequences in the /Calochroi & Fulvi clade of Cortinarius subg. Phlegmacium (Liimatainen & al. 2014) were selected and ITS sequences were retrieved from GenBank for phylogenetic analyses. The outgroup Cortinarius aureifolius and C. caerulescens were used to root the phylogeny (Froslev & al. 2005, Garnica & al. 2009). Although fewer in number, LSU sequences for genus Cortinarius were also selected to construct a phylogenetic tree, but the LSU-based phylogeny was not well resolved due to low bootstrap values and no phylogenetic separation at the subgenus level was observed for LSU regions. Our two constructed MP and ML phylogenetic trees showed similar topologies for each genomic region. We present only the ML tree to indicate phylogenetic relationship of the studied species based on the ITS region. The sequence from our Cortinarius specimen clustered with sequences derived from C. rapaceoides described from Italy and France (including the ex-holotype sequence KF732407) and C. caroviolaceus (EU057049) from Italy with high bootstrap support (Fic. 2). Liimatainen & al. (2014) suggested that C. caroviolaceus sequence actually represented C. rapaceoides based on their ITS sequence phylogeny; and our phylogeny also supports it as conspecific with C. rapaceoides. Discussion Cortinarius rapaceoides, originally described from France and collected under Quercus ilex in a deciduous forest with calcareous soil, is characterized by a cream to yellow color, smooth convex pileus with a rolled margin, Fic. 1. Cortinarius rapaceoides (GOPUF - Isik 798): A. basidiomata; B. basidia and basidioles; C. basidia; D. basidiospores. Scale bars: A = 3 cm; B-D = 10 um. 564 ... Sengiil Demirak & Isik cream to purple lamellae, blue, cream or purple colored cylindrical stipe with a marginate bulb, 10-13 x 6-7 um verrucose spores, and a hardwood mycorrhizal association (Boisselet 2012a, Tanchaud 2016). ‘The species is morphologically similar to C. aleuriosmus Maire [= C. caro- violaceus P.D. Orton] and C. saporatus Britzelm. These fungi may be confused with each other due to their similar ecological features; however, C. aleuriosmus can be distinguished from C. rapaceoides by its whitish pileus and lamellae, smaller, almond-to lemon-shaped rough spores, abundant white veil, and farinaceous taste and odor (Orton 1955, Moser 1983, Phillips 1981, 2013; Boisselet 2012a, 2012b; Tanchaud 2016). Cortinarius saporatus differs in its bigger pileus and stipe, dark brown colour changes with KOH on the pileus, its yellowish colour at the stipe base, and an odor that is initially fruity but later more sour (Phillips 1981, 2013). Although some spores of Turkish C. rapaceoides are larger, the morphological characters are very similar and agree with French C. rapaceoides (Boisselet 2012a, Tanchaud 2016). Sequence analyses also support identification of Turkish collection as C. rapaceoides. It belongs in Cortinarius subg. Phlegmacium, a polyphyletic subgenus (Peintner & al. 2004, Garnica & al. 2005). Although Cortinarius species are morphologically quite variable, their ITS sequences show a high sequence identity and are reliably used for species delimitation and identification (e.g. Froslev & al. 2005, 2007; Ortega & al. 2008; Garnica & al. 2009; Niskanen & al. 2009). We found the ITS region more useful than the LSU region for inferring the phylogenetic relationships and were able to determine independent phylogenetic lineages with high boostrap support. Our ITS sequence clustered in Cortinarius sect. Calochroi and showed 100% sequence identity with C. rapaceoides from France and Italy without any intraspecific sequence polymorphisms. Our first record of C. rapaceoides in Turkey is well supported by both morphological and molecular evidence. Acknowledgments We would like to thank Dr. Ibrahim Turkekul for critically reviewing this article. We also thank the referees, Drs Abdullah Kaya (Gazi University, Ankara, Turkey) Fic. 2. Phylogeny of selected Cortinarius species inferred from ITS sequences using the ML method. The diamond [@] indicates the Turkish C. rapaceoides identified in this study. Ex-type sequences are annotated with [T]. Cortinarius aureifolius and C. caerulescens represent the outgroup. Bootstrap support values =>50% from ML analysis are shown on the branches. Bar = 0.02 expected changes per site per branch. Cortinarius rapaceoides new for Turkey ... 565 67 Cortinarius caesiocinctus DQ663239 [T] Cortinarius caesiocinctus DQ663241 63 | Cortinarius cobaltinus KF673472 Cortinarius cobaltinus KF673471 Cortinarius cobaltinus KF673470 [T] Cortinarius bigelowii KF732265 [T] 97, Cortinarius metarius KF732347 [T] Cortinarius barbarorum DQ663236 Cortinarius flavipallens KF732554 [T] Cortinarius olympianus KF732364 [T] 74' Cortinarius olympianus KF732553 99 | Cortinarius sublilacinopes KF732561 [T] Cortinarius sublilacinopes DQ663434 Cortinarius pseudogracilior KF732394 [T] Cortinarius calojanthinus KF732272 [T] 73' Cortinarius corrosus DQ663281 Cortinarius arenicola KF732252 [T] Cortinarius frondosophilus KF732562 [T] 96! Cortinarius platypus KF732563 [T] Cortinarius magnivelatus EU056976 Cortinarius spectabilis DQ663425 Cortinarius spectabilis DQ663426 99 Cortinarius sannio KF732536 Cortinarius sannio KF732537 76, Cortinarius pseudoglaucopus DQ663395 97| | Cortinarius pseudoglaucopus AY669573 Cortinarius elotoides KF732300 [T] Cortinarius rapaceoides DQ663417 Cortinarius caroviolaceus EU057049 99| | Cortinarius rapaceoides KF732407 [T] 74 | Cortinarius rapaceoides NR130253 @ Cortinarius rapaceoides MN094878 Cortinarius cacodes KF732270 [T] 80 ; Cortinarius saxamontanus KF732421 [T] Cortinarius saxamontanus EU057026 Cortinarius cf. aureofulvus KF732544 Cortinarius cf. aureofulvus KF732545 Cortinarius cf. aureofulvus KF732543 Cortinarius sulphurinus var. fageticola DQ663439 : Cortinarius sulphurinus KC842431 Cortinarius sulphurinus DQ663437 Cortinarius luteicolor KF732546 Cortinarius luteicolor KF732547 Cortinarius luteicolor FJ717511 Cortinarius citrinipedes KF732281 [T] Cortinarius osmophorus DQ663368 Cortinarius evosmus KF732302 [T] 9 Cortinarius osmophorus AY174815 Cortinarius cupreorufus KF732548 Cortinarius cupreorufus KF732549 98 | Cortinarius cupreorufus KF732550 Cortinarius cupreorufus KF732294 [T] 96 Cortinarius flavobulbus EU057017 Cortinarius flavobulbus KF732305 [T] 98 , Cortinarius viridirubescens KF732476 [T] Cortinarius viridirubescens EU057007 Cortinarius dibaphus DQ663286 79, Cortinarius alnobetulae EU655672 Cortinarius alnobetulae KF732246 [T] Cortinarius amnicola KF732249 [T] Cortinarius subpurpureophyllus KF732450 [T] Cortinarius subpurpureophyllus KF732557 79| Cortinarius napus GU363492 Cortinarius napus KC842428 Cortinarius cf. meinhardii KF732551 96 | Cortinarius meinhardii AY174840 Cortinarius parafulmineus EF014269 99! Cortinarius parafulmineus KF732552 [T] Cortinarius caerulescens AY174863 Cortinarius aureifolius AF268893 566 ... Sengiil Demirak & Isik and Yusuf Uzun (Van Yuzuncu Yil University, Turkey), for their helpful comments and suggestions. We greatly appreciate Dr. Lorelei Norvell’s editorial review and Dr. Shaun Pennycook’s nomenclatural review. Literature cited Akata I, Kabaktepe S, Akgiil H. 2015. Cortinarius caperatus (Pers.) Fr., a new record for Turkish mycobiota. Kastamonu University Journal of Forestry Faculty 15: 86-89. https://doi.org/10.17475/kuofd.94670 Boisselet P. 2012a. https://www.mycocharentes.fr/pdf2/8%201152%201%20.pdf (accessed 14 May 2019). Boisselet P. 2012b. https://www.mycocharentes.fr/pdf2/965.pdf (a ccessed 14 May 2019). Breitenbach J, Kranzlin F. 2000. Fungi of Switzerland. Vol. 5. Verlag Mykologia, Lucerne, Switzerland. Froslev TG, Matheny PB, Hibbett DS. 2005. 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Sengiil Demirak & Isik Tanchaud P. 2016. https://www.mycocharentes.fr/pdf1/1338.pdf (accessed 14 May 2019). Vilgalys R, Hester M. 1990. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172(8): 4238-4246. https://doi.org/10.1128/jb.172.8.4238-4246.1990 White TJ, Bruns T, Lee SJ, 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. New York, NY, USA: Academic Press. https://doi.org/10.1016/B978-0- 12-372 180-8.50042-1 MYCOTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 July-September 2020—Volume 135, pp. 569-578 https://doi.org/10.5248/135.569 New records of Toninia from China CoNGCONG Mi1Ao*', MEIJIE SUN*’, XIAO ZHANG!, ZHAOJIE REN’, LING Hu? ' Key Laboratory of Plant Stress Research, College of Life Sciences, Shandong Normal University, Jinan, 250014, P. R. China ? Shandong Museum, Jinan, 250014, P. R. China 3 Institute of Environment and Ecology, Shandong Normal University, Jinan, 250014, P. R. China “ CORRESPONDENCE TO: ccmjy123@163.com ABSTRACT—Four Toninia taxa (T. albilabra, T: poeltii, T. tristis subsp. Arizonica, and T. tristis subsp. fujikawae) are reported for the first time from China. Descriptions with morphological and chemical characters and known distribution are given for each taxon. A key to the species of Toninia s.l. in China is also provided. KeEyYworps—lichen-forming fungi, Lecanorales, Ramalinaceae, taxonomy Introduction Toninia, widely distributed in drought and arid areas, is an important component of desert lichen communities (Timdal 1991, 2002). The current delimitation of the genus is based mainly on the features of the asci and paraphyses (Meijie & al. 2019) and molecular genetic characters (cf. Kistenich & al. 2018, Ekman 2001, Ekman & al. 2008). Toninia is characterized by a crustose to squamulose and epruinose to densely pruinose thallus; lecideine, epruinose to densely pruinose apothecia; dark brown to colourless hypothecium; usually colourless, grey, green, or brown epihymenium and exciple that changes colour in K and N; clavate, Biatora-type, 8-spored asci; and elliptical to spindle-shaped, colourless, 1-8-celled ascospores. Toninia * ConGCONG Miao & MEWIE SUN contributed equally to this work. 570 ... Miao, Sun & al. was previously placed in Lecideaceae (Massalongo 1852; Timdal 1991; Zahlbruckner 1890). With the advent of the molecular age, phylogenetic analysis of lichens is carried out in combination with their morphological and anatomical features; currently, Toninia is classified in Ramalinaceae (Timdal 2002; Ekman 2001). In this paper we contribute to the knowledge of Toninia in China and add accurate data for the Lichen Flora of China project by reporting on four taxa new to the country: T. albilabra, T: poeltii, T: tristis subsp. arizonica and T: tristis subsp. fujikawae. Materials & methods The specimens studied are preserved in the Lichen Section of Botanical Herbarium, Shandong Normal University, Jinan, China (SDNU) and the Lichen Herbarium of the Kunming Institute of Botany, Kunming, China (KUN). The morphological and anatomical characters of the specimens were examined under a COIC XTL7045B2 stereomicroscope and an Olympus CX41 polarizing microscope. Thallus and medulla were tested with K (a 10% aqueous KOH solution), Cl (a solution of aqueous NaOCl), and I (Lugol’s iodine) for identification. Lichen substances were identified using standardized thin layer chromatography (TLC) techniques with system C (Orange & al. 2010). Photographs were taken with Olympus SZX16 and BX61 microscope with a DP72 camera. Taxonomy Toninia albilabra (Dufour) H. Olivier, Bull. Géogr. Bot. 21: 196 (1911) Fic. 1 THALLUS squamulose, indeterminate. Squamules <2(-4) mm diam., scattered or contiguous to irregularly imbricate, orbicular or sometimes slightly lobed, weakly concave to weakly convex. Upper side reddish brown, usually epruinose, shiny, with regular and usually deep fissures in the cortex, pores and pseudocyphellae absent. Margin partly to entirely white pruinose. Underside white to pale brown. Algal layer continuous. Lower cortex poorly developed or absent. APOTHECIA $1.5(-2.5) mm diam., weakly concave to weakly convex, usually persistently (but narrowly) marginate, densely white pruinose or more rarely epruinose. Proper exciple reddish brown throughout or with an additional grey pigment near the rim, lacking crystals. Hypothecium medium brown in upper part, pale brown to colourless in lower part. Hymenium 60-70 um high; epithecium grey, K+ violet, N+ violet, containing crystals of calcium oxalate. Spores broadly to narrowly fusiform, 1-septate, 13.5-22.5 x 3-4 um. Toninia taxa new for China... 571 aN Baa Dh. fh « ao iy, ‘ > P Why rs Fic. 1. Toninia albilabra (15-49721 KUN). A. Thallus; B. Epihymenium; C. Purple K reaction of epihymenium; D. Ascospores. PYCNIDIA not seen. CHEMISTRY— Terpenoids detected by TLC. SPECIMENS EXAMINED—CHINA. TIBET, Baju County, on the way from Bangda to Bangda Airport, alt. 4129 m, 17 Sep. 2014, Wang Lisong & al. 14-47025 (KUN); Jiangda County, Tongpu Township, 317 national highway side slope, 31°38’33”N 98°26'10’E, alt. 3940 m, 5 Oct. 2016, Wang Lisong & al. 16-51356 (KUN). YUNNAN, Zhongdian, Benzilan Town, 28°10.47’N 99°23.01’E, alt. 2115 m, 27 Aug. 2006, Wang Li Song & al. 06-26674 (KUN); Deqin, Baima Snow Mountain, 28°19.47’N 99°05.13’E, 2 Nov. 2015, alt. 4350 m, Wang Lisong & al. 15-49721 (KUN). DisTRIBUTION—On rock or soil. Toninia albilabra has been reported in Europe, North Africa, Macaronesia, and the Middle East (Timdal 1991). New to China. COMMENTS— The Chinese material closely matches the previously published description by Timdal (1991). Toninia albilabra is morphologically similar to T: sedifolia, which differs mainly by having a smooth upper cortex, a thinner epinecral layer, and farinose pruina. The white edge of the squamules in 572 ... Miao, Sun & al. T. sedifolia is caused by pruina on the cortex surface and is not sharply delimited from the less pruinose inner part of the upper side. The white edge in T: albilabra, however, is caused by the white medulla being visible outside the sharply delimited, usually entirely epruinose, upper cortex (Timdal 1991). Fic. 2. Toninia poeltii (16-53317 KUN). A. Thallus and apothecia; B. Epihymenium; C. Purple K reaction of epihymenium; D. Ascospores. Toninia poeltii Timdal, Opera Bot. 110: 85 (1991) Fic. 2 Thallus squamulose, indeterminate. Squamules <1 mm diam. More or less continuous crust. Upper side dark greyish brown, epruinose, dull, with a few shallow fissures in the cortex, lacking pores and pseudocyphellae. Margin concolorous with upper side, epruinose. Upper cortex 30-80 um thick, lacking crystals. Algal layer continuous. Medulla lacking crystals. Lower cortex <40 um thick, brown pigment hyphae. Apothecia <0.8 mm diam., plane and indistinctly marginate when young, later more or less convex and immarginate, epruinose. Proper exciple olivaceous green in the rim, colourless in inner part, K+ violet or brown, N-, lacking crystals. Hypothecium colourless. Hymenium 60-70 um high; Toninia taxa new for China... 573 epithecium olivaceous green, K+ violet or brown, N-. Spores bacilliform, often slightly curved, 1-3-septate, 12-21.5 x 4-5 um. Pycnidia not seen. CHEMISTRY—Terpenoids detected by TLC. SPECIMENS EXAMINED—CHINA. TIBET, Mangkang, on the way from Lawushan to the town of Rumei, alt. 4290 m, 20 Sep. 2016, Wang Lisong & al. 16-53317 (KUN); Dingri County, Alpine meadow, alt. 5250 m, 22 Aug. 2007, Wang Lisong & al. 07- 28528 (KUN). YUNNAN, Deqin County, Sola Mountain, 28°38.19’N 98°36.30’E, alt. 4800 m, 10 Sep. 2012, Niu Dongling & al. 12-35865 (KUN). DiIsTRIBUTION—On soil or rock. Reported only in the Himalayas (Timdal 1991). New to China. COMMENTS— The Chinese material closely matches the previously published description by Timdal (1991). Toninia poeltii is morphologically similar to T. squalida (which has not been recorded for China), but differs in having an olivaceous green, K+ violet/brown, N- pigment in the epithecium and exciple rim. Toninia poeltii has at most 3-septate spores, but T. squalida has 3-7-septate spores (Timdal 1991). Toninia tristis subsp. arizonica Timdal, Opera Bot. 110: 112 (1991) Fia. 3 THALLUS squamulose, squamules <6(-8) mm diam., scattered to adjacent, rounded, bullate but often with an irregular depression. Upper surface castaneous brown to dark brown, epruinose, dull, smooth in the cortex, lacking pseudocyphellae. Upper cortex <160 um high, lacking crystals. APOTHECIA <6 mm diam., plane and weakly convex, distinctly marginate, epruinose. Proper exciple brown to dark brown, colourless in inner part. Epithecium brown, partly with a green tinge. Spores simple, narrowly ellipsoid to fusiform, 8-17 x 4-4.5 um. Orange pigment present in the upper part of the hypothecium and in the lumina of many paraphyses, asci, and spores; yellow pigment present in the upper part of the hypothecium. CHEMISTRY—Terpenoids and fatty acid detected by TLC. SPECIMENS EXAMINED—CHINA. INNER MONGOLIA, Urad Rear Banner, Huhe Bash GeTusheng, alt. 1600 m, 19 Aug. 2011, Tong Debao 20123675 (SDNU). NINGXIA, Jingyuan County, Migang Mountain, alt., 2300 m, 20 Jun. 2011, Cheng Yuliang 20116001 (SDNU). Sicnuan, Ningnan County, Baihetan Town, alt. 701 m, 18 Oct. 2013, Wang Lisong & al. 13-39450 (KUN); Jinsha River, alt. 1550 m, 20 Apr. 2014, Wang Lisong & al. 14-43367 (KUN). TIBET, Naqu County, 317 country road side slope, alt. 4680 m, 30 Sep. 2016, Wang Lisong & al. 16-51961 (KUN). YUNNAN, Yunmin County, Jinsha River Valley, alt. 920 m, 27 Nov. 2014, Wang Lisong & al. 14-46464 (KUN); Dongchuan, Sandstone from Dongchuan to Qiaojia Road, alt. 1080 m 11 May 2017, Wang Lisong & al. 17-55053 (KUN). 574 ... Miao, Sun & al. Fic. 3 Toninia tristis subsp. arizonica (16-53317 KUN). A. Thallus; B. Epihymenium; C. Ascus; D. Ascospores. DISTRIBUTION—On soil. Arizona, USA (Timdal 1991). New to China. ComMMENTS—The Chinese material closely matches the previously published description by Timdal (1991). Toninia tristis subsp. arizonica produces orange asci similar to T! tristis subsp. asiae-centralis, which differs in having 1-septate spores (Timdal 1991). In its possession of simple spores, the Chinese material closely matches previously published descriptions of T. tristis subsp. pseudotabacina, which differs in its absence of orange pigments (Timdal 1991). Toninia tristis subsp. fujikawae (M. Satd) Timdal, Opera Bot. 110: 113 (1991) Fic. 4 Thallus squamulose, squamules <4(-5) mm diam., continuous, bullate, often forming cushions. Upper side castaneous brown to dark brown, lacking pseudocyphellae. Margin concolorous with upper side, epruinose. Upper cortex not containing crystals. Algal layer continuous. Medulla Toninia taxa new for China... 575 lacking crystals. Lower cortex resembling upper cortex, but stainable layer often dark reddish brown. Apothecia <4 mm diam., plane and weakly convex, distinctly marginate, epruinose, Proper exciple brown to dark brown, colourless in inner part, K-, N-, lacking crystals. Hypothecium pale brown, 60-70 um high; epithecium olivaceous green to bright green, K+ violet or brown, N-. Spores simple, ellipsoid, 7-12 x 3-5 um. Orange and yellow pigments lacking. CHEMISTRY—Terpenoids and fatty acid detected by TLC. SPECIMENS EXAMINED—CHINA. SICHUAN, Huili County, Jinsha River, alt. 1550 m, 20 Apr. 2014, Wang Lisong & al. 14-43349; 14-43368 (KUN); TiBET, Wuqi County, next to the 214 state road, 31°09’51”N 96°37'54’E, alt. 3840 m, 2 Oct. 2016, Wang Lisong & al. 16-51390 (KUN); Baqing County, Laxi village, 317 national road side slope, 31°50’17”N 94°23’24’E, alt. 4180 m, 1 Oct. 2016, Wang Lisong & al. 16-53934 (KUN). YUNNAN, Luquan County, alt. 2540 m, 19 Apr. 2014, Wang Lisong & al. 14-43234 (KUN); Deqin County, Benzilan, 28°1135.54”N 99°21’08’E, alt. 2112 m,19 Aug. 2018, Wang Chunxiao & al. 20180326 (SDNU). DiIsTRIBUTION—On rock or soil. Japan (Timdal 1991). New to China. CoMMENTS— Toninia tristis subsp. fujikawae may be confused with T. tristis subsp. canadensis in having green epithecium, but the latter subspecies has mainly 1-septate ascospores (Timdal 1991). Although also characterized by simple spores, T! tristis subsp. arizonica is distinguished by a brown epithecium (Timdal 2002). Key to the species of Toninia s.|. in China Kistenich & al. (2018) place some species below in Thalloidima (T. opuntioides, T: physaroides, T. sedifolia) or other genera (e.g., T: gobica, T. poeltii). Here we follow Timdal (1991) and treat all species in Toninia s.1. ie Thalliis proinose (arinose.or-cranulaty .) wees lie ete scorn ae eels as ae 2 i thallusseprumeseto faintly prtimoses £2 4 5.53 2 5.x Alay ton eschg eo nes oaoraae eget 14 22 Epitheciom: prays PIO NG VAIOlet th. nres WN An Reh cba eieee MO sear ece 2 3 2. Epithecium brown, K+ red, or epithecium dark green to bright green, Ka, N+ Violet® oes iG cage ee eee! eens Hae bios ate ee ne eae eee 13 SRASGOSPOLeSzat LEASt AN Sep tate’ Seo see Steeirerd aca Shepton agit ree A aoa scl eRe or AR Biss 9s 4 3. ASCOSPOres OU ESeptate®.... oe eck che sah ile eee fend a eee ee Dee eae T. toniniana A) Aseospotes.maily. s-septater... Strachan s.r tet cc ohne get kes ARS Aen een ct S) Av Ascosporesiexclusively.lusepiate s+ %. 18 As dchaa sort. Set Ake, BB eee ne: Book 6 5. Ascospores 1-3-septate, 13.5-22.5 3.5-5 UM ..... 2. ce eee ee cece ee T. superioris 5. Ascospores mainly 3-septate, 23.5-33 x 3-4 Um... 6... eee eee eee T. alutacea 6. Thallts pruingse; pruinatarinose® 42.2 oni we ee ne eee sca pet we ee ees Yi 6. Thallus:prutnhose, pruina. granulate) 0-4 iu oh este sict wide SIE Pack andi 10 576 ... Miao, Sun & al. 7. Thallus rosulate, pruina exposed surface of thallus .................... T. candida 7: Thallusnot-rosulate, priuina-patehy.en thallus: ../ 5.2 eee5 an se ee ee 8 SeThalluswvithpsetidocyphellae %) 1.34.0 2048 oer sek bat teed T. physaroides Sathallusswithout pseudocyphellae ¢ .. GoW akg ete poe he Gear a eS 9 9. Squamules bullate and partly vertically flattened, more or less imbricate, containing an unknown substance (TLC solvent system C Rf.21) ... T. opuntioides 9. Squamules weakly convex to bullate but not vertically flattened or imbricate; not containing an unknown substance (TLC solvent system C Rf. 21) ... T. sedifolia 10. Squamules margin usually densely with white pruina ............... T. albilabra 10. Pruina not only on the edge of squamules ............... 00. e eee e eee eee 11 11. Ascosporeés: stall (835-13 4-5: (ith) att we ole PE ee ina eae es T. nordlandica LL-Ascospores larger(12—24-% 335 punt) ee ge Ae arte papa ah ped eka s Panam aiaite 12 12;-Thallus-rosulate; apothecia S4omime. aoe ky Been ee ae eee T. rosulata 12Atnallasnoetrosulate;apothecia S157 tien. sho: opt Max doar eee T. diffracta 13, Epithecttimred-brown, K+ red) ni. c. gered be Sedo Vee Pes ee ees T. lutosa 13. Epithecium green, K-, N+ purple; ascospores 3-septate ................ T. gobica 14, Thallus epruinose-or slighthypruinose <3 * 2; 2s cajos 53556652 ba Rees oS: 15 WAS. Phalitis<e premise” af Seen. eh Mea ents fein tiis eared Meta Ye tae tae Bae Rwy 17 15. Thallus pale brown; epithecium dark brown ..................... T: sculpturata 15. Thallus of different colour; epithecium not dark brown...................06- 16 16. Thallus crustose; epithecium gray, K+ purple, N+ purple ............. T. pennina 16. Thallus squamulose, epithecium olive green to bright green, K-, NEF ipurplesascosperes las-sepiate Mg... 2. s Cok ah maker Ges T aromatica 17. Epithecium olive green to bright green, K-, N+ purple ....................04. 18 17. Epithecium red-brown, K+ red; ascospores (1-)7(-9) -septate ........ T. ruginosa 18. ‘thallus :crustose:ascospores-luseptate: +... nee Be ee ee T. philippea 18. Thallus squamulose, or crustose to subsquamulose ............... 22.0020 eee 19 19. Thallus crustose to subsquamulose; epithecium olive green; ASCOSPOLES 3S + SCWUALE ac iso's, ote Cecio E es Shope Mites) oot ew Aten Lane T. coelestina 19. Thallus squamulose; epithecium olive green to bright green .................. 20 20. Ascospores simple to 1-septate; epithecium bright green to dark brown .. T. tristis 20. Ascospores 1 to more septate; epithecium olivaceous to bright green .......... 21 21. Epithecium olivaceous to bright green (K-), ascospores 1(—3)=septate s..sess02. Gs vas tees ee seee tom euesnes T’ cinereovirens 21. Epithecium olivaceous (K+ purple to brown), ascospores 1-(-3)-septate ... T. poeltii Key to the subspecies of Toninia tristis Ik -ASCUSIOLAM OC GE PEGE tote Metta totale Stas Phas Ue BRR OR MRE 51 .C cea ale Geet ah IRE se aE CP 2 LPASCUSHIO LOPAN CGPI oh aa. ov hge bers tee OS Sane Makes: Sng shne tees hy SMe Pa hah Sr cots 3 Toninia taxa new for China... 577 Zi ASCOSPOLeS L-SEPtate,. shea srs oihtetier as map Ue eleye a! ath wen odes subsp. asiae-centralis D SASEOSPOLES SUN pLeT ae. Y ye rik. toree 4g. C28 Se he sees eee oe ates subsp. arizonica J MEpit DE CWT AO FEEN Bc i at Te rguee eet Siete Ay eee See a ON dan ee Ue as 4 Deve PIC Ve CAUTION TA. ta heel = Leche iay ens coisas eater os bSK oheaeraaih sant Tae Males SEA» ene Ns nce 7 At ASCOSPOLES. Ue SEDIALE bo ah cttlaliee ek SAP ace te Sethe 8 PeRSO Nees, Lei bee We. /e SG subsp. canadensis Al PASCOS POLES SLIP agen give eo trend rer ase he ape te, ates Pe SE AR MB Cha 5 5, sxscospores largerd OES 5: WIT) tena Ms es anos Nae es subsp. pseudotabacina d. Ascosporesismall(Sa12-Minn): Wier ics kone. nee oe es ae eRe nee «Ae 6 G. No, chemical substance Ss. s.enzo se Seo ook oR a at. Seek oe subsp. thalloedaemiformis 6: Containine-chemical substance: e's ont ee hg ete co eons ie subsp. fujikawae FacNSCOSPOTES STAPLE Maes 42h. harks tee agers ten Saitek a GME e eke. aah subsp. scholanderi 7, EXSCOS POLES! | -SEPTAte a eo ule satan, sleet ics, as telns a edz “niet Beles ase, hn 0 Dhow de onde Gaagaet 8 Se OGM ATMS Se NCO IN TaNaIN 81a: PS Rt a ec dT es aT MI otc tetne dd tay tla subsp. tristis 8. Squamules <3(—4) mim! ps esas Feec dw ead Gee eee awa ee eee ae subsp. coahuilae Acknowledgments We thank Dr Edit Farkas (MTA Centre for Ecological Research, Vacratot, Hungary) and Dr Shou-Yu Guo (State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China) for presubmission review. The authors would also like to thank Lisong Wang and Xinyu Wang (Kunming Institute of Botany, CAS, China) for assistance during this study. This work was supported by the Emergency Management Project of National Natural Science Foundation of China (31750001), the National Natural Science Foundation of China (31900010). Literature cited Ekman S. 2001. Molecular phylogeny of the Bacidiaceae (Lecanorales, lichenized Ascomycota). Mycological Research 105(7): 783-797. https://doi.org/10.1017/s0953756201004269 Ekman S, Andersen HL, Wedin M. 2008. The limitations of ancestral state reconstruction and the evolution of the ascus in the Lecanorales (lichenized Ascomycota). Systematic Biology 57(1): 141-156. https://doi.org/10.1080/10635150801910451 Kistenich S, Timdal E, Bendiksby M, Ekman S. 2018. Molecular systematics and character evolution in the lichen family Ramalinaceae (Ascomycota: Lecanorales). Taxon 67(5): 871-904. https://doi.org/10.12705/675.1 Massalongo AB. 1852. Ricerche sull’autonomia dei licheni crostosi. Verona. Orange A, James PW, White FJ. 2010. Microchemical methods for the identification of lichens. 2nd edition. London, British Lichen Society. Scopoli JA. 1760. Flora carniolica. Wien, Sumptibus Joannis Thomae Trattner. Scopoli JA. 1772. Flora carniolica, ed. 2. Tom. 2. Wien, Impensis Ioannis Pauli Krauss. Sun MJ, Yan SK, Tang R, Wang CX, Zhang LL. 2019. New records of Bilimbia and Toninia from China. Mycotaxon. 134: 139-146. https://doi.org/10.5248/134.139 578 ... Miao, Sun & al. Timdal E. 1991. A monograph of the genus Toninia (Lecideaceae, Ascomycetes). Opera Botanica 110. 137 p. Timdal E. 2002. Toninia. 488-501, in: TH Nash & al. (eds). Lichen Flora of the Greater Sonoran Desert Region. Vol. 1. Tempe, Lichens Unlimited, Arizona State University. Zahlbruckner A. 1890. Prodromus einer Flechtenflora Bosniens. Annalen des Kaiserlich- K6niglichen Naturhistorischen Hofmuseums, Wien 5: 20-48. MYCOTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 July-September 2020—Volume 135, pp. 579-587 https://doi.org/10.5248/135.579 Serendipita sacchari sp. nov. from a sugarcane rhizosphere in southern China LING XIE?*, YAN- YAN LONG?”*, YAN ZHANG’”, YAN-Lu CHEN’”, WEN-LONG ZHANG?* 'Plant Protection Research Institute, Guangxi Academy of Agricultural Science, Nanning 530007, China ? Microbiology Research Institute, Guangxi Academy of Agricultural Science, Nanning 530007, China *CORRESPONDENCE TO: chris1933@126.com ABSTRACT—We isolated a new species, proposed here as Serendipita sacchari, from a sugarcane rhizosphere in Guangxi Province, China. This species is characterized by its unstable nucleus numbers (1-15) in its chlamydospores versus their regular distribution in hyphal cells. ITS rDNA and combined LSU+ TEFl-a sequence analyses also support the uniqueness of this new plant symbiont. Key worps—molecular phylogeny, Sebacinales, Serendipitaceae, taxonomy Introduction Serendipita P. Roberts (Basidiomycota, Sebacinales, Serendipitaceae), typified with S. vermifera (Oberw.) P. Roberts, originally comprised seven species (Roberts 1993). Two additional new species S. lyrica Trichiés (Trichiés 2003), and S. herbamans K. Riess & al. (Riess & al. 2014) have been proposed in this genus, and two anamorphic species in Piriformospora Sav. Verma & al. have been recombined as S. indica (Sav. Verma & al.) M. Weiss & al., and S. williamsii (Zuccaro & M. Weiss) M. Weiss & al. (Verma & al. 1998; Basiewicz & al. 2012; WeifS & al. 2016). Serendipita currently contains 11 species and DNA barcodes are widely accepted as an important tool in delineating species (Schoch & al. 2012; Riess & al. 2014). * LING XIE & YAN-YAN LonG contributed equally to this work. 580 ... Xie, Long & al. During an investigation of plant endophytes in sugarcane rhizosphere in Guangxi Province, China, an interesting strain has been morphologically and phylogenetically confirmed as a new species of Serendipita and is described herein. Materials & methods Soil samples were collected from a 5-15-cm sugarcane rhizosphere layer in Tanluo Town, Nanning City, Guangxi Province, China, in Feb 2011. Fungal isolates were obtained by baiting with Chinese cabbage as described by Narisawa & al. (1998), cultured on PDA media, and deposited in the China General Microbiological Culture Collection Center (CGMCC3.19906). A pure culture of the fungus was air-dried and frozen to prepare the holotype specimen, which is curated in the Mycological Herbarium, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China (HMAS). Observations and measurements were made with an Olympus BX53 Ci-L light microscope and Tescan-vega3 LMU scanning electron microscope (SEM). DNA extraction, amplification, sequencing Genomic DNA was extracted using the Sangon Biotech Rapid Fungi Genomic DNA Isolation Kit from mycelia grown at 28 °C for 10 d on oatmeal medium (OA, 10 g oatmeal, 10 g bacto agar, 1 g MgSO,e7H,O, 1.5 g H,PO,, 1 g NaNO,, for 1 L distilled water). The internal transcribed spacer of ribosomal DNA (ITS rDNA), large subunit of ribosomal DNA (LSU), and translation elongation factor 1-a (TEF1-a) were amplified with fungal specific primer pairs ITS1/ITS4, LROR/ LRS, and EF1-983f/EF1-2218r1, respectively (Vilgalys & Hester 1990, Basiewicz & al. 2012). PCR reaction mixture and conditions followed the protocol of 2xEasyTaq PCR SuperMix. The DNA was amplified in 50 uL volumes containing PCR buffer [20 mM KCl, 10 mM (NH,),SO,, 2 mM MgCl , 20 mM Tris-HCl, pH8.4], 200 uM of each dNTP, 15 pmol of each primer, 100 ng DNA template, and 2.5 units of Taq DNA polymerase (Biocolor BioScience & Technology). Thermal cycling began with 5 min of denaturation at 94 °C, followed by a 35 cycles of denaturation (94 °C for 40 s) + annealing (56 °C for 40 s) + extension (72°C for 60 s), and finalized with a final extension at 72 °C for 10 min. A negative control using sterilized distilled water instead of template DNA was included in the amplification process. The PCR products were examined by electrophoresis at 75 V for 2 h in 0.8 % (W/V) agarose gel in 1xTAE buffer (0.4 M Tris, 50 mM NaOAc, 10 mM EDTA, pH 7.8) and visualized under an ultraviolet light after staining with ethidium bromide (0.5 ug mL). The PCR products were purified using MultiScreen® PCRw96 Cleanup Filter Plates according to the manufacturer’s protocol. Purified PCR products were directly sequenced with primer pairs mentioned above in an ABI 3730-XL DNA sequencer. The sequences were deposited at GenBank (http://www.ncbi.nlm.nih. gov) and compared through a BLAST search. Serendipita sacchari sp. nov. (China) ... 581 TABLE 1. Taxa with GenBank ITS accession numbers SPECIES VOUCHER GENBANK NO. Cantharellus avellaneus 1217/ER KX857081 Chaetospermum camelliae MFLUCC12-0433 KF516965 C. chaetosporum CBS:154.59[T] KJ710461 Craterocolla cerasi TUB 020203 KF061265 Ditangium altaicum LE 231836 [T] NR 163760 Efibulobasidium albescens — AF384860 Globulisebacina rolleyi — AY509550 Helvellosebacina concrescens TUB 019706 JQ665516 H. helvelloides TUB 019983 KF000415 Paulisebacina allantoidea — AF490396 RoKi 179 KF061266 Sebacina candida TUB 020331 KF061278 S. pallida TUB 019650 JQ665562 Serendipita herbamans S1[T] KF061285 S. indica DSM 11827[T] KF061284 S. sacchari CGMCC3.19906 [T] KY496808 S. williamsii DAR 29830 KY509323 Tremellodendron ocreatum TH8577 KT339265 Tremelloscypha dichroa VB4212 KF061281 TABLE 2. Taxa with GenBank LSU and TEF1-a accession numbers GENBANK NO. SPECIES VOUCHER LSU TEF1-a Serendipita. indica DSMZ 11827[T] AY505557 AJ249911 S. sacchari CGMCC3.19906 [T] KY496809 MF196313 S. vermifera MAFF 305835 DQ983814 JN211111 MAFF 305838 DQ983816 JN211116 MAFF 305828 DQ520096 JN211115 S. williamsii DAR 29830 AY505556 JN211110 Phylogenetic analyses The three DNA loci were separately aligned using Clustal-X (1.83). Two neighbour-joining trees were derived from ITS1-5.8S-ITS2 and the combined LSU+ TEF1-a datasets using MEGA v. 4 (Tamura & al. 2007)(TABLES 1,2; Fics 2,3). Bayesian analyses of the aligned DNA sequence dataset were conducted with MrBayes v. 3.1.2 (Huelsenbeck & Ronquist 2001) following Sun & Guo (2010). The best-fit evolutionary model was determined for each dataset by comparing different evolutionary models via MrModeltest v. 2.3 (Nylander 2008). Four simultaneous Markov Chain Monte Carlo chains were run by starting from random trees and sampling every 100 generations. The analyses were halted at 4,000,000 generations after the calculation reached stationarity. Of the 40,000 trees generated, 25% were 582 ... Xie, Long & al. excluded as “burn in” when calculating the posterior probabilities. Bayesian posterior probabilities were obtained from the remaining 50% majority rule consensus trees. Significant support was defined by clades contained in >95% of the sampled trees. Taxonomy Serendipita sacchari L. Xie, Y.Y. Long & Y.L. Chen, sp. nov. Fig. 1 MB 836761 Differs from other Serendipita species by its variable nucleus numbers (1-15 nuclei) in the chlamydospore cells. Type: China. Guangxi Province: Nanning City, Tanluo Town, in sugarcane rhizosphere, 22°55’30"N 107°58’34’E, alt. 96 m, Apr. 2011 (holotype, HMAS 247074; ex-type culture CGMCC3.19906; GenBank KY496808, KY496809, MF196313). EtryMo.oey: sacchari, referring to the plant from whose rhizosphere the species was first collected. Colonies reaching 50 mm in diam on PDA medium at 28 °C after 2 weeks, light yellow, circular (or nearly circular), less aerial hyphae. Hyphae hyaline to light yellow-green, septate, 1.2-3.4 um (2.2 + 0.5 um, n = 50), each cell regularly containing 1-2 nuclei. Chlamydospores abundant, spherical to pear-shaped, singly on top of hypha, aseptate. Spores 11.1-17.6 um (13.5 + 1.3 um, n = 55) in diameter, variably containing 1-15 nuclei. Neither conidiophores nor sexual structures observed. Sequences & the phylogenetic analyses The obtained ITS (610 bp), LSU (945 bp), and TEF1-a (1131 bp) sequences were deposited in GenBank. To determine the phylogenetic position of Serendipita sacchari, all available ITS, LSU, and TEFl-a sequences from Serendipita and related genera were downloaded from GenBank (TABLES 12s The ITS sequences from one isolate of Serendipita sacchari, five Serendipitaceae, nine Sebacinaceae, three Sebacinales incertae sedis, and one outgroup Cantharellus avellaneus, were included in the phylogenetic analysis. In the alignment of these 19 sequences, the data matrix comprised 591 characters. The alignment dataset was analyzed using MrBayes, applying the GTR+G model selected by MrModeltest as the best-fit model. The prior probability density is a flat Dirichlet (all values = 278 1.0) with both Revmatpr and Statefreqpr as default settings. Posterior probability (BPP) and bootstrap (BS) values are shown on the branches of the ITS Bayesian tree (Fic. 2). The ITS phylogeny places S. sacchari in Serendipitaceae and in a clade with S. indica (BPP = 0.83; NJ support = 97%). Serendipita sacchari sp. nov. (China) ... 583 Fic. 1. Serendipita sacchari (ex-type, CGMCC3.19906). A: Colony; B, C: Bright field microscopic image of chlamydospores; D-F: Scanning electron microscopic image of chlamydospores; G, H: DAPI-stained chlamydospores; I: DAPI-stained hyphae. Scale bars: B-I = 10 um. A combined LSU+TEF1-a dataset comprising one Serendipita sacchari isolate, five Serendipita isolates, and the outgroup Sebacina incrustans was included in a second phylogenetic analysis. These seven sequence pairs (LSU+TEF1-a) aligned in a 1990-character data matrix. The alignment dataset was analyzed in MrBayes, applying the GTR+G model selected by MrModeltest as the best-fit model. The prior probability density is a flat Dirichlet (all values = 278 1.0) with both Revmatpr and Statefreqpr as default settings. Here also BPP and BS values are shown on the branches of the combined Bayesian tree (Fic. 3). 584 ... Xie, Long & al. Sebacina pallida JQ665562 0.86/88 0.97/81 | Tremelloscypha dichroa KF061281 Sebacina candida KF061278 0.89/* . : 1.00/100 Helvellosebacina concrescens JQ665516 0.90/* 0.85/* Helvellosebacina helvelloides KF000415 : Sebacinaceae Tremellodendron ocreatum KT339265 )-S1/* 4 men ee Globulisebacina rolleyi AY 509550 0.94/100 Craterocolla cerasi KF061265 Ditangium altaicum NR 163760 Paulisebacina allantoidea AF490396 1.00/100 0.97/99 Paulisebacina allantoidea KF061266 Serendipita herbamans KF061285 aT / . Serendipitaceae Serendipita sacchari KY 496808 0.83/97 Serendipita indica KF061284 1.00/100 Serendipita williamsii KY 509323 0.56/* Chaetospermum chaetosporum KJ710461 0.80/88 Efibulobasidium albescens AF384860 Sebacinales incertae sedis Chaetospermum camelliae KF516965 Cantharellus avellaneus KX857081 OL Fic. 2. Phylogenetic tree based on ITS sequences of Serendipita and related Sebacinales species. Cantharellus avellaneus was included as outgroup. The numbers at each branch represented Bayesian posterior probabilities (left) and bootstrap support calculated from 1000 replicates (right). The sequence derived from the proposed new species is in bold. Bar = 0.1 expected changes per site. In the LSU+TEF1-a phylotree, S. sacchari formed a clade with S. williamsii with 0.95 BPP support and 100% NJ support. A comparison of the ITS, LSU and, TEFl-a sequence dataset indicates that S. sacchari differs from S. williamsii in 12/530 bp (2.3%, ITS), 15/930 bp (1.6%, LSU), and 60/1068 bp (5.6%, TEF1-a); and from S. indica in 22/552 bp (4.0%, ITS), 21/938 bp (2.2%, LSU), and 63/1132 bp (5.6%, TEF1-a). COMMENTS. Four species in Serendipita—S. herbamans, S. indica, S. williamsii, and S. vermifera—were closely related to and allied with S. sacchari. Neither conidiophores nor sexual structures were observed in these four taxa. Serendipita sacchari can be easily distinguished from S. herbamans Serendipita sacchari sp. nov. (China) ... 585 Serendipita vermifera DQ983814+JN2 11111 Serendipita vermifera DQ983816+JN211116 Serendipita vermifera DQ520096+JN2 11115 Serendipita sacchari KY 496809+MF196313 Serendipita williamsti AY 505556+JN2 11110 1.00/100 Serendipita indica AY 505557+AJ2499 11 Sebacina incrustans FJ6445 13+KF3 13907 Q.'] Fic. 3. Phylogenetic tree based on the combined LSU+TEF 1-a sequence data of Serendipita species. Sebacina incrustans was designated as outgroup. The numbers at each branch point represented Bayesian posterior probabilities (left) and bootstrap support calculated from 1000 replicates (right). The sequence derived from the proposed new species is in bold. Bar = 0.1 expected changes per site. and S. vermifera by its bigger (11.1-17.6 um diam.) chlamydospores (3-5 um x 4-5 um in S. herbamans; approximately 8 um diam. in S. vermifera; Riess & al. 2014, Ray & Craven 2016). The main difference among S. sacchari, S. indica, and S. williamsii is the distribution and number of nuclei in the cells (Basiewicz & al. 2012). Nuclear number in the chlamydospores of Serendipita sacchari (1-15 nuclei) distinguishes the new species from S. indica (8-25 nuclei) and S. williamsii (<10 nuclei) (Basiewicz & al. 2012). Moreover, hyphal cells of S. sacchari have fewer nuclei and exhibit a more regular distribution compared with S. williamsii (2-6 nuclei irregularly distributed) (Verma & al. 1998; Basiewicz & al. 2012). Morphological traits and phylogenetic data both support the recognition of Serendipita sacchari as an independent species. Serendipita herbamans is a common species in plant roots and widely distributed across agricultural and grassland ecosystems. It has the potential to promote the growth of herbaceous plants (Riess & al. 2014). Serendipita 586 ... Xie, Long & al. indica (= Piriformospora indica) is widely distributed and well known as a symptomless root endophyte that colonizes bryophytes, pteridophytes, gymnosperms and angiosperms. It has been reported to occur in four continents, is extremely versatile in its mycorrhizal associations, and is known for its ability to promote plant growth (Varma & al. 2012). Serendipita williamsii (= Piriformospora williamsii) was isolated from the spore of arbuscular mycorrhizal fungal in Australia (Basiewicz & al. 2012). Serendipita vermifera was isolated from the Australian orchid Cyrtostylis reniformis (Warcup 1988). It is reported to induce beneficial effects on plant performance including growth promotion, increase nutrient uptake, enhance seed production, and increase resistance against different biotic and abiotic stresses (Ray & Craven 2016). Serendipita sacchari was from sugarcane rhizosphere soil, and symbiotic with Chinese cabbage and sugarcane as a symptomless root endophyte (data not shown). All five Serendipita species are plant symbiotic microbionts. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 31460016), Guangxi Natural Science Foundation (No. 2015GXNSFBA139083), and Basic Scientific Research Special Project of Guangxi Academy of Agricultural Sciences (No. 2015YT80). We appreciate the thoughtful reviews of Dr. Xiaoyong Liu (Institute of Microbiology, Chinese Academy of Sciences) and Dr. Jiwen Xia (College of Plant Protection, Shandong Agricultural University). Literature cited Basiewicz M, WeifS M, Kogel KH, Langen G, Zorn H, Zuccaro A. 2012. Molecular and phenotypic characterization of Sebacina vermifera strains associated with orchids, and the description of Piriformospora williamsii sp. nov. Fungal Biology 116: 204-213. https://doi.org/10.1016/j.funbio.2011.11.003 Huelsenbeck JP, Ronquist E 2001. MRBAYES: Bayesian inference of phylogeny trees. Bioinformatics 17: 754-755. https://doi.org/10.1093/bioinformatics/17.8.754 Narisawa K, Tokumasu S, Hashiba T. 1998. Suppression of clubroot formation in Chinese cabbage by the root endophytic fungus, Heteroconium chaetospira. Plant Pathology 47: 206-210. https:// doi.org/10.1046/j.1365-3059.1998.00225.x Nylander JAA. 2008. MrModeltest 2.3 REAME. Accessed 22 May 2008: http://www.abc.se/nylander/mrmodeltest2/mrmodeltest2.html. Ray P, Craven KD. 2016. Sebacina vermifera: a unique root symbiont with vast agronomic potential. World Journal of Microbiology & Biotechnology 32: 1-10. https://doi.org/10.1007/s11274-015-1970-7 Riess K, Oberwinkler F, Bauer R, Garnica S. 2014. Communities of endophytic Sebacinales associated with roots of herbaceous plants in agricultural and grassland ecosystems are dominated by Serendipita herbamans sp. nov. Plos One 9: e94676. https://doi.org/10.1371/journal.pone.0094676 Serendipita sacchari sp. nov. (China) ... 587 Roberts P. 1993. Exidiopsis species from Devon, including the new segregate genera Ceratosebacina, Endoperplexa, Microsebacina, and Serendipita. Mycological Research 97: 467-478. https://doi.org/10.1016/S0953-7562(09)80135-4 Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA, Chen W & al. 2012. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences, USA 109: 6241-6246. https://doi.org/10.1073/pnas.1117018109 Sun X, Guo LD. 2010. Micronematobotrys, a new genus and its phylogenetic placement based on rDNA sequence analyses. Mycological Progress 9: 567-574. https://doi.org/10.1007/s11557-010-0664-7 Tamura K, Dudley J, Nei M, Kumar S. 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24: 1596-1599. https://doi.org/10.1093/molbev/msm092 Trichiés G. 2003. Serendipita lyrica sp. nov., Achroomyces lotharingus sp. nov. et quelques autres hétérobasidiés notables de Lorraine (France). Bulletin trimestriel de la Société 118 (4): 351-379. Verma A, Varma S, Rexer KH, Hassel A, Kost G, Sarbhoy A, Bisen P, Bittehorn B, Franken P. 1998. Serendipita indica, gen. et sp. nov., a new root-colonizing fungus. Mycologia 90: 896-903. https://doi.org/10.2307/3761331 Varma A, Bakshi M, Luo B, Hartmann A, Oelmueller R. 2012. Serendipita indica: a novel plant growth-promoting mycorrhizal fungus. Agricultural Research 1: 117-131. https://doi.org/10.1007/s40003-012-0019-5 Vilgalys R, Hester M. 1990. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: 4238-4246. Vohnik M, Panek M, Fehrer J, Selosse MA. 2016. Experimental evidence of ericoid mycorrhizal potential within Serendipitaceae (Sebacinales). Mycorrhiza 26: 831-846. https://doi.org/10.1007/s00572-016-0717-0 Warcup JH. 1988. Mycorrhizal associations of isolates of Sebacina vermifera. New Phytologist 110: 227-231. https://doi.org/10.1111/j.1469-8137.1988.tb00256.x WeifS M, Waller F, Zuccaro A, Selosse MA. 2016. Sebacinales - one thousand and one interactions with land plants. New Phytologist 211: 20-40. https://doi.org/10.1111/nph.13977 MYCOTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 July-September 2020—Volume 135, pp. 589-612 https://doi.org/10.5248/135.589 Five Nolanea spp. nov. from Brazil FERNANDA KARSTEDT*, SARAH E. BERGEMANN?, MARINA CAPELARI’ * Nucleo de Pesquisa em Micologia, Instituto de Botanica, Caixa Postal 68041, 04045-902 Sao Paulo, SP, Brazil * Biology Department, Middle Tennessee State University, PO Box 60, Murfreesboro, TN 37132, USA " CORRESPONDENCE TO: fernanda.karstedt@gmail.com ABSTRACT—Five new species of Nolanea (Entolomataceae, Agaricales)—N. albertinae, N. atropapillata, N. pallidosalmonea, N. parvispora, and N. tricholomatoidea—were collected from the Sao Paulo metropolitan region. Phylogenetic analyses based on partial sequences of the mitochondrial small subunit (mtSSU), nuclear large subunit (nLSU) and second largest RNA polymerase subunit (rpb2), confirm that all these species belong to Nolanea. Morphological descriptions, comments, illustrations, scanning electron microscopy and color photos are included. Key worps—basidiomycetes, Entoloma, Neotropical fungi, South America, taxonomy Introduction Currently, Nolanea has two main circumscriptions, one according to Largent (1994) and Henkel & al. (2014) as Nolanea (Fr.) P. Kumm., and the other according to Noordeloos (1992, 2004) and Noordeloos & Gates (2012) as Entoloma subg. Nolanea (Fr.) Noordel. Both circumscriptions agree that Nolanea comprises species often mycenoid with the pileus surface typically hygrophanous, usually translucent-striate and with angled basidiospores with five, six or seven angles in profile view, which can be subisodiametric or heterodiametric. The main difference between the accepted circumscriptions of the genus Nolanea is in the pileipellis structure. Largent (1994) considers Nolanea to encompass only species that have a pileipellis composed of two layers, with a suprapellis composed of a cutis or a repent entangled hyphae 590 ... Karstedt, Bergemann, Capelari layer overlying a subpellis of inflated hyphae, whereas Noordeloos accepts species in Entoloma subg. Nolanea in which the repent, cylindrical hyphae of the pileipellis often (but not necessarily always) overlie a distinct differentiated subpellis of inflated cells. In general, the few phylogenetic studies that include Nolanea species do not define which circumscription is the most appropriate for the group; additionally, they suggest Nolanea can be polyphyletic (Co-David & al. 2009, Baroni & Matheny 2011). However, Nolanea species are always in the Nolanea- Claudopus clade, independent of the combination of loci used: mtSSU, nLSU, and rpb2 (Co-David & al. 2009, Baroni & Matheny 2011) or ITS, nLSU, and rpb2 (Kinoshita & al. 2012). Also, it is usually possible to visualize two separate branches within the Nolanea—Claudopus clade, with a clade of Nolanea species separate from a clade comprising Claudopus species. More recent molecular studies of Entoloma subg. Nolanea (Vila & al. 2013, Raj & al. 2014, Raj & Manimohan 2016) examined Nolanea species using only ITS sequences, with special emphasis on taxa delimitation and new taxa proposition. The phylogeny produced in these studies supports, even with only ITS sequences, the recognition of Nolanea as a good phylogenetic group and would seem to support Nolanea as an autonomous genus in the Entolomataceae. Nolanea (with about 250 names in MycoBank and Index Fungorum) is considered a common and speciose genus, especially in temperate regions (Noordeloos 1992, 2004, Largent 1994, Horak 2008), but it is also present in tropical and subtropical regions (Romagnesi & Giles 1979). In Brazil of the 23 Nolanea species recorded (Rick 1919, 1920, 1930, 1961, Bresadola 1920, Singer 1953, Horak 1977, Capelari 1989, Putzke & Cavalcanti 1997, Pegler 1997, de Meijer 2001, 2006, Rosa 2002), eleven have been cited and/or described by Rick (1919, 1920, 1930, 1961, Singer 1953) and are considered as incertae sedis (Horak 1977). Many other species have been reported in dissertations (Capelari 1989, Rosa 2002), an identification key (Pegler 1997), and lists (Singer 1953, de Meijer 2001, 2006), but only three have been published with a full description (Putzke & Cavalcanti 1997). In this work, we describe five new species found in the Sao Paulo metropolitan region accompanied by molecular information and phylogenetic analyses. Materials & methods Basidiomata collection and morphological observations The studied materials were collected mainly at the Parque Estadual da Cantareira and the Parque Estadual das Fontes do Ipiranga in Sao Paulo City and Reserva TABLE I. Collections used in the phylogenetic analyses Cl. = Claudopus; E. = Entoloma; I. = Inocephalus; L. = Leptonia; P. = Pouzarella; Nolanea spp. nov. (Brazil) ... 591 Rh. = Rhodocybe; R. = Richoniella; T. = Trichopilus; Sequences generated in this study in bold. NCBI spECIES NAME Cl. minutoincanus Cl. viscosus E. abortivum E. albidoquadratum E. bloxamii E. cephalotrichum E. conferendum E. cf. conferendum E. gasteromycetoides E. haastii E. hebes E. nidorosum E. pallideradicatum E. parasiticum E. porphyrescens E. procerum E. pygmaeopapillatum Entoloma readiae E. sericatum E. sericionitidum E. serrulatum E. sinuatum E. strictius E. strictius var. isabellinus E. turbidum E. undatum RPB2 HQ731517 HQ731518 GU384642 GQ289222 GQ289223 GQ289226 GQ289229 GQ289231 GQ289235 GQ289238 GQ289241 GU384643 GQ289247 GQ289248 GQ289253 GQ289254 GQ289256 GQ289257 GQ289260 EF421016 GQ289263 GQ289264 GU384641 GU384656 GQ289270 LSU HQ731514 HQ731516 GU384616 GQ289150 GQ289151 GQ289154 GQ289157 GQ289160 JQ320115 GQ289164 GQ289167 GQ289170 GU384617 GQ289176 GQ289177 GQ289182 GQ289183 GQ289185 GQ289186 GQ289189 AF261315 GQ289192 GQ289193 AF042620 GU384618 GU384630 GQ289202 GENBANK ACC. NO. mtSSU HQ731511 HQ731513 GU384595 GQ289290 GQ289291 GQ289294 GQ289297 GQ289300 GQ289304 GQ289307 GQ289310 GU384596 GQ289316 GQ289317 GQ289322 GQ289323 GQ289325 GQ289326 GQ289329 EF421098 GQ289332 GQ289333 GU384594 GU384603 GQ289342 VOUCHER DLL 9871 DLL 9788 TB6693 den Bakker 92 Manimohan 667-T Noordeloos 200442 Ulje 1997-08-01 Noordeloos 200313 HKAS 48953 Gates E2031 Noordeloos 2004055 Hartman 1992- 10-28 TB9971 Hausknecht Noordeloos 200330 Noordeloos 2004113 Noordeloos 2004070 Noordeloos 200364 Noordeloos 2004050 Noordeloos 200328 TB7144 Noordeloos 2004062 Wisman 2003- 09-19 M96/10 1TB7710 TB6949 Noordeloos 200327 ORIGIN Australia, NSW Australia, QLD USA, NY Canada India, KL Austria Netherlands Belgium China, SC Australia, TAS Australia, TAS Netherlands USA, NY Austria Belgium Australia, TAS Australia, TAS Slovakia Australia, TAS Slovakia USA, NY Australia, TAS Netherlands USA, TN USA, NY Belgium 592 ... Karstedt, Bergemann, Capelari GENBANK ACC. NO. NCBI sPECIES NAME RPB2 LSU mtSSU VOUCHER ORIGIN E. valdeumbonatum GQ289271 GQ289203 GQ289343 Meusers E4565 [T] Germany I. murrayi GU384637 GU384620 GU384590 VHAs02.02 — I. sp. MCA2479 GU384640 GU384622 GU384593 MCA2479 — L. serrulata GU384634 GU384624 GU384588 VHAs01.02 — L. sp. MCA1486 GU384635 GU384623 GU384589 MCA1486 — N. albertinae — KF738937 KF738925 FK1731 Brazil, RJ KF771345 KF738938 KF738926 FK1732 Brazil, RJ KF771344 KF738936 KF738924 FK0935-T Brazil, SP N. atropapillata — KF738940 KF738929 FK0898-T Brazil, SP N. cetrata KF771346 KEF738942 KF738927 DLL9531 USA, CA — AF261319 — TB7382 USA, NY N. conferenda KF771351 KF738946 KEF738935 ~—11CA014 USA, CA N. conferenda — AF261321 — TB7660 USA, NY N. hirtipes — AF261320 — K1171992 N. pallidosalmonea — KF738941 KF738930 _—_‘-FK0891 Brazil, SP N. parvispora KF771348 KF738943 KF738931 FK1140 Brazil, SC — — KF738932 FK2135 Brazil, SC N. sericea DQ367435 DQ367423 EF421099 VHAs 03/02 — GQ289262 GQ289191 GQ289331 Noordeloos Slovakia 200329 KF771349 KF738944 KF738933 11CA055 USA, CA KF771350 KF738945 KF738934 11CA056 USA, CA — AF261318 —_— TB6506 USA, CA N. strictior EF421017 — EF421100 DUKE-JM96/10 N. tricholomatoidea KF771347 KF738939 KF738928 FK1049-T Brazil, SP P. albostrigosa HQ876513 HQ876535 HQ876557 __Largent 9641 Australia P lasia HQ876507. HQ876529 HQ876555 _Largent 9662 Australia P. nodospora _ AF261308 — TB5716 USA, SC P. setiformis HQ876503 HQ876525 HQ876525 _ Largent 9809 Australia Rhodocybe trachyspora GU384658 GU384629 GU384605 TB5856 USA, CA Richoniella asterospora JF706311 JF706310 — PBM3268 USA, TN iG dar ee Le AF261290 — TB6957 USA, NC OUTGROUP Clitocybe dealbata DQ825407 = AF223175 AF357138 IE-BSG-HC95cp3 — Lyophyllum IE-BSG- DQ367434 AF223202 AF357101 = leucophaeatum HAe251.97 Nolanea spp. nov. (Brazil) ... 593 Bioldgica de Paranapiacaba in Santo André City, in remnants of the Atlantic Forest in Sao Paulo State, Southeast Brazil. Revived sections of tissues from dried basidiomata mounted in 5% KOH, 5% NH,OH, or 1% aqueous Congo Red dye used to stain hyaline structures were examined microscopically. All the micrographs were drawn with the aid ofa drawing tube. ‘Q’ represents the minimum and maximum length/width quotient, and ‘Qn represents the mean length/width quotient of the total sample of measured spores. The pileipellis structure was examined from the middle portion of the pileus and not the disc or margin. The specimens were deposited in the herbarium of the Instituto de Botanica (SP) and of the Royal Botanic Gardens (K). The samples were prepared for SEM following Baroni (1981) and scanning electron micrographs (SEMs) were made using a LEO 435 VP scanning electron microscope. DNA extraction, amplification, sequence analyses The DNA was extracted with hexadecyltrimethylammonium bromide (CTAB) (Ferreira & Grattapaglia 1995) using lyophilized tissues from basidiomata ground to a fine power in liquid nitrogen. The sample was resuspended in 50 uL of TE and stored at —20 °C. The partial sequences of the mitochondrial small subunit (mtSSU) were amplified via the polymerase chain reaction (PCR) primed by MS1 and MS2 (White & al. 1990); the 28S nuclear large subunit (nLSU) rDNA was amplified using LROR and LR5 (Moncalvo & al. 2000); the ITS1-5.8S-ITS2 was amplified using ITS1-F and ITS4 (White & al. 1990, Gardes & Bruns 1993); and the second largest subunit of the RNA Polymerase II (rpb2) using rpb2-i6F and rpb2-i7R (Co- David & al. 2009) or rpb2-F1 and rpb2-R1b (Largent & al. 2013). The PCRs were performed in 25-50 uL reaction volumes, where the reaction concentration was 1x for PCR Buffer, 2 mM for MgCl2, 0.2 mM for each dNTP, 0.5 uM for each forward and reverse primer, and 0.025 U of Taq polymerase. Products from multiple reactions were pooled for weak PCR amplifications. The reaction was performed using the following cycling parameters: 94 °C for 2 min, 34 cycles at 94 °C for 45 s, 50 °C (54C? for rpb2) for 1 min and 10 s and 72 °C for 2 min, and then 72 °C for 10 min. PCR amplifications products were purified using the AxyPrep PCR Clean-up Kit (Axygen Biosciences, Union City, USA) or alternatively, using the ExoSapIT kit following the protocols outlined in Largent & al. (2011). DNA sequencing reactions were performed with BigDye Terminator v3.1 Cycle Sequencing Kit. The sequences were run in a ThermoFisher 3730 DNA Analyzer using Sequencing Analysis 5.3.1 and Base Caller KB. The forward and reverse sequences for each sample were aligned and edited using CodonCode Aligner or Sequencher. The phylogenetic analysis was conducted using sequences obtained in this study and sequences found in GenBank of species traditionally accepted as Nolanea [Nolanea—Claudopus clade sensu Co-David & al. (2009) and Baroni & Matheny (2011)] (TaBLeE I). Lyophyllum leucophaeatum (P. Karst.) P. Karst. and Clitocybe dealbata (Sowerby) P. Kummer were selected as outgroups based on phylogenetic analyses performed by Co-David & al. (2009). The sequences were manually aligned using Se-Al Sequence Alignment Editor; spliceosomal introns in the rpb2 594 ... Karstedt, Bergemann, Capelari were delimited using Augustus ver. 2.4 webserver (Stanke & al. 2008) and omitted along with ambiguous sites in mtSSU. For these phylogenetic analyses, sequences from the three genes including missing data were assembled. The final alignment after the exclusion of introns and ambiguous sites was 3162 bp including gaps: 592 bp for the mtSSU, 1390 bp for the nLSU and 1180 bp for the rpb2 (Tree-BASE database ID14850). Phylogenetic analyses were performed using maximum likelihood (ML), Bayesian Inference (BI), and maximum parsimony (MP). For the BI, the best-fit model of nucleotide substitution was performed using the Akaike information criterion (AIC) in TOPALI v.2 (Milne & al. 2009). ML analysis was performed using RAXML 7.0.4 with a GTR + G model according to recommendations (Stamatakis 2006, Stamatakis & al. 2008). Support for nodes was tested using 1000 bootstrap replicates. For the BI phylogeny, the best-fit model of nucleotide substitution for each gene partition and codon for the rpb2 (GTR + G for LSU, mtSSU and codons and 3 of rpb2, and GTR + G + I for first codon of rpb2). The BI was performed with MrBayes 3.2.2 (Huelsenbeck & Ronquist 2001, Ronquist & Huelsenbeck 2003) specifying four chains (one cold, three heated) for 2,000,000 generations (sampling trees every 1000 generations), with a burn-in of 30,000 generations. MP heuristic searches were performed using the program TNT (Goloboff 1999, Nixon 1999, Goloboff & al. 2008). Tree searches were conducted with equal weights and treating gaps as a fifth character state. Branch support was calculated with 1000 bootstrap replicates. A bootstrap value of 70% was considered significant for ML and MP. The Bayesian posterior probability (BPP) statistical support values of branches were considered informative if = 0.95. Phylogenetic results The DNA sequences obtained, including partial sequences of the nLSU, mtSSU,andrpb2maresummarizedin TABLE I. Additionally, sequences obtained from Nolanea albertinae (FK0912: KF679349, FK0935 [holotype]: KF679348), N. atropapillata (FK0898 [holotype]: KF679354), N. pallidosalmonea (FK0891 [holotype]: KF738923), N. parvispora (FK1140 [holotype]: KF679353) and N. tricholomatoidea (FK1049 [holotype]: KF679352) were deposited in GenBank. The phylogenetic analyses placed all five species studied into /Nolanea- Claudopus (Co-David & al. 2009, Baroni & Matheny 2011, Kinoshita & al. 2012), an unsupported clade based on ML, MP, and BPP. The phylogenies place all five species into one of two strongly supported internal clades (Fie. 1) in the /Nolanea clade with other species of Nolanea. Taxonomy Five new species are described and illustrated with line drawings, macroscopic photographs and SEMs of basidiospores. Nolanea spp. nov. (Brazil) ... 595 Entoloma cf.conferendum HKAS48953 Nolanea cf. conferenda11CA014 Entoloma conferendum MEN200313 Nolanea conferendaTB7660 Nolanea albertinae FK1731 Nolanea albertinae FK1732 Nolanea albertinae FK0935 Nolanea tricholomatoideaFK1049 Entoloma cephalotricum CU19970801 Nolanea atropapillata FK0898 Nolanea pallidosalmoneaFK0891 Entoloma strictus var.isabelina TB7710 Nolanea cetrata DLL9531 Nolanea cetrata TB7382 Entoloma pallideradicatumWU189010 Nolanea parvispora FK1140 Nolanea parvispora FK2135 Entoloma pygmaeopapillatum MEN200364 Nolanea sericea VHAs03/02 Entoloma strictius M9610 lI Nolanea sericea MEN200329 "/ Nolanea sericea 11CA055 Nolanea sericea11CA056 Entoloma valdeumbonatum MME4565 sya Nolanea sericea TB6506 Nolanea hebes CH19921028 Nolanea hirtipes K1171992 Nolanea strictior DUKEJM9610 Entoloma readiae MEN2004050 Entoloma arbortivum TB6693 Entoloma abortivum Bakker92 Entoloma sericeonitidumTB7144 Entoloma undatum MEN200327 Claudopus minutoincanus DLL9871 Entoloma parasiticum MEN200330 Claudopus viscosusDLL978 Claudopus Inocephalus sp. MCA2479 Inocephalus murrayi VHAs02/02 Entoloma procerum MEN2004070 Entoloma albidoquadratum PM667 Entoloma porphyrescens MEN2004113 Trichopilus porphyrophaeusTB6957 Entoloma gasteromycetoides GGE2031 Entoloma serrulatum MEN2004062 Leptonia sp. MCA1486 Leptonia serrulata VHA01/02 Inocephalus-Cyanula Pouzarella albostrigosa DLL9641 Pouzarella lasiaDLL9662 Pouzarella setiformis DLL9809 Pouzarella nodospora 1B5716 Pouzarella Richoniella asterospora PBM3268 adie *t00°_ Entoloma nidorosum 1B9971 100 100 / 100) Entoloma sinuatum JW20030919 J ae ae sericatum MEN200328 Entoloma s.s. / Rhodopoloid Entoloma haastii MEN2004055 Entoloma turbidum TB6949 | Rhodocybe trachyspora TB5856 Entoloma bloxamii MEN200442 Clitocybe dealbata HC95cp3 Lyophyllum leucophaeatum HAe25197 Nolanea-Claudopus 93/81 100 100 497. 100 Fic. 1. Phylogram of maximum likelihood (ML) phylogenetic reconstruction using rpb2, nLSU, and mtSSU sequences. Support is indicated with ML/MP bootstrap percentage above and Bayesian posterior probabilities (BPP) below branches. The names of species studied in this paper are in boldface. I. Species with heterodiametric basidiospores. II. Species with iso- to subisodiametric basidiospores. 596 ... Karstedt, Bergemann, Capelari Nolanea albertinae Karstedt & Capelari, sp. nov. FIGS. 2A-E, 3, 8A,B MB826784 Differs from Entoloma belouvense by its smaller basidiospores and presence of refractive hyphae. Type—Brazil, Sao Paulo State, Santo André: Reserva Bioldgica de Paranapiacaba, 23°46’S 46°18’W, trail 15, 18.V.2006, F. Karstedt & M. Capelari FK0935 (Holotype: SP; GenBank KF771344, KF738936, KF738924). EtymMoLoGy—an honour to Albertina Teresinha Karstedt. PitEus 14-18 mm diam., broadly conic, conico-convex, convex, plano-convex or plane, often slightly papillate, rarely with the center plane or depressed, pale brown, brown, dark brown (chocolate-colored), or gray-brown, center often darker than the margin, surface glabrous or with a few appressed, concolorous radial fibrils, when humid strongly translucent-striate, when dry, opaque, hygrophanous, margin slightly or strongly eroded. PILEUS CONTEXT very thin, whitish. LAMELLAE adnexed or sinuate, pale pinkish brown, subdistant, with 1-5 lamellulae (3 series), margin slightly eroded. ST1PE 36-71 x 1-4 mm, often cylindrical, rarely tapered above or subbulbous over base, brownish beige, pale brown, gray-brown or ocher brown, rarely yellowish brown or concolorous with pileus, apex always paler than the base, sometimes beige or whitish, surface glabrous, often with the apex slightly pruinose, fibrillose- twisted, hollow, base of stipe often with a white tomentum. SPORE PRINT not recorded. BASIDIOSPORES 7.5-10(-11.2) x 5-7.5 um (Q = 1.16-1.75, Qm = 1.36), heterodiametric, strongly angled, with 5 or 6 angles in profile, slightly pinkish brown, thin-walled. Basip1A 23-37 x 8.7—15 um, clavate, hyaline, thin-walled, tetrasporic. LAMELLAE EDGES heterogeneous. CHEILOCYSTIDIA always present, dispersed or present [in portion of] on the lamellae edges near the stipe, 26-68 x 5-7.5 um, cylindrical, often apex capitate, hyaline, thin-walled. PLEUROCYSTIDIA absent. LAMELLAR TRAMA subparallel, with hyphae 5-40 um diam., cylindrical or fusiform, hyaline, thin-walled; subhymenium ramose. PILEITRAMA composed of radially arranged and parallel hyphae, with hyphae 8.7-25 um diam., inflated or fusiform, hyaline, thin-walled. PILEIPELLIs a cutis, made up of cylindrical hyphae, with brown intracellular pigment, hyphae thin-walled, 3.7-7.5 um diam., with cylindrical or cylindrical-clavate terminal elements; subpellis made up of inflated or fusiform, hyaline, thin- walled hyphae, 10-37 um diam. STIPITIPELLIs a cutis of straw yellow, thin- walled hyphae 5-15 um diam. CAULocysTIDIA absent. CLAMP CONNECTIONS present, but inconspicuous. REFRACTIVE HYPHAE present at pileipellis. Nolanea spp. nov. (Brazil) ... 597 Fic. 2. Basidiomata. Nolanea albertinae: a. FK735; b. FK883; c. FK912; d. FK935 (holotype); e. FK1111. N. atropapillata: f. FK649; g. FK898 (holotype). N. pallidosalmonea: h. (FK892, holotype). N. parvispora: i. FK664 (holotype); j. FK689; k. FK794. N. tricholomatoidea: |. (FK1049, holotype). Scale bars = 1 cm. Photos: a, b, d-j, 1: F. Karstedt; c, k: M. Capelari. Hasitat—Solitary or sometimes gregarious, on litter or in soil with fragmented organic material. ADDITIONAL SPECIMENS EXAMINED—BRAZIL, SAO PauLo STATE: Sao Paulo, Parque Estadual da Cantareira, Nucleo Engordador, 23°20’S 46°41’W, Macuco trail, 27.1V.2006, EF Karstedt & M. Capelari FK630 (SP); 29.VI.2006, F. Karstedt & M. Capelari FK0690 (SP); 19.IX.2006, E Karstedt & M. Capelari FK0735 (SP); Cachoeira trail, 20.III.2007, F. Karstedt & al. FK0883 (SP); 29.V.2007, F. Karstedt & al. FK0912 (SP); 13.III.2008, F. Karstedt FK1101 (SP); Parque Estadual das Fontes do Ipiranga, 23°39’S 46°37’W, Nascente trail, 20.V1.2006, F. Karstedt & al. FK0682 (SP); 28.VIII.2008, FE. Karstedt & K. Patekoski FK1111 (SP); Santo André, Reserva Bioldgica de Paranapiacaba, 23°46’S 46°18’W, trail 15, 18.V.2006, F. Karstedt & M. Capelari FK0651 (SP), FK0652 (SP); trail 2, 18.V.2006, F. Karstedt & M. Capelari FK0656 (SP); trail 3, 22.VI.2006, F. Karstedt FK0685 (SP); trail 1, 23. VIII.2006, F. Karstedt FK0727 598 ... Karstedt, Bergemann, Capelari (SP). SANTA CATARINA STATE: Gaspar, #1111 Pedro Schmitt Street, hill behind the Joao Pedro Lenfers’ house, 02.VIII.2008, F. Karstedt & A.T. Karstedt FK1110 (SP). RIO DE JANEIRO STATE: Parque Nacional do Itatiaia, 11.1.2011, EF Karstedt, PB. Schwartsburd & J.B. Pereira FK1731 (SP; GenBank KF738937, KF738925), FK1732 (SP; GenBank KF771345, KF738938, KF738926). CoMMENTS—WNolanea albertinae apparently isa common species in the Atlantic Forest of southern and southeastern Brazil; additionally it is usually solitary and almost undetectable in the litter, considering that both have similar color. Nolanea albertinae is similar to N. atripes Dennis and N. pseudopapillata Pegler, which are both described from Central America (Horak 1977, Pegler 1983). It also resembles Entoloma dissimile (Singer) E. Horak (described from Argentina; Singer 1969, Horak 1977), N. fuscifolia (Peck) Sacc. (described from the United States (Peck 1874, Hesler 1963) but also found in Argentina; Singer 1969) and Entoloma novum E. Horak, (described from Chile; Horak 1977). These species have a similar stature including the mycenoid habit; a pileus that is brownish, conical to plane, papillate and hygrophanous; a similar pileipellis with brown pigmented repent hyphae; and similarly shaped and sized basidiospores, 7-10 x 5-7.5 um. All also can be separated morphologically besides lacking the cylindrical to capitate cheilocystidia that characterize N. albertinae. Nolanea atripes has a darker pileus, no cheilocystidia, and pileipellis with a vacuolar pigment (Pegler 1983). Nolanea pseudopapillata, Entoloma dissimile and Entoloma novum differ (from N. albertinae) by the absence of cheilocystidia and clamp connections and by the presence of encrusted pileipellis hyphae (Singer 1969, Horak 1977, Pegler 1983). Nolanea fuscifolia grows on wood (instead soil or litter), has a dark brown pileus, lacks cheilocystidia, and has clamp connections; additionally, N. fuscifolia was originally described from New York (Peck 1874, Hesler 1963). Nolanea fuscifolia sensu Singer (1969) from Argentina, is closer to Nolanea albertinae in its very infrequent cheilocystidia and not (or only slightly) encrusted pigmentation; however it is distinguished by its growth on dead wood and by the presence of fusoid- ventricose caulocystidia. The species most closely related to Nolanea albertinae is Entoloma belouvense Noordel. & Hauskn. from the Republic of Seychelles, an island archipelago in the Indian Ocean off the east coast of Africa (Noordeloos & Hausknecht 2007). Entoloma belouvense has similar macroscopic features and scattered cheilocystidia that are cylindrical and often capitate. However, in addition to its different distribution, N. albertinae has distinctly smaller basidiospores (7.5-10 x 5-7.5 um versus 10-12 x 6-7,5 um) and the presence of refractive hyphae at pileipellis. Nolanea spp. nov. (Brazil) ... 599 Fic. 3. Nolanea albertinae (holotype, FK935): a. Basidiospores; b. Basidia; c. Cheilocystidia; d. Pileipellis. Scale bars: a-c = 10 um; d = 20 um. 600 ... Karstedt, Bergemann, Capelari Nolanea atropapillata Karstedt & Capelari, sp. nov. FIGS. 25,G, 4, 8C,D MB805603 Differs from N. albertinae, N. atripes, N. pseudopapillata, and Entoloma dissimile by its bisporic basidia, by its intracellular and encrusted pigment in pileipellis hyphae, and by the absence of clamp connections. Type—Brazil, Sao Paulo State, Santo André: Reserva Bioldgica de Paranapiacaba, 23°46’S 46°18’W, Estrada Frederico Carlos Hoehne, 12.IV.2007, F. Karstedt & al. FK0898 (Holotype: SP, GenBank KF738940, KF738929). EtryMoLocy—refers to the color of the papillae that are darker than the pileus. PILEus 9-25 mm diam., convex, plano-convex or plane, papillate or umbonate, brown or dark brown, darker in the center compared to the edge, turning grayish brown or pale gray-brown with blackish papilla, surface smooth or sometimes slightly squamulose-fibrillose at center, striate-translucent, strongly hygrophanous then silky, margin slightly eroded or split and upturned with age. PILEUS CONTEXT very thin. LAMELLAE almost free, narrowly adnexed, pink or pink-brown, subdistant, with 1-5 lamellulae (2 tiers), margin smooth or slightly eroded. St1pzE 20-55 x 1-7 mm, cylindrical, with subbulbous base or slightly tapering upwards from swollen base, nearly white, pale beige or very pale brown, apex that is paler than the base, fibrillose-twisted, hollow, base of stipe often with a white tomentum. SPORE PRINT not recorded. BASIDIOSPORES 8.7-12.5 x 6.2-8.7 um (Q = 1.16-1.6, Qm = 1.4), heterodiametric, strongly angled, with 5-6 angles in profile, slightly brownish pink, thin-walled. Basrp1a 20-35 x 7.5-10 um, clavate, hyaline, thin-walled, bisporic. LAMELLAE EDGE fertile. CHEILOCYSTIDIA and PLEUROCYSTIDIA absent. LAMELLAR TRAMA made up of regular cylindrical or fusiform, hyaline, thin-walled hyphae, 6.2-26 um diam.; subhymenium ramose. PILEITRAMA composed of radially arranged and parallel cylindrical or fusiform, hyaline, thin-walled hyphae, 3.7-37 tm diam. PILEIPELLIS a cutis, suprapellis made up of cylindrical hyphae, with brown intracellular pigment, sometimes with brown encrusted pigment, hyphae thick or thin-walled, 2.5-18 um diam.; terminal elements repent or anticlinal and then almost a trichoderm, cylindrical or cylindrical-clavate at center of pileus; subpellis composed of inflated or fusiform, hyaline or slightly straw yellow, thin-walled hyphae, 13.7-44 um diam. STIPITIPELLIS a cutis made up of hyaline or slightly straw yellow, thin-walled hyphae, 2.5-10 um diam. CAULOCysTIDIA absent. CLAMP CONNECTIONS absent. REFRACTIVE HYPHAE not observed. HasBitaT—Solitary or dispersed in litter. ADDITIONAL SPECIMENS EXAMINED—BRAZIL, SAO PAuLo State, Santo André: Reserva Bioldgica de Paranapiacaba, 23°46’S 46°18’W, trail 12, 18.V.2006, F. Karstedt Nolanea spp. nov. (Brazil) ... 601 Fic. 4. Nolanea atropapillata (holotype, FK898): a. Basidiospores; b. Basidia; c. Pileipellis. Scale bars: a, b = 10 um; c = 20 um. & M. Capelari FK0649 (SP), FK0650 (SP); trail 6, 18.V.2006, F. Karstedt & M. Capelari FK0655 (SP); Estrada Frederico Carlos Hoehne, 12.V.2007, E. Karstedt & al. FK0899 (SP). CoMMENTS—Nolanea atropapillata resembles N. albertinae, N. atripes, N. pseudopapillata, and Entoloma dissimile from South America and N. fuscifolia from the United States. All of these species have papillate, pale brown pilei with a darker center and a translucent-striate surface and heterodiametric basidiospores in the 8-13 x 6-9 um range. However, all differ from N. atropapillata in producing tetrasporic basidia and clamp connections. Nolanea atripes is further distinguished by its slender basidioma, membranaceous pileus, and black stipe (Dennis 1961). The description of N. atripes by Pegler (1983) differs from the Dennis's type description, in which Dennis (1961) emphasized the contrast between the black stipe and gray- brown pileus, while Pegler (1983) described the stipe as pale brown to dark, and the basidiomata illustrated is much more robust. Nolanea albertinae also differs by the presence of cheilocystidia and absence of encrustations in the pileipellis hyphae, and Entoloma dissimile differs by the pileipellis as a cutis without a differentiated subpellis. Nolanea fuscifolia is distinguished by its subnodulose basidiospores (Hesler 1963, Singer 1969) and N. pseudopapillata by its strongly heterodiametric basidiospores (Q = 1.61, Pegler 1983). 602 ... Karstedt, Bergemann, Capelari Nolanea pallidosalmonea Karstedt & Capelari, sp. nov. FIGS. 2H, 5, 8E,F MB805604 Nolanea pallidosalmonea differs from other Nolanea spp. by its delicate and slender basidiomata, by a pale pinkish salmon pileus with salmon pink striations and slightly brownish center, by 5-6-angled heterodiametric basidiospores, by a pileipellis composed of repent hyphae, and by the presence of refractive hyphae. Type—Brazil, Sao Paulo State, Santo André: Reserva Bioldgica de Paranapiacaba, 23°46’S 46°18’W, trail 1, 23.11.2007, F. Karstedt & al. FK0892 (Holotype, SP; isotype K). EtryMoLoGy—the name refers to the pale salmon pink coloration of the basidioma. PitEus 4-15 mm diam., convex, slightly papillate, when young with more pronounced papillae, very pale salmon pink or whitish salmon pink, darker at the center and strongly translucent-striate when in fresh condition, hygrophanous and then whitish from the margin towards the center and finally whitish salmon pink and when dry, sometimes slightly brownish at center, translucent-striate, after opaque and silky-fibrillose, margin entire. PILEUS CONTEXT thin, translucent. LAMELLAE adnexed or sinuate, pale pink, ventricose, subdistant, with 2 tiers of lamellulae. Stipe 25-54 x 1-3 mm, cylindrical or slightly tapering, pale salmon pink or light brownish, hollow, fibrous, with a small amount of white tomentum at the base. SPORE PRINT not recorded. BasIDIosPpoREs 8.7-10 x 6.2-7.5 um (Q = 1.16-1.6; Qm = 1.41), heterodiametric, strongly angled, with (5)6-7 angles in profile view, hyaline or slightly pinkish brown, thin-walled. Basip1a 25-37 x 8.7-11.2 um, clavate, hyaline, thin-walled, tetrasporic. PLEUROCYSTIDIA and CHEILOCYSTIDIA absent. LAMELLAE EDGE fertile. LAMELLAR TRAMA subparallel, hyphae with 5-20 um diam., cylindrical or slightly inflated, sometimes fusiform, hyaline, thin-walled; subhymenium ramose. PILEITRAMA trama composed of regular cylindrical, inflated or fusiform, hyaline, thin-walled hyphae, 11.2-26 um diam. PILEIPELLIS a cutis made up of cylindrical, hyaline, thin-walled hyphae, 2.5-8.7 um diam.; subpellis made up of inflated or fusiform, hyaline, thin-walled hyphae, 10-21 um diam. STIPITIPELLIs a cutis of hyaline, thin-walled hyphae, 3.7-12.5 um diam. CauLocysTipia not observed. CLAMP CONNECTIONS absent. REFRACTIVE HYPHAE abundant in pileipellis. HaBITAT— Gregarious or scattered, on soil. ADDITIONAL SPECIMEN EXAMINED—BRAZIL, SAo PAULO STATE, Santo André: Reserva Bioldgica de Paranapiacaba, 23°46’S 46°18’W, trail 1, 23.11.2007, FE. Karstedt & al. FK0891 (SP; GenBank KF738941, KF738930). ComMMENTS—Nolanea pallidosalmonea resembles Entoloma quadratum (Berk. & M.A. Curtis) E. Horak, cited from Borneo, Singapore, Costa Rica, United Nolanea spp. nov. (Brazil) ... 603 JOYSHOLOHOEGOOLY Fic. 5. Nolanea pallidosalmonea (holotype, FK892): a. Basidiospores; b. Basidia; c. Pileipellis. Scale bars: a, b = 10 um; c = 20 um. States, Madagascar, Malaysia and Papua New Guinea, in its conical pileus and a smooth surface (a cutis) that is salmon colored. However, E. quadratum is clearly separated from N. pallidosalmonea by its cuboidal basidiospores and clavate cheilocystidia (Horak 1975, 1977, Baroni & Halling 2000). Nolanea minuta P. Karst., described from Europe, is another species with pale and pinkish basidiomata, a silky-fibrillose pileus surface, heterodiametric basidiospores, and absence of cheilocystidia. However, N. minuta differs from N. pallidosalmonea in basidiospores with 5-7 angles in profile view, and a pileitrtama composed of hyphae with intracellular and encrusted pigments (Noordeloos 1980). Nolanea parvispora Karstedt & Capelari, sp. nov. FIGS. 21-K, 6, 8G-I MB805605 Nolanea parvispora differs from other Nolanea spp. by its lignicolous habitat, pale beige pileus with dark center, stipe with a dark base and pale apex, sinuate lamellae, small (6.2-7.5 x 6.2-7.5 um) isodiametric basidiospores, a lack of cystidia, and a pileipellis composed of repent hyphae with dispersed groups of anticlinal hyphae. TypE—Brazil, Sao Paulo State, Santo André: Parque Estadual da Cantareira, 23°20’S 46°41’W, Nucleo Engordador, Cachoeira trail, 25.V.2006, E Karstedt & M. Capelari FK0664 (Holotype, SP). EtyMoLoGcy—refers to the small size of the basidiospores. PiLteEus 13-23 mm diam., convex, plano-convex or plane, sometimes with a slightly or prominent papilla or slightly depressed at the center, whitish beige 604 ... Karstedt, Bergemann, Capelari or pale brownish beige or light pinkish beige, brown at the center (insertion of stipe), smooth and sometimes pruinose at the center, glabrous, translucent- striate, margin revolute, even, or sometimes lobate. PILEUS CONTEXT very thin. LAMELLAE sinuate, white or pinkish beige, abundant, with 5-7 lamellulae (2 series), margin even. STIPE 25-42 x 2-4 cm, cylindrical or flattened, brown or brownish translucent, beige at the apex, surface smooth, base with white tomentum. SPORE PRINT pinkish. BASIDIOSPORES 6.2-7.5 x 6.2-7.5 um (Q = 1-1.2; Qm = 1.1), isodiametric, with 5-6 angles in profile view, slightly pinkish, thin-walled. Bastp1a 27-43 x 8.7-10 um, clavate, hyaline, thin-walled, tetrasporic. PLEUROCYSTIDIA and CHEILOCYSTIDIA absent. LAMELLAE EDGE fertile. LAMELLAR TRAMA subregular, cylindrical, inflated or fusiform, hyaline, thin-walled hyphae 3.7-12.5 um diam.; subhymenium ramose. PILEITRAMA regular, made up of cylindrical, inflated or fusiform, hyaline, thin-walled hyphae, 3.7-25 um diam. PILEIPELLIS a cutis composed of cylindrical, hyaline or pale straw yellow, thin-walled hyphae, 6.2-13.7 um diam., with dispersed or clustered thin-walled cylindrical or clavate, hyaline or pale straw yellow anticlinal terminal elements; subpellis made up of inflated or fusiform, hyaline, thin-walled hyphae 10-20 um diam. STIPITIPELLIs a cutis, made up of hyaline or pale straw yellow, sometimes with encrusted (external) yellow-brown pigment, thin or thick-walled hyphae, 6.2-16.2 um diam. CAULOCYSTIDIA rare, cylindrical, clavate or ventricose, with pale straw yellow plasmatic pigment, thin-walled, 28-75 x 2.5-7.5 um. CLAMP CONNECTIONS absent. REFRACTIVE HYPHAE present in pileipellis. HasitatT—Solitary or gregarious, on hardwood. ADDITIONAL SPECIMENS EXAMINED—BRAZIL, SAO PAULO STATE, Sao Paulo: Parque Estadual da Cantareira, Nucleo Engordador, 23°20’S 46°41’W, Macuco trail, 29.V1.2006, F. Karstedt & M. Capelari FK689 (SP); Cachoeira trail, 29.VI.2006, F. Karstedt & M. Capelari FK0696 (SP); 25.VII.2006, E. Karstedt & M. Capelari FK0723 (SP); 24.X.2006, E. Karstedt & M. Capelari FK794 (SP); 30.X.2007, E Karstedt & al. FK0926 (SP). SANTA CATARINA STATE, Joinville: Reserva Particular de Patrimdénio Natural Caetezal, 02.1].2009, F. Karstedt FK1140 (SP; GenBank KF771348, KF738943, KF738931); Corupa, Reserva Particular de Patriménio Natural Emileo F. Battistella, falls route, 12.XII.2011, FE Karstedt FK2135 (FLOR; GenBank KF738932). CoMMENTS—Rhodophyllus avellanicolor Romagn. & Gilles, Nolanea mazophora (Berk. & Broome) Pegler, Entoloma myceliosum E. Horak, and Entoloma imbecille (E. Horak) E. Horak ex Segedin & Pennycook [= E. fragile E. Horak, nom. illegit.] are among the few species that resemble Nolanea parvispora based on pale basidiomata and isodiametric basidiospores measuring c. 7 x 7 um. Rhodophyllus avellanicolor, described from Africa, and Nolanea spp. nov. (Brazil) ... 605 506909HQGO6O Fic. 6. Nolanea parvispora (holotype, FK664): a. Basidiospores; b. Basidia; c. Caulocystidia; d. Pileipellis. Scale bars: a-c = 10 um; d = 20 um. Nolanea mazophora, described from Sri Lanka, are terrestrial. Rhodophyllus avellanicolor has clamp connections (Romagnesi & Gilles 1979) and N. mazophora has a convex pileus with prominent papillae (Pegler 1977, Horak 1980). Entoloma myceliosum, described from Chile, is also lignicolous but differs from N. parvispora by having a white pileus and stipe and a pileus surface densely covered by white fibrils (Horak 1977). Entoloma imbecille, found in New Zealand (Horak 1973, as “E. fragile”) and Argentina (Horak 1977, as “E. fragile”), differs by a pileus that is initially brown and then turns beige, brown encrusted pigments in the pileipellis hyphae, and abundant clamp connections. 606 ... Karstedt, Bergemann, Capelari Nolanea tricholomatoidea Karstedt & Capelari, sp. nov. FIGS. 2L, 7, 8J,K MB805607 Nolanea tricholomatoidea differs from other Nolanea spp by its robust stature, a mostly beige pileus that turns brownish beige at the center; its whitish beige stipe, its often pentagonal basidiospores; its basidial basal clamp connections at the base of the basidia; and the encrusted pigment in the subpellis hyphae. Type—Brazil, Sao Paulo State, Santo André: Parque Estadual da Cantareira, Nucleo Engordador, 23°20’S 46°41’W, Cachoeira trail, 31.1.2008, F. Karstedt, L.A. Silva Ramos & M. Capelari FK1049 (Holotype, SP; GenBank KF771347, KF738939, KF738928). ETyMoLoGy—the name refers to the tricholomatoid stature. PiLEuS 80-92 mm diam., conico-campanulate or plane, umbonate, pale beige, beige or brownish beige at center, surface smooth or slightly pruinose at center, opaque, hygrophanous, translucent-striate at margin. PILEUS CONTEXT white. LAMELLAE adnexed (almost free), beige, ventricose, subdistant, with 2 tiers of lamellulae. Stipe 100-116 x 8-12 mm, and 39 mm at base, subclavate, beige or whitish, fibrous and fibrillose, with white basal mycelium. Opor indistinct. SPORE PRINT not recorded. BASIDIOSPORES 8.7-10 x 7.5-8.7 um (Q = 1-1.33; Qm = 1.2), iso- diametric or subisodiametric, strongly angled, with 5-6 angles in profile view, with an obvious hilar appendix, hyaline or slightly brownish pink, thin-walled. Basrp1a 37-51 x (8.7—)10-15 um, clavate with a narrow base, hyaline, thin-walled, tetrasporic. LAMELLAE EDGE fertile. PLEUROCYSTIDIA and CHEILOCYSTIDIA absent. LAMELLAR TRAMA regular and hyaline, with hyphae 3.7-17.5 um diam., cylindrical or slightly inflated, hyaline, thin- walled, septate; subhymenium ramose. PILEITRAMA regular, composed of cylindrical or inflated, hyaline, thin-walled hyphae 3.7—27 um diam. PILEIPELLIS a cutis, made up of cylindrical, hyaline, thin-walled hyphae 2.5-8.7 um diam., with rare anticlinal elements; subpellis composed of inflated, hyaline, thin-walled hyphae 16.2-46 x 50-120 um diam. STIPITIPELLIS a cutis made up of straw-yellow, hyaline, thin-walled hyphae, 3.7-7.5 um diam. CAULOCYSTIDIA absent. CLAMP CONNECTIONS present in the hymenium. REFRACTIVE HYPHAE present in the pileipellis. HABITAT—Solitary, on soil. ADDITIONAL SPECIMEN EXAMINED—BRAZIL. Rio DE JANEIRO STATE, Nova Iguacu: Reserva Bioldgica de Tingua, Ouro road, 8 Dec 2004, M.P. Albuquerque & A.A. Carvalho-Jr. 29-VI (RB415680). COMMENTS—Nolanea tricholomatoidea was collected in Rio de Janeiro State but reported as Entoloma lividoalbum (Kihner & Romagn.) Kubicka Nolanea spp. nov. (Brazil) ... 607 0O9HCO0G000 Fic. 7. Nolanea tricholomatoidea (holotype, FK1049): a. Basidiospores; b. Basidia; c. Pileipellis. Scale bars: a, b = 10 um, c = 20 um. (Albuquerque & al. 2007). The robust stature is reminiscent of a feature common to Entoloma sensu stricto, and based mainly on its stature, its umbonate pileus that is sometimes translucent-striate, and its white stipe, N. tricholomatoidea might seem related to species of Entoloma s.s., such as E. lividoalbum and E. prunuloides (Fr.) Quél. Entoloma lividoalbum, described from Europe and recorded from the United States, differs in its pileus that varies from dark brown, dark yellowish brown, pale yellowish white to pale grayish beige, and clamp connections that are present in all tissues (Largent 1994, Noordeloos & Polemis 2008). Entoloma prunuloides, recorded from Europe, differs in its smaller basidiospores (6.5-8 x 6.5-8 um), an ixocutis-type pileipellis, and the presence of clamp connections in all tissues (Noordeloos 1992). Although a tricholomatoid stature is uncommon in Nolanea, the pileipellis structure in N. tricholomatoidea, comprising a subpellis with inflated elements and a suprapellis with thin elements, is a feature shared among many Nolanea spp. 608 ... Karstedt, Bergemann, Capelari Discussion The phylogenetic analyses as supported by ML, MP, and BPP cluster the species described here and other species traditionally accepted as Nolanea within a single clade, the /Nolanea clade (Fia. 1). Four of the species described in this paper—Nolanea albertinae, N. atropapillata, N. pallidosalmonea, and N. tricholomatoidea—occur in litter or soil and have heterodiametric basidiospores. They cluster in clade I. Nolanea parvispora, the only lignicolous species and characterized by isodiametric basidiospores, clusters in clade II. Species that are placed in Nolanea often have a mycenoid habit (Henkel & al. 2014, Largent 1994, Noordeloos 1992, 2004, Noordeloos & Gates 2012). Nolanea tricholomatoidea described herein and the two species classified as Entoloma s.s. (E. pallideradicatum Hauskn. & Noordel. and E. valdeumbonatum Noordel. & Meusers) have a tricholomatoid habit and yet are included in the /Nolanea clade. These species share with Nolanea species a pileipellis with a subpellis composed of inflated hyphae and heterodiametric basidiospores. Curiously, all species studied and almost all species (that have been published with a detailed description of the pileipellis) in the /Nolanea clade have a pileipellis composed of a suprapellis with narrow hyphae and a subpellis with inflated hyphae. We recommend paying careful or close attention to this character in the future to determine if these features are phylogenetically informative. Acknowledgments The authors are grateful to Dr. David L. Largent and Ms. Kerri Kluting for the donation of collections that were included in DNA amplification and the phylogenetic analyses. A special thanks to Dr. David L. Largent and Dr. Genevieve M. Gates for their suggestions, and Dr. Timothy J. Baroni and Dr. Felipe Wartchow for the reviews. This study was supported by the grants FAPESP 2006/58549-4 to Fernanda Karstedt (master’s grant) and FAPESP 2004/04319-2 to Marina Capelari provided by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo. M. Capelari also thanks the CNPg - Conselho Nacional de Desenvolvimento Cientifico e Tecnoldgico. Literature cited Albuquerque MP, Carvalho Jr. AA, Pereira AB. 2006. Novas ocorréncias de Agaricales (Basidiomycota) para o Brasil. R Bras Bioci 5: 1143-1145. Baroni TJ. 1981. 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Academic Press, Inc., New York. https://doi.org/10.1016/B978-0- 12-372180-8.50042-1 MYCOTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 July-September 2020—Volume 135, pp. 613-616 https://doi.org/10.5248/135.613 First record of Trappea darkeri from Turkey YASIN UZUN', OSMAN BERBER’, ABDULLAH KAYA? 2 Department of Biology, Science Faculty, Karamanoglu Mehmetbey University, 70100, Karaman, Turkey * Department of Biology, Science Faculty, Gazi University, 06560, Ankara, Turkey “ CORRESPONDENCE TO: kayaabd@hotmail.com ABSTRACT—The truffle-like basidiomycete genus Trappea is reported as a new record from Turkey and western Asia, based on a collection of T: darkeri from Nigde province. This is also the first record of the family Trappeaceae for the country. A brief description and photographs of the collection is provided. Key worps—Basidiomycota, biodiversity, Hysterangiales, taxonomy Introduction Trappea Castellano is a truffle-like fungi genus in the family Trappeaceae (Kirk & al. 2008). The genus was proposed by Castellano (1990), who segregated T: darkeri and T: phillipsii from Hysterangium Vittad. based on their smooth bacilloid basidiospores and layer of sterile locules below the peridium. Trappea species are characterized by subglobose to irregularly lobed fruit bodies with one or more rhizomorphs emerging from the base; a thin white peridium covering a zone of sterile chambers; an olive- brown to bright olive-green gleba with small empty chambers and a dendroid, gelatinous to cartilaginous columella; and ellipsoid to oblong basidiospores. Index Fungorum (www.indexfungorum.org accessed 25 June 2019) lists four Trappea species, none of which has been reported from Turkey (Acar & al. 2019, Sesli & Denchev 2014, Solak & al. 2015, Turkekul 2017). Here we contribute to the Turkish mycobiota and report T: darkeri as the first representative of the genus and family in Turkey. 614 ... Uzun, Berber, Kaya Materials & methods The Trappea specimen was collected from Ulukisla district, Nigde province, in 2019. The basidiomata were photographed in their natural habitat, where ecological and morphological observations were taken. Dried material was examined and photographed microscopically in the fungarium using a Nikon Eclipse Ci-S trinocular compound microscope with Nikon DS-Fi2 camera and a Hitachi SU5000 scanning electron microscope. The specimen was identified by consulting the literature (Castellano 1990, Desjardin & al. 2014, Gémez-Reyes & al. 2014, Montecchi & Sarasini 2000, Ruini 1990, Zeller 1939). The collection is preserved in the fungarium of Department of Biology, Science Faculty, Karamanoglu Mehmetbey University, Karaman, Turkey (KMU). Taxonomy Trappea darkeri (Zeller) Castellano, Mycotaxon 38: 3 (1990) FIG. 1 BasIDIOMA 40 mm in diam, hypogeous to semi-hypogeous, globose, surface glabrous, with basal rhizomorphs at the base, whitish, light brownish when rubbed. PERIpIUM whitish in section, not separable, membranous- leathery. GLEBA firm, bright olive-green, olive gray to light olive-brown, white near the peridium, spongy gelatinous. COLUMELLA distinct, gelatinous, translucent, typically dendroid and reaching almost throughout the sporocarp. Odor unpleasant. Basip1A 19-25 x 5-8 um, irregularly cylindrical to clavate, (4-)6(-8)-spored, clamp connections present. CysTipIA not observed. BASIDIOSPORES 4-5 x 2-3 um, ellipsoid to cylindrical, smooth, hyaline, sessile or with a short pedicel. SPECIMEN EXAMINED—TURKEY, NiGDE: Ulukisla, Ciftehan village, 37°30’N 34°47’E, 930 m, in soil among needle-litter in pine-oak mixed forest, 23.03.2019, O.Ber 342 (KMU). CoMMENTS— Trappea darkeri has a hypogeous to semi-hypogeous habit with long rhizomorphs at the base and is distributed in high altitudes of 1000- 3000 m (Castellano 1990, Desjardin & al. 2015, Gomez-Reyes & al. 2014). It is distinguished by a layer of sterile locules just under the peridium, and small, cylindrical spores (G6mez-Reyes & al. 2014). Morphologically it may be confused with Hysterangium species with rubbery-gelatinous olive-green gleba, which differ in their easily separable peridia, absence of sterile locules next to the peridium and larger, spindle-shaped spores (Desjardin & al. 2015, Siegel & al. 2019). Ecologically and morphologically similar, Trappea pinyonensis States can easily be distinguished from T. darkeri by the staining reactions of the peridium and rhizomorphs and its longer (5-6.2 x 2-2.5 um) spores (States 1991). Trappeaceae newly recorded for Turkey... 615 . ow ; Fic. 1. Trappea darkeri (KMU - Ber 342). a. Basidiomata; b. Basidia and basidiospores; c-e. Basidiospores (SEM). Scale bars: b = 10 um; c-e = 3 um. Although our specimen was collected at a slightly lower elevation (930 m) than cited elsewhere, its morphological characters agree with those cited in the literature (Castellano 1990, Desjardin & al. 2015, Gomez-Reyes & al. 2014, Montecchi & Sarasini 2000, Ruini 1990, Zeller 1939). Acknowledgments The authors would like to thank Dr. Ali Keles (Yuziincti Yil University, Van, Turkey), Prof. Dr. [brahim Tiirkekul (Gaziosmanpasa University, Tokat, Turkey) and Nomenclature Editor Dr. Shaun Pennycook for their helpful comments and careful review. Literature cited Acar I, Uzun Y, Keles A, Dizkirci A. 2019. Suillellus amygdalinus, a new species record for Turkey from Hakkari Province. Anatolian Journal of Botany 3(1): 25-27. https://doi.org/10.30616/ajb.514778 616 ... Uzun, Berber, Kaya Castellano MA. 1990. The new genus Trappea (Basidiomycotina, Hysterangiaceae), a segregate from Hysterangium. Mycotaxon 38: 1-9. Desjardin DE, Wood MG, Stevens FA. 2014. California mushrooms, the comprehensive identification guide. London: Timber Press. Gomez-Reyes VM, Gomez-Peralta M, Terrén-Alfonso A, Guevara-Guerrero G. 2014. Description of Trappea darkeri (Trappeaceae: Hysterangiales) from Mexico. Revista Mexicana de Biodiversidad 85: 1265-1268. https://doi.org/10.7550/rmb.43995 Kirk PM, Cannon PF, Minter DW, Stalpers JA. 2008. Dictionary of the fungi. 10th ed. Wallingford, UK: CAB International. Montecchi A, Sarasini M. 2000. Fungi ipogei d’ Europa. Vicenza: Centro Studi Micologici. Ruini S. 1990. Un nuovo gasteromicete ipogeo: Trappea darkeri (Zeller) Castellano var. lazzarii Ruini v. nov. Rivista di Micologia 33(3): 322-330. Sesli E, Denchev CM. 2014. Checklists of the myxomycetes, larger ascomycetes, and larger basidiomycetes in Turkey. 6th ed. Mycotaxon Checklists Online. 136 p. http://www.mycotaxon.com/resources/checklists/sesli-v106-checklist.pdf Siegel N, Vellinga EC, Schwarz C, Castellano MA, Ikeda D. 2019. A field guide to the rare fungi of California’s National Forests. Bookmobile: Minneapolis, MN. 313 p. Solak MH, Isiloglu M, Kalmus E, Alli H. 2015. Macrofungi of Turkey, checklist, vol. 2. Izmir, Turkey: Universiteliler Ofset [in Turkish]. States JS. 1991. A new false truffle in the genus Trappea (Hysterangiaceae). Mycotaxon 41(1): 127-133. Trappe M, Evans E Trappe J. 2007. Field guide to North American truffles. Berkeley: Ten Speed Press. Tirkekul I. 2017. New Calbovista, Mycena, Rhizopogon, Stictis, and Symphyosirinia records from Turkey. Mycotaxon 132: 503-512. https://doi.org/10.5248/132.503 Zeller SM. 1939. New and noteworthy gasteromycetes. Mycologia 31(1): 1-32. https://doi.org/10.2307/3754429 MYCOTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 July-September 2020—Volume 135, pp. 617-622 https://doi.org/10.5248/135.617 Mesocorynespora sinensis gen. & sp. nov. from southern China ZHAO-HUAN XU’, KAI ZHANG’, YOU-QIANG LUO’, Xi1u-Guo ZHANG?, RAFAEL F. CASTANEDA-RuiIz‘, JIAN Ma‘* ' College of Agronomy, Jiangxi Agricultural University, Nanchang, Jiangxi 330045, China ? Department of Landscaping, Shandong Yingcai University, Jinan, Shandong 250104, China > Shandong Provincial Key Laboratory for Biology of Vegetable Diseases and Insect Pests, College of Plant Protection, Shandong Agricultural University, Taian, Shandong 271018, China ‘Instituto de Investigaciones Fundamentales en Agricultura Tropical Alejandro de Humboldt (INIFAT), Académico Titular de la Academia de Ciencias de Cuba, Calle 1 Esq. 2, Santiago de Las Vegas, C. Habana, Cuba, C.P. 17200 * CORRESPONDENCE TO: majian821210@163.com; jxaumj@126.com ABSTRACT—A new anamorphic genus and species, Mesocorynespora sinensis collected on decaying culms of bamboo in China, is described and illustrated. The fungus is distinguished by short, unbranched, clavate conidiophores with monotretic, conidiogenous cells that produce solitary, acrogenous, obclavate, euseptate conidia. A key to Mesocorynespora and morphologically similar genera is provided. KEY worps—asexual fungi, hyphomycetes, taxonomy Introduction Lushan Mountain, located in the northern part of Jiangxi Province, China, covers approximately 302 km’ and exhibits a unique geography with complex terrains and unusual climate. The mountain's distinctive vegetation has favored the survival and multiplication of various microbial species, yet only relatively little information has been published on saprobic hyphomycetes recorded in China (e.g., Ma & Zhang 2015, Ma 2016, Ma & al. 2016, Xu & al. 2017, Ai & al. 2019). During our continuing surveys of asexual fungi in this region, an 618 ... Xu &al. interesting fungus was collected that differed remarkably from all previously described hyphomycetes (Seifert & al. 2011). Thus, it is described here as a new genus and species. Taxonomy Mesocorynespora Jian Ma, X.G. Zhang & R.F. Castaneda, gen. nov. MB 836511 Differs from Solicorynespora by its short, determinate, clavate conidiophores and multi- euseptate conidia; and from Corynespora by its euseptate conidia. TYPE SPECIES: Mesocorynespora sinensis Jian Ma & al. ErymMo.Loey: Greek, meso- meaning middle- + Latin, -corynespora, referring to the genus Corynespora. CONIDIOPHORES short, single or in groups, clavate, unbranched, brown. CONIDIOGENOUS CELLS monotretic, integrated, terminal, clavate, determinate. Conidial secession schizolytic. Conrp1A solitary, acrogenous, dry, obclavate, multi-euseptate. Mesocorynespora sinensis Jian Ma, X.G. Zhang & R.F. Castafieda, sp. nov. Fics 1, 2 MB 836512 Differs from Solicorynespora spp. by its short, determinate conidiophores with multi- euseptate conidia, and from Corynespora spp. by its euseptate conidia. Type: China, Jiangxi Province: Lushan Mountain, on decaying culms of bamboo, 9 November 2014, J. Ma (holotype, HJAUP M0296). ETyMoOLoGy: refers to China where the fungus was collected. COLONIES on natural substrate effuse, brown to dark brown. Mycelium superficial and immersed, composed of branched, septate, pale brown to brown, smooth-walled hyphae. ConipiopHorgs short, single or in groups, erect, straight or flexuous, clavate, unbranched, brown, smooth, 1-3-septate, 25-40 x 3.5-5 um. CONIDIOGENOUS CELLS monotretic, integrated, terminal, determinate, clavate, brown, smooth, 9-14.5 x 5-6.5 um. Conidial secession schizolytic. Conrp14 solitary, dry, acrogenous, obclavate, smooth, brown to dark brown, rounded and golden yellow at the apex, truncate at the base, 20-35-euseptate, 72-171 x13.5-16 um, tapering to 7-10.5 um diam. at the apex, 4-5 um diam. at the base. Discussion Mesocorynespora sinensis is unique in having short, clavate, unbranched conidiophores, and solitary, acrogenous, obclavate, multi-euseptate conidia Mesocorynespora sinensis gen. & sp. nov. (China) ... 619 B wig] Fic. 1. Mesocorynespora sinensis (holotype, HJAUP M0296). A. Developing conidia; B. Conidiophores with developing conidia; C. Conidiophores, and conidiogenous cells. 620 ... Xu &al. B 20um 20um 20um Fic. 2. Mesocorynespora sinensis (holotype, HJAUP M0296). A-C. Conidiophores, conidiogenous cells, and conidia; D. Conidia. that secede schizolytically from the monotretic, integrated, terminal, determinate conidiogenous cells. We initially thought of assigning it to the genus Solicorynespora R.F. Castafteda & W.B. Kendr. (Castafeda-Ruiz & Kendrick 1990), but Solicorynespora species have long, cylindrical to obclavate conidiophores with percurrently extending conidiogenous cells and conidia with fewer septa. Several other genera including Corynespora Giissow, Corynesporella Munjal & H.S. Gill, Hemicorynespora M.B. Ellis, Corynesporopsis P.M. Kirk, and Corynesporina Subram. (Giissow 1905, Munjal & Gill 1961, Ellis 1972, Kirk 1981, Gams & al. 2009) have the same conidiogenesis as Mesocorynespora, but Mesocorynespora differs in having clavate, determinate conidiogenous cells Mesocorynespora sinensis gen. & sp. nov. (China) ... 621 formed from short, clavate, unbranched conidiophores; and differs further from Corynespora, Corynesporella, and Corynesporina by its euseptate conidia and from Corynesporopsis by its solitary conidia. Conidia in Hemicorynespora are 0-1-septate, an obvious difference from Mesocorynespora. Key to Mesocorynespora and morphologically similar genera 1. Conidiophores short, conidiogenous cells Wwithoutpercurtent extension § 2.25. 51.5 aed ng ee eee Mesocorynespora 1. Conidiophores long, conidiogenous cells with percurrent extension............ 2 2sConidig-aseptate oreuseptate 6 hic. wie tae ok ete whom ce ery ee pA eg 3 DAG QIN IC AGH SEOSCDLALEY tsi age ber sel gia cite ages ses soneet vara a wtbeneed ac seet astt 9 wiica byeUe Ey arise AAs ae 5 8 Conidia-catenates si 4 ous. trons Hd nh Fed Wee Ek ee OREO Corynesporopsis SiS onidia-solitaryes tan te shaes bot atethe ue ae kone ane tena ee ec eaece 4 4. Conidia aseptate or with a single septum ..................... Hemicorynespora A ONIdia tWO*.t6-pIMPISeptaler sce. anesthe a ants cs Ee oh Solicorynespora 5. Conidiogenous cells formed as terminal and lateral branchlets On The CONIMIGPROTES srecse.t, sto. ste str somese neat ob Hen ge ae eeaE ste taba cae Corynesporella 5. Conidiogenous cells integrated, terminal on the conidiophores ................ 6 6. Gonidia formed'singlé or in.acropetal chains’ 2: ..0.25 4.5 orc mesees Corynespora 6. Conidia solitary and in basipetal chains .....................4.. Corynesporina Acknowledgments The authors express gratitude to Dr. Patricia Oliveira Fiuza (Programa de Pos- graduacao em Sistematica e Evolu¢gao, Universidade Federal do Rio Grande do Norte, Brazil) and Dr. De-Wei Li (The Connecticut Agricultural Experiment Station Valley Laboratory, USA) for serving as pre-submission reviewers and to Dr. Shaun Pennycook for nomenclatural review and Dr. Lorelei L. Norvell for editorial review. This project was supported by the National Natural Science Foundation of China (Nos. 31970018, 31360011, 31870016), and the Education Department of Jiangxi Province of China (No. GJJ160357). Literature cited Ai CC, Ma J, Zhang K, Castaneda-Ruiz RF, Zhang XG. 2019. Cordana meilingensis and C. lushanensis spp. nov. from Jiangxi, China. Mycotaxon 134: 329-334. https://doi.org/10.5248/134.329 Castafieda-Ruiz RE, Kendrick B. 1990. Conidial fungi from Cuba: II. Univ. University of Waterloo Biology Series 33. 61 p. Ellis MB. 1972. Dematiaceous hyphomycetes. XI. Mycological Papers 131. 25 p. Gams W, Seifert KA, Morgan-Jones G. 2009. New and validated hyphomycete taxa to resolve nomenclatural and taxonomic issues. Mycotaxon 110: 89-108. https://doi-org/10.5248/110.89 Giissow HT. 1905 [”1904”]. Notes on a disease of cucumbers, II. Journal of the Royal Agricultural Society of England 65: 271-272. 622 ... Xu &al. Kirk PM. 1981. New or interesting microfungi IH. Dematiaceous hyphomycetes from Esher Common, Surrey. Transactions of the British Mycological Society 77: 279-297. https://doi.org/10.1016/S0007-1536(81)80031-9 Ma J. 2016. Corynesporopsis obclavata and Stanjehughesia jiangxiensis spp. nov. from Lushan Mountain, China. Mycotaxon 131: 583-588. https://doi.org/10.5248/131.583 Ma J, Zhang XG. 2015. A preliminary report of dematiaceous hyphomycetes from dead branches in Jiangxi Province. Biological Disaster Science 38(4): 290-293. https://doi.org/10.3969/j.issn.20953704.2015.04.003 Ma J, Zhang XG, Castafieda-Ruiz RE 2016. Podosporiopsis, a new genus of synnematous hyphomycetes from China. Mycotaxon 131: 773-780. https://doi.org/10.5248/131.773 Munjal RL, Gill HS. 1961. Corynesporella: a new genus of hyphomycetes. Indian Phytopathology 14(1): 6-9. Seifert K, Morgan-Jones G, Gams W, Kendrick B. 2011. The genera of hyphomycetes. CBS Biodiversity Series 9. 997 p. Xu ZH, Hu DM, Luo YQ, Ma J. 2017. Three species of Linkosia and Spadicoides new to China. Mycotaxon 132: 243-250. https://doi.org/10.5248/132.243 MYCOTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 July-September 2020—Volume 135, pp. 623-630 https://doi.org/10.5248/135.623 Corynesporopsis hainanensis sp. nov., a bambusicolous fungus from southern China ZHAO-HUAN Xu’, XU-GEN SHI’, WEI-GANG KUANG’, X1u-Guo ZHANG’, RAFAEL F. CASTANEDA-RuiIz}, JIAN Ma'* ' College of Agronomy, Jiangxi Agricultural University, Nanchang, Jiangxi 330045, China ? Department of Plant Pathology, Shandong Agricultural University, Taian, Shandong 271018, China 3 Instituto de Investigaciones Fundamentales en Agricultura Tropical Alejandro de Humboldt (INIFAT), Académico Titular de la Academia de Ciencias de Cuba, Calle 1 Esq. 2, Santiago de Las Vegas, C. Habana, Cuba, C.P. 17200 * CORRESPONDENCE TO: majian821210@163.com jxaumj@126.com ABSTRACT—A new anamorphic species, Corynesporopsis hainanensis, is described and illustrated from dead bamboo culms collected in the tropical mountain rainforest of Jianfengling, Hainan, China. The fungus is characterized by its terminal monotretic conidiogenous cells with catenate, obclavate, brown to pale brown, smooth, (3-)5-6(-10)-euseptate conidia. A dichotomous key and a synoptic table to Corynesporopsis species are provided. KEY worps—asexual fungi, hyphomycetes, saprobes, taxonomy, Xylariales Introduction Taking into account its large area, varied geographical conditions and advantageous natural environments, China is considered an important reservoir of the original bamboo diversity (Yang & Xue 1998). Its bamboo forest ecosystems offer favorable habitats for survival and multiplication of bambusicolous fungi. However, our knowledge of bambusicolous fungi is scant, and relatively little information about their taxonomy has been published in China (e.g. Zhou & al. 2001, Xu & al. 2006, 2007, Ma 2016). During our ongoing surveys of saprobic microfungi associated with plant debris, we 624 ... Xu &al. collected on dead bamboo culms an interesting hyphomycete with the typical morphological features of Corynesporopsis P.M. Kirk (Kirk 1981a). The fungus, significantly different from previously described taxa, is proposed here as new to science. Materials & methods Samples of litter were placed in paper and plastic bags, taken to the laboratory, and prepared according to Castafeda-Ruiz & al. (2016). Mounts were prepared in PVL (polyvinyl alcohol and lactic acid) and measurements were made at a magnification of x1000. Micrographs were obtained with a Nikon Eclipse 80i microscope equipped with bright field and Nomarski interference optics. The type specimen was deposited in the Herbarium of Jiangxi Agricultural University, Nanchang, Jiangxi, China (HJAUP). Taxonomy Corynesporopsis hainanensis Z.H. Xu, Jian Ma, X.G. Zhang & R.F. Castafieda, sp. nov. Fig. 1 MB 836568 Differs from Corynesporopsis curvularioides, C. iberica, and C. rionensis by its (3-)5-6(-10)-euseptate obclavate conidia; from C. iberica by its wider conidia; and from C. curvularioides and C. rionensis by its longer and narrower conidia. Type: China, Hainan Province, Jianfengling National Nature Reserve, on dead culms of bamboo, 18 April 2014, J. Ma (Holotype, HJAUP M0377). EryMo.oey: refers to the province where the type was collected. CoLonigs on dead wood effuse, dark brown to black, hairy. Mycelium partly superficial, partly immersed in the substratum, composed of branched, septate, pale brown to brown, smooth hyphae. ConrpiopHor:s differentiated, single, erect, unbranched, straight or slightly flexuous, brown to dark brown, smooth, septate, 45-62 x 5.5-6.5 um, thick-walled. CONIDIOGENOUS CELLS monotretic, integrated, terminal, determinate, cylindrical, brown, smooth, 10-13 um long, 4.5-5.5 um wide. Conidial secession schizolytic. CONIDIA acrogenous, catenate, in unbranched, acropetal chains, obclavate, straight or curved, brown to pale brown, smooth, (3-)5-6(-10)-euseptate, 17-48 um long, 6.5-8 um diam. in the broadest part, 2-3 um diam. at the truncate base. Dissussion Corynesporopsis was established by Kirk (198la) with Corynespora quercicola Borowska [= Corynesporopsis quercicola (Borowska) P.M. Kirk] as the type species, and was mainly characterized by acrogenous, catenate, euseptate conidia seceding schizolytically from monotretic, integrated, Corynesporopsis hainanensis sp. nov. (China) ... 625 20 pm Fic. 1. Corynesporopsis hainanensis (holotype, HHAUP M0377). A, B. Conidiophores, conidiogenous cells, and conidia; C. Conidiophores; D. Conidia. terminal, determinate or rarely percurrently extending conidiogenous cells. It is similar to Corynespora Giissow, Corynesporella Munjal & H.S. Gill, Hemicorynespora M.B. Ellis, Solicorynespora R.F. Castaheda & W.B. Kendr., and Corynesporina Subram. in having monotretic conidiogenous cells (Giissow 1905, Munjal & Gill 1961, Ellis 1972, Castafeda-Ruiz & Kendrick 1990, Gams & al. 2009). However, Corynesporopsis differs from Hemicorynespora and Solicorynespora by its catenate conidia and from Corynespora, Corynesporella, 626 ... Xu &al. uteds 10 eqny puryod Pury Pury eqn) eIpul niog yesnji0g euryO euly elperjsny euly 0 eqn) Pury KLITVOOT AdA, poom peep pesiourqns SARI] Ped saypouRig Us}}0yY poom 8utde0q sayoueig peaq sayoueiq peag gjoned ured u3}0y SIM] prod gjoned ured u9}}0y yred soyourig prog sayoueig prod Ppoom prog sayoueig prod poom prog SARI] Peo poom prog IVLIGV] gqeize,, UIIOFIU) WIIOJIUL) aiqeise,, wWIIOJIUL) UIIOFIU) wWIIOJIUL) wWIIOFIUL) aiqerse,, UWIIOFIU) UWLIOJIUL) UWIIOFIU) UIIOFIU) UIIOFIU) UIIOFIU) gqeise,, UIIOFIU) AOTOD) aye}dasna-| ‘Teao 0} urro0zrAd -qns eoripurAo “suopqo-qns ‘a}eaepoqo “prosdiyy[q ayeydasnoa-T| ‘prosdiyytq ayeydasna-(¢-)p—-¢ ‘Teplosdiyjo Jo WUIIOFIsn} peosq “WIOFISN,] ayejdasna-z ‘festpulpAd 0} prosdiyyja ATpeorg ayejdasna-6-¢ ‘ayearpqo ayeydasna-| ‘prosdiyja 0} waospNITAeN ajeydasna-| “WIIOJIPNITALU 9}eBUOTE IO UTIOFISHy ayesuoyy aye}dasna-T| ‘proaogo ATpeosgq 0} prosdiyyyq ayejdasna-| ‘a}earpyqO ayeydasna-/-¢(-Z) ‘yestupulyAD a}eydasna-(QT-)9-G(—-¢) ‘ayVARPGOC ayejdasno-¢-] qeopurpé+ ayeydasna-(7-)T qeorpurpA) ayejdasna-¢-]| ‘eprosdiyja 0} ayearppqo ayejdasno-z Apsour JeorpurpAy ayejdasna-(9-)s(-¢) Jeprosdiyq aye}dasno-| ‘spua 94} Je popunos ATpeosg ‘fedtpuTfAd 0} Teprosdiy[q NOLLVLdadS GNV dd VHS VIGINO‘) ‘PLOT Te 29 Odessay-zopueusIey or II86T HM cr ‘986T PATAIS Opeozay 2B PAOYAL-PAOGNTOH y “VTSGT ATEN er ‘9TOT PW cr ‘ZTOT Te 28 PN 11 *Z86T PAOYDA[-PAOGNIOH 0; ‘E861 YEA « ‘E66T PUMTYSNSIeYY ¢ ILOT [P29 ZINY-epourysey , ‘POT [E28 PIX, “6861 UOUNS . ‘E10T Te LIX, ‘8S6T SAUO{-URSIOW , ‘0661 YUIPUSY 2 ZIMY-epourysey ; ‘G{OZ IOUYSsITY , ‘Woy VIE 8-S x OI-TI L£-G X 9I-CI (01-6 Apsour) II-8 X 9€-¥7 (6-)8-9 X 8I-F1(-Z1) ZI-9 X 19-LZ S°S-S'€ X S‘9I-OT V9-F X SEP-LU(-¥7) (PI-)ZI-8 x (LZ7-)P7-L1(-¥1) S'S-P X S7-LI D-€ X (65-)8P-ST 8-S'°9 X SP-LI S'0I-S'L X OF-ST S°L-9 X S07-S°7I LI-Z X S€-ST 6-L X €€-8T 8-S X (€€-)8T-1Z (S°S-)S(-S'F) X €T-II(-OT) (um) azIs or SI[IQUIADA “2 <p DIDIdasIUN *D 71 SISUUOLL *D) ep bjoot4anb “4 2, VIVAVIIGO *D 11 SHvquippinby “D or 2VINJAQUSI *D) « PIIPUL “D bIvidasinbavut *D , botdagt “2D sisuauvurvy *D 9 91409909 *D) ¢ DILApurfAd “D , SOPIOLADINAAN *F+) € vjvjdasiq 0) - BUDyyIUD “D 7 PVIIVDID *) SaIOddS ‘JUOJ PJOq UT Jas st satdads Mau ay], ‘sodeds sisdosodsaud10+ Jo saye1jsqns pure eIpruod Jo suostieduroy 1 ATAVI, Corynesporopsis hainanensis sp. nov. (China) ... 627 and Corynesporina by its euseptate conidia. Corynesporopsis is also similar to Heteroconium Petr. (Petrak, 1949) in having catenate, euseptate conidia, but Heteroconium is separated by monoblastic conidiogenesis. Although Corynesporopsis was placed in Xylariales (Sordariomycetes) based on ITS and LSU rDNA based phylogenetic studies of C. acaciae R. Kirschner, its family remains incertae sedis due to its tretic conidiogenesis, previously unrecorded for Xylariales (Kirschner 2015). Teleomorphs and phylogenetic affinities with other Corynesporopsis species are not yet known. Among the known species of Corynesporopsis, only C. curvularioides J.W. Xia & X.G. Zhang (Xia & al. 2013), C. iberica R.F. Castafieda & al. (Castaneda- Ruiz & al. 2011), and C. rionensis Hol.-Jech. (Holubova-Jechova & Mercado Sierra 1986) are comparable to C. hainanensis. However, C. curvularioides differs in its obclavate to ellipsoidal, shorter and wider conidia with 1-5 eusepta; C. iberica differs in its cylindrical, narrower conidia with (2-)3-7 eusepta; while C. rionensis differs in its wider fusiform, broadly fusiform, or ellipsoidal conidia with 3—4(-5) eusepta. With the addition of C. hainanensis, Corynesporopsis currently contains 17 species. A synopsis for comparisons of conidia and substrates of these accepted species is presented in TABLE 1. The conidia of these 17 Corynesporopsis species are mostly ellipsoidal, cylindrical, obclavate, or fusiform, but conidia may also be obovoid, naviculiform, sub-oblong, sub-pyriform, or oval. The conidia are mostly unicolored, but C. antillana R.F. Castaheda & W.B. Kendr., C. inaequiseptata Matsush., C. quercicola, and C. variabilis R.F. Castaneda & al. produce versicolored conidia. Eight species produce predominately 1-septate conidia, usually thickened and darkened at the septa—C. acacia, C. cylindrica B. Sutton, C. indica P.M. Kirk, C. isabelicae Hol.-Jech., C. liquidambaris Jian Ma & X.G. Zhang, C. uniseptata P.M. Kirk, C. inaequiseptata, and C. variabilis— while the other Corynesporopsis species produce conidia with more septa. All species were found as saprobes on bark, decaying leaves, rotten wood, or dead palm rachides in terrestrial environments, except for C. variabilis on submerged dead wood. Fourteen species definitely produce smooth-walled conidia; the conidial ornamentation of the other three species (C. uniseptata, C. curvularioides and C. excoecariae J.W. Xia & X.G. Zhang) is inexplicably omitted in their original descriptions (Kirk 1981b, Xia & al. 2013, 2014). Keys to species of Corynesporopsis have been provided by Castafieda- Ruiz & al. (2011) and Hernandez-Restrepo & al. (2014), based on conidial morphology. The following expanded key includes all 17 accepted Corynesporopsis species. 628 ... Xu &al. Key to Corynesporopsis species lConidiazpredomirianthyslvetiseptat]e mo icnst etree Bl Me inte ge Pee ls kos 2 t.. Conidia: predominantly 2 or more septas a... nx esses eu ekg Adages aed wits 9 2. Conidia versicoloraus 1... benaeahoaceles dee hee bodes ee Se ee oe 3 ae Conidia cOnCOlOKOUS *. dasisrect othe eristhy tds oryp raat agrees: Setincapeds So aghast 4 3. Conidia obclavate, 17-25 x 4-5.5 um ....... eee eee eee C. inaequiseptata 3. Conidia variable ellipsoid, obclavate, sub-oblong, cylindrical, sub-pyriform to-ovakl 1-16 x 5-8-2 ets 2 sages Ae C. variabilis 4, Conidia (14—)17-24(-27) x 8-12(-14) um ..... eee eee C. indica 4, COnidia-Mot.exceecin et: Ziaafltiy CIA. 04,5 ween Ra thane Game cae ee Re ees 5 5. Conidia (24-)27-43.5 x 4-6.4 um, elongate fusiform or elongate naviculiform ....................-. C. isabelicae 5; Gonidia net exceedine 20%s:1itin lone: 8:2 534/22 Renee a Ss ee Age Soe Feasts St 6 6; Gonidia stricthreyiindnicaltorcellipsoidal *: .e Sacg td non pate ae Ad «oes 7 6. Conidia ellipsoidal to cylindrical, or naviculiform to ellipsoid .................. 8 7: Gonidia.cylindrical, 12-5520. 56 —Z, Sat eects ag cess ata PAS Coane C. cylindrica 7. Gonidiaellipsoidal, 12-16 537M «bso ot ox eels fee eae C. uniseptata 8. Conidia ellipsoidal to cylindrical, (10-)11-13 x (4.5-)5(-5.5) um ...... C. acaciae 8. Conidia naviculiform to ellipsoid, 10-16.5 x 3.5-5.5 um ......... C. liquidambaris OF OMIA VEESICOlOTOUS Ss. bet oc. 2 percus dete cee P es oe oa scene eee SC Pu, ye 2 es. AR 10 9 Conidia concolorous. .4)..2 je eres eget epee beg ee ee ee 11 10. Conidia ellipsoidal, 21-28(-33) x 5-8 um, (3-)5(-6)-euseptate ...... C. antillana 10. Conidia broadly ellipsoid to cylindrical, (12-)14-18 x 6-8(-9) um, 2-euseptate ............... 0... eee C. quercicola WiGonidiacylindri¢al™ 2.52.6. hae tse ee tae twee He ee ee crows es 12 11. Conidia fusiform, ellipsoidal, obclavate, or obclavate to ellipsoidal ............ 14 12. Conidia 15-48(-59) x 3-4 um, (2-)3-7-euseptate ................00.. C. iberica 2 Conidia-aVleastfpmiewide, 2.2 Filet ce feta en hse ide, Wet eh Pane ence ik eee 13 13. Conidia 18-33 x 7-9 um, mostly 2-euseptate ...................0.. C. biseptata 13. Conidia 15-40 x 7.5-10.5 um, 1-3-euseptate .................... C. excoecariae 14:-Conidia strictly obclavate: «+3 tesco nay thtmadia ds apie angccedempa eae ghneme 4 15 14. Conidia fusiform, ellipsoidal, or obclavate to ellipsoidal ..................... 16 15. Conidia 17-48 x 6.5-8 um, (3-)5-6 (-10)-euseptate .............. C. hainanensis 15. Conidia 27-61 x 6-12 um, 5-9-euseptate .......... eee ee eee C. obclavata 16. Conidia obclavate to ellipsoidal, 1539: 7511 um, J SFetseptate: 8.6 era ee Fa thane vale ones C. curvularioides 16. Conidia fusiform or ellipsoidal, 24-36 x 8-11 (mostly 9-10) um, 3-4(-5)-euseptate .............. C. rionensis Corynesporopsis hainanensis sp. nov. (China) ... 629 Acknowledgments The authors express gratitude to Dr. De-Wei Li (The Connecticut Agricultural Experiment Station Valley Laboratory, USA) and Dr. Silvana Santos Da Silva (Departamento de Ciéncias Bioldgicas, Laboratério de Micologia, Universidade Estadual de Feira de Santana, Brazil) for serving as pre-submission reviewers and to Dr. Lorelei L. Norvell for final editorial review and Dr. Shaun Pennycook for nomenclatural review. This project was supported by the National Natural Science Foundation of China (Nos. 31970018, 31760513, 31360011) and the Education Department of Jiangxi Province of China (No. GJJ160357). Literature cited Castafieda-Ruiz RE, Kendrick B. 1990. Conidial fungi from Cuba: II. University of Waterloo Biology Series 33. 61 p. Castafieda-Ruiz RF, Silvera-Sim6n C, Gené J, Guarro J, Minter DM, Stadler M, Saikawa M. 2011 [“2010”] A new species of Corynesporopsis from Portugal. Mycotaxon 114: 407-415. https://doi.org/10.5248/114.407 Castaneda-Ruiz RF, Heredia G, Gusmao LFP, Li DW. 2016. Fungal diversity of Central and South America. 197-217, in: DW Li (ed.). Biology of Microfungi. Springer International Publishing. https://doi.org/10.1007/978-3-319-29137-6_9 Ellis MB. 1972. Dematiaceous hyphomycetes. XI. Mycological Papers 131. 25 p. Giissow HT. 1905 [“1904”]. Notes on a disease of cucumbers, II. Journal of the Royal Agricultural Society of England 65: 271-272. Gams W, Seifert KA, Morgan-Jones G. 2009. New and validated hyphomycete taxa to resolve nomenclatural and taxonomic issues. Mycotaxon 110: 89-108. https://doi.org/10.5248/110.89 Hernandez-Restrepo M, Gené J, Castafieda-Ruiz RF, Kirk PM, Guarro J. 2014. A new species of Corynesporopsis from Spain. Mycotaxon 127: 155-160. https://doi.org/10.5248/127.15 Holubova-Jechova V. 1987. Studies on hyphomycetes from Cuba VI. New and rare species with tretic and phialidic conidiogenous cells. Ceska Mykologie 41(2): 107-114. Holubova-Jechova V, Mercado Sierra A. 1986. Studies on hyphomycetes from Cuba IV. Dematiaceous hyphomycetes from the Province Pinar del Rio. Ceska Mykologie 40(3): 142-164. Kirk PM. 1981la. New or interesting microfungi II. Dematiaceous hyphomycetes from Esher Common, Surrey. Transactions of the British Mycological Society 77: 279-297. https://doi.org/10.1016/S0007-1536(81)80031-9 Kirk PM. 1981b. New or interesting microfungi III. A preliminary account of microfungi colonizing Laurus nobilis leaf litter. Transactions of the British Mycological Society 77: 457-473. https://doi.org/10.1016/S0007-1536(81)80093-9 Kirk PM. 1983. New or interesting microfungi VII. Corynesporopsis indica sp. nov. Mycotaxon 17: 405-408. Kirschner R. 2015. Phylogenetic placement of a new species of Corynesporopsis from dead acacia wood indicates occurrence of tretic conidiogenesis within Xylariales. Phytotaxa 192 (1): 24-34. https://doi.org/10.11646/phytotaxa.192.1.3 Ma J. 2016. Corynesporopsis obclavata and Stanjehughesia jiangxiensis spp. nov. from Lushan Mountain, China. Mycotaxon 131(3): 583-588. https://doi.org/10.5248/131.583 630 ... Xu & al. Ma J, Ma LG, Zhang YD, Castafieda-Ruiz RE, Zhang XG. 2012. New species and record of Corynesporopsis and Hemicorynespora from southern China. Nova Hedwigia 95(1-2): 233-241. https://doi.org/10.1127/0029-5035/2012/0030 Matsushima T. 1993. Matsushima Mycological Memoirs No. 7. Published by the author, Kobe, Japan. Morgan-Jones G. 1988. Notes on hyphomycetes. LVII. Corynespora biseptata, reclassified in Corynesporopsis. Mycotaxon 31(2): 511-515. Munjal RL, Gill HS. 1961. Corynesporella: a new genus of hyphomycetes. Indian Phytopathology 14: 6-9. Petrak F. 1949. Neue hyphomyzeten-gattungen aus Ekuador. Sydowia 3: 259-266. Sutton BC. 1989. Notes on deuteromycetes. II. Sydowia 41: 330-343. Xia JW, Ma LG, Ma YR, Castafieda-Ruiz RF, Zhang XG. 2013. Corynesporopsis curvularioides sp. nov. and new records of microfungi from southern China. Cryptogamie, Mycologie 34(3): 281-288. https://doi.org/10.7872/crym.v34.iss3.2013.281 Xia JW, Ma YR, Zhang XG. 2014. New species of Corynesporopsis and Lylea from China. Sydowia 66 (2): 241-248. https://doi.org/10.12905/0380.sydowia66(2)2014-0241 Xu MQ, Dai YC, Fan SH, Jin LX, Lu Q, Tian GZ, Wang LF. 2006. Records of bamboo diseases and the taxonomy of their pathogens in China (I). Forest Research 19(6): 692-699. Xu MQ, Dai YC, Fan SH, Jin LX, Lu Q, Tian GZ, Wang LF. 2007. Records of bamboo diseases and the taxonomy of their pathogens in China (II). Forest Research 20(1): 45-52. Yang YM, Xue JR. 1998. Bamboo resources and their utilization in China. In: Proceedings of the Workshop Bamboo Conservation, Diversity, Ecogeography, Germplasm, Resource Utilization and Taxonomy. Kunming and Xishuangbanna, Yunnan, China. Zhou DQ, Hyde KD, Wu XL. 2001. New records of Ellisembia, Penzigomyces, Sporidesmium and Repetophragma species on bamboo from China. Acta Botanica Yunnanica 23(1): 45-51. MYCOTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 July-September 2020—Volume 135, pp. 631-635 https://doi.org/10.5248/135.631 Hymenochaete longisterigmata sp. nov. from India NAVPREET KAUR, AVNEET PAL SINGH*, GURPAUL SINGH DHINGRA Department of Botany, Punjabi University, Patiala-147002, Punjab, India. *CORRESPONDENCE TO: avneetbot@gmail.com ABSTRACT—A new hymenochaetoid species, Hymenochaete longisterigmata, is described from Himachal Pradesh, India. Keyworps—Basidiomycota, Hymenochaetales, Hymenochaetaceae, Himalaya Introduction During the 2014 fungal forays conducted in the Narkanda area of district Shimla, Himachal Pradesh, India, Navpreet & Dhingra collected a fungal specimen on the branch of a living tree of Picea smithiana. This fungus was again collected (2016) in the Jalori Pass area of district Kullu, Himachal Pradesh, India, by Navpreet & Avneet on the branch of a living Quercus semecarpifolia. Based on the comparison of morphological characters (Cunningham 1963, Sharma 1995 & 2012, Natarajan & Kolandavelu 1998, Bernicchia & Gorjon 2010, Dai 2010, Kaur & al. 2015, Sharma & Mishra, 2015, Mycobank 2019), we referred the specimens to Hymenochaete Lév. (Hymenochaetaceae) and close to H. corrugata (Fr.) Lév. based on its resupinate, adnate basidiomata, heavily cracked hymenial surface, and presence of abundant setae with encrusted apices. We now propose the fungus as a new species H. longisterigmata, named in reference to its exceptionally long sterigmata. Materials & methods The present study is based on fungal samples collected during the field trips conducted in Narkanda (District Shimla) and Jalori Pass (District Kullu) forest areas of Himachal Pradesh during September 2014 and 2016. Colours terms follow Kornerup 632 ... Kaur, Singh, Dhingra & Wanscher (1978). After removing any extraneous matter, we dried the specimens in the sun or with a portable electric drier. The dried specimens were deposited at the internationally recognized herbarium of Department of Botany, Punjabi University, Patiala, India (PUN). The arrangement, size and shape of hyphae, sterile structures, basidia, and basidiospores were studied by making crush mounts and free hand sections in water, KOH (3%/5%/10%), 1% Congo red, 1% phloxine, cotton blue (15 in Lactophenol), and Melzer’s reagent. We examined the material microscopically at 100x, 400x and 1000x magnifications using a compound microscope and drew structural line diagrams on white paper sheets with the aid of a camera lucida. Photomicrographs of important structures were taken using a Nikon 80i Eclipse microscope. Taxonomy Hymenochaete longisterigmata Nav. Kaur, Avn.P. Singh & Dhingra, sp. nov. PLATE 1 MB 832678 Differs from Hymenochaete corrugata by its thick-walled hyphidia, its larger basidia with long sterigmata, and its broadly ellipsoid basidiospores. Type: India, Himachal Pradesh, Shimla, Narkanda, about 3 Km from Narkanda towards Baghi, on branch of living tree of Picea smithiana (Wall.) Boiss., 4 September 2014, Navpreet & Dhingra 9689 (Holotype, PUN). Etrymo.oey: The specific epithet refers to the long sterigmata. Basidiomata annual, resupinate, <2 mm thick in section, adnate, soft and corky when collected, hard and corky after drying; hymenial surface smooth to somewhat aculeate under lens, cracked, greyish orange to greyish brown when collected, no significant change after drying; margins thinning, indeterminate, paler concolorous, sterile portion <0.5 mm. Hyphal system monomitic. Subicular hyphae brown, heavily encrusted, up to 6.3 um wide; subhymenial hyphae subhyaline to yellowish brown, up to 3.4 um diam. Setae subulate, apices straight with crystalline encrustation, dark brown, abundant, thick-walled, 68-92 x 11.4-20 um; arising from the subhymenium, extending through hymenium, projecting <52 um above the hymenium. Hyphidia subfusiform with moniliform apices, yellowish brown, abundant, thick-walled, 20-34 x 4.2-5.2 um; arising from the top of subhymenium and base of hymenium. Basidia narrowly clavate to subcylindrical, subhyaline, thin-walled, tetrasterigmate, 22-30 x 2.8-4.6 um; sterigmata <17.2 um long. PLATE 1. Hymenochaete longisterigmata (holotype, PUN - Navpreet & Dhingra 9689). 1. Basidiocarp showing hymenial surface; 2. Basidiospores; 3. Vertical section through basidiocarp showing: a. basidia; b. hyphidia; c. setae; d. subhymenial hyphae; 4. Subicular hyphae; 5. Basidium; 6. Setae. Hymenochaete longisterigmata sp. nov. (India) ... 633 ~S el [p> ow ty NX Ey. xX) [= S N/Zty YP “9 634 ... Kaur, Singh, Dhingra Basidiospores broadly ellipsoid, subhyaline, smooth, thin-walled, 4-5.1 x 2.8- 3.4 um, inamyloid, acyanophilous. ADDITIONAL SPECIMEN EXAMINED: INDIA, HIMACHAL PRADESH, Kullu, about 3 km from Jalori Pass towards Shoja, on branch of living tree of Quercus semecarpifolia Sm., 3 September 2016, Navpreet & Avneet 9688 (PUN). Discussion Hymenochaete (Hymenochaetaceae) is a widespread genus with c. 350 published species names and c. 150 currently accepted species (Kirk & al. 2008, Index Fungorum 2019, MycoBank 2019). The earliest record of this genus from India was by Montagne (1842), who described Stereum rheicolor [= H. rheicolor| from Gudalur, Nilgiri Hills, Tamil Nadu. Until our report, 40 Hymenochaete taxa were reported from India. Wagner & Fischer (2002) inferred phylogenetic relationships from DNA sequence data for several Hymenochaete species. Their phylogeny supporting traditional Hymenochaete species in two genera, they described the new genus Pseudochaete T. Wagner & M. Fisch. The two genera are not easily distinguished by morphology alone. The new species described here has not been sequenced and is therefore referred to Hymenochaete s.1. Acknowledgments Thanks are due to Head, Department of Botany, Punjabi University, Patiala, for providing research facilities and University Grants Commission, New Delhi, for financial support under UGC DRS-SAP, DSA-I programme. We also thank Dr. Nils Hallenberg (Professor Emeritus, University of Gothenberg) for expert comments and peer review and Prof. B.M. Sharma (Department of Plant Pathology, COA, CSKHPAU, Palampur, India) for peer review. Literature cited Bernicchia A, Gorjoén SP. 2010. Corticiaceae s.l. Fungi Europaei 12. Edizioni Candusso. Alassio, Italia. 1008 p. Cunningham GH. 1963. The Thelephoraceae of Australia & New Zealand. Bulletin of the New Zealand Department of Scientific and Industrial Research 145. 359 p. Dai YC. 2010. Hymenochaetaceae (Basidiomycota) in China. Fungal diversity 45: 131-343. https://doi.org/10.1007/s13225-010-0066-9 Index Fungorum. 2019. Search Index Fungorum. Accessed September, 2019: http://www.indexfungorum.org/names/Names.asp Kaur N, Sharma J, Singh AP, Dhingra GS. 2015. Additions to genus Hymenochaete Lév. from Himachal Pradesh. International Journal of Advanced Research 3(5): 836-843. Kirk PM, Cannon PEF, Minter DW, Stalpers JA. 2008. Ainsworth & Bisby’s dictionary of the fungi, 10" ed. CAB International, Wallingford, UK. 770 p. https://doi.org/10.1079/978085 1998268.0000 Hymenochaete longisterigmata sp. nov. (India) ... 635 Kornerup A,Wanscher JH. 1978. Methuen’s handbook of colour, 3rd ed. Methuen & Co. Ltd. London. 252p. Montagne JPFC. 1842. Cryptogamae Nilgherienses. Annales des Sciences Naturelles, Botanique sér., 18: 12-23. Mycobank. 2019. Hymenochaete. Website accessed 11/03/2019: http://www.mycobank.org/Biolomics.aspx? Table=Mycobank&Rec=56728&Fields=All Natarajan K, Kolandavelu K. 1998. Resupinate Aphyllophorales of Tamil Nadu, India. Centre for Advanced Study in Botany, University of Madras, Chennai. 133 p. Sharma JR, Mishra D. 2015. A synoptic mycoflora of wood-rotting fungi of Andaman. Nelumbo. 57: 1-30. Sharma JR. 1995. Hymenochaetaceae of India. Botanical Survey of India, Ministry of Environment & Forests, Calcutta. 219 p. Sharma JR. 2012. Aphyllophorales of Himalaya (Auriscalpiaceae - Tremellodendropsis). Botanical Survey of India, Ministry of Environment & Forests, Kolkata. 590 p. https://doi.org/10.20324/nelumbo/v57/2015/87118 Wagner T, Fischer M. 2002. Classification and phylogenetic relationships of Hymenochaete and allied genera of the Hymenochaetales, inferred from rDNA sequence data and nuclear behavior of the vegetative mycelium. Mycological Progress 1: 93-104. https://doi.org/10.1007/s11557-006-0008-9 MYCOTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 July-September 2020—Volume 135, pp. 637-647 https://doi.org/10.5248/135.637 Entoloma conferendum, Hygrocybe coccineocrenata, and Hypholoma ericaeum new to Montenegro ILINKA CETKOVIG ', ZDENKO TKALGEC 2’, SNEZANA DRAGICEVIG *, ANTUN ALEGRO 3, VEDRAN SEGOTA 3, MARGITA JADAN 2, NEVEN MATOCGEC ?, IVANA KUSAN 2, ZELJKO ZGRABLIG 4, ARMIN MESIG 2 ' Natural History Museum of Montenegro, Trg Vojvode Becir bega Osmanagica 16, 81000 Podgorica, Montenegro ? Ruder Boskovicé Institute, Bijenicka cesta 54, 10000 Zagreb, Croatia > Department of Botany, Faculty of Science, University of Zagreb, Marulicev trg 20/II, 10000 Zagreb, Croatia * Croatian Research Forest Institute, Research Centre for Forest Ecosystem Goods & Services Josip Ressel, 154. brigade Hrvatske vojske 2, 52000 Pazin, Croatia * CORRESPONDENCE TO: Ztkalcec@irb.hr ABSTRACT—As a result of the first research on macrofungi on peat moss habitats in Montenegro, three species new to the Montenegrin mycobiota are presented and described: Entoloma conferendum, Hygrocybe coccineocrenata, and Hypholoma ericaeum. The descriptions are accompanied by photographs of the basidiomata on site and some microscopic characters. Additionally, the second Montenegrin record of Bovista paludosa is presented and described, for the first time with locality and habitat data. Species identifications were checked by comparing ITS rDNA sequences from samples with the sequences in GenBank. Key worps—Agaricales, Basidiomycota, biodiversity, taxonomy Introduction Sphagnum (peat moss) is a genus with several hundred species that live in wet habitats and can store water in their cells. The most important habitats are peat bogs, where due to constant water availability and oxygen reduction, peat sediments form by the accumulation of the partially decomposed plant remnants. Peat bogs are characterised by acidic soils, low nutrient content, 638 ... Cetkovié & al. and dominance of peat mosses. These habitats, which form in humid and cold climates, are widely distributed in northern parts of Asia, Europe, and North America. However, despite a still favourable climate, natural peatlands are vanishing from northern Europe due to human activities such as peat extraction, drainage, and land development. In the western Balkans, primarily due to unfavourable climate conditions, peat bogs are exceptionally rare and relict habitats, occupying only small highly isolated areas. They are particularly sensitive to climate changes, which can cause them to dry out and become overgrown by other vegetation types. Therefore, peat bogs are one of the most endangered ecosystems in Montenegro. Some macrofungal species live exclusively in this type of habitat, but a certain number of fungi with a wider habitat tolerance can also be found in peat bogs. Peat mosses can also live in wet, acidic habitats without forming peat, mostly in coniferous forests. Due to the small number of researchers, Montenegrin fungi, including agarics, are rather poorly explored. Kasom (2013) lists 839 macro-basidiomycete species for Montenegro, 444 of which belong to the Agaricales. Bubak (1915), who conducted the first mycological research on Montenegrin peat bogs in 1904, reported several microfungi from the Barno jezero peat bog (Mt. Durmitor). However, until our research, no macrofungi had been reported in peat bogs of Montenegro. During the summer and autumn of 2017 and 2018 we surveyed macrofungi of mountain peat bogs and peat moss carpets on Mt. Sinjajevina, Mt. Durmitor, and Mt. Hajla. We describe three Agaricales species new to Montenegrin mycobiota, as well as one previously reported rare species with the first data on its locality and habitat in Montenegro. Materials & methods Collected basidiomata were photographed on the site, described, preserved by drying and deposited in the mycological collections of Natural History Museum of Montenegro, Podgorica (NHMP) and/or Croatian National Fungarium in Zagreb (CNE). The morphological descriptions are entirely based on the collected material. Microscopic features are described from dried material mounted in 2.5% potassium hydroxide (KOH) and observed with a light microscope under magnification up to 1500x. Basidiospore measurements were made by Motic Images Plus 2.0 software on 30 randomly chosen mature basidiospores from calibrated digital images. Arithmetic means of basidiospore length and width are shown in italic font in the centre of the measurements. The length/width ratio of all measured basidiospores is given as the “Q” value (min. — av. — max.). Genomic DNA was extracted from dried specimens using DNeasy Plant Mini kit (Qiagen) according to the manufacturer’s instructions. PCR primers ITS1F and Three agarics newly recorded for Montenegro ... 639 ITS4 (Gardes & Bruns 1993) were used for ITS region amplification and sequencing purposes. The ITS rDNA regions were amplified in a thermocycler (Eppendorf) with a final volume of 25 uL containing approximately 50 ng DNA template, 0.2 uM of each primer, 0.2 mM dNTP, 1.5mM MgCl, 1x buffer, and 1 unit of Taq polymerase (Invitrogen). The cycling protocol was: 94 °C for 5 min, 30 cycles (94 °C for 45, 60 °C for 45 s, 72 °C for 45 s), followed by a final extension of 72 °C for 10 min. The PCR products were resolved on a 1% agarose gel stained with ethidium bromide and viewed under ultraviolet light. PCR products were purified and sequenced by Macrogen Inc. (The Netherlands). Sequences were aligned in both directions and edited using Sequencher v.4.1.4 and submitted to GenBank. Taxonomy Hygrocybe coccineocrenata (P.D. Orton) M.M. Moser, Rohrlinge & Blatterpilze, 3. Aufl.: 68 (1967). Fic. 1 A,B PILEUS 6-23 mm broad, convex at first, later applanate to concave with a depressed centre and mostly inflexed and somewhat crenate margin, scarlet red, orange red or reddish orange, densely covered with small squamules or tufts which are reddish brown to grey towards the centre and concolorous towards the margin, not translucently striate, dry. LAMELLAE decurrent, distant, broad, white when young, wax yellow at maturity. STIPE 25-70 x 2-5 mm, cylindrical or tapering downwards, scarlet red to orange red, orange near the base, whitish in the base, glabrous, hollow, dry. CONTEXT thin, pale orange red. SMELL and TASTE indistinct. SPORE-PRINT white. BASIDIOSPORES 9.2-11-12.1 x 5.3-6.1-6.8 um, Q = 1.51-1.81-2.13, ellipsoid to oblong, often slightly phaseoliform in side view, sometimes with slight median constriction, thin-walled, hyaline. Basip1a 4-spored, narrowly clavate. CysTIDIA absent. PILEIPELLIS at centre and in squamules a trichoderm composed of ascending hyphae constricted at septa, with brown intracellular pigment. CLAMP CONNECTIONS present. SPECIMEN EXAMINED—MONTENEGRO, KOoLaSIN MUNICIPALITY, Mt. Sinjajevina, Semolj saddle, 42.9092°N 19.2731°E, 1580 m as.l, in peat bog, among dense Sphagnum subsecundum Nees peat moss with Carex lasiocarpa Ehrh. sedge, 18 July 2017, leg. S. Dragicevi¢ (NHMP 368/6986; GenBank MN082022). ECOLOGY & DISTRIBUTION—his waxcap occurs mostly in peat bogs among or near peat moss, but also in wet meadows. Widespread (but generally rare) in Europe, known also from North America and Asia (Tkalc¢ec & al. 2008, Boertmann 2010). In the surrounding countries, H. coccineocrenata is recorded in Croatia and Serbia, and it is a strictly protected species in both countries. New to Montenegro. 640 ... Cetkovié & al. CoMMENTS—Hygrocybe coccineocrenata is characterised by decurrent lamellae, predominantly red pileus and stipe (at least in young basidiomata), darker small squamules on the pileal surface, and by living in open, wet habitats (mostly peat bogs). It has often been confused with H. turunda (Fr.) P. Karst., which also has darker squamules on pileus, but which differs in its orange to orange yellow pileus and stipe ground colours and growth in rather dry grasslands and heathlands (Boertmann 2010). Our material fits well with the modern descriptions (e.g., Boertmann 2010, Ludwig 2012) of H. coccineocrenata. The GenBank BLAST analysis shows that our ITS sequence is identical with one unpublished sequence (FM208899, as “H. turunda;” Query Cover 87%), and very close to another sequence from the same authors (FM208865, Identity > 99%, Query Cover 87%; deposited as “H. cantharellus, but quite different from the other H. cantharellus (Schwein.) Murrill sequences in the database). We assume that both sequences were derived from misidentified samples. Our Montenegrin sequence is the only GenBank entry deposited as H. coccineocrenata. Hypholoma ericaeum (Pers.) Kihner, Bull. Trimest. Soc. Mycol. Fr. 52: 23 (1936) FIG. 1 C-G PiLEUs 17-60 mm broad, paraboloid to hemispherical at first, later convex to plano-convex, often subumbonate or with central papilla, hygrophanous, not translucently striate, brown to dark reddish brown when moist, orange brown to ochre brown on drying, often paler near margin in young basidiomata, surface dry to greasy, often with fine, whitish, fibrillose veil remnants near margin in young basidiomata. LAMELLAE narrowly adnate, subventricose to ventricose, moderately crowded, broad, pale brownish grey when young, then mottled grey brown, finally mottled dark purplish brown, edge whitish. STIPE 35-78 x 2-7 mm, subcylindrical, often with broadened base (up to 10 mm), solid to fistulose, pale to medium brown, whitish at apex in young basidiomata, surface dry, pruinose at apex, fibrillose-furfuraceous below, mostly with white basal tomentum. CONTEXT brown in the narrow zone beneath the pileus surface and above the lamellae, pale brown in the inner part of the pileus, whitish to dark red brown in the stipe. SMELL fungoid. TasTE slightly bitter. SPORE-PRINT violaceous brown. BASIDIOSPORES 12.7—13.7-15 x 7.5-8.2-8.9 um, Q = 1.56-1.67-1.83, ellipsoid, moderately thick-walled to thick-walled, smooth, with distinct and central germ-pore, yellow brown in KOH, light red brown in H,O. Basrp1a 4-spored, narrowly clavate or Three agarics newly recorded for Montenegro ... 641 Fic. 1. Hygrocybe coccineocrenata (NHMP 368/6986). A. basidiomata; B. basidiospores. Hypholoma ericaeum (NHMP 614/9034). C. basidiomata; D. basidiospores; E. cheilocystidia; FE, G. pleurocystidia. Scale bars: A = 10 mm; C = 20 mm; B, D-G = 10 um. 642 ... Cetkovié & al. with median constriction. CHEILOCYSTIDIA (leptocystidia type) densely packed, forming a sterile lamellar edge, 20-45 x 5-12 um, lageniform, narrowly utriform or subcylindrical (often with somewhat broadened apex), sometimes forked, thin-walled, hyaline. PLEUROCYSTIDIA (chrysocystidia type) abundant, 30-56 x 11-18 um, clavate, mostly with mucronate apex, thin- to moderately thick-walled, hyaline with yellowish amorphous body in alkali solution. PILEIPELLIS a cutis composed of thin- walled, hyaline, repent hyphae. CLAMP CONNECTIONS present. SPECIMENS EXAMINED—MONTENEGRO, KOLASIN MUNICIPALITY, Mt. Sinjajevina, Semolj saddle, 42.9074°N 19.2758°E, 1572 ma.s.L, among Sphagnum moss and grass on the edge of the peat bog, 15 September 2018, leg. Z. Tkaléec & al. (CNF 1/7623, NHMP 614/9034; GenBank MN082025); (CNF 1/7628). ECOLOGY & DISTRIBUTION— The species is saprotrophic and occurs on peaty acid soil (mostly among mosses) and in wet poor grasslands and moist heaths (Watling & Gregory 1987, Noordeloos 2011, Vesterholt & Rald 2012). It is widespread all over the boreal and temperate regions of Europe and North America (Noordeloos 2011), but usually (rather) rare. In the surrounding countries, H. ericaeum is recorded only in Croatia (Tkaléec & MeSi¢ 2003). New to Montenegro. CoMMENTS—Hypholoma ericaeum is characterised by long basidiospores (>13 um) and a hygrophanous pileus that is neither translucently striate nor viscid. Only three other European Hypholoma species have similarly long basidiospores (H. eximium (C. Laest.) Rald, H. myosotis (Fr.) M. Lange, and H. udum (Pers.) Quél.), but their spore lengths reach at least 17 um and their pilei are viscid or subviscid. Our collections correspond relatively well with recent descriptions of H. ericaeum (Watling & Gregory 1987, Ludwig 2001, Noordeloos 2011, Vesterholt & Rald 2012). However, the maximum stipe width of 7 mm exceeds the 4.5 mm maximum reported by Watling & Gregory (1987), the maximum cheilocystidia width of 12 um exceeds the 7.5 um maximum reported by Noordeloos (2011), and the maximum pleurocystidia size of 56 x 18 um exceeds the 45 x 15 um maximum reported by Noordeloos (2011). Moreover, Watling & Gregory (1987) described distinctly narrower cheilocystidia (30-40 x 4-5.5 um) and pleurocystidia (30-40 x 8-10 um). Noordeloos (2011) described somewhat flattened basidiospores, but the spores in our collections and in the descriptions of other cited authors are not flattened. According to the BLAST analysis, our ITS sequence does not match any sequence in GenBank database. Three agarics newly recorded for Montenegro ... 643 The closest sequence (96.91% identity) is derived from H. dispersum Quél. (HQ604746). The only GenBank ITS sequence annotated as H. ericaeum (MH856009) is genetically much more distant (84% identity). It is possible that MH856009 was sequenced from a misidentified sample, but it is also possible that two morphologically close species are not yet recognized as different; further research is required. Entoloma conferendum (Britzelm.) Noordel., Persoonia 10: 446 (1980) FIG. 2 A,B = Entoloma nothofagi G. Stev., Kew. Bull. 16: 234 (1962) = Entoloma staurosporum (Bres.) E. Horak, Sydowia 28: 222 (1976) PiLEus 10-30 mm broad, paraboloid at first, expanding to plano-conical or plano-convex with subumbonate centre, hygrophanous, brown with black centre when young, later greyish brown at centre and gradually fading to light brown towards the margin when moist, pale brownish when dry, translucently striate up to 2/3 of the radius when moist, surface dry, glabrous. LAMELLAE free, ventricose, broad, moderately crowded, white when young, pink at maturity. StrpE 35-85 x 2-4 mm, cylindrical with slightly to distinctly broadened base, densely silvery striate on pale to light grey brown background, dry. CoNTExT thin and fragile, light grey brown. SMELL and TASTE farinaceous. SPORE-PRINT dirty pink. BAsIDIosPORES 9.3-10.8-11.8 x 8.1-9.4-10.4 um, Q = 0.95-1.15-1.31, cruciform-stellate, thin-walled, hyaline to pinkish. Basrp1A 4-spored, narrowly clavate. CysT1p1A absent. PILEIPELLIS a cutis with transition to a trichoderm at centre, with intracellular brown pigment. CLAMP CONNECTIONS absent. SPECIMEN EXAMINED—MONTENEGRO, Rozaje MuNICcIPALITY, Mt. Hajla, right bank of the Ibar river, 42.7981°N 20.1083°E, 1170 m a.s.l, on dense carpet of peat moss Sphagnum quinquefarium (Lindb.) Warnst., on very steep (almost vertical) siliceous rocks, surrounded by mixed forest of spruce (Picea abies), fir (Abies alba) and beech (Fagus sylvatica), 7 September 2017, leg. A. Alegro (NHMP 426/7230; GenBank MN082023). ECOLOGY & DISTRIBUTION— This species occurs in various habitats from the lowlands to arcto-alpine zone, including grasslands, marshes, peat bogs, damp places in deciduous and coniferous forests. It is a very common saprotroph with a wide distribution, including most temperate zones on both hemispheres (Noordeloos 1992). In the surrounding countries, E. conferendum is recorded only in Croatia (Mesi¢ & Tkaléec 2003). New to Montenegro. CoMMENTS—Entoloma conferendum is characterised by a_ brown translucently striate pileus, cruciform-stellate basidiospores (unique among 644. ... Cetkovié & al. European Entoloma species), and absence of cystidia and clamp connections. Noordeloos & Gates (2012) place it in E. sect. Staurospora in E. subg. Nolanea with species having predominantly cuboid basidiospores. Entoloma staurosporum is currently accepted as a synonym of E. conferendum. Horak (1976, 1980) synonymised E. nothofagi, described from New Zealand, with E. staurosporum, but subsequently reverted to his original opinion (Horak 1971, 1973), treating E. nothofagi as a separate species (Horak 2008). Noordeloos & Gates (2012), who later revised the type of E. nothofagi, found only minimal morphological differences from E. conferendum, and therefore regarded the two species as probably conspecific. This conclusion is supported by our GenBank BLAST analysis showing the RPB2 sequence of Slovakian collection of E. conferendum (KC710191) as almost identical with the RPB2 sequence of E. nothofagi from New Zealand (MH190134), with only a single base pair difference between them. There is no ITS sequence available for E. nothofagi. Another similar species is E. brevispermum G.M. Gates & Noordel. from Australia, which differs by shorter (<9 um long) basidiospores. Our material fits well with the modern descriptions of the species (Noordeloos 1992, Ludwig 2007, Noordeloos & Gates 2012), and our ITS sequence matched well (identity > 99%) with most E. conferendum sequences in GenBank. Bovista paludosa Lév., Ann. Sci. Nat., Bot., Sér. 3, 5: 163 (1846) FIG. 2 Cc, D BASIDIOMATA pyriform, 23-48 mm high and 16-24 mm broad, sometimes with white rhizoids. ExoPERIDIUM when young white and smooth or with small, very thin, adherent, greyish, polygonal patches, later breaking up into irregular, rather large plates. ENDOPERIDIUM smooth, white at first, becoming brown to olivaceous. GLEBA white when young, then yellowish brown, becoming olive brown at maturity. SUBGLEBA well developed, <15 mm high, compact, white when young, olive to grey brown at maturity. SPORE-PRINT Olive brown. BASIDIOSPORES 4.6-5.1-5.6 x 4.2-4.7-5.4 um, Q = 1-1.07-1.19, globose to subglobose (rarely broadly ellipsoid), almost smooth to asperulate, moderately thick-walled, pale yellow brown in KOH, with sterigmal remnant <15 um long. CAPILLITIUM Bovista-type, non- poroid, thick-walled, rarely septate. SPECIMEN EXAMINED—MONTENEGRO, ZABLJAK MUNICIPALITY, Mt. Durmitor, Barno jezero lake, 43.1582° N, 19.0932° E, 1495 m a.s.l, among Aulacomnium palustre (Hedw.) Schwagr., Bryum pseudotriquetrum (Hedw.) P. Gaertn. & al., and Campylium stellatum (Hedw.) C.E.O. Jensen mosses on the outer zone of the peat bog (less acidic), 9 July 2017, leg. I. Cetkovié (NHMP 425/7229; GenBank MN082024). Three agarics newly recorded for Montenegro ... 645 Fic. 2. Entoloma conferendum (NHMP 426/7230). A. basidiomata; B. basidiospores. Bovista paludosa (NHMP 425/7229). C. basidiomata; D. basidiospores. Scale bars: A, C = 10 mm; B, D= 10 um. ECOLOGY & DISTRIBUTION— This puffball lives as a saprotroph in a rather wide range of open wet mossy habitats, most often in alkaline to neutral fens, bogs, mires, wet meadows, and heaths, but avoiding oligotrophic and strongly acidic mires (Kaltucka 2019). The species is rather widespread but rare in Europe (Red Listed in most countries where reported), known also from Asia (China, Russia, India) and North America (Canada) (Jeppson 2018). The only earlier 646 ... Cetkovié & al. Montenegrin record of B. paludosa from 1904 (Bubak 1915) was published without locality and habitat data. In the surrounding countries, it is recorded only in Serbia (Katucka 2019). COMMENTS—Bovista paludosa is characterised by its occurrence on wet mossy habitats and by the presence of a well-developed subgleba, long sterigmal remnants on basidiospores, and a Bovista-type capillitium lacking pores. The most similar species, B. cretacea T.C.E. Fr., differs in a very poorly developed (<2 mm high) subgleba and regularly septate capillitium. Our material agrees with the modern descriptions (Pegler & al. 1995, Sarasini 2005, Jeppson 2018). According to the BLAST analysis, our ITS sequence matched well with all three sequences of B. paludosa in GenBank database (Identity > 99%). Acknowledgements We are very grateful to Dr. André Fraiture (Botanic Garden Meise, Belgium) and Dr. Vladimir Antonin (Moravian Museum, Brno, Czech Republic) for their critical review of the manuscript. The research received financial support from the Montenegrin Ministry of Science and Croatian Ministry of Science and Education through the project “Inventory of biodiversity and conservation status of the representative peat bogs on the area of Montenegro and Croatia.” Also, this work has been partially supported by Croatian Science Foundation under the project ForFungiDNA (IP-2018-01-1736). Literature cited Boertmann D. 2010. The genus Hygrocybe, 2nd revised edition. Fungi of Northern Europe 1. Svampetryk, Tilst. Bubak FE. 1915. Dritter Beitrag zur Pilzflora von Montenegro. Botanikai K6zlemények 14(3-4): 39-83. Gardes M, Bruns TD. 1993. ITS primers with enhanced specificity for 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 Horak E. 1971. A contribution towards the revision of the Agaricales (Fungi) from New Zealand. New Zealand Journal of Botany 9: 403-462. https://doi.org/10.1080/0028825x.1971.10430193 Horak E. 1973. Fungi agaricini Novazelandiae I-V. Beihefte zur Nova Hedwigia 43. J. Cramer, Lehre. Horak E. 1976. On cuboid-spored species of Entoloma (Agaricales). Sydowia 28: 171-236. Horak E. 1980. Entoloma (Agaricales) in Indomalaya and Australasia. Beihefte zur Nova Hedwigia 65. J. Cramer, Vaduz. Horak E. 2008. Agaricales of New Zealand 1: Pluteaceae - Entolomataceae. The fungi of New Zealand 5. Fungal Diversity Research Series, vol. 19. Fungal Diversity Press, Hong Kong. Jeppson M. 2018. Puffballs of northern and central Europe. SMF, Goteborg. Katucka IL. 2019. Bovista paludosa Lév. The Global Fungal Red List. Viewed online on 22 May 2019: http://iucn.ekoo.se/iucn/species_view/209589/ Three agarics newly recorded for Montenegro ... 647 Kasom G. 2013. Basidiomycota macrofungi of Montenegro. PhD ‘Thesis, University of Montenegro, Faculty of Science and Mathematics, Podgorica. (in Montenegrin). https://fedora.ucg.ac.me/fedora/get/0:334/bdef:Content/get (viewed online on 22 May 2019) Ludwig E. 2001. Pilzkompendium, Band 1. IHW-Verlag, Eching. Ludwig E. 2007. Pilzkompendium, Band 2. Fungicon-Verlag, Berlin. Ludwig E. 2012. Pilzkompendium, Band 3. Fungicon- Verlag, Berlin. MeSi¢ A, Tkaléec Z. 2003. Preliminary checklist of Agaricales from Croatia IV: Families Bolbitiaceae, Coprinaceae, Entolomataceae and Pluteaceae. Mycotaxon 87: 283-309. Noordeloos ME. 1992. Entoloma s.l. Fungi Europaei 5. Libreria editrice Giovanna Biella, Saronno. Noordeloos ME. 2011. Strophariaceae s.1. Fungi Europaei 13. Edizioni Candusso, Alassio. Noordeloos ME, Gates GM. 2012. The Entolomataceae of Tasmania. Springer, New York - London. https://doi.org/10.1007/978-94-007-4679-4 Pegler DN, Lzessge T, Spooner BM. 1995. British puffballs, earthstars and stinkhorns. Royal Botanic Gardens, Kew. Sarasini M. 2005. Gasteromiceti epigei. A.M.B., Trento. Tkaléec Z, Mesi¢ A. 2003. Preliminary checklist of Agaricales from Croatia V: Families Crepidotaceae, Russulaceae and Strophariaceae. Mycotaxon 88, 279-314. Tkaléec Z, MeSi¢ A, Matocéec N, KuSan I. 2008. Red book of Croatian fungi. Ministry of Culture - State Directorate for Protection of Nature, Zagreb (in Croatian). Vesterholt J, Rald E. 2012. Hypholoma (Fr.) P. Kumm. 942-946, in: H Knudsen, J Vesterholt (eds). Funga Nordica, Nordsvamp, Copenhagen. Watling R, Gregory NM. 1987. Strophariaceae & Coprinaceae p.p.: Hypholoma, Melanotus, Psilocybe, Stropharia, Lacrymaria and Panaeolus. British Fungus Flora: Agarics and Boleti 5. Royal Botanic Garden, Edinburgh. MYCOTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 July-September 2020—Volume 135, pp. 649-655 https://doi.org/10.5248/135.649 First Pakistani report of Erysiphe betae on the invasive weed Chenopodium ambrosioides AYESHA ANWAR™, NAJAM UL SEHAR AFSHAN’, AAMNA ISHAQ3, MarRIA RIAz?, ABDUL NASIR KHALID’, SIRAJ UDDIN' ' Department of Botany, University of Peshawar, 25120, Pakistan ? Department of Botany, University of the Punjab, Quaid-e-Azam Campus, Lahore, 54590, Pakistan > Department of Botany, Faculty of Fisheries and Wildlife, University of Veterinary and Animal Sciences-Lahore, Ravi Campus, Pattoki, 55300, Pakistan " CORRESPONDENCE TO: Ayeshaanwar556@gmail.com AxssTRACT—During September-October 2017, powdery mildew symptoms were observed on both surfaces of leaves of Chenopodium ambrosioides in Abbottabad, Malakand, and Upper Dir districts of Pakistan. The causal agent was identified as Erysiphe betae, based on its asexual morphology and chasmothecia, and its identity was confirmed by molecular data. This is the first Pakistani report of Erysiphe betae on this host. Key worps—Amaranthaceae, Erysiphaceae, mexican tea, new record Introduction Fungi belonging to Erysiphales are widely distributed all over the world and cause serious economic damage on wild and cultivated plants. This order comprises the single family Erysiphaceae (powdery mildews) represented by 17 genera and c. 820 species (Braun & Cook 2012). Powdery mildews infect 9839 angiosperm species in 1617 genera, 196 families, and 44 orders (Amano 1992, Takamatsu 2004), including numerous economically important plants. Various powdery mildews may cause serious economic damage on wild and cultivated plants in Pakistan (Burni & al. 2010, Mukhtar & al. 2012, 650 ... Anwar & al. 2013). Nineteen of the 56 powdery mildew species reported from Pakistan represent the most common genus, Erysiphe, which infects 52 hosts in 19 plant families (Ahmad & al. 1997, Burni & al. 2010, Mukhtar & al. 2013). This includes Erysiphe betae on Chenopodium botrys L. and Spinacia oleracea L. (Amaranthaceae; Amano 1986). In 2017, a powdery mildew appeared on the leaves of Chenopodium ambrosioides in the districts Abbottabad, Malakand, and Upper Dir, Pakistan. The infected leaf surfaces were fully covered with white powdery mycelial masses, asexual conidia, and sexual chasmothecia indicating progressed stages of infection and severity. Materials & methods Sample collection During phytopathological surveys during September-October 2017, we observed powdery mildew infections on leaves and stems of Chenopodium ambrosioides in the districts Abbottabad, Malakand, and Upper Dir, Khyber Pakhtunkhwa, Pakistan. The infected plants were shade dried on blotting paper and protected in brown envelopes for future use. The specimens are deposited at the herbarium of the University of Peshawar, Khyber Pakhtunkhwa, Pakistan (PUP) and Department of Botany, University of the Punjab, Lahore, Punjab, Pakistan (LAH). Morphology We first examined infected leaves under a Labomed CSM2 stereomicroscope, and then prepared using lactic acid. We examined the hyphae on the host; hyphal appressoria; conidial, conidiophore, and chasmothecial shapes and sizes; and ascospores and asci under a Nikon YS 100 microscope. Measurements (20 repetitions per structure) were recorded using a Lomo Filar Eyepiece Micrometer AM9-2-15x on a Zeiss microscope. Micrographs were made using a HDCE-5X digital camera. DNA extraction, PCR amplification, phylogenetic analysis Dried powdery mildew infections were scraped off from fresh fungal specimens with sterile razor blades, ground in liquid nitrogen, and stored in Eppendorf tubes at -18 °C. DNA was extracted using GeneJET Plant Genomic DNA Purification Mini Kit #K0791 according to the manufacturer’s instructions. The Internal Transcribed Spacer (ITS) region was amplified using PMITS1/PMITS2 primers (Cunnington & al. 2003) and then commercially sequenced by Tsingke in China. Raw sequence data were edited using BioEdit (Hall 1999). The ITS sequences were BLAST searched against the GenBank database (www.ncbi.nlm.nih.gov) and aligned using Muscle E multiple alignment tool within MEGA v. 7.0 (Kumar & al. 2016). Twenty ITS sequences were analyzed using the maximum likelihood (ML) method based on the Tamura 3-parameter model (Tamura 1992). The initial trees for the heuristic search were obtained by applying the Neighbor-Joining method to a matrix of pairwise Erysiphe betae new on Chenopodium ambrosioides in Pakistan ... 651 KY660905 Erysiphe ludens 35| KY660901 Erysiphe ludens KY660739 Erysiphe trifoliorum KY660903 Erysiphe trifoliorum KJ845646 Erysiphe sp. KJ845645 Erysiphe sp. KY926844 Erysiphe castaneigena 59 89' KY926843 Erysiphe castaneigena KY660931 Erysiphe cruciferarum KY660884 Erysiphe cruciferarum KY660750 Erysiphe pisi LC270861 Erysiphe viciae-unijugae 79 KY660922 Erysiphe buhrii KY660881 Erysiphe buhrii 82] | @ MN368297 Erysiphe betae 71'LC009946 Erysiphe betae MG938639 Erysiphe berberidicola MG938640 Erysiphe berberidicola AB080464 Phyllactinia angulata LC108847 Phyllactinia leveilluloides 67 0.05 Fic. 1. Phylogenetic analysis of the ITS region for 18 Erysiphe sequences, with Phyllactinia angulata and P. leveilluloides as outgroup. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model. The tree with the highest log likelihood (-470.6292) is shown, and the amplified sequence from Pakistan is denoted by ¢. distances estimated using the Maximum Composite Likelihood (MCL) approach. Evolutionary analyses were conducted in MEGA6 (Tamura & al. 2013). in the phylogenetic analyses. After elimination of gaps and missing data, the final dataset comprised 134 positions, and Phyllactinia angulata (E.S. Salmon) S. Blumer and P leveilluloides Moreno-Rico & U. Braun served as outgroup. All sequences were aligned and trimmed at conserved sites from both 5’ and 3’ ends. The phylogenetic tree with the highest log likelihood (-470.6292) is shown in Fic. 1; the percentage of trees in which the associated taxa clustered together is shown next to the branches, and the tree is drawn to scale, with branch lengths measured in the number of substitutions per site. 652 ... Anwar & al. Fic. 2. Erysiphe betae (PUP Bot.01). A. Infected plant, Chenopodium ambrosioides; B. Fungal mycelium under stereomicroscope; C. Conidia; D. Conidiophore; E. Chasmothecium; E Germinating Conidium; G. Asci with ascospores; H. Ascospores; I. Chasmothecial appendage. Seale‘bars: A= 1 cm, B =2am,'C, D, BG= 15pm; EH, L = 10 pm. Erysiphe betae new on Chenopodium ambrosioides in Pakistan ... 653 Taxonomy Erysiphe betae (Vanha) Weltzien, Phytopathol. Z. 47: 127 (1963) BIG 72 Mycelium amphigenous, forming dense, thick, white patches; hyphae hyaline, smooth, thin-walled; hyphal appressoria nipple-shaped to somewhat lobed, 2-5 um diam. Conidiophores arising from the top of mother cells, 43-87 x 3.5-7.5 um; foot cells cylindrical, straight or sometime curved, 22-61 x 4.5-8 um, constricted at the basal septum, followed by 1-2 shorter cells; conidia formed singly, ellipsoid-ovoid to cylindrical-doliiform, 14.5-51 x 7-12 um; germ tubes arising from one end, tips slightly swollen; Chasmothecia scattered to gregarious, globose to subglobose, light to dark brown, 48.5-104 um diam.; peridium cells small, 12-27 um diam., irregularly polygonal; appendages numerous, mycelioid, irregularly branched, septate, thin-walled, at first hyaline but pigmented when chasmothecia mature, 29-119 x 2.5-4.5 um; asci 3-5, ellipsoid-obovoid, 28-49.5 x 19.5-29.5 um, stalked, 4-5-spored; ascospores globose-ellipsoid to ovoid, 8.5-18.5 x 5.5-10.5 um, colorless. SPECIMENS EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Malakand Division, Malakand district, 844 ma.s.l., on Chenopodium ambrosioides L. (Amaranthaceae), 19 September 2017, asexual morph, A. Anwar AA#4 (PUP Bot.01; GenBank MN368297); Upper Dir district, 844 m a.s.l., on C. ambrosioides, 29 October 2017, asexual and sexual morphs, A. Anwar AA#5 (PUP Bot.02); Hazara Division, Abbottabad district, Ayubia National Park, 2438 m a.s.l, on C. ambrosioides, 30 October 2017, asexual morph, N.S. Afshan NSA#34 (LAH 35658). Phylogenetic results The NCBI BLASTn analysis showed that our ITS sequence (MN368297 Erysiphe betae) closely matched (99.54% identity) a sequence of E. betae (LC009946) on Beta vulgaris from Japan (Takamatsu & al. 2015). The 20 nucleotide sequences used in the phylogenetic analysis included Phyllactinia angulata (AB080464) and P leveilluloides (LC108847) as outgroup. The final aligned data set contained 818 positions of which 567 were conserved, 202 variable and parsimony uninformative, and 49 parsimony informative sites. Maximum likelihood (ML) analysis clustered the Pakistan E. betae collection (MN368297) with the Japanese E. betae collection (LC009946) with a 71 bootstrap value. Based on these results, we assigned the name Erysiphe betae to the powdery mildew pathogen on Chenopodium ambrosioides in Pakistan. 654 ... Anwar & al. Discussion Worldwide, three Erysiphe spp. (including E. betae), two Leveillula spp., and three anamorphic powdery mildew species have been reported on Chenopodium ambrosioides (Fungus-Host Database https://nt.ars-grin.gov, 29 May, 2018, Braun & Cook 2012). From Pakistan, only “Erysiphe communis (Wallr.) Schltdl.” (nom. rej.) on Chenopodium botrys and Spinacia oleracea and Leveillula cylindrospora U. Braun and L. taurica (Lév.) G. Arnaud on Chenopodium murale L. have been reported. Chenopodium ambrosioides is reported here as a new host record for Erysiphe betae in Pakistan. Previously, only asexual morphs of Erysiphe have been reported on Chenopodium species from Asia and Europe, whereas sexual fruiting bodies (chasmothecia) have been reported only from India and Japan (Braun & Cook 2012). We now report both asexual and sexual morphs of Erysiphe betae from Pakistan. We found only the asexual morph in the Abbottabad and Malakand districts, but additionally collected the chasmothecia in the Upper Dir district. Previous reports from Pakistan of Erysiphe on Chenopodium botrys (as “E. communis;” Amano 1986) is probably allocable to E. betae. Francis & al. (2007), who analysed ITS rDNA sequences retrieved from European (UK) and North American (USA) collections of E. betae on sugar beet, confirmed the involvement of a single recognizable Erysiphe species more closely allied to Erysiphe heraclei DC. than to E. polygoni DC. Takamatsu & al. (2015) included in their phylogenetic analyses a sequence obtained from E. betae on Beta vulgaris L. with corresponding results, but sequences retrieved from E. betae on Chenopodium ambrosioides are not yet available and still in need of molecular confirmation. Our research represents the first phylogenetic analysis of E. betae on Chenopodium ambrosioides, which clusters our ITS sequence in a strongly supported clade with a sequence of E. betae on Beta vulgaris (Fic. 1). Acknowledgements We are really thankful to Prof. Dr. Uwe Braun (Martin-Luther-Universitat, Institut fiir Biologie, Germany) for his help in the identification of Erysiphe betae on Chenopodium ambrosioides and peer review of the manuscript. The authors are also thankful to Dr. Abdul Rehman Khan Niazi, Department of botany, University of Punjab, Lahore, for his valuable suggestions to improve this manuscript. Literature cited Ahmad §, Iqbal SH, Khalid AN. 1997. Fungi of Pakistan. Sultan Ahmad Mycological Society of Pakistan, Lahore, PK. Amano K. 1986. Host range and geographical distribution of the powdery mildew fungi. Japan Scientific Societies Press, Tokyo. Erysiphe betae new on Chenopodium ambrosioides in Pakistan ... 655 Amano K. 1992. Notes on the host range and geographical distribution of Podosphaera. Transactions of the Mycological Society of Japan 33: 139-148. Braun U, Cook RTA. 2012. Taxonomic manual of the Erysiphales (powdery mildew). CBS Biodiversity Series 11. 707 p. Burni T, Jamil F, Jabeen M. 2010. Erysiphales of Peshawar valley, Khyber Pakhtunkhwa, Pakistan. Pakistan Journal of Plant Sciences 16(1): 11-14. Cunnington JH, Takamatsu S, Lawrie AC, Pascoe IG. 2003. Molecular identification of anamorphic powdery mildews (Erysiphales). Australasian Plant Pathology 32: 421-428. https://doi.org/10.1071/AP03045 Francis SA, Roden BC, Adams MJ, Weiland J, Asher MJ. 2007. Comparison of ITS sequences from UK and North American sugar-beet powdery mildews and the designation of Erysiphe betae. Mycological Research 111(2): 204-212. https://doi.org/10.1016/j.mycres.2006.10.010 Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95-98. https://doi.org/10.1017/S0953756203008517 Kumar S, Stecher G, Tamura K. 2016. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33(7): 1870-1874 https://doi.org/10.1093/molbev/msw054. Mukhtar I, Khurram R, Hannan A, Hayat Z. 2012. First report of powdery mildew of Cucurbita moschata Duch. caused by Golovinomyces cichoracearum DC. in Neelum Valley, Azad Kashmir. Plant Disease 96(6): 906. https://doi.org/10.1094/PDIS-08-11-0642-PDN Mukhtar I, Mushtaq S, Khokhar I. 2013. First report of powdery mildew on Dahlia (Dahlia variabilis) caused by Golovinomyces cichoracearum in Lahore, Pakistan. Australasian Plant Disease Notes 8(1): 1-3. https://doi.org/10.1007/s13314-011-0025-7 Takamatsu S. 2004. Phylogeny and evolution of the powdery mildew fungi (Erysiphales, Ascomycota) inferred from nuclear ribosomal DNA sequences. Mycoscience 45: 147-157. https://doi.org/10.1007/s10267-003-0159-3 Takamatsu S, Ito H, Shiroya Y, Kiss L, Heluta V. 2015. First comprehensive phylogenetic analysis of the genus Erysiphe (Erysiphales, Erysiphaceae) 1. The Microsphaera lineage. Mycologia 107: 475-489. https://doi.org/10.3852/15-007 Tamura K. 1992. Estimation of the number of nucleotide substitutions when there are strong transition-transversion and G + C-content biases. Molecular Biology and Evolution 9: 678-687. https://doi.org/10.1093/oxfordjournals.molbev.a040752 Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution 30: 2725-2729. https://doi.org/10.1093/molbev/mst197 MYCOTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 July-September 2020—Volume 135, pp. 657-663 https://doi.org/10.5248/135.657 Crustose lichens new to India RUPJYOTI GOGOT’, SILJO JOSEPH?, MANAS PRATIM CHOUDHURY’, SANJEEVA NAYAKA’, FARISHTA YASMIN’* ' Department of Botany, Nowgong College, Nagaon, Assam - 782001, India ? Lichenology Laboratory, CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow - 226001, India * CORRESPONDENCE TO: farishtayasmin4rs@gmail.com ABSTRACT—Bacidia pycnidiata (Ramalinaceae), Malmidea nigromarginata (Malmideaceae), Porina malmei and P. nuculastrum (Porinaceae), and Pyrenula laetior and P. wrightii (Pyrenulaceae) are reported for the first time from India. The specimens were collected from the state of Assam. Taxonomic descriptions, distributions, and illustrations are provided for each species. Key worps—biodiversity, lichenized Ascomycota, Nagaon, taxonomy, Tezpur Introduction Lichens constitute a significant part of India’s biodiversity, and 2714 lichen species are known from the country (Sinha & al. 2018). Although lichens occur everywhere in India, ecological studies reveal that Northeast India, Western Ghats, Western Himalaya, and Andaman & Nicobar Islands are the major centres of diversity (Nayaka & Asthana 2014). Assam, a state of Northeast India, possesses the heterogeneous climatic conditions that make it suitable for rich floral, faunal, and fungal diversity. Over 325 species (in 83 genera and 27 families) have been reported from Assam (Gupta & Sinha 2018, Gogoi & al. 2019). Here we record six additional lichen species (in Malmideaceae, Porinaceae, Pyrenulaceae, and Ramalinaceae) new to Assam and to India. 658 ... Gogoi & al. Materials & methods The present study is based on the freshly collected lichen samples from Assam. The lichen specimens were dried, preserved, and deposited in the herbarium at CSIR- National Botanical Research Institute, Lucknow, India (LWG). The lichen thallus was morphologically examined under a Leica EZ4 stereo zoom microscope. Thin hand-cut sections of ascus and thallus were mounted in distilled water, Lactophenol Cotton Blue (LCB), 5% KOH, or Lugol’s iodine solution for observation under a Leica DM 2500 compound microscope. The thallus and ascomatal tissue were spot-tested with K (5% KOH aqueous solution), C (aqueous Ca(ClO)2 solution) and P (0.5 g of para-phenylenediamine dissolved in 5 ml of ethanol) following Orange & al. (2001). Thin Layer Chromatography was performed in solvent system C (toluene: acetic acid; 85: 15 ml) following Orange & al. (2001). Taxonomy Bacidia pycnidiata Czarnota & Coppins, Lichenologist 38: 407. 2006 Pie ika Thallus epilithic, pale yellowish to greenish, slightly granular, cracked. Apothecia few, 0.2-0.4 mm diam. Excipulum prominent, colourless, 90-140 um wide. Hymenium hyaline, 40-55 um high, I+ blue. Asci cylindrical, Bacidia-type, 35-41 x 4.5-5.8 um. Ascospores acicular, 3(-—7)-septate, 25-38 x 1.2-2.8 um. Pycnidia very few, having round flask shaped bottom with long neck, 0.1-0.2 mm diam., constricted at base, concolorous with apothecia, neck slightly paler than bottom, ostiole present at the tip of the beak; conidia hyaline, 3-5-septate, straight to slightly curved, filiform, 37-62 x 0.4-1.0 um. CHEMISTRY— Thallus K-, C-, P-, UV-; no chemicals detected in TLC. SPECIMEN EXAMINED—INDIA, Assam, Tezpur, Ouguri Hills, 26°37’03”N 92°46'37”E, elev. 113 m, on rock, 16 May 2015, Manas P. Choudhury 15027001 (LWG 35885). REMARKS— The ascospore length of the Indian specimen is slightly shorter than that given in the protologue of Bacidia pycnidiata (35-52 um; Czarnota & Coppins 2006). Further, the Indian specimen had occasional 7-septate ascospores rather than the usual 3-septate, which has not been reported previously for B. pycnidiata. In all other aspects the Indian specimen agrees with the published description of B. pycnidiata. DISsTRIBUTION—Belgium, Czech Republic, Estonia, Finland, Lithuania, Mordovia, North Caucasus, Poland, and Ukraine (Urbanavichene & Urbanavichus 2014). This is a first report of B. pycnidiata for India. Malmidea nigromarginata (Malme) Licking & Breuss, Lichenologist 47: 19. 2015. Pr lb Thallus crustose, epiphloeodal, smooth, grayish black, medulla ochraceous, K+ yellow; borderline blackish. Apothecia mostly solitary, Bacidia, Malmidea, Porina & Pyrenula spp. new for India ... 659 PLATE 1. Habit. a. Bacidia pycnidiata (LWG 35885); b. Malmidea nigromarginata (LWG 35887); c. Porina malmei (LWG 35886); d. Porina nuculastrum (LWG 35888); e. Pyrenula laetior (LWG 35889); f. Pyrenula wrightii (LWG 35890). Scale bars = 1 mm. 660 ... Gogoi & al. rarely 3—4 aggregated, margin black, disc flesh coloured. Excipulum without algal cells, lacking medullary layer, hyphae radiating and conglutinated. Hymenium hyaline, 70-90 um high, I+ blue. Hypothecium dark brownish to brown black, 70-100 um thick. Asci 6-8-spored. Ascospores simple, hyaline, 8.9-13.4 x 4.5-5.4 um. CHEMISTRY—Thallus K—, C—, P-, UV-; no lichen substances detected im ELE. SPECIMEN EXAMINED—INDIA, AssaM, Nagaon district, Samaguri, Chapanalla, 26°19'13”N 92°54’16’E, elev. 119 m, on bark, 18 February 2018, Rupjyoti Gogoi NCLH 030 (LWG 35887). REMARKS—The ascospore sizes of the Indian specimen fit well within the range of Malmidea nigromarginata (10-14 x 4-6 um) as mentioned in the world key to the genus by Breuss & Licking (2015). DIsTRIBUTION—Nicaragua (Breuss & Liicking 2015) and Puerto Rico (Mercado-Diaz & al. 2015). This is a first report of M. nigromarginata for India. Porina malmei P.M. McCarthy, Biblioth. Lichenol. 52: 70. 1993. Baeale Thallus saxicolous, medium to dark grey, areolate, ecorticate. Perithecia emergent, hemispherical to subglobose, dull black, 0.1-0.3 mm diam. Ostioles apical, conspicuous. Involucrellum brownish, continuous with the exciple. Excipulum black, 20-50 um thick. Centrum 0.15-0.25 um wide. Hymenium clear. Asci elongate-cylindrical. Ascospores hyaline, fusiform, (3-)6-7-septate, 20-27 x 4-6 um. Pycnidia not seen. CHEMISTRY— Thallus K-, C-, P-, UV-; no chemicals detected in TLC. SPECIMEN EXAMINED—INDIA, Assam, Tezpur, Rudrapad Temple, bank of River Brahmaputra, 26°36’55”N 92°46’10”E, elev. 65 m, on rock, 16 March 2015, Manas P. Choudhury 15027003 (LWG 35886). REMARKS— The Indian specimen agrees with the description of Australian Porina malmei in most features, and ascospore lengths fit well within the 17-30 um range cited in the protologue (McCarthy 1993). DisTRIBUTION—Brazil, north-eastern Australia (McCarthy 2000). This is a first report of P malmei for India. Porina nuculastrum (Mill. Arg.) R.C. Harris, More Florida Lichens, incl. 10 Cent Tour Pyrenol. (New York): 174. 1995. Bred Thallus crustose, epiphloeodal, olivaceous, slightly rimose, prothallus black. Perithecia erumpent to + immersed, mostly solitary, rarely grouped into 2-3, perithecia concolorous with the thallus except in the peri-ostiolar and ostiolar regions, convex to sub-globose 0.4-0.65 mm diam.; ostiole inconspicuous or Bacidia, Malmidea, Porina & Pyrenula spp. new for India ... 661 in a shallow depression; peri-ostiolar area 0.12-0.18 mm diam. Involucrellum enclosing the exciple yellowish to blackish. Exciple ca. 30 um thick. Hymenium clear. Asci elongate-cylindrical. Ascospores narrowly ellipsoidal to fusiform, submuriform to muriform with 6-10 transverse septa and (0-)1(-3) longitudinal septa, 49.8-67.6 x 13.5-22.2 um. Pycnidia not seen. CuHEMISTRY— Thallus K-, C—, P-, UV-; no chemicals detected in TLC. SPECIMEN EXAMINED—INDIA, Assam, Nagaon district, Samaguri, Suang Reserve Forest, 26°19’20”N 92°54’18’E, elev. 142 m, on bark, 18 February 2018, Rupjyoti Gogoi NCLH 031 (LWG 35888). ReMARKS—'The Indian specimen closely matches the Australian and Vietnamese descriptions of Porina nuculastrum (McCarthy 2001, Joshi & al. 2019), except that our ascospores have fewer transverse septa. DIsTRIBUTION—Neotropics, Madagascar, the Philippines, Hong Kong and neighbouring countries (McCarthy 2001), Vietnam (Joshi & al. 2019). This is a first report of P nuculastrum for India. Pyrenula laetior Mill. Arg., Bot. Jahrb. Syst. 6: 413. 1885. PL. le Thallus crustose, corticate, epiphloeodal, yellow, without external pigments. Perithecia black, solitary, 0.4-0.9 mm diam. Ostiole apical without pigment. Hamathecium inspersed with oil droplets. Asci 8-spored. Ascospores brown, 3-septate, eciliate, distoseptate, lumina in a straight line, 13.8-19.2 x 5.8-7.7 um, central lumina not strongly elongated, terminal lumina separated from the exospore wall by endospore thickenings, old ascospores without orange oil. CHEMISTRY— Thallus K—, C—, P-, UV-; no chemicals detected in TLC. SPECIMEN EXAMINED—INDIA, Assam, Nagaon district, Samaguri, Suang Reserve Forest, 26°19’20”N 92°54’18’E, elev. 143 m, on bark, 18 February 2018, Rupjyoti Gogoi NCLH 032 (LWG 35889). REMARKS—Aptroot (2012) cites ascospores ranging from 13-18 um for Pyrenula laetior, while those of the Indian specimen are slightly longer. The Indian material agrees with P Jaetior in all other characters. DISTRIBUTION—Neotropics (Aptroot 2012), Vietnam (Joshi & al. 2018). This represents a first report of P laetior for India. Pyrenula wrightii (Mill. Arg.) R.C. Harris, More Florida Lichens, incl. 10 Cent Tour Pyrenol.: 111. 1995. Prt Thallus corticolous, epiphloeodal, olive green with pseudocyphellae. Perithecia black, without external pigments, solitary to mostly aggregated with separated ostioles, 0.2-0.5 mm diam. Ostiole apical without pigment. 662 ... Gogoi & al. Hamathecium not inspersed with oil droplets. Asci 8-spored. Ascospores brown, 2-3-septate, 27.3-33.2 x 12.5-14.5 um, lumina in a straight line, external lumina separated from the exospore wall by endospore thickening. CHEMISTRY— Thallus K—, C—, P-, UV-; no lichen substance detected bia es bs oe SPECIMEN EXAMINED—INDIA, Assam, Nagaon district, Doboka Circle, Jamunamukh, 26°05'54’"N 92°44’44”E, elev. 40 m, on bark, 5 February 2017, Rupjyoti Gogoi NCLH 033 (LWG 35890). REMARKS— The Indian specimen agrees with the published description of Pyrenula wrightii by having pseudocyphellae and in ascospore characteristics (Aptroot 2012). The ascospores of the Indian specimen are slightly longer than the measurements provided in the basionym protologue (27-30 um; Miller 1885). DisTRIBUTION—Cuba (Aptroot 2012). This represents a first report of P. laetior for India and the first outside the type country. Acknowledgments The authors thankfully acknowledge the financial support of Department of Biotechnology (DBT, Govt. of India) through Institutional Biotech Hub (No. BT/04/ NE/2009) for establishing the Lichenology Laboratory at Nowgong College, Assam, to provide laboratory facilities for this study. The authors thank the Director of CSIR- NBRI for providing the laboratory and herbarium facilities and Dr. D.K. Upreti for his encouragement and help with the identification of the Pyrenula and Porina specimens. We thank Drs. G.P. Sinha (Botanical Survey of India, Allahabad, India) and Udeni Jayalal (Sabaragamuwa University of Sri Lanka, Belihuloya, Sri Lanka) for reviewing the manuscript. One of the authors (SJ) is grateful to DST-SERB for providing financial assistance under the NPDF scheme (PDF/2016/002054). Literature cited Aptroot A. 2012. A world key to the species of Anthracothecium and Pyrenula. Lichenologist 44(1): 5-33. https://doi.org/10.1017/s0024282911000624 Breuss O, Liicking R. 2015. Three new lichen species from Nicaragua, with keys to the known species of Eugeniella and Malmidea. Lichenologist 47(1): 9-20. https://doi.org/10.1017/S0024282914000565 Czarnota P, Coppins BJ. 2006. A new Bacidia with long-necked pycnidia from Central Europe. Lichenologist 38(5): 407-410. https://doi.org/10.1017/s0024282906005986 Gogoi R, Joseph S, Nayaka S, Yasmin F. 2019. Additions to the lichen biota of Assam State, India. Journal of Threatened Taxa 11(6): 13765-13781. https://doi.org/10.11609/jott.4642.11.6.13765-13781 Gupta P, Sinha GP. 2018. Lichen flora of Assam. Bishen Singh Mahendra Pal Singh, Dehra Dun, India. Bacidia, Malmidea, Porina & Pyrenula spp. new for India ... 663 Joshi S, Upreti DK, Hur JS. 2018. Key to the lichen families Pyrenulaceae and Trypetheliaceae in Vietnam, with eight new records. Mycotaxon 132(4): 957-969. https://doi.org/10.5248/132.957 Joshi S, Upreti DK, Hur JS. 2019. Lichen genus Porina in Vietnam. Korean Journal of Mycology 47(4): 303-311. https://doi.org/10.4489/KJM.20190035 McCarthy PM. 1993. Saxicolous species of Porina Mull. Arg. (Trichotheliaceae) in the Southern Hemisphere. Bibliotheca Lichenologica 52: 1-134. McCarthy PM. 2000. Key to the saxicolous taxa of Porina. Lichenologist 32(1): 1-13. https://doi.org/10.1006/lich.1999.0213 McCarthy PM. 2001. Trichotheliaceae. 105-157, in: PM McCarthy (ed.). Flora of Australia, Volume 58A, Lichens 3. ABRS/CSIRO Australia, Melbourne. Mercado-Diaz JA, Gould WA, Gonzalez G, Liicking R. 2015. Lichens in Puerto Rico: an ecosystem approach. General Technical Report IITF-GTR-46. San Juan, PR: US. Department of Agriculture, Forest Service, International Institute of Tropical Forestry. https://doi.org/10.2737/iitf-gtr-46 Miller J. 1885. Pyrenocarpeae Cubenses a cl. C. Wright lectae. Botanische Jahrbiicher fiir Systematik, Pflanzengeschichte und Pflanzengeographie 6: 375-421. Nayaka S, Asthana S. 2014. Diversity and distribution of lichens in India vis a vis its lichenogeographic regions. 79-96, in: T Marimuthu & al. (eds). Biodiversity Conservation — Status, Future and Way Forward. National Academy of Biological Science, Chennai, India. Orange A, James PW, White FJ. 2001. Microchemical methods for the identification of lichens. British Lichen Society, London. Sinha GP, Nayaka S, Joseph S. 2018. Additions to the checklist of Indian lichens after 2010. Cryptogam Biodiversity and Assessment, Special Volume: 197-206. https://doi.org/10.21756/cab.esp16 Urbanavichene I, Urbanavichus G. 2014. Bacidia pycnidiata discovered in European Russia. Folia Cryptogamica Estonica 51: 109-111. https://doi.org/10.12697/fce.2014.51.12 MYCOTAXON ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2020 July-September 2020—Volume 135, pp. 665-718 https://doi.org/10.5248/135.665 Chromelosporium re-evaluated, with Chromelosporiopsis gen. nov. and Geohypha stat. nov. GREGOIRE L. HENNEBERT Rue de l'Elevage 32, B 1340 Ottignies-Louvain-la-Neuve, Belgium CORRESPONDENCE TO: hennebertg@scarlet.be ABSTRACT—Chromelosporium-like asexual morphs of the subterraneous Pezizaceae in the /Pachyphlodes clade are not congeneric with the type of Chromelosporium. Some Chromelosporium morphs are of species in the /Pezizaceae clade (the /Peziza clade), and the genus Chromelosporium as it has been defined is polyphyletic. A diagnostic character that distinguishes these two groups is the presence or absence of conidiophore fasciculation. Mononematous conidiophores characterize Chromelosporium and species related to the /Peziza clade. Synnematous conidiophores characterize Pachyphlodes and other asexual species and define the new genus Chromelosporiopsis, to be excluded from Chromelosporium. Hyphelia terrestris, long misapplied to Chromelosporium, is revaluated, lectotypified, redescribed and recombined as Geohypha terrestris. Keyworps—hyphomycete, nomenclature, taxonomy Introduction Collections of Chromelosporium-like hyphomycetes made by R.P. Korf around Ithaca, NY, which he tentatively named Glischroderma because of their production of gel (Korf 1994), were revaluated by Healy & al. (2015) and identified as conidial morphs in the truffle genus Pachyphlodes. Fresh Chromelosporium-like specimens collected on bare soil in woods in Belgium, identified by DNA sequencing as Pachyphlodes nemoralis and P. citrina, provided the necessary asexual material to compare with and segregate from the asexually typified species, Chromelosporium ochraceum (Hennebert & Decock 2020). 666 ... Hennebert The main distinction that emerged between the asexual morphs in C. ochraceum and the two Pachyphlodes species is conidiophore fasciculation. This criterion, applied to species already named in Chromelosporium (Hennebert 1973), segregates them into two genera. It seems generally accepted that these fungi are asexual morphs of Pezizales. As long as their type species are not known to be congeneric with species with a known sexual morph, however, it seems sensible to keep these genera separate. Mononematous species define Chromelosporium. Synnematous species are excluded and are accommodated in the new genus Chromelosporiopsis. Names such as Botrytis epigaea, Phymatotrichum species, and others are discussed as doubtful taxa. The conidial ornamentation observed in the numerous herbarium specimens that might contribute to the diagnosis of unidentified Chromelosporiopsis species is also analysed. Hyphelia terrestris, currently misclassified in Chromelosporium, is re- evaluated and recombined as the type of Geohypha [= Hyphelia sect. Geohypha elevated to generic rank]. The asexual morph of Plicaria endocarpoides, previously supposed to show similarities with Chromelosporium, is described. The illustrated descriptions of the asexual morphology of these taxa provide the rationale for the present paper. Materials & methods Species descriptions are based on observations made on herbarium material borrowed from many herbaria and some fresh collections made during 1960-62 while working as a postdoctoral researcher under the late Dr S.J. Hughes at the Agriculture & Agri-Food mycology group in Ottawa (DAOM). This material is complemented with some contemporary collections from Europe. A 1960 Olympus FH microscope equipped with Olympus positive low phase contrast objectives and an adapted Wild drawing tube was used for the microscopic examinations. All samples were mounted in lactic acid with Cotton blue. Slides made from the examined specimens are preserved in the Canadian National Mycological Herbarium, Ottawa, Canada (DAOM) and/or Mycotheque, Université Catholique de Louvain, Louvain-la-Neuve, Belgium (MUCL), and accession numbers are listed for each specimen in square brackets. Information provided in the Examined specimen sections duplicates the herbarium labels with bold numbers separating each specimen. Taxonomy Chromelosporium This section includes the mononematous species considered morphologically congeneric with the type species Chromelosporium ochraceum, which currently Chromelosporiopsis gen. nov. & Geohypha stat. nov. ... 667 delimits the genus Chromelosporium. In addition, one Peziza and one Plicaria species with similar asexual morphs are each described. Chromelosporium Corda, in Sturm’s Deutschl. Fl., Abt. 3 (Pilze), 3(13): 81, 1833. TYPE SPECIES: Chromelosporium ochraceum Corda?? Ascomycota, Pezizaceae, sexual morph unknown. HyPHAE septate, creeping on the substratum. CONIDIOPHORES mononematous, erect, septate, apically branching in successive symmetrical dichotomies, septate, apically obsuse. CONIDIOGENUS CELLS subterminal and terminal branches forming synchronous conidia on denticles along their lengths, collapsing after rhexolytic secession of conidia. Conrp14_ holoblastic, one-celled, globose or subglobose, with wall ornamented from finely to coarsely verrucose, colored in mass. HasitTat: on plant fragments. COMMENTS—Hennebert (1973) made a clear distinction between Chromelosporium and Ostracoderma Fr., so Chromelosporium should not be placed as a synonym of Ostracoderma as cited in the 10" edition of Ainsworth & Bisby’s Dictionary of the Fungi (Kirk & al. 2008) and in Index Fungorum. Chromelosporium is also incorrectly listed as a synonym of Peziza in Baral’s contribution to Jaklitsch & al. (2016). Such synonymy is indeed impossible— Peziza being clearly polyphyletic and Chromelosporium an “orphan” genus. Pfister & al. (2016) did not mention the case when evaluating competing sexually and asexually typified names. Indeed, as the type species is not connected to any sexual morph, the generic name Chromelosporium cannot be competing so far. Also, Chromelosporium should not have been ignored in the recent “Outline” of fungal genera (Wijayawardene & al. 2020). It is possible that the four species described in the genus below, beside the type species, might also be revealed as polyphyletic. Chromelosporium ochraceum Corda, in Sturm’s Deutschl. Fl., Abt. 3 (Pilze), 3(13): 81, 1833. FIGS 1, 2 = Sporotrichum ochraceum (Corda) Sacc. Syll. Fung. 4: 105, 1886. = Ostracoderma ochraceum (Corda) S. Hughes, Canad. J. Bot. 36: 792, 1958. = Botrytis dichotoma Corda, Icon. Fung. 1: 18, 1837. = Campsotrichum dichotomum (Corda) Bonord., Handb. Mykol.: 102, 1851. = Botrytis isabellina Preuss, Linnaea 25: 75, 1852. = Ostracoderma isabellinum (Preuss) S. Hughes, Canad. J. Bot. 36: 792, 1958. 668 ... Hennebert Fig. 1. Chromelosporium ochraceum. A. Conidiophore with conidiogenous cells (drawing by S.J. Hughes, DAOM 83330). B, D. Conidiophore and conidia (holotype, PRM 155414), C. conidiophore after secession of the conidiogenous cells (DAOM74695, Mycoth. Ven. 1080). Scale bar = 10 um. = Polyactis crystallina Bonord., Abh. Naturf. Ges. Halle 8: 95, 1864. = Botrytis crystallina (Bonord.) Sacc., Syll. Fung. 4: 135, 1886. = Botrytis spectabilis Harz, Bull. Soc. Imp. Nauralistes Moscou 44(1): 144, 1871. = Clonostachys spectabilis (Harz) Oudem. & Sacc., Ned. Kruid. Arch., 2. ser., 4: 539, 1886. Chromelosporiopsis gen. nov. & Geohypha stat. nov. ... 669 Type: Chromelotrichtm [crossed out] sporium ochraceum Ca. ramulis furcis, Myrinema generis” [scr. et del. A.C.J. Corda]. On Allium dead stem, Prague, Czechoslovakia. Herb. Corda (Holotype: PRM 155414; isootypes: [DAOM 40946, MUCL.2895] CoLonigs in small patches, <10 mm diam., velvety, at first white, then ochraceous when mature. HyPpHAE 3-5 um wide, creeping in the substratum. CONIDIOPHORES mononematous, erect, emerging from hyphae in substratum, with a bulbous basal cell, 28-35 x 12-15 um, ochraceous, extending into a cylindrical, septate stipe, 100—400 um long, pale ochraceous, the individual cells 40—95 x 10-14 um, terminally repeatedly branched with up to five dichotomies, each branch with a basal septum and a second septum before the subsequent dichotomy, the length of branch decreasing from one dichotomy to the next, usually the first dichotomy 40-90 x 9-10 um, the second 30—70 x 6-10 um, the third 30—50 x 6—9 um and so on, but in some cases dichotomies being very short decreasing from 20 to 10 um, the angle of the dichotomies ranging from 30° to 45°. CONIDIOGENOUS CELLS comprising the last four and five terminal dichotomies, together 70-100 x 6—9 um, apically clavate, <11 um, covered with synchronous conidia borne on a denticle, and collapsing after conidial release, seceding or (rarely) remaining as a frill on the last conidiophore cell. Conrp1A holoblastic, borne simultaneously on denticles 2 x 1 um, one- celled, globose or subglobose, ochraceous to salmon-colored in mass, 4—6.5 um, wall 0.5 um thick, cyanophilic, verrucose, warts prominent 0.3 um high and blunt, with 12—14 warts in median view, seceding rhexolytically. HasirtaT: on plant fragments. DISTRIBUTION: Europe: Italy, France, Belgium, England, Africa: Maroc, Uganda, Asia: Sumatra. CoMMENTS— The name Chromelosporium remained unused until Hughes (1958) brought the genus to light. Corda’s 1833 description of an overmature collection was so unclear that even Corda (1837) himself did not recognize the conspecificity of his later taxon, Botrytis dichotoma. His figure (Fic. 3A) shows globose ochraceous conidia accumulated along the sides and the top of the obtuse dichotomous branches. Korf & Hennebert (1975) stated that Botrytis spectabilis was a synonym of Chromelosporium ochraceum based on an original illustration from Harz reproduced here (Fics. 3B-C) and preserved in MUCL 1072. The names Botrytis spectabilis and Polyactis crystallina (another synonym of Chromelosporium ochraceum) have been misapplied to the asexual morph 670 ... Hennebert Fig. 2. Chromelosporium ochraceum (DAOM 46357). A. Conidiophores under low magnification. B. Dichotomies of conidiophore. C-D. Conidiogenous dichotomies. E. Conidiophore after secession of conidiogenous cells. F Verrucose conidia. Chromelosporiopsis gen. nov. & Geohypha stat. nov. ... 671 =. — i 2 ” * . i 7 b = é _ 4B. dicholemie. A Nes Fig. 3. Illustrations of A. Botrytis dichotoma Corda, Icon. Fung. 1: 18, fig. 244, 1837. B-C. Botrytis spectabilis Harz, original drawings (MUCL 1072). of Peziza ostracoderma Korf (Hennebert & Korf 1975). For that reason, both Botrytis spectabilis and Polyactis crystallina have been erroneously listed as synonyms of Peziza ostracoderma in Index Fungorum. Chromelosporium ochraceum is also erroneously cited as a synonym of C. fulvum (Link) McGinty& al., typified by the asexual morph of Peziza ostracoderma in Seifert & al. (2012, p. 139). The lectotype collection of Botrytis isabellina and the authentic collection distributed in Rabenhorst’s KLoTzscHi] HERBARIUM VIVUM MYCOLOGICUM N° 1570, were described in sufficient detail by Preuss (1853) as “Floccis erectis crassis repetito ramosis dichotomis vel trichotomis, apice verrucosis sporis irregulariter accumulatis, globosis, hilo instructo, episporio verrucoso” to confirm its synonymy with Chromelosporium ochraceum. SPECIMENS EXAMINED TYPE & AUTHENTIC MATERIAL. EUROPE: Czecuia: (2) Botrytis dichotoma, Herb.Corda (PRM 155393 type), [DAOM 84706, MUCL 2896]. GERMANY: (3) Botrytis isabellina Preuss 1850, auf [rotten] nadelholz, Herb Preuss 451 (B) (designated here as lectotype MBT 392406) [MUCL 5999]. (4) Botrytis isabellina Pr., ad lignum abietinum humi jacentem, prope Hoyerswerda [Germany], Preuss [1851], 672 ... Hennebert Rabenh., Klotzschii Herb. Viv. Mycol. 1570 (B, PR, BM, BR, PAV, L) (isolectotypes) [DAOM 51762, MUCL 1810]. (5) Botrytis spectabilis Harz, original drawing by Harz (isolectotype 1975) (MUCL 1072). OTHER SPECIMENS. EUROPE: ITALY: (6) Botrytis epigaea Link f. ochracea Sacc, ad terram argillosam in horto botanico, Padova. Aug. 1902. D. Saccardo, Mycotheca italica Mucedinaceae. [1903, Cent. XII] n° 1178 (PR 184863, BM, L 910.224-158) [DAOM 74898, MUCL 1803]. [The name has been listed in Michelia I :102, 1877]. (7) Botrytis dichotoma Corda, in sarmentis, foliis, capsulis putrescentibus, in Horto botanico ticinensi. Autumno, coll. Cavara. Micol. Lomb. n° 137, tab. 1, fig. 20-24 [1894]. Cavara, Fungi Longobardiae exsiccate. 195, (PAD, BM, PAV, BPI, NY, B, L) [DAOM 83330, MUCL 1801]. (8) Botrytis fulva Link. in scapis Allii cepae, Consiglio (Treviso). Sept. 1897. D. Saccardo Mycotheca italica Mucedinaceae. Cent. II.186, (K- M) [DAOM 74699, MUCL 1802]. (9) Botrytis fulva Lk. Polyactis fulva Bon. in truncis putridis. Selva (Treviso) Oct. 1876. Saccardo PA. Mycotheca veneta. Cent. XI. 1897. n°1080 (K-M) [DAOM 74695, MUCL 1812]. (10) Botrytis fulva Link, ad corticem ramorum, Valtaro, Sept. 1923, coll. Renji, det. G. Bresadola, (BPI) [DAOM 83884, MUCL 2413]. (11) Botrytis fulva Link f. argillacea, ad Salicem petandram, Eichler, Polachia, 1894. [scr. Bresadola] Herb. Bresadola, [In Bresadola Fungi polonici a cl. Viro B. Eichler lecti. Ann. Mycol. 1, 2: 97-131, p. 127(1903), forma argillacea of Botrytis fulva Link is not described] (S) [MUCL2531]. UK: (16) Botrytis fulva Link. Queen’s Cottage grounds, Kew, Nov. 1898. G.E. Massee. (K-M) [MUCL 2295]. (17) Phymatotrichum, on Fagus sylvatica rotten wood, Sheepleas, Horsley, Surrey, R.W.G. Dennis, March 2 1952. (K-M-IMI 49481) [MUCL 3547]. (18) Botrytis splendida Schwein., on mossy Salix bark, Wheatfen Broad, Norfolk, E.A. Ellis , (K-M-IMI 26983) [MUCL 3568]. (16) Phymatotrichum, on Petasites ovalis, Forge Valley, Yorkshire, coll. I.M.I., det. S.J. Hughes. (K-M-IMI 8160) [MUCL 3581]. (19) Botrytis (Phymatotrichum), on Heracleum, Forge Valley, Yorkshire, Sept. 16 1946, coll. I.M.L., det. S.J. Hughes. (K-M-IMI 6851) [MUCL3592]. (20) Botrytis, Phymatotrichum, on Petasites dead petioles, Forge Valley, Yorkshire, Oct. 1945, coll. I.M.L, det. S.J. Hughes, (K-M-IMI 1586) [MUCL 3595]. (21) Phymatotrichum, on Eupatorium cannabinum, Farwath, Newton Dale, Pikering, Yorkshire, Aug. 6 1960, W. Bramley. (K-M-IMI 83016a) [MUCL 3622]. (22) Phymatotrichum, on Heracleum, Forge Valley, Yorkshire, Sept. 16 1946, coll. I.M.L., det. S.J. Hughes. (K-M-IMI 7341) [MUCL 3582]. FRANCE: (12) Botrytis fulva Link, in ligno putrido, Céte d’Or, Sept. 1889, coll Fautrey n°9 (PC) [DAOM 83390, MUCL 2378]. (13) Botrytis fulva Bon., sur tricot de coton pourri pres dun lavoir, Lyon, coll. J.J. Therry. Cryptogames du Lyonnais. J.J. Therry n° 6474, (G) [MUCL 2398]. (14) Botrytis carnea Schum., ad culmos Paludi, Meudon, Nov. 3 1893, coll. E. Roussel]. (PC) [MUCL 2706]. BELGrum: (23) Phymatotrichum, on herbaceous stems and leaves of compositae, Forét de Soignes, Sept. 16 1956, coll. M.B. Ellis. (K-M-IMI 69956) [MUCL 3542]. AFRICA: Morocco: (24) Phymatotrichum, sur souche de Quercus ilex, Forét dAzrou, Moyen Atlas, Maroc, 17 Nov. 1965, G. Malencon. Herb. Cryptog. G. Malencgon 5805, (PC) [MUCL 8041]. UGANDA: (25) Botrytis, on dead wood, Kampala, 4000; May 1936, G. Hansford. Herb. Mycol. Dep. Agric. Uganda n° 1769 (K-M-IMI 7899) [MUCL 3613]. Chromelosporiopsis gen. nov. & Geohypha stat. nov. ... 673 ASIA: INDONESIA: (26). Botrytis? op aarde, bladeren, takjes, Sumatra, Kampong Baroe bij Medan, Juli 1927, K.B. Boedijn. n° 4619 (Bot. Museum Utrecht 179892) [MUCL 5381]. Fig. 4. Chromelosporium herbicola (isotype NY, MUCL 5889). A-C. Conidiophores missing the seceded conidiogenous cells. D. Verrucose conidia. Scale bar = 10 um. Chromelosporium herbicola (Ellis & Dearn.) Hennebert, comb. nov. Fic. 4 MB 833990 = Rhinotrichum herbicola Ellis & Dearn., Proc. Canad. Inst., n.s. 1: 90, 1897 [(as “herbicolum” |. Type: Rhinotrichum herbicolum E|llis] & Dearness, on Solidago virga-aurea golden rod stems, Aug. 13 1895, D.V.S. Valley, London, Canada, J. Dearness. Herb. Dearness 2314 (Holotype: DAOM [MUCL2321]; isotype NY [MUCL 5889]). 674 ... Hennebert COLONIES in small patches, 3-5 mm wide, about 1 mm high, ochraceous. HyPpHAE 5—15 um wide, creeping on the substratum. CONIDIOPHORES mononematous, erect, emerging single from creeping hyphae, as a stipe 100-150 um high, 9-15 um wide, ochraceous, slightly constricted at septa in cells 25-35 um long, terminally branching in successive dichotomies, at 10-45° angles, branches septate at the base and sometimes over their length, first dichotomies 30-50 um long, second dichotomies 20—25 um long, CONIDIOGENUS CELLS presumably the third and fourth dichotomies collapsed and fallen away, not observed in the material but similar to those of Chromelosporium fulvum after Sumstine (1911). Conip1a presumably holoblastic, borne simultaneously all along the conidiogenous cell as described by the original authors, globose to obovate, one-celled, (6—)7—9(—11) um, ochraceous, thick-walled and verrucose, with 18—20 warts in median view, seceding rhexolytically. Hasirat: On decaying herbaceous plant stems, similar to Solidago. DISTRIBUTION: Canada: Ontario. CoMMENTS — The original description of Rhinotrichum herbicola by Ellis & Dearness reads: “Effuse, light yellow, becoming brown in the center. Hyphae coarse, septate, branched, nearly hyaline, 8—10 u, thick, Fertile hyphae sub- undulate above, tips swollen and bearing the globose, sub-hyaline, finely echinulate, 7—9 u, conidia. On dead stems of Solidago canadensis, London, Can., Aug. 1895. Herb. Dearness n°2314 Sumstine (1911) examined the isotype specimen in NY and observed that the “spores clustered at the apex of the fertile branches” Although “the specimen proved to be poor’, he concluded that “it resembles in many respects Botrytis fulva Link.’ This comment joined to the observed dichotomous branching and the verrucose globose conidia leads to transfer the species to Chromelosporium. The species differs from Chromelosporium ochraceum by its shorter conidiophores and conidial size and ornamentation. Chromelosporium canadense Hennebert, Persoonia 7: 196, 1973. FIGS 5, 6 = Rhinotrichum carneum Ellis & Everh., J. Mycol. 1(7): 93, 1885 (non Chromelosporium carneum (Pers.) Hennebert TyPE: Chromelosporium canadense Hennebert, on rotten wood in forest, Gatineau Park, Gatineau, Quebec, Canada, 25.11.1960, leg. S.J. Hughes & G.L. Hennebert, (Holotype: DAOM 71947; isotype: MUCL 1689). Chromelosporiopsis gen. nov. & Geohypha stat. nov. ... 675 Fig. 5. Chromelosporium canadense (holotype, DAOM 71947). A. Young conidiogenous cells on terminal dichotomies. B. Primordia of the conidiogenous cells. C. Conidiogenous cells. D. Dichotomous branching of the conidiophore. E. Mature conidia. Scale bar = 10 um. 676 ... Hennebert COLONIES in patches <15 mm wide and <3 mm thick, effuse, loose, first white, then tawny to cinnamon, composed of mixed hyphae and branched conidiophores bearing clusters of conidia. HypuHAe on substrate, thin, 4-7 um, evanescent. CONIDIOPHORES mononematous, erect, single, tawny, stipes 100-300 x 6-10 um, then branching in regular and repeated dichotomies of different lengths, branches divergent at 20—60° angle, the basal ones 160-200 um long, the distal ones shorter (16—48 um), 10—12(—18) um wide, the stipe and each branch septate at its base, the last dichotomy bearing two clusters of 4—8 primordial conidiogenous cells. CONIDIOGENUS CELLS formed as two groups of quadri-furcate terminal primordia that elongate up to 30—45 x 12—22 um, slightly clavate, becoming conidiogenous over their entire length. Conip1A4 holoblastic, borne simultaneously on denticles, one-celled, globose, (3.5—)4—5(—7) um, tawny, verrucose with 10-18 warts in median view, seceding rhexolytically. HABITAT: on rotten wood and bark, branches, mosses, plant stems and herbs, in gardens and forests. DISTRIBUTION: Canada and USA. CoMMENTS—Growth on PDA was obtained from young conidiophore hyphae taken after 3 days at 4°C from fresh material of four collections (MUCL 1689 T, 1690, 1691, 1692) but not from conidia. Mycelium in culture is effuse, ochre, forming dense dispersed patches; hyphae are regular, septate, branching at an angle of c. 45°, or very irregular, closely septate in swollen cells. In two cases, small clusters of dark brown cells were produced, suggesting primordia of apothecia. Herbarium specimens often lack the conidiogenous cells which are collapsed or absent; their presence is inferred from the branching pattern and the conidial characters. SPECIMENS EXAMINED: TYPE & AUTHENTIC MATERIAL: USA: OREGON: (2) Rhinotrichum carneum Ell. & Ev., on bark, Oregon, 1885, W.S. Carpenter n° 125 [scr. Ellis] (NY).) [DAOM 84659, MUCL 2694]. (holotype) NEw York: (3) Rhinotrichum carneum Ell. & Everh., on rotten wood, Macoun [scr. Ellis]. Ellis Collection (NY) [DAOM 84677, MUCL 2839]. (4) Rhinotrichum carneum Ell. & Everh. [no substrate, no date], Macoun [scr. J.B. Ellis]. Ellis Collection (NY) [MUCL 2840]. OTHER SPECIMENS: CANADA, Ontario (5) Hyphelia terrestris, on Tilia, Nashville, York Co., Oct. 25 1953, coll. det. RF. Cain (TRT 30178) [DAOM 84684, MUCL 2858]. (6) Hyphelia terrestris, on hardwood, Nashville, Nov. 14 1954, coll. Chromelosporiopsis gen. nov. & Geohypha stat. nov. ... 677 Fig. 6. Chromelosporium canadense (holotype, DAOM 71947). A. Conidiophores at low magnification. B-D. Young stages of conidiogenous cells. C. Half and fully mature conidioma. E. Wide angle of the dichotomies. F. Terminal bunch of mature conidiogenous cells. det. R.E. Cain (TRT 31311) [DAOM 84683, MUCL 2857]. (7) Hyphelia terrestris, on Populus Nashville, Oct. 22 1955, coll. det. RE Cain (TRT 31699, BPI) [MUCL 2419]. (8) Hyphelia terrestris, on decayed log, New Durham, Brant Co., Oct. 14 1956 (TRT 678 ... Hennebert 32870) [DAOM 84688, MUCL 2862]. (9) Botrytis fulva Link, on dead wood, Oct. 20 1896, Macoun [Scr. J.B. Ellis]. Herb. Ellis (BPI) [DAOM 83886, MUCL 2415]. (10) Botrytis (Phymatotrichum) fulva Link, on decayed wood, N. Bathurst street Toronto, Nov. 17 1934, H.S. Jackson, det. D.H. Linder, Herb. R.E Cain 8207 (TRT 8065, FH, DAOM 81409) [MUCL 2889)] (11) Botrytis (Phymatotrichum) fulva Link, on rotten wood, S. Aurora, Oct. 23 1932, H.S. Jackson, det. D.H. Linder. Herb. R.E. Cain 8210 (TRT 8060, DAOM 81410) [MUCL 2890]. (12) Ostracoderma, on soil of lawn, North Toronto, June 17 1960, S.J. Hughes. (DAOM 83325) [MUCL 2255]. (13) Chromelosporium, on bark and plant decay, South March, April 10 1962, G.L. Hennebert (MUCL 3067). QUEBEC (14) Ostracoderma on bark, Gatineau Park, Nov. 25 1960, S.J.Hughes. (DAOM 71946) [MUCL 1688]. (15) Ostracoderma, on wood, Gatineau Park, Nov. 25 1960, S.J.Hughes. (DAOM 71948) [MUCL 1690]. (16) Ostracoderma, on wood, Gatineau Park, Nov. 2(5, 1960, S.J.Hughes. (DAOM 71949) [MUCL 1691]. (17) Ostracoderma, on wood, Gatineau Park, Nov. 25, 1960, S.J. Hughes. (DAOM 71950) [MUCL 1692]. (18) Ostracoderma, on wood and bark, Gatineau Park, Oct. 31, 1960, S.J. Hughes. (DAOM 71399) [MUCL 1689]. (19) Ostracoderma, on stem of Veratrum viride, Yoho Valley, Aug. 8 1960, S.J. Hughes. (DAOM71326) [MUCL 1513]. (20) Chromelosporium, on Betula papyrifera rotten wood, Gatineau Park, Oct. 31 1960, G.L.Hennebert. (DAOM 71545, MUCL 1590). (21) Chromelosporium, on rotten wood, Gatineau Park, Oct. 7 1960, G.L.Hennebert. (DAOM 71419, MUCL 1505). MANITOBA (22) Botrytis near phymatotricha Sacc. prov. cfr. lateritia, C.E. Fairman suggests Botrytis carnea Schum, on old board, Winnipeg, Sept. 16 1928, G.R. Bisby 4140 [scr. Dearness]. Herb. Dearness 6732 (DAOM 84789) [MUCL 3070]. USA, OunI0 (23) Mycobiota of Ohio. Botrytis isabellina Preuss, on rotten wood, The 7 Caves, Highland Co., coll. det. D-H. Linder 2667, Sept. 6 1933, Herb. W. Bridge Cooke (NY, FH, DAOM 84679) [MUCL 2847]. Iowa (24) Hyphelia laxa Schw., on decorticated conifer wood. (IA) [DAOM 83317, MUCL 2260]. SouTH CAROLINA: (25) Botrytis epigaea Link var. on clay surface after rain, Sept. 1883, H.W.R. [scr. H.W. Ravenel]. Herb. U.W. Ravenel, (K-M, DAOM 83332) [MUCL 2281]. MASSACHUSETTS (26) Botrytis? Hyphelia spectabilis Harz, on coniferous bark, York, Sept. 14 1891. Herb. Thaxter 137 (FH) [DAOM 84702, MUCL 2886]. (27) Botrytis fulva Link, on hardwood branches on the ground, Beaver Brook reserve, Belmont, Oct. 3 1937, coll. G.D. Darker 64-90, det. D.H. Linder. (FH) [DAOM 84703, MUCL 2888]. NEw YorRK (28) Ostracoderma canadense, on stem of fern in a pond, Forest Home, Ithaca, May 1 1962, G.L. Hennebert. (MUCL 3120). NEw JERSEY (29) Botrytis epigaea Pers., on old pine boards partly buried in the ground, Newfield, N.J., Sept. 1881, Ellis. Ellis North American Fungi 827 (WIS, CUP, K-M, B, G, L) [MUCL 1804]. Chromelosporium arenosum Hennebert, Persoonia 7: 196, 1973. FIG. 7 Type: Flora Venezuelae 2474, on rotten wood of Espeletia, 3550 m, alt., Mucudaji, Sanide Santo Domingo, Estada Mesida, July 22 1958, coll. R.W.G. Dennis. (K-M) (Holotype: MUCL 2298 [DAOM 83359]; isotype IMI 75582 [MUCL 5889]). EryMo.Locy: from the Latin arenosus or sand-like, suggesting the spreading granulose aspect of the sporulating conidiophores of ochraceous color seen under low magnification. Chromelosporiopsis gen. nov. & Geohypha stat. nov. ... 679 Fig. 7. Chromelosporium arenosum (holotype, DAOM 83359). A. Young conidiophore branching. B. Erect conidiophore with conidiogenous cells. C. Mature conidiophore with conidia borne on aerial hypha. D. Conidiophore after secession of conidiogenous cells. E. Conidia. Scale bar = 10 um. COLONIES superficial, sparse, delicate, cobweb-like white mycelium, becoming arenose ochraceous with mature conidiophores appearing like minute, scattered grain. Hypuae repent, hyaline, single, 7—9 um wide, regular, straight, branched, thin-walled, smooth, septate with cells 7-12 um long, sometimes regenerating after collapse from septum by internal narrow hyphal proliferations 3—4 um wide. CONIDIOPHORES arising singly, mononematous, as erect branches of the repent hyphae, short, 70-100 um high, 8—9 um wide, stipes with the basal cell bulbous 11—13 x 6—8 um,, constricted at septa, cells 15-50 um, apically branching in dicho- or trichotomies forming 2—8 terminal cells 40-50 um long, obtuse, clavate up to 13 um wide and conidiogenous. 680 ... Hennebert Conip1a holoblastic, borne simultaneously on denticle 1 x 0.5 um, one- celled, globose or ovoid, 3.5—5.5 x 3.5—6.5(—8.1) um, mostly 4.5 um wide, 0.5—1 um thick-walled, the outer wall hyaline, the inner wall subhyaline to ochraceous and coarsely verrucose, with 6—10 globoid or elongate warts in median view, seceding rhexolytically. HABITAT: on rotten bark of Espeletia sp. in the Tropics. DISTRIBUTION: Venezuela. Chromelosporium macrospermum Hennebert, Persoonia 7: 197, 1973. Fic. 8 Type: living and dried cultures from Peziza sp. ind. on sterilized soil in greenhouse, Heverlee, Belgium, 2.04.1960, leg. G.L. Hennebert (Holotype: MUCL 1116; isotype DAOM 67492 = dried culture of MUCL 1116). COLONIES on malt agar substrate thin, white to rust-color when sporulating, comprising interwoven and prostrate hyphae, hyaline to subhyaline, producing laterally large spherical cells, 40-50 um diam, thin-walled. CONIDIOPHORES mononematous, erect, pale to rust-colored, septate, stipes 400-600 x 15-18 um, apically bi- or quadri-furcate, branches 20 um long, few septate, the distal ones erect, radiate, cylindrical to clavate 130-160 x 20-25 um, conidiogenous. Conip1A holoblastic, borne on a denticle, more or less simultaneous, globose, rust-colored, 15-23 (av. 19.7) um, thin-walled, finely punctate, seceding rhexolytically. Hapsirat: sterilized greenhouse or nature soil DISTRIBUTION: Europe: Belgium, Oceania: Australia. ComMENnts — This unique strain MUCL 1116 was found in one ascospore germinating from several apothecia then identified as Peziza ostracoderma, collected in a tropical greenhouse of the Faculty of Agronomy UCL in Heverlee, Belgium in 1960. Korf differentiated the size and ornamentation of the ascopores preserved in lactic acid mounted slides of the original Peziza apothecia (Hennebert & Korf 1975) but in the absence of the single original apothecium, he could not circumscribe the species. The same species has been isolated from soil in South Australia by Dr. G.C. Hansford at the University of Adelaide in 1953 and preserved as living culture IMI 54710 in the culture collection at Egham and as dried culture in K-M—IMI 54710 under the name Phymatotrichum tax. sp. 7. The culture is C. macrospermum mixed with conidia of P. ostracoderma and unidentified hyaline chlamydospores. The species might be a mutant Peziza ostracoderma or a distinct undetermined species of Peziza. Chromelosporiopsis gen. nov. & Geohypha stat. nov. ... 681 Fig. 8. Chromelosporium macrospermum (ex holotype, MUCL 1116). A-B. Conidiophores at diverse stages. B. Punctate conidia. D. Swollen cells in culture. Scale bar = 10 um. 682 ... Hennebert Species of Pezizaceae with known sexual morphs and similar mononematous conidiophores and conidiogenesis Peziza ostracoderma Korf, Mycologia 52: 650, 1961 [“1960”]. Fic. 9 = Plicaria fulva R. Schneid., Zentralbl. Bakteriol. Parasitenk. Infektionskrankh. Hyg., 2. Abt., 108: 147, 1954 [non Peziza fulva Pers. 1822, nom. sanct.]. = Dematium ollare Pers., Syn. Meth. Fung. 2: 697, 1801. = Botrytis fulva Link, Spec. Plant., ed. 4, 6(1): 58, 1824, nom. illeg. [superfluous]. = Sporotrichum fulvum Fr., Syst. Mycol. 3: 418, 1832, nom. sanct. [not “(Link) Fr’; non Link 1809]. = Trichosporum fulvum (Fr.) Fr., Summa Veg. Scand. 2: 492, 1849. = Polyactis fulva (Fr.) Bonord., Handb. Mykol.: 115, 1851 [not “(Link) Bonord.”]. = Chromelosporium fulvum (Fr.) McGinty, Hennebert & Korf, Mycologia 67: 216, 1975 [not “(Link) McGinty et al.”]. = Chromelosporium ollare (Pers.) Hennebert, Persoonia 7: 197, 1973. = Haplaria nitens Delacr., Bull. Soc. Mycol. France 6: 140, 1890. = Botrytis luteobrunnea Krzemien. & Badura, Acta Soc. Bot. Poloniae 23: 727, 1955 (1954). = Mycotypha dichotoma F.A. Wolf, J. Elisha Mitchell Sci. Soc. 71: 217, 1955. = Ostracoderma dichotomum<