MYCOTAXON
THE INTERNATIONAL JOURNAL OF FUNGAL TAXONOMY & NOMENCLATURE
VOLUME 129(1) JULY-SEPTEMBER 2014
Ypsilomyces elegans gen. & sp. nov. (Almeida & Gusmao— Fie. 2, p. 184) Davi AUGUSTO CARNEIRO DE ALMEIDA, artist
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MYCOTAXON
THE INTERNATIONAL JOURNAL OF FUNGAL TAXONOMY & NOMENCLATURE
VOLUME 129(1)
OCTOBER-DECEMBER 2014
EDITOR-IN-CHIEF
LORELEI L. NORVELL
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NOMENCLATURE EDITOR
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CONSISTING OF I-X + 214 PAGES INCLUDING FIGURES
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Iv ... MYCOTAXON 129(1)
MY COTAXON
VOLUME ONE HUNDRED TWENTY-NINE(1) — TABLE OF CONTENTS
COVER SECTION
TTRT 5. oa Beng tock APOE AEs rah ee Pak, tates sted th 5 re es Led eee eke vi PCV IW CNS A occu ches od ahaae aed oh ted pede Prkce ose Meek aae vad ena aed peice cereale tale iad east 4 vii SUDIMISSION PrOCCUIT Cs 2 S245. ieh ea tag to DR ORAS AT Ee ep he cada Viii TBO NOATE CLE OE wi ate Mel con So aber PSRTGL dl cP tet Nea, Sod op Where Pade Gea st tees ix
RESEARCH ARTICLES
Beltraniella species associated with leaf litter of the Atlantic Forest
in southern Bahia, Brazil Marcos VINI{cIUS OLIVEIRA Dos SANTOS, FLAVIA RODRIGUES BARBOSA, DILZE MARIA ARGOLO MAGALHAES,
EDNA DorA MARTINS NEWMAN LUZ, & JOSE LUIZ BEZERRA Neofomitella polyzonata gen. et sp. nov., and N. fumosipora and N. rhodophaea transferred from Fomitella Hal-J1ao Li, XING-CHUN LI, JOSEF VLASAK, & YU-CHENG DAI A new species of Uromyces from Turkey ZELIHA BAHCECIOGLU Cladosporium species from hypersaline environments as endophytes in leaves of Cocos nucifera and Vitis labrusca RAFAEL JOSE VILELA DE OLIVEIRA, THAIS EMANUELLE FEIJO DE LIMA, GLADSTONE ALVES DA SILVA, & MARIA AUXILIADORA DE QUEIROZ CAVALCANTI
Aschersonia narathiwatensis sp. nov. from southern Thailand SUCHADA MONGKOLSAMRIT, ARTIT KHONSANIT, WASANA NOISRIPOOM, PAGMADULAM BALDOR], & J. JENNIFER LUANGSA-ARD South Florida microfungi: Linkosia longirostrata, a new hyphomycete on paurotis palm GREGORIO DELGADO Taxonomy and phylogeny of Heterobasidion in South Korea YEONGSEON JANG, SEOKYOON JANG, YOUNG WOON LIM, CHANGMU KIM, & JAE-JIN KIM Two new combinations and a new record of Zasmidium from China FENGYAN ZHAI, W.H. HsIgH, YINGJIE Liv, & Y.L. Guo New reports of Gymnopus from Pakistan based on ITS sequences M. SABA & A.N. KHALID First report of Callistosporium luteoolivaceum from Western Himalaya, Pakistan M. SaBA & A.N. KHALID Gastroboletus thibetanus: a new species from China SHU-RONG WANG, Qi WANG, DE-LI WANG, & YU LI
21
33
41
47
DL
63
ies
#9
JULY-SEPTEMBER 2014... V
Biogeographical patterns in pyrenomycetous fungi and their taxonomy. 4. Hypoxylon and the southern United States LARISSA N. VASILYEVA & STEVEN L. STEPHENSON 85
Rhizophagus natalensis, a new species in the Glomeromycota JANUSZ BLASZKOWSKI, GERARD CHwaT, ANNA GORALSKA, & BRUNO T. GOTO 97
A new species of Nawawia from Malaysia, with a synopsis of the genus TEIK-KHIANG Gou, WalI-Yip Lau, & KAH-CHENG TEO 109
The Entolomataceae of the Pakaraima Mountains of Guyana 6: ten new species and a new combination in Nolanea Terry W. HENKEL, M. CATHERINE AIME, DAVID L. LARGENT, & TIMOTHY J. BARONI 119
Two new species of Xylaria and X. diminuta new to China Gu Huang, LIN Guo, & NA Liu 149
Erysiphe magnoliicola, a new powdery mildew on Magnolia SUNG-EUN CHO, SUSUMU TAKAMATSU, JAMJAN MEEBOON, & HYEON-DONG SHIN 153
Phoma recepii sp. nov. from the Caloplaca cerina group in Turkey MEHMET GOKHAN HALICcI, MEHMET CANDAN, MituHat GULLU, & AHMET OZCAN 163
Geastrum from the Atlantic Forest in northeast Brazil — new records for Brazil JULIETH DE OLIVEIRA Sousa, BIANCA DENISE BARBOSA DA SILVA, & IURI GOULART BASEIA 169
Ypsilomyces, a new thallic genus of conidial fungi from the semi-arid Caatinga biome of Brazil Davi AUGUSTO CARNEIRO DE ALMEIDA & Luis FERNANDO PASCHOLATI GUSMAO 181
Agaricus taeniatus sp. nov., a new member of Agaricus sect. Bivelares from northwest China Sal-Fer Li, Ya-Li X1, Cal-X1A QI, QIAN-QIAN LIANG, SHENG-LONG WEI, GUO-JIE LI, DoNG ZHAO, SHAO-JIE Li, & HUA-AN WEN 187 Macrolepiota distribution extends to the montane temperate forests of Pakistan MUHAMMAD FIAz, SANA JABEEN, AMNA IMRAN, HaBIB AHMAD, & ABDUL NASIR KHALID 197 Hyphodontia dhingrae sp. nov. from India SAMITA, S.K. SANYAL, & G.S. DHINGRA 209 NOMENCLATURAL NOVELTIES AND TYPIFICATIONS PROPOSED IN MYCOTAXON 129(1) 25.3
v1 ... MYCOTAXON 129(1)
ERRATA FROM PREVIOUS VOLUMES
VOLUME 128
p.99, line 11 FOR: Penicillium citreonigrum p.100, Table 2, line 9 FOR: MTCC 3019 p.100, Table 2, line 11 FOR: Penicillium citreonigrum
p.114, bottom line For: C,D=50 wm
READ: Penicillium chrysogenum READ: MTCC 3017 READ: Penicillium chrysogenum
READ: C, D, F = 50 um
PUBLICATION DATE FOR VOLUME ONE HUNDRED TWENTY-EIGHT MYCOTAXON for APRIL-JUNE, VOLUME 128 (I-1x + 1-208) was issued on August 21, 2014
JuLYy-SEPTEMBER 2014... VII
REVIEWERS — VOLUME ONE HUNDRED TWENTY-NINE (ONE)
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 of MycoTaxon 129.
Vladimir Antonin J.L. Azevedo
José Luiz Bezerra Wolfgang von Brackel Uwe Braun
Mehmet Candan R.E Castaneda-Ruiz Michael Castellano Santiago Chacon Johannes C. Coetzee Yu-Cheng Dai Kanad Das
Antonio Hernandez Gutiérrez Ian Robert Hall
Nils Hallenberg Richard A. Humber Bryce Kendrick Kerry Knudsen
T.K. Arun Kumar David P. Lewis De-Wei Li
Shuyan Liu
Hayato Masuya
Juan Luis Mata Wieslaw Mulenko Lorelei L. Norvell Fritz Oehl
Clark L. Ovrebo Shaun R. Pennycook Ronald H. Petersen Amy Y. Rossman Alvaro Figueredo Dos Santos B.M. Sharma Gladstone Alves da Silva Michal TomSovsky Clement Tsui
Larissa Vasilyeva
Else C. Vellinga Nadja Santos Vitoria Felipe Wartchow Anthony J.S. Whalley Bayram Yildiz Rui-Lin Zhao
Li-Wei Zhou
vill ... MYCOTAXON 129(1)
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JULY-SEPTEMBER 2014 ... IX
FROM THE EDITOR-IN-CHIEF
THE ‘ISSUE’ ISSUE — Mycotaxon has always prided itself on being able to deliver complete volumes quarterly, making it an exception in the world of periodical journals. We based our nomenclature on the fact that any individual publication containing over 400 pages merited the name ‘volume. In the past year or so, however, what the editorial office calls a ‘perfect storm’ of misfortune — bad health, the need for page charges, and proliferation of competing online alternatives — has produced a temporary publications bottleneck leading to fewer pages. In an effort to speed up our quarterly delivery, we have decided to publish every three months regardless of size, assigning issue numbers to smaller publications as needed. Welcome, therefore, to Mycotaxon 129(1). We hope to deliver the remainder of this volume [Mycotaxon 129(2)] shortly!
INSTRUCTIONS TO AUTHORS: SIMPLIFIED! — Instructions are the bane of author and editor alike. Authors too often find them picky and/or incomprehensible, while editors despair because few follow the guidelines that they have spent too much time fine- tuning. At the moment, we all agree that MycoTaAxon instructions are too long to help anyone. The blame for this rests (alas) squarely on my editorial shoulders, which are now straightening to chop, cut, and simplify so that our authors can spend far less time changing font sizes and margins and much more time on research. Stay tuned for further developments.
MYCOTAXON 129(1) contains 22 papers by 82 authors (representing 15 countries) and revised by 45 expert reviewers.
Within its pages are two new genera (Neofomitella from China and Ypsilomyces from Brazil) and 30 species new to science representing Agaricus, Gastroboletus, and Xylaria from China; Aschersonia from Thailand; Erysiphe from South Korea and Japan; Hyphodontia from India; Hypoxylon and Linkosia from the U.S.A.; Nawawia from Thailand; Nolanea from Guyana; Phoma and Uromyces from Turkey; and Rhizophagus from Brazil.
In addition to new combinations in Neofomitella and Zasmidium, we also offer range extensions of Callistosporium, Gymnopus, and Macrolepiota species to Pakistan and Geastrum species to Brazil as well as conclusions from an excellent taxonomic and phylogenetic study of Heterobasidion in South Korea.
Warm regards,
Lorelei L. Norvell (Editor-in-Chief) 11 November 2014
ISSN (print) 0093-4666 © 2014. Mycotaxon, Ltd. ISSN (online) 2154-8889
MY COTAXON
http://dx.doi.org/10.5248/129.1 Volume 129(1), pp. 1-6 July-September 2014
Beltraniella species associated with leaf litter of the Atlantic Forest in southern Bahia, Brazil
MARCOS VIN{CIUS OLIVEIRA DOS SANTOS’ , FLAVIA RODRIGUES BARBOSA’, DILZE MARIA ARGOLO MAGALHAES?, EpNnA DorA MARTINS NEWMAN Luz’, & JOSE LuIzZ BEZERRA*™
‘Departamento de Micologia, Universidade Federal de Pernambuco, Av. Prof. Nelson Chaves, s/n°, Recife, PE, 50670-901, Brasil
?Instituto de Ciéncias Naturais, Humanas e Sociais, Universidade Federal de Mato Grosso, Avenida Alexandre Ferronato, 1200, Setor Industrial, Sinop, MT, 78557-267, Brasil
*Setor de Fitopatologia, Centro de Pesquisas do Cacau, Comissao Executiva do Plano da Lavoura Cacaueira, Rod. Ilhéus-Itabuna, km 22, Ilhéus, BA, 45662-000, Brasil
‘Centro de Ciéncias Agrarias e Biolégicas, Universidade Federal do Recéncavo da Bahia, Rua Rui Barbosa, 710, Cruz das Almas, BA, 44.380-00, Brasil
* CORRESPONDENCE TO: marcosvos@ymail.com
ABSTRACT — Two species of Beltraniella, B. botryospora and B. portoricensis, were found to be associated with the leaf litter of representative plants of the Atlantic Forest (Inga thibaudiana, Myrcia splendens, Pera glabrata) in the Reserva Biolégica de Una, municipality of Una, Bahia State, Brazil. A description and illustration are provided for B. botryospora, reported here for the first time from the Americas. A key to Beltraniella species cataloged in Brazil is also presented.
Key worps — conidial fungi, taxonomy, Fabaceae, Myrtaceae, Peraceae
Introduction
Beltraniella was proposed in 1952 by Subramanian with the type species B. odinae Subram., found in India on leaf litter of Odina wodier Roxb. (Subramanian 1952). The genus is characterized by setiform conidiophores and polyblastic sympodial denticulate conidiogenous cells. Conidia are turbinate or biconic and often caudate (Ellis 1971).
Beltraniella species are typically found on decomposing fallen leaves on the ground and other natural substrates (Polishook et al. 1996, Marques et al. 2007, Shirouzu et al. 2009, Magalhaes et al. 2011) in tropical and subtropical
2 ... Santos & al.
regions (Kirk 1981). Some species correspond to the anamorphic stage of the ascomycetes Pseudomassaria Jacz. or Leiosphaerella Hohn. (Kirk et al. 2008).
Currently, approximately 20 species are described in Beltraniella (Shirouzu et al. 2010, Priya et al. 2011), of which four have been cataloged in Brazil: B. amoena R.F. Castaneda et al., B. japonica Matsush., B. portoricensis, and B. fertilis Heredia et al. (Gusmao et al. 2001, Marques et al. 2007, Magalhaes et al. 2011). Beltraniella portoricensis and B. fertilis have been previously listed as associated with plants endemic to the Atlantic Forest (Magalhaes et al. 2011). The present study aimed to identify Beltraniella species associated with the leaf litter from three common representative plant species from the Atlantic Forest in the Reserva Bioldgica de Una, municipality of Una, Bahia State, Brazil.
Materials & methods
The materials were collected in September 2011 and April 2012 in the Reserva Biologica de Una (an Atlantic Forest Conservation Unit), municipality of Una, Bahia State, Brazil. Five specimens of three representative Atlantic Forest tree species were marked and identified in the field as Inga thibaudiana DC. (Fabaceae), Myrcia splendens (Sw.) DC. (Myrtaceae), and Pera glabrata (Schott) Poepp. ex Baill. (Peraceae). The leaf litter of these trees was collected at different stages of decomposition, stored in Kraft paper bags, and sent to the laboratory of Fungal Diversity of the Centro de Pesquisas do Cacau (CEPEC), Comissao Executiva do Plano da Lavoura Cacaueira (CEPLAC), Ilhéus, Bahia State, Brazil.
In the laboratory, the samples were placed in pre-perforated plastic containers and washed gently for one hour in running water so that the water did not hit them directly, thus allowing impurities to be removed. The washed samples were placed in moist chambers, observed after 72 h with a stereomicroscope, and monitored periodically for 30 days. The reproductive structures of the microfungi were removed with a histological needle and placed in a permanent mounting medium containing polyvinyl-lacto- glycerol (PVLG) resin (Silva & Grandi 2011).
The prepared fungal slides were observed using a light microscope, and the species were identified by morphological comparison with specific literature (Ellis 1971, Seifert et al. 2011). Photomicrographs were taken with a Sony Cyber-shot 16.2 megapixel digital camera. Subsequently, the permanent slides were deposited in the Mycological Collection of CEPEC.
Taxonomy
Beltraniella botryospora Shirouzu & Tokum., Fungal Diversity 43: 88 (2010) PLATE 1
CoLonligs amphigenous, effused, dark-brown. MycELIuM immersed in the substrate. SETAE subulate, arising from lobed basal cells, single, erect, straight or slightly curved, simple, septate, verrucose, dark-brown, 112-380 x 4-8 um. CONIDIOPHORES macronematous, mononematous, with two forms: long conidiophores arising from lobed basal cells, setiform, single, erect, straight
Beltraniella in Brazil ... 3
\|| ¥ |
PLaTE 1. Beltraniella botryospora. A, B. Verrucose setae; C. Long conidiophore; D, E. Short conidiophores; F—H. Conidia; I, J. Separating cells. Scale bars: A-C = 10 um; D-J = 5 um.
or slightly curved, simple or rarely branched in the apical region, septate, verrucose, dark-brown, lighter in the apical region, 170-432 x 5-9.4 um, and short conidiophores single or in groups with long conidiophores, erect, straight or slightly curved, simple or branched, septate, smooth, pale brown, 8-20 x 4-6 um. CONIDIOGENOUS CELLS terminal, cylindrical, 6-12 x 4-6 um, polyblastic, integrated, sympodial, denticulate, pale brown. SEPARATING CELLS ellipsoid to subglobose, 7-10 x 2.6-4 um, smooth, subhyaline, with one denticle on each end. Conip1a originated directly from conidiogenous cells in the long conidiophores and from separating cells in short conidiophores, turbinated, obovate to obpyriform, subhyaline, 0-septate, 18-24.6 x 4.6-9 um, with a supraequatorial subhyaline transverse band.
SPECIMENS EXAMINED — BRAZIL. Banta: Una, on decaying leaves of Myrcia splendens, 22/TX/2011, M.V.O.dos Santos 1360 (CEPEC 2361); on decaying leaves of
4 ... Santos & al.
Pera glabrata, 22/1X/2011, M.V.O.dos Santos 1361 (CEPEC 2362); on decaying leaves of Inga thibaudiana, 2/1V/2012, M.V.O.dos Santos 1362 (CEPEC 2363).
GEOGRAPHICAL DISTRIBUTION — Japan (Shirouzu et al. 2010), Brazil (this paper).
Comments — Beltraniella botryospora was previously described as associated with living and dead leaves of Quercus acuta Thunb. and Q. salicina Blume in Japan (Shirouzu et al. 2010). In Brazil, B. botryospora is now being reported in association with the leaf litter of three trees belonging to different families, none closely related to Fagaceae, clearly indicating its adaptability to a wide range of substrates.
The specimen’s characters are consonant with the original description (Shirouzu et al. 2010). However, in the original description, the setae were larger (140-550 um), the conidiophores were shorter (12-30 um), and the conidiogenous cells were larger (10-20 um), while the separating cells (5 um diam.) and conidia (7.5-10 um diam.) were narrower. Young and immature structures were not considered for measuring the dimensions of the setae.
Beltraniella botryospora is similar to B. fertilis due to the presence of setae, two types of conidiophores (long setiform conidiophores and short non- setiform conidiophores) with fertile apices, and turbinate conidia (Heredia et al. 2002). However, the conidiophores of B. fertilis branch more than once at the apex, whereas branching is rare in B. botryospora (Shirouzu et al. 2010); also, B. fertilis has smaller setae and narrower conidia (Heredia et al. 2002). Beltraniella botryospora is also similar to B. portoricensis, which differs by producing only one type of conidiophore (short non-setiform conidiophores; Pirozynski & Patil 1970).
This is the first record of Beltraniella botryospora in the Americas.
Beltraniella portoricensis (F. Stevens) Piroz. & S.D. Patil, Can. J. Bot. 48: 575 (1970) DESCRIPTION AND ILLUSTRATION: Ellis (1971, as Ellisiopsis gallesiae), Gusmao & Grandi (1996).
SPECIMENS EXAMINED — BRAZIL. BAutA: Una, on decaying leaves of Inga thibaudiana, 22/1X/2011, M.V.O.dos Santos 1363 (CEPEC 2364); on decaying leaves of Pera glabrata, 22/1X/2011, M.V.O.dos Santos 1364 (CEPEC 2365); on decaying leaves of Myrcia splendens, 22/1X/2011, M.V.O.dos Santos 1365 (CEPEC 2366).
GEOGRAPHICAL DISTRIBUTION — Cosmopolitan.
ComMMENTS — No previous records were found of associations between B. portoricensis and the three plants studied, and this is the first record of this taxon for the county of Una. Beltraniella portoricensis was found only in the first sampling. This species is commonly found in the leaf litter of various plants occurring in tropical, subtropical, and temperate regions (Grandi & Gusmao 2002) and appears to be adaptable regarding substrate (Hyde et al. 2007).
Beltraniella in Brazil ... 5
Key to Beltraniella species cataloged in Brazil
bas Setaé.and separating CEUs presente soko n whee urn Mice ite dee ote ie 8 oe ie eS oad 2, IbeSetae and-separatinia-céll oa bSentee. i. he chon Me dogs date eit de aonpeertee B. japonica Day SeuLOriMcONnidiopHhOres ADSeMN bd Ly ala'y Manele sm ibbly wh bhig nh beig oh dace Beeaans Ricci B 3 PBs SeueOrm CONIMIOPMOFeS: PTESEN TEs hs cet Al set NT cle h treet een tate edna ees vata - 3a. Setae smooth; separating cells clavate, often remaining attached on the conidial (BEER eh die ie. ein aetie ares tie civ roe A averse ler geet eh comes Cov B. amoena 3b. Setae verrucose; separating cells oval to fusiform ................ B. portoricensis
4a. Conidiophore, simple or rarely branched at apex, with branching occurring once at most, up to 560 um long; setae up to 550 um long; separating cells SUDSIODOSE 10 OM OIG 4.6 seieg db wntondrh sabe arte aeheg drt peda dels pobed dle 2cdad doh Sekt B. botryospora 4b. Conidiophore, branched at the apex more than once, up to 328 um long; setae up to 202 um long; separating cells obovoid.................... B. fertilis
Acknowledgements
We thank the Coordenagao de Aperfeigoamento de Pessoal de Nivel Superior (CAPES) for the grant awarded to the first author and the Conselho Nacional de Desenvolvimento Cientifico e Tecnolégico (CNPq) for grants awarded to the last two authors; the Reserva Bioldgica de Una, the Programa de Pés-Graduag¢ao em Biologia de Fungos (PPGBF) of the Universidade Federal de Pernambuco (UFPE) and José Lima da Paixao for assisting us in the sampling; and the Comissao Executiva do Plano da Lavoura Cacaueira (CEPLAC) for providing the laboratory to conduct the research. The authors also thank Drs. Alvaro Figueredo dos Santos and Nadja Santos Vitoria for the pre-submission review for our manuscript.
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Marques MFO, Barbosa FR, Gusmao LFP, Castafieda-Ruiz RF, Maia LC. 2007. Conidial fungi from the semi-arid Caatinga biome of Brazil. Cubasina microspora sp. nov., a note on C. albofusca, and some new records for South America. Mycotaxon 102: 17-23.
Pirozynski KA, Patil SD. 1970. Some setose hyphomycetes of leaf litter in south India. Canadian Journal of Botany 48: 567-581.
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Priya SW, Nagaveni HC, Kunwar IK, Manoharachary IK. 2011. A new pathogenic species of Beltraniella from India. Journal of Mycology and Plant Pathology 41: 20-23.
Seifert K, Morgan-Jones G, Gams W, Kendrick B. 2011. The genera of hyphomycetes. CBS Biodiversity Series 9. 997 p.
Shirouzu T, Hirose D, Fukasawa Y, Tokumasu S. 2009. Fungal succession associated with the decay of leaves of an evergreen oak, Quercus myrsinaefolia. Fungal Diversity 34: 87-109.
Shirouzu T, Hirose D, Tokumasu S, To-Anun C, Maekawa N. 2010. Host affinity and phylogenetic position of a new anamorphic fungus Beltraniella botryospora from living and fallen leaves of evergreen oaks. Fungal Diversity 43: 85-92. http://dx.doi.org/10.1007/s13225-010-0037-1
Silva P, Grandi RAP. 2011. A newspecies of Thozetella (anamorphic fungi) from Brazil. Cryptogamie, Mycologie 32: 359-363. http://dx.doi.org/10.7872/crym.v32.iss4.2011.359
Subramanian CV. 1952. Fungi imperfecti from Madras—III. Beltraniella gen. nov. Proceedings of the Indian Academy of Sciences, Section B, 36: 223-228.
ISSN (print) 0093-4666 © 2014. Mycotaxon, Ltd. ISSN (online) 2154-8889
MY COTAXON
http://dx.doi.org/10.5248/129.7 Volume 129(1), pp. 7-20 July-September 2014
Neofomitella polyzonata gen. et sp. nov., and N. fumosipora and N. rhodophaea transferred from Fomitella
Hat-Jr1Ao Li”, XING-CHUN LI’, JOSEF VLASAK3, & YU-CHENG Dal'*
' Institute of Microbiology, Beijing Forestry University, Beijing, 100083, China ? National Institute of Occupational Health and Poison Control,
Chinese Center for Disease Control and Prevention, Beijing, 100050, China ° Biology Centre of the Academy of Sciences of the Czech Republic,
Branisovské 31, CZ-370 05 Ceské Budéjovice, Czech Republic
*CORRESPONDENCE TO: daiyucheng2013@gmail.com
ABSTRACT — Phylogenetic analysis based on ITS, nLSU, and RPB2 sequences revealed that Fomitella in the current sense belonged to two distantly related subclades in the core polyporoid clade. Fomitella in a narrow sense is proposed for the type species, F. supina, and Neofomitella gen. nov. is proposed for Fomitella fumosipora and F. rhodophaea and a new species N. polyzonata. Neofomitella differs from Fomitella by its distinctly crusted basidiocarps with the cuticle developing from base to margin. Illustrated descriptions of the new genus and species are provided. The main morphological differences between Fomitella, Neofomitella, and related genera are discussed, and an identification key to Neofomitella is also provided.
Key worps — phylogeny, Polyporaceae, Polyporales, Basidiomycota, taxonomy
Introduction
Fomitella Murrill, typified by E supina (Sw.) Murrill, was erected as a monotypic genus by Murrill (1905). Subsequently, F fumosoavellanea (Romell) Murrill [ Trichaptum fumosoavellaneum (Romell) Rajchenb. & Bianchin.], E fumosipora, FE. rhodophaea, and F. malaysiana (Corner) T. Hatt. & Sotome were transferred to the genus (Murrill 1908; Hattori 2005; Hattori & Sotome 2013). Hattori (2005) emended the generic concept of Fomitella as follows: basidiocarps annual to perennial, effused-reflexed to distinctly pileate, pileal surface glabrous to minutely tomentose, context firm-fibrous to corky, light orange to pale brown, with a dark agglutinated crust; hyphal system trimitic with clamped generative hyphae, skeletal and binding hyphae well differentiated, negative in Melzer’s reagent; cystidia absent; basidiospores
8 ... Li & al.
ellipsoid to cylindrical, colorless, thin-walled, smooth, and negative in Melzer’s reagent; causing a white rot.
On-going studies on the diversity of wood-rotting fungi in eastern China have produced several new species (Dai & Cui 2005; Cui & Dai 2008a,b; Cui et al. 2007, 2008; He & Dai 2012; Wang et al. 2009, 2011; Cao et al. 2012), and additional study on the specimens from this area recently revealed an undescribed species. Phylogenetic analysis clustered our undescribed species with Fomitella fumosipora and FE. rhodophaea as a clade separated from E supina. Therefore, we propose a new genus Neofomitella to accommodate the new species and two new combinations from Fomitella.
Materials & methods
Morphological studies
The studied specimens were deposited at the herbaria of the Institute of Microbiology, Beijing Forestry University (BJFC) and Institute of Applied Ecology, Chinese Academy of Sciences (IFP). The microscopic procedure follows Cui & Zhao (2012). To present the basidiospore size variation, 5% of measurements were excluded from each end of the range and were given in parentheses. In the text the following abbreviations were used: IKI = Melzer’s reagent, IKI- = both inamyloid and non-dextrinoid, KOH = 5% potassium hydroxide, CB = Cotton Blue, CB- = acyanophilous, L = mean spore length (arithmetic average of all spores), W = mean spore width (arithmetic average of all spores), Q = variation in the L/W ratios between the specimens studied, n = number of spores measured from given number of specimens. Special colors follow Petersen (1996).
Molecular study and phylogenetic analysis
Dried specimens were used for molecular study. CTAB rapid Plant genome extraction kit-DN14 (Aidlab Biotechnologies Co. Ltd, Beijing) was used to extract DNA, according to the manufacturer's instructions with some modifications. ITS region was amplified with primer pair ITS5 and ITS4 (White et al. 1990), nLSU region with primer pair LROR and LR7 (http://www.biology.duke.edu/fungi/mycolab/primers.htm), and RPB2 with primer pair fRPB2-f5F and bRPB2-7.1R (Liu et al. 1999; Matheny 2005). The PCR procedures were: (1) for ITS — initial denaturation at 95°C for 3 min, followed by 35 cycles at 94°C for 40 s, 54°C for 45 s and 72°C for 1 min, and a final extension of 72°C for 10 min; (2) for nLSU — initial denaturation at 94°C for 1 min, followed by 35 cycles at 94°C for 30 s, 50°C for 1 min and 72°C for 1.5 min, and a final extension of 72°C for 10 min; and (3) for RPB2 — initial denaturation at 94°C for 2 min, followed by 10 cycles at 94°C for 40 s, 60°C for 40 s and 72°C for 2 min, then followed by 37 cycles at 94°C for 45 s, 55°C for 1.5 min and 72°C for 2 min, and a final extension of 72°C for 10 min. The PCR products were directly sequenced in Beijing Genomics Institute, China, with the same primers.
Other reference sequences for our phylogenetic analysis were selected from Binder et al. (2005), Justo & Hibbett (2011), Miettinen & Rajchenberg (2012), and BLAST searches in GenBank (TABLE 1). These sequences were sampled from Polyporales with
Neofomitella gen. nov. ... 9
TABLE 1. Polyporalean and outgroup taxa used in phylogenetic analysis. (Sequences generated in this study are in bold.)
SPECIES SAMPLE NUMBER ITS NLSU RPB2 Abundisporus pubertatis Dai 11927 KC867398 KC867494 KF274654 Abundisporus violaceus MUCL38617 FJ411100 FJ393867 = Climacodon septentrionalis AFTOL-ID 767 AY854082 AY684165 AY780941 Coriolopsis aspera Cui 6702 KC867353 KC867476 KF274658 Cui 6725 KC867356 KC867477 KF274659 Coriolopsis brunneoleuca Dai 12087 KC867416 KC867435 KF274656 Dai 12180 KC867414 KC867432 KF274655 Coriolopsis byrsina FP-105050-Sp JN165001 JN164788 JN164871 Coriolopsis cf. caperata CR22 JN164999 JN164789 JN164870 Ryvarden 45481 KC867399 KC867428 KF274657 Coriolopsis retropicta Dai 9870 KC867404 KC867443 KF274653 Coriolopsis rigida BJFC12680 KC867381 KC867454 KF274664 Coriolopsis sanguinaria Cui 5444 KC867387 KC867463 — Dai 9314 KC867390 KC867467 — Coriolopsis sp. BRFM1125 JX082370 — — BRFM1126 JX082371 — — Coriolopsis strumosa Dai 10642 JX559278 JX559303 JX559312 Dai 10657 KC867371 KC867491 KF274650 Daedaleopsis confragosa Cui 9732 JX569731 JX569748 KEF274647 Daedaleopsis sinensis Dai 11431 JX569732 JX569749 KF274648 Datronia mollis RLG6304sp JN165002 JN164791 JN164872 Datronia scutellata RLG9584T JN165004 JN164792 JN164873 Dentocorticium sulphurellum T609 JN165015 JN164815 JN164875 Donkioporia expansa P188 HM536087 HM536052 HM536102 Earliella scabrosa PR1209 JN165009 JN164793 JN164866 Fomes fomentarius Cui 8020 JX290073 JX290070 — Fomitella supina JV0610 KF274645 KF274646 —_ Nunez 1183 KF274644 _— — Ryvarden 39027 KF274643 — _ Fomitopsis pinicola AFTOL-ID 770 AY854083 AY684164 AY786056 Funalia gallica BJFC12697 KC867379 KC867453 = RLG-7630-sp JN165013 JN164814 JN164869 Funalia trogii RLG-4286-Sp JN164993 JN164808 JN164867 Ganoderma tsugae AFTOL-ID 771 DQ206985 AY684163 DQ408116 Grifola sordulenta AFTOL-ID 562 AY854085 AY645050 AY786058 Hexagonia apiaria Cui 6447 KC867362 KC867481 KF274660 Hexagonia glabra Cui 8468 JX559277 JX559302 JX559311 Dai 10991 JX569733 JX569750 KF274649 Lignosus rhinocerotis PEN94 JQ409359 AB368074 AB368132 Lopharia cinerascens FP-105043-sp JN165019 JN164813 JN164874 Megasporia major Cui 10253 JQ314366 JQ780437 JX559314 Megasporoporiella subcavernulosa Cui 9252 JQ780378 JQ78041 JX559315 Microporus affinis Cui 7714 JX569739 JX569746 KF274661 Microporus flabelliformis Dai 11574 JX569740 JX569747 KF274662 Microporus xanthopus Cui 8284 JX290074 JX290071 JX559313 Neofomitella fumosipora Cui 8816 JX569734 JX569741 — Dai 10777 JX569735 JX569742 — Neofomitella polyzonata Dai 10419 JX569738 JX569745 KF274663 Dai 10420 JX569736 JX569743 — Dai 11360 JX569737 JX569744 _— Neofomitella rhodophaea TFRI 414 EU232216 EU232300 — Perenniporia corticola Cui 1465 JN048759 JN048779 KF274651 Perenniporia tenuis Wei 2783 JQ001858 JQ001848 KF274652 Phlebia radiata FPL6140 AY854087 AF287885 AY218502 Polyporus grammocephalus WD2343 AB587626 AB368089 AB368146 Polyporus varius WD2347 AB587636 AB368111 AB368168 Pseudofavolus cucullatus WD2157 AB587637 AB368114 AB368170 Trametes betulina HHB-9942-sp JN164983 JN164794 JN164860 Trametes elegans FP-105679-sp JN164944 JN164799 JN164861 Trametes polyzona BKW-004 JN164978 JN164790 JN164856 Trametes sanguinea PR-SC-95 JN164982 JN164795 JN164858 Trametes suaveolens FP-102529-sp JN164966 JN164807 JN164853 Trametes versicolor FP-135156-sp JN164919 JN164809 JN164850 Trametopsis cervina TJV-93-216-sp JN165020 JN164796 JN164877 Boletopsis leucomelaena AFTOL-ID 1527 DQ484064 DQ154112 GU187820 Hydnellum geogenium AFTOL-ID 680 DQ218304 AY631900 DQ408133
10... Li &al.
the aim of providing representatives for all major clades within the order, concluded by Larsson et al. (2004), Binder et al. (2005) and Larsson (2007). Boletopsis leucomelaena (Pers.) Fayod and Hydnellum geogenium (Fr.) Banker were selected as outgroup (Justo & Hibbett 2011). Phylogenetic analysis for each single gene was carried out, and we got similar topologies to combined dataset in both MP and Bayes analyses. So in the paper we showed the result from the combined dataset that received higher support values. This combined dataset was aligned using Clustalx 1.83 (Chenna et al. 2003) and manually edited as necessary. Sequence alignment was deposited at TreeBase (submission ID 14418).
Maximum parsimony (MP) analysis was performed using PAUP* 4.0b10 (Swofford 2002) with gaps treated as missing data. Trees were generated using 100 replicates of random stepwise addition of sequence and tree-bisection reconnection (TBR) branch- swapping algorithm. All characters were given equal weight. Branch support for all parsimony analysis was estimated by performing 1 000 bootstrap (BP) replicates (Felsenstein 1985) with a heuristic search of 10 random-addition replicates for each bootstrap replicate.
The best-fit model of nucleotide substitution was selected by hierarchical likelihood ratio tests (hALRT, Huelsenbeck & Crandall 1997; Posada & Crandall 2001) implemented in the MrModelTest 2.2 (Posada & Crandall 1998; Nylander 2004). Following this model, MrBayes 3.1.2 (Ronquist & Huelsenbeck 2003) was used for Bayesian inference (BI). Eight Markov chains were run from random starting tree for 3,000,000 generations and sampled every 100 generations. The first one-fourth of the trees, which represented the burn-in phase of the analysis, were discarded, while the last three-fourths were used for calculating Bayesian posterior probabilities (BPPs) in the consensus tree. Confident branch support is defined as BPPs not less than 0.95 and bootstrap values above 50%.
Results
Molecular phylogeny
The combined dataset included 66 samples with 66 ITS, 62 nLSU, and 51 RPB2 sequences, representing 50 species and two collections not identified to species level from the main clades of Polyporales, as well as two species from Thelephorales as outgroup. MP analysis yielded eight equally parsimonious trees (tree length = 9 153, CI = 0.283, RI = 0.520, RC = 0.147, HI = 0.717). The best model for the alignment was estimated as “GTR+1+G”. BI resulted in an average standard deviation of split frequencies 0.005127. The topologies from MP and BI were similar, and only the BI tree was presented along with the BP from MP analysis (Fic. 1). In the phylogenetic analysis, sampled species of Fomitella formed two subclades within the core polyporoid clade. One subclade comprised Fomitella supina, the generic type, and two unidentified collections (labelled as Coriolopsis) from French Guiana (BPP = 1.00, MP = 100%; Fic. 1), while the second subclade included E fumosipora, F. rhodophaea and our undescribed species (BPP = 1.00, MP = 96%; Fia. 1). Taken the morphology into consideration together, we propose a new genus, Neofomitella, to accommodate
Neofomitella gen. nov.... 11
1.00/1007 HexagoniaglabraDai 10991 core polyporoid clade 1.00/10 © Hexagonia glabra Cui 8468 i . Daedaleopsis confragosa Cui 9732 Daedaleopsis sinensis Dai 11431 Coriolopsis strumosa Dai 10642 1.00/100| Coriolopsis strumosaDai 10657 1.00/100; Coriolopsis aspera Cui 6702 .00/ Coriolopsis aspera Cui 6725 Hexagoniaapiaria Cui 6447 Foes fomentarius Cui 8020 EarliellascabrosaPR1209 1.00/82) Coriolopsis sp BRFM1125 . sey Coriolopsis sp BRFM1126 |Fomitetta ABER Y Fomitella supinaNunez 1183 ; L Foinitellasupina Ryvarden 39027 Fomitella supina Fomitella supinaJV0610 Coriolopsis rigida BIFC 12680 Coriolopsis sanguinaria Cui 5444 Coriolopsis sanguinaria Dai 9314 Funalia gallica RLG7630sp 1.00/74 ; Funaliatrogii RLG4286sp 1.00100 FimaliagallicaBJFC 12697 0.97/92 Neofomitella fiimosipora Dai 10777 1.00/96] | 1.00/100|! Neofomitella finosipora Cui 8816
1.00/10 Neofomnitellarhodophaea TFRI414 2 . , C | Neofomitella polyzonataDai 10420 Neofomitella 1.00/8 CM! Neofomitella polyzonataDai 11360
0.92/- Neofomitella polyzonataDai 10419 } 1.00/100; Microporus affinis Cui 7714
= Mier c >Hiformis Dai 1157- Toortod Microporus flabelliformis Dai 11574
Microporus xanthopus Cui 8284 Lignosus rhinocerotis PEN94 1.00/100 Coriolopsis cf. caperata CR22 1.00/72 Coriolopsis cf. caperata Ryvarden 45481
1.00/-} 1.00/70) Coriolopsis brunmeoleucaDai 12180
1.00;.[ |!000 Coriolopsis brunmeoleucaDai 12087 i Coriolopsis retropicta Dai 9870 1.00/4 Megasporiamajor Dai 10253 ie | 1.90/95 — Abundisports violacetis MUCL38617
1.00/5 1.00/94
Abundisporus pubertatis Dai 11927 ay Coriolopsis byrsina FP-105050-sp LT Perenniporiacorticola Cuil465 sll Peremiporia tenuis W ei 2783 Ganoderina tsugae AFTOL-ID 771 DonkioporiaexpansaP 188 0.98/67 1.00/9 Datronia mollis RLG6304sp 1.00/100 DatroniascutellataRLG9584T Polyporus varitts WD2347 00/58 Pseudofavolus cucullattis WD2157 Polyporus grammocephalis WD2343 Megasporoporiellasubcavernulosa Cui 9252
1.00/1Q0 0.99!
0.99/64
0 ai- fn .00/100 Trametes suaveolens FP-102529-sp 1.00/8 qd Trametes versicolor FP-135156-sp
pee Trametes betulina HHB9942sp 0.99/98 S Trametes polyzonaBKW 004
Trametes elegans FP-105679-sp Trametes sanguinea PRSC95
DentocorticiumsulphurelhinT609 : core polyporoid clade
aC? 1.00/100 Lopharia cinerascens FP-105043-sp Grifola sordulenta AFTOL-ID 562 IGrifolaclade Polyporales 0.99/63 Climacodon septentrionalis AFTOL-ID 767 1.00/100 00/100 Trametopsis cervina TIV-93-216-sp | Phlebioid clade PhiebiaradiataFPL6140 Fomitopsis pinicola AFTOL-ID 770 lAntrodiacdade
Hydnellium geogenitim AFTOL-ID 680 Boletopsis leucomelaenaAFTOL-ID 1527
Fic. 1. Phylogram of polyporalean taxa obtained from Bayesian inference of the combined dataset of ITS, nLSU, and RPB2. Bayesian posterior probabilities more than 0.95 and bootstrap values above 50% are indicated above or below the branches. The scale bar represents estimated number of changes per site.
the second subclade; we transfer the two Fomitella species to the new genus and describe our new species as N. polyzonata. Phylogenetically, the Neofomitella subclade clusters with a Microporus subclade (Fie. 1).
12°. Li Sak Taxonomy
Neofomitella Y.C. Dai, Hai J. Li & Vlasak, gen. nov. MycoBank MB 804799 Differs from Fomitella by its distinctly crusted basidiocarps with the cuticle developing from base to margin and from Microporus by its buff, yellowish brown, brown to pale grey context. TYPE SPECIES — Polyporus rhodophaeus Lév.
EryMoLocy — Neofomitella (Lat.): referring to the morphological similarity to Fomitella.
Basidiocarps annual or perennial, pileate, sessile or effused-reflexed. Pileal surface yellowish-brown, brown, orange-brown, reddish-brown, fuscous to almost black, usually concentrically zonate or sulcate, glabrous to velutinate. Context buff, yellowish brown, brown to pale grey, corky to hard corky, with a dark agglutinated crust developing from base to margin. Pore surface usually white, cream to pale buff when fresh, pale brown to yellowish-brown when dry. Hyphal system trimitic with clamped generative hyphae, skeletal and binding hyphae well differentiated, negative in Melzer’s reagent. Cystidia absent. Basidiospores oblong ellipsoid to cylindrical, hyaline, thin-walled, smooth, IKI-, CB-, tissue turn into black in KOH. Growing usually on angiosperm wood and causing a white rot.
Neofomitella fumosipora (Corner) Y.C. Dai, Hai J. Li & Vlasak, comb. nov. Fic. 2a MycoBank MB 804803
=Trametes fumosipora Corner, Beih. Nova Hedwigia 97: 106 (1989). =Fomitella fumosipora (Corner) T. Hatt., Mycoscience 46: 309 (2005).
SPECIMENS EXAMINED — CHINA. GUANGDONG PROVINCE, SHIXING COUNTY, Chebaling Nature Reserve, fallen angiosperm trunk, 25 Jun 2010, B.K. Cui 8816 (BJFC 7756); 23 Nov 2010, B.K. Cui 8715, 8717 (BJFC 7657, 7659); HAINAN PROVINCE, CHANGJIANG County, Bawangling Nature Reserve, fallen angiosperm trunk, 8 May 2009, Y.C. Dai 10777 (BJFC 5021). MALAYSIA. NEGERI SEMBILAM, Pasoh For. Res., 5 Dec 1998, T. Hattori (BJFC: ex. TFM ex. FE 19017); PENANG, Penang Hill, alt. 500-750 m, 13 Dec 2002, T. Hattori (BJFC: ex. TFM ex. F. 20477).
Neofomitella polyzonata Y.C. Dai, Hai J. Li & Vlasak, sp. nov. Fras 2b,<c, 3 MycoBank MB 804804
Differs from Neofomitella fumosipora and N. rhodophaea by its distinctly velutinate pileal surface and larger pores.
Type — China, Jiangxi Province, Fenyi County, Dagang Mountain, on fallen trunk of Cyclobalanopsis blakei (Skan) Schottky (Fagaceae), 18 Sep 2008, Y.C. Dai 10419 (holotype, BJFC 4668).
ETyMoLocy — polyzonata (Lat.): referring to the multiple zones on the pileal surface.
Neofomitella gen. nov. ... 13
Fic. 2. Neofomitella and Fomitella basidiocarps. a: N. fumosipora. b, c: N. polyzonata. d, e: N. rhodophaea. f: FE. supina.
FruiITBopy — Basidiocarps annual, pileate, sessile, usually imbricate, without odor or taste when fresh, hard corky to woody hard and light in weight upon drying. Pilei applanate, semicircular to dimidiate, up to 6 cm long, 10 cm wide and 6 mm thick at base. Pileal surface buff-yellow, curry-yellow, cinnamon, orange-brown to reddish brown, with one or more vinaceous brown, dark blue
14... Li &al.
[pr \ Ve A
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tella polyzonata (drawn from the holotype). es. b: Basidia and basidioles. c: Cystidioles.
e from trama. e: Hyphae from context.
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Fic. 3. Neofom a: Basidiospo
a 3
d: Hyph:
y velutinate, concentrically zonate; white to cream eading from the base with age. Margin cream, buff e or slightly wavy. Pore surface cream to buff when
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to almost black zones, fine
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outgrowth occasionally sp
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Neofomitella gen. nov. ... 15
fresh, buff to yellowish brown when dry or bruised; sterile margin indistinct, white to cream, up to 0.5 mm wide; pores round, 3-4 per mm; dissepiments thin, entire. Context buff to yellowish brown, hard corky, azonate, up to 3 mm thick, a more or less dark agglutinated crust present as black zones in context towards upper surface. Tube layer concolorous with pore surface, up to 3 mm long.
HyPHAL STRUCTURE — Hyphal system trimitic; generative hyphae bearing clamp connections; skeletal and binding hyphae IKI-, CB-; tissue turning into black in KOH.
CONTEXT — Generative hyphae in context infrequent, colorless, thin- walled, moderately branched, 1.5-3 um in diam; skeletal hyphae in context dominant, colorless to pale yellowish brown, thick-walled with a narrow lumen to subsolid, occasionally branched, straight, more or less regularly arranged, 3-5 um in diam; binding hyphae in context abundant, colorless to pale yellowish brown, thick-walled with a narrow lumen to subsolid, flexuous, frequently branched, interwoven, 1-2.5 um in diam.
TuBEs — Generative hyphae infrequent, colorless, thin-walled, moderately branched, 1.2-2 um in diam; skeletal hyphae in trama dominant, colorless to pale yellowish brown, thick-walled, occasionally branched, more or less straight, interwoven, 2.4-4 um in diam; binding hyphae in trama colorless to pale yellowish brown, thick-walled with a narrow lumen to subsolid, flexuous, frequently branched, interwoven, 1-2 um in diam. Cystidia absent, while cystidioles present, fusoid to tubular, sometimes tips of cystidioles branched and septate, colorless, thin-walled, 22-34 x 2.5-4.5 um; basidia clavate, bearing four sterigmata and a basal clamp connection, 18-24 x 3.5-5 um; basidioles in shape similar to basidia, but distinctly smaller.
Spores — Basidiospores cylindrical, colorless, thin-walled, smooth, IKI-, CB-, (3.8-)3.9-5 x (1.8-)1.9-2.1(-2.5) um, L = 4.34 um, W = 2.04 um, Q = 2.13 (n = 9/1).
TYPE OF ROT —White rot.
ADDITIONAL SPECIMENS EXAMINED — CHINA. FUJIAN PROVINCE, Wuyi Mountains, Longfenggu Forest Park, on fallen angiosperm trunk, 27 Aug 2006, B.K. Cui 4124 (BJFC 485, IFP 1196); Wuyishan Nature Reserve, Taoyuanyu, on fallen angiosperm trunk, 24 Aug 2006, Y.C. Dai 7376 (IFP 11887); HUNAN PROVINCE, SHIMEN COUNTY, Hupingshan Nature Reserve, on fallen angiosperm trunk, 16 Sep 2009, Y.C. Dai 11360
(BJFC 7283); JIANGXI PROVINCE, FENy1I County, Dagang Mountain, on fallen trunk of Cyclobalanopsis blakei, 18 Sep 2008, Y.C. Dai 10420 (BJFC 4669).
Neofomitella rhodophaea (Lév.) Y.C. Dai, Hai J. Li & Vlasak, comb. nov. Fics 2d,e MycoBank MB 804800 = Polyporus rhodophaeus Lév., Ann. Sci. Nat., Bot., 3e Sér., 2: 190 (1844). = Fomitella rhodophaea (Lév.) T. Hatt., Mycoscience 46: 305 (2005).
16... Li &al.
SPECIMENS EXAMINED — JAPAN. KAGOSHIMA PREF., KIMOTSUKI-GuN, Uchindura, Oct 1962, K. Aoshima (BJFC: ex. O 10866); Tokyo, Meguro, 15 Oct 1943 (BJFC: ex. TFM F-10861); MALAYSIA. NEGERI SEMBILAN, Pasoh, Sembilan For. Nat. Res., 25 Dec 1997, T. Hattori (BJFC: ex. TFM 18313).
OTHER SPECIMENS EXAMINED — Fomitella supina (Fic. 2f): BRAZIL. ALAGOAS STATE, PILAR MuNICcIPALITY, Reserva Particular do patrimonio Natural (PPPN) Sao Pedro, on dead hardwood, Oct 2000, B. Tatiana, N. Gibertoni 357 (BJFC: ex. O 10849). COLOMBIA. DEPT. DE ANTIOQUIA, MUNICIPIO CALDAS FINCA, alt. 1700 m, 25 Jun 1978, L. Ryvarden 16 590 (BJFC: ex. O 10769). GUATEMALA. Lago Atitlan, on hard wood, J. Vlasak JV0610. PANAMA. Ensenada de Santa Cruz. Parque Nac. de Coiba, 17 Nov 1996, M. Nunez 1183 (BJFC: ex. O 10770). PUERTO RICO. Toro Negro, Commonwealth For., on deciduous wood, 24 Jun 1996, L. Ryvarden 39 027 (BJFC: ex. O 10772).
Discussion
The new genus Neofomitella is composed of two distinct lineages, one comprising Neofomitella rhodophaea and N. fumosipora and the other N. polyzonata, newly described from China. The main morphological characters of Fomitella supina and the three Neofomitella species are presented in TABLE 2. Like Neofomitella polyzonata, N. fumosipora has more or less brown basidiocarps; however, it has a glabrous pileal surface and smaller pores (7-9 per mm; Hattori 2005). All three Neofomitella species show distinctly crusted basidiocarps with a cuticle that develops from base to margin (Fic, 2a-e). In F. supina the cuticle also develops from the base but does not usually extend to the very margin (Fie. 2f).
Neofomitella is phylogenetically close to several Microporus species (Fic. 1). Although Microporus also has a trimitic hyphal system, colorless, thin- walled, smooth, non-dextrinoid, inamyloid basidiospores, its species usually produce stipitate basidiocarps with a white to cream context (Gilbertson & Ryvarden 1986; Nunez & Ryvarden 2001). Its type species, M. perula P. Beauv. [= M. xanthopus (Fr.) Kuntze], also has an encrusted pileal surface, but its centrally or laterally stipitate and usually infundibuliform basidiocarps, distinctly small pores, and white context (NUufiez & Ryvarden 2001) clearly differentiate it from Neofomitella species.
Morphologically, Coriolopsis shares many features with Fomitella and Neofomitella, such as a more or less brown context, trimitic hyphal system with clamped generative hyphae, colorless basidiospores, and causing a white rot (Gilbertson & Ryvarden 1986; Nufez & Ryvarden 2001; Hattori 2005). However, phylogenetic analysis shows Coriolopsis as polyphyletic with its type species, Polyporus occidentalis Klotzsch [= Coriolopsis occidentalis (Klotzsch) Murrill; = Trametes polyzona (Pers.) Justo], clustering within the Trametes clade and distinctly separated from Fomitella and Neofomitella (Fic. 1).
Neofomitella gen. nov. ... 17
TaBe 2. Main morphological characters of Fomitella and Neofomitella species.
BASIDIOCARPS* PORES BASIDIOSPORES CYSTIDIOLES? DISTRIBUTION* SPECIES (/mm) (um) F supina A/P, 5-7 6.5-9 x 2.4-3.5, - T/S, V then G C America, Africa N. fumosipora A/P,G 6-10 3-4 x 1.7-2.2, + T/S, C/OE Asia N. polyzonata A, V 3-4 3.9-5 x 1.9-2.1, + S/WT, C China N. rhodophaea = A/P,G 7-8 3.5-4.5 x 2.5-3, - T, also S/WT, OE Asia, Africa
"A = annual, P = perennial, G = glabrous, V = velutinate; *C = cylindrical, OE = oblong-ellipsoid; 34 = presence, — = absence; *T = tropics, S = subtropics, WT = warm-temperate.
The transfer of this taxon to Trametes was suggested by Corner (1989) and validated by Justo & Hibbett (2011). Coriolopsis strumosa (Fr.) Ryvarden usually has encrusted basidiocarps that are similar to Neofomitella, but C. strumosa has a soft corky, olivaceous-brown, umber or hazel-brown context and distinctly larger basidiospores (7-10 x 3-4 um; Nunez & Ryvarden 2001; Li 2013).
Funalia species cluster in the same clade with Fomitella. Both genera share trimitic hyphal system and more or less similar basidiospores, but Funalia species usually produce a strongly tomentose to hispid pileal surface, a white, cream to straw colored context, and cyanophilous skeletal hyphae (Niemela et al. 1992; Dai 1996).
Hexagonia has pileate brown basidiocarps, tissues that darken in KOH, a trimitic hyphal system, and colorless thin-walled basidiospores similar to those in Fomitella and Neofomitella (Nuitez & Ryvarden 2001). However, Hexagonia species usually have larger hexagonal pores and distinctly larger basidiospores (usually longer than 10 um; Gilbertson & Ryvarden 1986; Nufiez & Ryvarden 2001).
No sequences of Fomitella malaysiana are available at present. The distinctly encrusted pileal surface (Hattori & Sotome 2013) indicates that this species may belong to Neofomitella. Further molecular studies are needed to resolve its taxonomic and phylogenetic position.
Key to species of Neofomitella
LMP ORSS SAA APSA y bs, Fests aes aee .aoea ap aes wote See Sone Shs Sune See Sane a N. polyzonata TL POKES OS LOsP CEN IN “ie tle sew at as Semel as Seas Senn ey ail nn Ney selon Noein eae tech at nabeny als 2 2; Basidiospores hy7 2 2M WIS ah it20 itn Pied Wee Eee eT ae N. fumosipora
2; Basidiospores2;5=3 pind WIE is i.e. 4 gd ice gd nace dg Heer ed Heer Ed Heer dd Hat N. rhodophaea
18 ... Li &al.
Acknowledgments
We express our gratitude to Drs. Michal TomSovsky (Mendel University in Brno, Czech Republic) and Li-Wei Zhou (IFP, China) who reviewed the manuscript. Great thanks to Drs. Hai-Sheng Yuan (IFP, China) and Shuang-Hui He (BJFU, China) for help in field collections. Special thanks are due to Dr. Tsutomu Hattori (FFPRI, Japan), Prof. Leif Ryvarden (O, Norway) and Dr. Tatiana B. Gibertoni (URM, Brazil) for loan of specimens. The research is supported by the National Natural Science Foundation of China (Project No. 31093440), the Program for New Century Excellent Talents in University (NCET-11-0585), and the institutional support RVO: 60077344 of the Czech Academy of Sciences to J. Vlasak.
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ISSN (print) 0093-4666 © 2014. Mycotaxon, Ltd. ISSN (online) 2154-8889
MY COTAXON
http://dx.doi.org/10.5248/129.21 Volume 129(1), pp. 21-23 July-September 2014
A new species of Uromyces from Turkey
ZELIHA BAHCECIOGLU
Department of Biology, Faculty of Science and Art, Inonu University, TR 44280, Malatya, Turkey *CORRESPONDENCE TO: Zeliha. bahcecioglu@inonu.edu.tr
ABSTRACT — A new species of Uromyces on Myosotis sp. (Boraginaceae) is described from Turkey. As far as is known, no Uromyces has previously been described on this host genus. Uromyces myosotidis sp. nov. is described and illustrated in this paper.
Key worps —Anatolia, new taxon, Puccineaceae, Basidiomycota
Introduction
Among the approximately 351 species of rust fungi recorded for Turkey, 74 species and one variety of Uromyces have been reported (Bahcecioglu & Kabaktepe 2012). Recently, an unknown Uromyces specimen was collected in the country on Myosotis; it is described here as a new species, U. myosotidis.
Materials & methods
The host specimen was collected from Adiyaman Province in 2012, prepared according to established herbarium techniques, and identified from the relevant botanical literature (Davis 1965, 1978; Davis et al. 1988; Grau 1978). Spores were scraped from the dried host specimen and mounted in lactophenol. The preparations were examined with an Olympus CX31 light microscope. Analysis LS Starter software was used to measure at least 30 spores for each spore state. The specimen is preserved in the herbarium of Inonu University (INU), Turkey.
Taxonomy
Uromyces myosotidis Bahc. sp. nov. Fic. 1 MycoBank 804880 Differs from Uromyces tairae by its smaller teliospores and urediniospores.
Type: Turkey, Adryaman Province, 10-12 km between Adiyaman and Celikhan, 1500-1600 m, on Myosotis sp. (Boraginaceae), 6 June 2012, Zeliha Bahcecioglu 3888 (Holotype, INU).
ErymMo_oey: from Myosotis, the host genus.
22 ... Bahcecioglu
10 um
Fic. 1. Uromyces myosotidis (holotype). Teliospores and urediniospore.
Pycnia and aecia not seen. Uredinia and telia often mixed together, hypophyllous, covered by epidermis, small, scattered, brown. Urediniospores 14-16 x 13-16 um globoid, subgloboid, walls 1.5-2 um thick, brown, smooth or finely sparsely echinulate, with 2-3 equatorial pores. Teliospores 14-22 x 12-18 um, globoid, subgloboid; walls <5-8 um apiculus, dark brown, smooth. Pedicels <56 um long, broken, hyaline.
Discussion
As far as is known, no Uromyces has previously been described on Myosotis. Uromyces myosotidis differs from Uromyces spp. determined on other genera of Boraginaceae in teliospore and urediniospore form and size. Uromyces tairae Hirats. f. on Messerschmidia (= Tournefortia) has larger teliospores (25-38 x 20-28 um) and urediniospores (22-30 x 17-25 um; Hiratsuka 1940). Uromyces permeritus Cummins and U. dolichosporus Dietel & Holw. on Tournefortia have ellipsoid, long fusiform, or long clavate teliospores (Holway 1897; Cummins 1940). Uromyces heliotropii Sred. on Heliotropium has larger teliospores (21-25 x 18-22 um) with an apical pore (Ul’yanishchev et al. 1985; Kuprevich & Ul’yanishchev 1975).
Acknowledgments
This work was supported by the BAP of Inonu University, project numbers 2010/104. Thus many thank BAP of Inonu University, also thanks Prof. Dr. Bayram Yildiz Balikesir University, Balikesir (Turkey), for help with identification of host and Dr. Mehmet Candan for presubmission expert reviews and Dr. Brian Spooner (Kew garden) for helpful suggestions.
Uromyces myosotidis sp. nov. (Turkey) ... 23
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Bahcecioglu Z, Kabaktepe S. 2012. Checklist of rust fungi in Turkey. Mycotaxon 119: 494. http://www.mycotaxon.com//resources/checklists/Bahcecioglu-v119-checklist.pdf
Cummins GB. 1940. Uredinales of New Guinea. Mycologia 32: 32-375. http://dx.doi.org/10.2307/3754317
Davis PH. 1965. Flora of Turkey and the East Aegean Islands. Vol. 1. Edinburgh. University Press.
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Grau J. 1978. Myosotis L. 264-280, in: PH Davis (ed.). Flora of Turkey and the East Aegean Islands. Vol. 6. Edinburgh: University Press.
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ISSN (print) 0093-4666 © 2014. Mycotaxon, Ltd. ISSN (online) 2154-8889
MY COTAXON
http://dx.doi.org/10.5248/129.25 Volume 129(1), pp.25-31 July-September 2014
Cladosporium species from hypersaline environments as endophytes in leaves of Cocos nucifera and Vitis labrusca
RAFAEL JOSE VILELA DE OLIVEIRA ', THAIS EMANUELLE FEIJO DE LIMA, GLADSTONE ALVES DA SILVA, & MARIA AUXILIADORA DE QUEIROZ CAVALCANTI”
Departamento de Micologia, Universidade Federal de Pernambuco, Rua Nelson Chaves, s/n, Cidade Universitaria, Recife, 50670-901, Brazil
* CORRESPONDENCE TO: ' rafaelvilela87@gmail.com, ? xiliamac@gmail.com
ABSTRACT — Cladosporium dominicanum and C. halotolerans were isolated in Pernambuco, Brazil, from healthy leaves of Cocos nucifera and Vitis labrusca, respectively. This is the first report of these species as endophytes and the first record of C. dominicanum for Brazil.
Key worps —ITS, phylogenetic analysis, tropical plants
Introduction
Cladosporium was originally described by Link in 1816, and Cladosporium herbarum was selected from amongst Link's original species by Clements & Shear (1931) as the generic type (see also Bensch et al. 2010). With over 772 taxa, Cladosporium is one of the largest hyphomycete genera (Dugan et al. 2004). Bensch et al. (2012), who analyzed the genus phylogenetically, created an identification key and described 169 species. Cladosporium species are commonly found as plant and human pathogens and are also decomposers of food, paints, textiles, and organic matter (Ellis, 1971, 1976, Kwon et al. 2001; Liu et al. 2001). They occur as endophytes in plants of tropical regions (Costa et al. 2012) and are the most frequent endophytic fungi in some plants (Bezerra et al. 2012).
Cladosporium halotolerans is saprobic on various substrates and has been isolated in hypersaline water from subtropical regions, indoor environments, arctic ice, human and animal lesions, plants, rock, window frames, and mycorrhizal roots (Bensch et al. 2012). Cladosporium dominicanum was first isolated from hypersaline water and was also found as a saprobe on the surface of fruits (Bensch et al. 2012).
26 ... Oliveira & al.
Here we report the first records of C. dominicanum as an endophyte in leaves of Cocos nucifera (and its first occurrence from Brazil) and of C. halotolerans as an endophyte in the leaves of Vitis labrusca.
Materials & methods
During February 2010 (dry season), healthy mature leaves of Vitis labrusca were collected from forest areas of the municipalities of Sao Vicente Férrer, Pernambuco, and in May 2012 (dry season), healthy mature leaves of Cocos nucifera were collected from forest areas of the municipalities of Goiana, Pernambuco.
Sterilization, isolation, and identification
In the laboratory, each leaf was washed gently in running water and soap. Leaf discs were cut with a sterile metallic cork punch (6 mm diam.), decontaminated with 70% alcohol for 30 sec and sodium hypochlorite solution (NaOCl) at 2% for 2.5 min, and twice washed with sterilized distilled water in order to remove the hypochlorite excess (Petrini 1996; modified technique). Six surface sterilized discs were transferred in triplicate to each Petri dish containing malt extract agar (MEA) + chloramphenicol (50 mg.L"') to prevent bacterial growth. The plates were incubated at room temperature (28 + 2°C) and observed daily during 15 days for colony development. For asepsis control, 50 uL of water, used to remove hypochlorite, was plated in MEA to confirm surface disinfection (Pereira et al. 1993). Species identification was based on macro- and microstructural characteristics of the colony, according to Bensch et al. (2012). For each species (Cladosporium halotolerans and C. dominicanum), one isolate was deposited in the URM Culture Collection of the Universidade Federal de Pernambuco.
Molecular analyses
The fungi biomass was obtained from cultures grown on malt agar contained in test tubes and kept at 28°C for up to six days. All mycelium was removed from the test tube with the aid of a platinum loop; the material was transferred to 2 ml micro-tubes with screw caps to which were added 0.5 g of glass beads with two different diameters in the 1:1 ratio (acid-washed, 150-212 um and 425-600 um; Sigma, U.S. sieve). The material was crushed by stirring at high speed in a FastPrep.
Genomic DNA was extracted according to Gées-Neto et al. (2005). The material was washed with chloroform : isoamyl alcohol (24:1) and following homogenization of the material in CTAB buffer at 2%, besides isopropanol precipitation, washing in 70% ethanol, and re-suspended in 50 uL of ultrapure water.
Primers ITS1 and ITS4 (White et al. 1990) were used to amplify the ITS region. PCR reactions were carried out in 50 wL volumes containing 75 mM Tris-HCl pH 8.8, 200 mM (NH,),SO,, 0.01% Tween 20, 2 mM MgCl, 200 uM each dNTPs, 1 uM of each primer, and 2 units of Taq DNA polymerase (Fermentas, Maryland, USA); cycling parameters were 5 min at 95°C (1 cycle), 45s at 94°C, 1 min at 60°C, 1 min at 72°C (39 cycles), and a final elongation of 7 min at 72°C.
The final amplicons were purified with the PureLink PCR Purification Kit (Invitrogen). Sequencing was provided by the Human Genome Research Center (Sao Paulo, Brazil). Sequence data were compared to gene libraries (EMBL and GenBank) using BLASTn. The new sequences deriving from the species were deposited in the NCBI database under the accession numbers KJ000286 and KJ000287.
Cladosporium spp. in Cocos nucifera & Vitis labrusca (Brazil) ... 27
Phylogenetic analyses
The phylogeny was reconstructed by sequences of the ITS1+5.8s+ITS2 rDNA gene. The fungal sequences were aligned in ClustalX (Larkin et al. 2007) and edited with the BioEdit program (Hall 1999). Prior to phylogenetic analysis, the model of nucleotide substitution was estimated using Topali 2.5 (Milne et al. 2004). Bayesian analysis (two runs over 1 x 10° generations with a burnin value of 2500) were performed in MrBayes 3.1.2 (Ronquist & Huelsenbeck 2003), and maximum likelihood analysis (1000 bootstrap) was performed in PhyML (Guindon & Gascuel 2003), launched from Topali 2.5, using the GIR + I+ G model. Neighbor-joining (established with the models cited above) and maximum parsimony analyses were performed using PAUP*4b10 (Swofford 2003) with 1000 bootstrap replications.
The phylogenetic tree described in Zalar et al. (2007) was used as reference.
Results
Taxonomy
Cladosporium halotolerans Zalar, de Hoog & Gunde-Cim., Stud. Mycol. 58: 172. 2007.
PotaTo DEXTROSE AGAR: Colonies after 14 days of culture at 25°C, presenting a growth of 53 mm, dark grayish green, reverse dark green. Conidiophores erect, lateral or terminal, olive to brown, septate, smooth, branched, 10-77.5 x 2.5-5 um. Conidia in tandem, smooth, olive to brown, non-septate, globose, subglobose, or shortly limoniform (majority), slightly rough, 1.25-3.75 x 2.5-3.75 um. Conidiogenous cells undifferentiated. Branch-primary conidia rare, with <2 distal scars; branch-secondary conidia, 0-1 septum (no majority), with <3 distal scars, 7.5-27.5 x 2.5-3.75 um.
Matt AGar: Colonies after 14 days of culture at 25°C, presenting a growth of 59 mm, dark green grayish yellowish to dark green, reverse dark green. Conidiophores erect, lateral or terminal, olive to brown, septate, smooth, branched, 13-190 x 2.5-6.25 um. Conidia in tandem, smooth, olive to brown, non-septate, globose to subglobose, 2.5-5 x 1.25-2.5 um. Conidiogenous cells undifferentiated. Branch-primary conidia (2.5 x 2.5-3 um) with up to 2 distal scars; branch-secondary conidia, 0-1 septum (no majority), with <2-3 distal scars, 3-22.5 x 2.5-3.75 um.
MALT + 5% NACL: Colonies after 14 days of culture at 25°C, presenting a growth of 50 mm, dark grayish green, reverse dark green, and with little sporulation. Conidiophores erect, lateral or terminal, olive to brown, septate, smooth, branched, 15-212.5 x 2.5 um. Conidia in tandem, smooth, olive to brown, non-septate, globose (majority) to limoniform, rough 2.5-5 x 1.25-3.75 uum. Conidiogenous cells undifferentiated. Branch-primary conidia present; branch-secondary conidia, 0-1 septum, with up to 3 (mostly 2) distal scars, 10-25 x 3.75 um.
28 ... Oliveira & al.
SPECIMEN EXAMINED: BRAZIL. PERNAMBUCO: Sao Vicente Férrer, in healthy mature leaves of Vitis labrusca L. cv. Isabel, Feb 2010, T.E.R Lima (URM6963; GenBank KJ000286). Notes: Cladosporium halotolerans is known from Africa, Arctic, Asia, Australasia, Europe, North America, and Central and South America. This is the first recorded occurrence of the species as an endophyte.
Cladosporium dominicanum Zalar, de Hoog & Gunde-Cim., Stud. Mycol. 58: 169. 2007.
PotaTo DEXTROSE AGAR: Colonies after 14 days of culture at 25°C, presenting a growth of 31 mm, green olive, velvety, reverse dark gray. Conidiophores erect, lateral or terminal, olive to brown, slightly verrucose, septate, branched or unbranched, 36-245 x 2-2.5 um. Conidiogenous cells undifferentiated. Conidia in tandem, slightly verrucose, light brown, not septate, ovoid, 3.7-5.58 x 2.7-3.2 um. Ramoconidia rarely formed; secondary cylindrical ramoconidia, 0-1 septate 14-28 x 2.5-3 um.
MALT AGAR: Colonies after 14 days of culture at 25°C, presenting a growth of 34 mm, dark green, velvety, grooved, reverse dark gray. Conidiophores erect, lateral or terminal, olive to brown, slightly verrucose, septate, branched or unbranched, 50-160 x 2-2.5 um. Conidiogenous cells undifferentiated. Conidia in tandem, slightly verrucose, light brown, non-septate, ovoid, 3.7-5.58 x 2.7-3.2 um. Ramoconidia rarely formed; secondary cylindrical ramoconidia, 0-1 septate, 12-19 x 2.5-3 um.
MALT + 5% NACL: Colonies after 14 days of culture at 25°C, presenting a growth of 40 mm, light green, grooved, reverse dark gray. Conidiophores erect, lateral or terminal, olive to brown, slightly verrucose, septate, branched or unbranched, 45-107.5 x 2-2.5 um. Conidiogenous cells undifferentiated. Conidia in tandem, slightly verrucose, light brown, non-septate, ovoid, 3.7-4.8 x 2.7-3.2 um. Ramoconidia rarely formed, secondary cylindrical ramoconidia, 0-1 septate, 10-22.5 x 2.5-3 um. SPECIMEN EXAMINED: BRAZIL. PERNAMBUCO: Goiana, in healthy mature leaves of Cocos nucifera L., May 2012, R.J.V. Oliveira (URM6962; GenBank KJ000287). Notes: Cladosporium dominicanum was previously known from Asia (Iran) and Central America (Dominican Republic). This is the first recorded occurrence of the species for South America (Brazil) and as an endophyte.
Molecular & phylogenetic analysis
The sequences generated from our fungal isolates grouped firmly in Cladosporium. Our isolate URM6962 (from leaves of Cocos nucifera) formed a clade with sequences from C. dominicanum, and isolate URM6963 (from leaves of Vitis labrusca) formed a clade with sequences from C. halotolerans (Fic. 1).
Cladosporium spp. in Cocos nucifera & Vitis labrusca (Brazil) ... 29
: C. cladosporioides DQ780408/EXF-321 199) C. cladosporioides DQ780409/EXF-780 oe °°! ©. cladosporioides AY213640/CBS 170.54 neotype ee C. spinulosum DQ780406/EXF-334 1.00! C. subinflatum DQ780405/EXF-343 —| C. tenuissimum DQ780397/EXF-452 PY C. tenuissimum DQ780398/EXF-563 54 C. oxysporum DQ780393/EXF-710 3] 24 C. oxysporum DQ780392/EXF-697
4.00 32 0.98 & C. oxysporum DQ780391/EXF-711 e5 28) C. psychrotolerans DQ780387/EXF-326 4 099} 121. psychrotolerans DQ780386/EXF-391
99 | C: fusiforme DQ780390/CBS 452.71 C. fusiforme DQ780389/EXF-397 "IC. fusiforme DQ780388/CBS 119414 991 C. velox DQ780361/CBS 119417 1.90 C. velox DQ780360/EXF-471 C. dominicanum DQ780355/EXF-720 C. dominicanum DQ780357/CPC 11683 holotype
0.80
87 1001 C. dominicanum KJ000287/URM6962 83 87 °°! C. dominicanum DQ780354/EXF-727
C. dominicanum DQ780353/CBS 119415 C. sphaerospermum DQ780343/CBS 193.54 neotype 891 C. sphaerospermum DQ780350/CBS 109.14
71 100 Be C. sphaerospermum DQ780348/EXF-738
C. sphaerospermum DQ780344/EXF-739 C. halotolerans DQ780364/CBS 119416 holotype
C. halotolerans DQ780368/EXF-380 93 | C. halotolerans KJ000286/URM6963 95 7 C. halotolerans DQ780366/EXF-646 ; C. halotolerans DQ780367/EXF-703
Fa C. salinae DQ780375/EXF-322
Re C. salinae DQ780374/CBS 119413
Cercospora beticola AY840527/CPC 11557
Change 0.02
Fic. 1. Phylogenetic reconstruction of the sphaerospermum complex in Cladosporium obtained from sequences of the ITS region. Sequences are labeled with their GenBank accession numbers. Support values (from top) are from neighbor-joining (NJ), maximum parsimony (MP), maximum likelihood (ML) and Bayesian analyses. Only bootstrap values of at least 50% are shown.
In their review of Cladosporium, Bensch et al. (2012) showed that C. halotolerans and C. dominicanum belong to the C. sphaerospermum complex and are grouped close together.
30 ... Oliveira & al.
In the Blastn analysis, our C. dominicanum sequence (KJ000287) showed 100% identity with sequence DQ780353 of the C. dominicanum ex-holotype culture (EXF752 = CBS 119415) and with two sequences (KC763352, KC845931) from Chinese isolates labelled “C. sphaerospermum”. However, as sequence DQ780343 of the C. sphaerospermum neotype (CBS 193.54) showed only 97% identity with the sequence of our isolate URM6962, we conclude that sequences KC763352 and KC845931 in fact represent C. dominicanum.
In the Blastn analysis, our C. halotolerans sequence (KJ000286) showed 100% identity with sequence DQ780364 of the C. halotolerans ex-holotype culture (EXF572 = CBS 119416) and with 24 databank sequences labelled either “C. cladosporioides” (EF577236, AY361968, AB456576) or “C. sphaerospermum” (AB572909, AB572908, AB572903, AB572897, JN084018, AY625063, AM182174, AM182171, AM182168, AM176749, AM176685, EU759978, EU823317, JX156365, KC009836, JX839460, HQ263345, HQ248189, GU017501, JN253512, JN253512). However as sequence HM148003 of the C. cladosporioides neotype (CBS 17054) showed only 98% identity and the C. sphaerospermum neotype sequence showed only 96% identity with the sequence of our isolate URM6963, we conclude that the 24 database sequences probably represent C. halotolerans and not C. cladosporioides or C. sphaerospermum.
Acknowledgments
The authors thank Dr. José Luiz Bezerra and Dr. Jodo Lucio Azevedo for presubmission critical review. Special acknowledgements to Dr. Shaun Pennycook and Dr. Lorelei Norvell for their additional important corrections and suggestions to the manuscript. The authors also acknowledge the financial support provided by Conselho Nacional de Desenvolvimento Cientifico e Tecnoldgico (CNPq).
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Bensch K, Braun U, Groenewald JZ, Crous P.W. 2012. The genus Cladosporium. Studies in Mycology 72. 401 p. http://dx.doi.org/10.3114/sim0003
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ISSN (print) 0093-4666 © 2014. Mycotaxon, Ltd. ISSN (online) 2154-8889
MY COTAXON
http://dx.doi.org/10.5248/129.33 Volume 129(1), pp. 33-40 July-September 2014
Aschersonia narathiwatensis sp. nov. from southern Thailand
SUCHADA MONGKOLSAMRIT”™, ARTIT KHONSANIT', WASANA NOISRIPOOM'’, PAGMADULAM BALDOR)’, & J. JENNIFER LUANGSA-ARD*
‘BIOTEC, NSTDA Science Park, 113 Paholyothin Road, Klong 1, Klong Luang, Pathum Thani, Thailand ?Institute of Biology, Mongolian Academy of Sciences, Ulaanbaatar-51, Mongolia
* CORRESPONDENCE TO: suchada@biotec.or.th
ABSTRACT — Aschersonia narathiwatensis was isolated from whitefly nymphs (Hemiptera) collected at Hala-Bala Wildlife Sanctuary in Narathiwat Province, Thailand. This species has circular flattened-discoid brown to dark brown stroma and non-fragmenting ascospores. A comparison of macro-and microscopic characters among similar species is given. Phylogenetic analyses of nuclear ribosomal large subunit (LSU) and translation elongation factor 1-a (TEF-1la) support recognizing this fungus as a new species.
Key worps — morphology, phylogenetics, taxonomy
Introduction
Aschersonia Mont. is a genus of entomopathogenic fungi that attack scale insects (Coccidae, Hemiptera) or white flies (Aleyrodidae, Hemiptera) of which several species are reported from tropical and subtropical regions (Montagne 1848; Petch 1921; Mains 1959a,b; Chaverri et al. 2008; Mongkolsamrit et al. 2009, 2011; Qiu et al. 2009, 2010; Qiu & Guan 2010). Research on entomopathogenic fungi has been conducted under the biodiversity studies program to further our knowledge of these fungi in Thailand. Here we describe a new Aschersonia species from a tropical rainforest in southern Thailand.
Materials & methods
Surveys and collections were made during the rainy season in Hala-Bala Wildlife Sanctuary, which is located at the southernmost tip of peninsular Thailand on the international border with Malaysia. Pure cultures were made from isolations from the teleomorphic state following Mongkolsamrit et al. (2009). Sections of the stroma prepared by using a freezing microtome were mounted in distilled water and in cotton blue in lactophenol (Heritage et al. 1996). Observation of the microscopic characters
34 ... Mongkolsamrit & al.
(e.g., perithecia, ascospores and asci) were made using a light microscope. The color of fresh specimens and cultures were compared with the colors from standard code of Kornerup & Wanscher (1962). A voucher specimen and culture were deposited in BIOTEC Bangkok Herbarium (BBH) and BIOTEC Culture Collection, Thailand. Genomic DNA was extracted from each sample with cetyltrimethyl-ammonium bromide (CTAB) according to Mongkolsamrit et al. (2009). PCR amplification was done
TABLE 1. List of fungi used in molecular analyses. (Newly generated sequences set in bold.)
GENBANK SPECIES VOUCHER/ISOLATE LSU EF1-a Aschersonia badia BBH 14255 = BCC 8105 DQ518752 KC713630 BBH 32386 = BCC 55524 — KF016991 A. calendulina *BBH 17344 = BCC 20309 GU552154 KF016993 BBH 17325 = BCC 20306 GU552148 KF016994 *BCC 9483 DQ384938 DQ384962 A. luteola *BBH 13011 = BCC 19360 GU552155 GU552147 BBH 14277 = BCC 9481 DQ384945 DQ384974 BBH 14078 = BCC 7865 DQ384946 DQ384975 A. minutispora BBH 17373 = BCC 20635 GU552149 DQ552143 BBH 14143 = BCC 7959 GU552150 — BBH 12951 = BCC 17487 GU552151 GU552144 A. narathiwatensis *BBH 31120 = BCC 49495 KC713633 KC713628 *BBH 31120 = BCC 49498 KF016998 — *BBH 31120 = BCC 49499 KF016999 KF016992 A. samoensis BBH 14540 = BCC 8775 DQ384944 DQ384972 *BCC 8237 DQ384937 DQ384977 *NHJ 4093 AF327381 — Balansia henningsiana GAM 16112 AY489715 AY489610 Epichloe elymi C. Schardl 760 AY986924 AY986951 Moelleriella phyllogena PC. 554 = CUP 067784 EU392609 EU392673 P.C. 555 = CUP 067785 EU392610 EU392674 M. raciborskii 193-901b = ARSEF 7661 EU392611 EU392676 M. umbospora PC. 457 = CUP 067816 AY986904 AY986929 Samuelsia geonomis P.C. 614 = CUP 067857 EU392638 EU392692 S. mundiveteris BBH 24706 = BCC 31750 GU552157 GU552142 BBH 26961 = BCC 40021 GU552152 GU552145 BBH 26961 = BCC 40022 GU552153 GU552146 S. rufobrunnea P.C. 613 = CUP 067858 AY986918 AY986944
* Living cultures isolated from ascospores.
Aschersonia narathiwatensis sp. nov. (Thailand) ... 35
in 50 ul volumes consisting of 1x PCR buffer, 200 uM of each of the four dNTPs, 2.5 mM MgCl2, 1 U Taq DNA polymerase (Promega, Madison, Wisconsin), and 0.5 uM of each primer. The amplification of the partial gene region of the large subunit nuclear ribosomal DNA (LSU) used primers LRORf and LR5r (Vilgalys & Sun 1994) and the translation elongation factor 1-a (TEF-la) was amplified with primers EF-la: 983f (Carbone & Kohn 1999) and 2218r (Rehner 2001). After amplification in a MJ Research
é A. calendulina BCC20309 99 |L A. calendulina BCC20306
A. calendulina BCC9483 A, narathiwatensis BCC49495
95
98_| 4. narathiwatensis BCC49499 A. narathiwatensis BCC49498
<50
99 A, badia BCC55524 <50
A. badia BCC8105 68 A. samoensis BCC8237
My 64 |¢ A. samoensis NHJ4093
Aschersonia (teleomorph A. samoensis BCC8775 Hypocrella sensu lato) Ny 97 (4: luteola BCC19360
100 97| | A. luteola BCC7865 A. luteola BCC9481 71 | 4: minutispora BCC7959
98 A. minutispora BCC17487
a A, minutispora BCC20635 96 S. mundiveteris BCC40021 100 |' S. mundiveteris BCC40022
100 S. mundiveteris BCC31750 91
87 S. geonomis P.C.614 S. rufobrunnea P.C.613 100 |4 phyllogena P.C.555 100 M. phyllogena P.C.554 69 M. umbospora P.C.457 M. raciborskii 193-901b Epichloe elymi
Balansia henningsiana
10 changes
PLATE 1. Phylogenetic relationship of Aschersonia narathiwatensis and related species based on maximum parsimony analysis of the LSU and EF-1 a. Numbers above each branch represent bootstrap support from 1000 replicates. [S. = Samuelsia; M. = Moelleriella]
36 ... Mongkolsamrit & al.
DNA Engine ALD1244 thermal cycler following the procedure described in Sung et al. (2001), the PCR products were purified with a QIAquick PCR Purification Kit (Qiagen GmbH, Hilden, Germany), following the manufacturer’s instructions. Purified PCR products were sent to Macrogen Inc. Korea for sequencing. Sequences were proofread manually and assembled using BioEdit v. 7.0.4 (Hall 1999), and deposited in GenBank (TABLE 1). Sequences were aligned with Clustal W incorporated in BioEdit, and alignments were refined manually by direct examination. Maximum parsimony analysis was performed on the combined dataset of LSU and tef-1a sequences in PAUP 4.0b10 using random addition sequence (10 replications) where gaps were treated as missing data. Bootstrap analysis was performed using maximum parsimony criterion in 1000 replication samples. Balansia henningsiana GAM 16112 and Epichloe elymi C. Schardl 760 were used as outgroup taxa.
Results
Molecular analysis
LSU sequences were obtained from three isolates, and tef-la sequences from six isolates. Twenty-four LSU-related sequences and 19 TEF-1la-related sequences from GenBank were used to construct a phylogenetic tree. Of the 1652 characters in the combined alignment, 268 characters were parsimony informative. Maximum parsimony analyses of this data set yielded one parsimonious tree (tree length 698; CI = 0.610, RI = 0.797, RC = 0.486, HI = 0.390) as shown in PLATE 1. The phylogenetic tree shows that three teleomorphic isolates assigned to our new species (BCC 49495, BCC 49498 and BCC 49499) belong to the genus Aschersonia with a strong bootstrap support of 100%.
Taxonomy
Aschersonia narathiwatensis Mongkols., Khonsanit & Luangsa-ard, sp. nov. MycoBank MB 803933 PLATE 2
Differs from A. calendulina by its brown to dark brown stromata and shorter ascospores.
Type — ‘Thailand: Narathiwat Province, Headquarter Trail, Hala-Bala Wildlife Sanctuary, on whitefly nymphs (Hemiptera) on underside of dicotyledonous leaf, 24 Aug. 2011, A. Khonsanit (Holotype, BBH 31120; ex-holotype cultures BCC 49495 [GenBank, KC713633, KC713628], BCC 49498 [GenBank, KF016998], BCC 49499 [GenBank, KF016999, KF016992].
ErymMo.ocy — referring to Narathiwat Province, the collection location.
TELEOMORPH: Hypocrella
STROMATA circular, flattened-discoid, brown to dark brown, 5E7, <1-3 mm diam and 0.5-1 mm high, margin undulate, base slightly constricted; internally hyaline to whitish-coloured. Hypothallus membranous, slight or thickened, 0.5-1 mm thick, pale yellow. Perithecia crowded, immersed, 200-430 x 100-200 um, elongate flask-shaped, ostioles slightly projecting, translucent
Aschersonia narathiwatensis sp. nov. (Thailand) ... 37
PLATE 2. Aschersonia narathiwatensis: A-C. Stromata on infected whitefly nymph hosts; D. Cross section of stroma showing perithecia; E, F Mature ascus with developing asci; G. Whole ascospores; H. Sporulating colony on PDA at 20°C after 4 wk.
dark-brown. Asci 8-spored, <200 x 10-12 um. Ascospores whole, non- fragmenting, 80-100 x 1.5-2 um, blunt at upper end tapering to base.
CULTURAL CHARACTERISTICS Ascospores germinating within 24 h on PDA. Colonies on potato dextrose agar (PDA) slow-growing, attaining a diam of 5 mm in 4 wk. Optimal temperature 20-25°C, with no growth at <5°C and >35°C. Stromatic colonies pale brown, forming moderately compact stromata. Conidial masses pale brown, 4C6, appearing as abundant slimy masses from immersed pycnidia scattered over surface.
ComMENnts: In this study, the anamorphic generic name Aschersonia is used based on the recent major changes in fungal nomenclature requiring only one scientific name per fungal species (Hawksworth 2011; Taylor 2011; Gams et al. 2012; McNeill et al. 2012). Described earlier in 1848, Aschersonia has priority over the teleomorphic generic name, Hypocrella Sacc. (1878). In this study, Hypocrella is treated as the teleomorph of Aschersonia.
38 ... Mongkolsamrit & al.
Aschersonia narathiwatensis was been found only at Hala-Bala Wildlife Sanctuary in southern Thailand. Despite several attempts to find it in different seasons of the year, the anamorphic state of this species was not found in the field. The teleomorphic characters are presented and compared with other Thai species representing Hypocrella sensu lato (TABLE 2). The teleomorphic state of A. narathiwatensis is similar to that of A. calendulina reported from Thailand by Mongkolsamrit et al. (2009) in having flattened discoid stromata but differs in the color and size of the stromata. Aschersonia narathiwatensis has brown to dark brown stromata while those of A. calendulina are bright orange. The ascospores of A. narathiwatensis are somewhat shorter than those of A. calendulina (TABLE 2). In the phylogenetic analysis, A. narathiwatensis and A. calendulina form a clade with 95% support. Both morphological characters and phylogenetic analysis of the partial gene regions of LSU and tef-1a support A. narathiwatensis as a new species.
TABLE 2. Morphological comparison of Aschersonia teleomorphs in Thailand.
STROMATAL STROMATAL ASCI ASCOSPORES REFERENCE SPECIES SHAPE COLOUR (um) (um) A. calendulina Flattened Bright <180 x 130-150 x Mongkolsamrit discoid orange 10-12 1.5-2 et al. (2009) A. luteola Discoid to Yellow <170 x 75-120 x Mongkolsamrit stud-shaped 75-8 2 et al. (2009) A. narathiwatensis Flattened Brown <200 x 80-100 x This study discoid 10-12 1.5-2 Abana a Discoid to Rust 140-180 60-90 x Hywel-Jones & : stud-shaped orange x 8 4-5 Evans (1993)
Acknowledgments
The authors would like to thank Dr. Amy Y. Rossman (Systematic Mycology & Microbiology Laboratory, Beltsville, MD, USA) and Dr. Richard A. Humber (USDA- ARS Biological Integrated Pest Management Research Unit, Ithaca, NY, USA) for their comments and suggestions to improve this manuscript. We also would like to thank Prof. Morakot Tanticharoen, Dr. Kanyawim Kirtikara, and Dr. Lily Eurwilaichitr for their support of the program, ‘Biodiversity studies of entomopathogenic fungi in Thailand.” We are grateful to Mr. Soonthorn Todam and the staff at Hala-Bala Wildlife Sanctuary for their kind cooperation.
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http://dx.doi.org/10.5248/129.41 Volume 129(1), pp. 41-46 July-September 2014
South Florida microfungi: Linkosia longirostrata, a new hyphomycete on paurotis palm
GREGORIO DELGADO
EMLab Pe&K North Phoenix, 1501 West Knudsen Drive, Phoenix, AZ 85027, U.S.A.
* CORRESPONDENCE TO: gdelgado@emlabpk.com
Asstract — Linkosia longirostrata sp. nov. is described and illustrated from rachides of dead leaves of Acoelorrhaphe wrightii collected in southeastern Florida, U.S.A. The fungus is distinct in having no or very reduced 1-2-septate conidiophores, smooth or slightly verrucose determinate or occasionally percurrent conidiogenous cells, and narrowly obclavate to long obclavate rostrate finely roughened 9-18-distoseptate conidia with 0-2 dark brown constrictions and a long slender straight rostrum with 0-5 intercalary nodular swellings. Differences and similarities with morphologically similar Linkosia species are discussed.
Key worps — anamorphic Ascomycota, palm fungi, Sporidesmium, taxonomy
Introduction
While examining collections of dead plant debris from south Florida, several interesting Sporidesmium-like taxa were found colonizing decaying parts of native or introduced palm trees. One clearly fits within Linkosia A. Hern.-Gut. & B. Sutton (Hernandez-Gutiérrez & Sutton 1997, Wu & Zhuang 2005) in having distoseptate conidia and very reduced or absent conidiophores. After a detailed comparison with previously described Linkosia species, the fungus was found to differ in several morphological features and therefore is described here as new.
Materials & methods
Samples of dead leaves of Acoelorrhaphe wrightii (Griseb. & H. Wendl.) H. Wendl. ex Becc. (paurotis palm; Arecaceae) were collected from a forested area in central Broward County, Florida, U.S.A., in 2010. The samples were cut into small pieces and placed in plastic bags for later processing and examination according to Cannon & Sutton (2004). Fungal structures were mounted in lacto-cotton blue and 100 measurements were made at 1000x magnification whenever possible. Minimum, maximum, 5th and 95th percentiles were calculated for all measurements using Microsoft Excel 2007, with
42 ... Delgado
extreme values given in parentheses when different from percentiles. Microphotographs were taken using an Olympus BX-45 microscope and edited using Adobe Photoshop. The type specimen and other specimen including semi-permanent slides are deposited in the Herbarium of the U.S. National Fungus Collections (BPI).
Taxonomy
Linkosia longirostrata G. Delgado, sp. nov. PLATE 1 MycoBank MB809951 Differs from Linkosia coccothrinacis in having very reduced 1-2-septate conidiophores, occasionally percurrent conidiogenous cells, and larger finely roughened long obclavate- rostrate conidia with more distosepta and a rostrum with 0-5 intercalary nodular swellings.
Type — U.S.A. Florida, Broward Co., Plantation, Plantation Heritage Park, Anne Kolb Memorial Trail, 26°06’25”N 80°13’19”W, on rachides of dead leaves of Acoelorrhaphe wrightii, 30.V.2010, coll. G. Delgado (Holotype: BPI 884152H).
EryMoLocy — longirostrata, from the Latin longus (long) and rostratus (beaked),
referring to the long conidial rostrum COLONIES effuse, hairy, brown. MyceLium superficial, composed of branched, septate, pale brown to brown hyphae, 2-3 um wide. CONIDIOPHORES absent or very reduced, erect, straight, 1-2-septate, dark brown, often with a lobed base, 19-30(-37) um long, 7-15 um wide at base. CONIDIOGENOUS CELLS monoblastic, integrated, terminal, determinate or occasionally percurrent, solitary, simple, subcylindrical or lageniform, smooth or slightly verrucose, tapering toward the truncate apex, brown to dark brown, 13-22 x 6-11 um, 3.5-5 um wide at the apex, with 0-1 ampulliform, brown, rarely 1-septate proliferation. CONIDIAL SECESSION schizolytic. Conip14 holoblastic, straight or slightly curved, narrowly obclavate to long obclavate rostrate, 9-18-distoseptate with 0-4 pigmented distosepta, pale brown, darker toward the apex, sometimes with 0-2 dark brown constrictions, finely rough, (73-)91-158(-172) um long (including rostrum), 6.5-12 um wide; basal cell cylindrical or conico-truncate, dark brown to blackish brown, 6-11 x 3.5-5 um; rostrum slender, straight, pale brown to brown, subhyaline at the tip, <82 um long, with 0-5 intercalary, nodular swellings, 3-5 um wide. TELEOMORPH unknown.
ADDITIONAL SPECIMEN EXAMINED — U.S.A. Florida, Broward Co., Plantation, Plantation Heritage Park, Anne Kolb Memorial Trail, 26°06’25”N 80°13’19’W, on rachides of dead leaves of Acoelorrhaphe wrightii, 30.V.2010, coll. G. Delgado (BPI 884154H).
Discussion
Hernandez-Gutiérrez & Sutton (1997) introduced Linkosia in the context of a morphology-based reassessment of Sporidesmium Link that emphasized conidial septation, conidiophore presence or absence, and conidiophore proliferation (Subramanian 1992). The type species, Sporidesmium coccothrinacis
Linkosia longirostrata sp. nov. (U.S.A.) ... 43
PLaTE 1. Linkosia longirostrata (holotype, BPI 884152H): A. Conidia. B. Conidiophores, conidiogenous cells with or without proliferations, and mature and immature conidia. Scale bars = 30 um.
A
44 ... Delgado
A. Hern.-Gut. & J. Mena (= Linkosia coccothrinacis; Hernandez-Gutiérrez & Sutton 1997), is a peculiar hyphomycete characterized by conidiophores reduced to a single monoblastic conidiogenous cell and distoseptate conidia (Hernandez-Gutiérrez & Mena 1994). Subsequently, nine additional species have been described or transferred to the genus based on differences in conidial morphology including shape, number of distosepta, dimensions, wall texture and presence or absence of apical appendages (Almeida et al. 2014, Castaneda et al. 2000, Ma et al. 2011, Wu & Zhuang 2005, Zhang et al. 2009). Multigene- sequence data indicate that Linkosia, as well as other morphologically circumscribed Sporidesmium-like genera, are polyphyletic (Shenoy et al. 2007, Iturriaga et al. 2008). The few phylogenetic studies conducted on this generic complex have revealed the diverse or uncertain affinities of Linkosia species within the Sordariomycetes (Shenoy et al. 2006, Yang et al. 2010). The genus needs to be redefined in the light of molecular data but very few sequences are currently available in GenBank and one (attributed to L. fusiformis W.P. Wu) is apparently a contaminant (Summerbell et al. 2011). For that reason and in the absence of a culture isolate and molecular data, the present fungus is placed in Linkosia for diagnostic purposes, following the traditional morphological approach.
Linkosia longirostrata is morphologically unique among Linkosia species. Conidiogenous cells, which are slightly verrucose or smooth, are usually determinate but sometimes proliferate percurrently at least once to form an ampulliform proliferation at the apex which can be either 0- or 1-septate, a feature not previously seen in any other Linkosia species but present in the morphologically similar genus Stanjehughesia Subram. (Castaneda & Kendrick 1990, McKenzie 1995). The conidia are narrowly obclavate but mostly long obclavate rostrate with a long slender straight rostrum where up to 5 nodular swellings form at intervals along its length. These swellings apparently relate to different stages of rostrum elongation. The conidial tip, usually tapering to 1.5-2 um, widens to 3-5 um diam. and is at first rounded, after which the rostrum elongates to a certain length, gradually tapers, darkens, and widens again at a further point to form a new swelling, giving the rostrum a knotty appearance. One or two constrictions with a distinct dark brown band are often present at some distosepta, and sometimes the conidium between the constriction and basal cell turns dark brown as well. Also a few distosepta (up to four in certain conidia) appear pigmented and darker than the others. Younger conidia are smooth, but older ones are finely roughened with basal cells that are cylindrical or conico-truncate and distinctly dark brown to blackish brown. Linkosia coccothrinacis (A. Hern.-Gut. & J. Mena) A. Hern.-Gut. & B. Sutton (Hernandez-Gutiérrez & Sutton 1997), a saprobe on dead leaves of a palm tree found in Cuba, is morphologically similar, sharing dark brown constrictions, a
Linkosia longirostrata sp. nov. (U.S.A.) ... 45
few pigmented distosepta, and conico-truncate darker basal cells. Its conidia, however, are smaller (43.7-71.5 x 7-10.6 um) and smooth and have only 4-7 distosepta and a short rostrum without swellings.
Additionally, conidiophores in L. longirostrata are absent or present, and when present they are very reduced, usually 1- but occasionally 2-septate and often with a lobed base. Three Linkosia species were originally described as producing short 0-2-septate conidiophores: L. ponapensis (Matsush.) R.F. Castafieda et al. (Matsushima 1981, as Sporidesmium ponapense), L. refugia (B. Sutton & Pascoe) D.A.C. Almeida & Gusmao, and L. canescens (B. Sutton & Pascoe) D.A.C. Almeida & Gusmao (Sutton & Pascoe 1988, as Janetia refugia and J. canescens), while the remaining species have only lageniform or ampulliform conidiogenous cells that form directly on the superficial mycelium (Santa Izabel et al. 2013, Wu & Zhuang 2005). Linkosia ponapensis is clearly separated from L. longirostrata by its cylindrical conidiogenous cells and shorter (34-50(-70) um) naviculiform conidia with (3—)5-7(-9) distosepta and an apical 6-25 um long subulate appendage. Linkosia refugia is distinguished from L. longirostrata by its very rarely branched conidiophores and smaller (31-37 x 7-8 um) obpyriform 4-6-distoseptate conidia that gradually taper towards an obtuse paler apex but are not distinctly rostrate. In L. canescens, conidiogenous cells are mono- or polyblastic, bearing a single or 2-3 denticulate conidiogenous loci per cell; therefore, despite the presence of distoseptate conidia, this fungus is not considered congeneric with Linkosia as presently circumscribed and is better retained in Janetia M.B. Ellis (Ellis 1976, Goh & Hyde 1996).
Acknowledgments
I am grateful to Drs. A. Hernandez-Gutiérrez (Universidade Federal do Para) and De-Wei Li (Connecticut Agricultural Experiment Station) for serving as presubmission reviewers and their many helpful comments on the manuscript. Thanks are also due to Dominick Shannon (USDA) for depositing specimens in BPI and Elsa Delgado for assistance in the field. Joshua Cox and Dr. Kamash Ramanathan (EMLab P&K) are gratefully acknowledged for provision of laboratory facilities and financial support.
Literature cited
Almeida DAC, Miller AN, Gusmao LFP. 2014. New species and combinations of conidial fungi from the semi-arid Caatinga biome of Brazil. Nova Hedwigia 98: 431-447. http://dx.doi.org/10.1127/0029-5035/2013/0162
Cannon P, Sutton B. 2004. Microfungi on wood and plant debris. 217—239, in: G Mueller et al. (eds). Biodiversity of fungi: inventory and monitoring methods. Elsevier Academic Press, Burlington.
Castafieda RE, Kendrick B. 1990. Conidial fungi from Cuba: I. University of Waterloo Biol. Ser. 33.61 p.
Castaneda RF, Decock C, Saikawa M, Gené J, Guarro J. 2000. Polyschema obclaviformis sp. nov., and some new records of hyphomycetes from Cuba. Cryptog. Mycol. 21: 215-220. http://dx.doi.org/10.1016/S0181-1584(00)01051-4
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Ellis MB. 1976. More dematiaceous hyphomycetes. Commonwealth Mycological Institute, Kew.
Goh TK, Hyde KD. 1996. Janetia curviapicis, a new species, and an emended description of the genus. Mycologia 88: 1014-1021. http://dx.doi.org/10.2307/3761066
Hernandez-Gutiérrez A, Mena J. 1994. Sporidesmium coccothrinacis Hernandez & Mena, sp. nov. Bol. Soc. Micol. Madrid 19: 313-314.
Hernandez-Gutiérrez A, Sutton BC. 1997. Imimyces and Linkosia, two new genera segregated from Sporidesmium sensu lato, and redescription of Polydesmus. Mycol. Res. 101: 201-209. http://dx.doi.org/10.1017/S0953756296002419
Iturriaga T, Hawksworth DL, Crane JL. 2008. ‘Sporidesmium lichenicola’ sp. nov., a new lichenicolous fungus on Leptogium from Venezuela. Mycologia 100: 392-396. http://dx.doi.org/10.3852/06-166R
Ma J, Ma LG, Zhang YD, Zhang XG. 2011. Three new hyphomycetes from southern China. Mycotaxon 117: 247-253. http://dx.doi.org/10.5248/117.247
Matsushima T. 1981. Matsushima mycological memoirs no. 2. Published by the author, Kobe.
McKenzie EHC. 1995. Dematiaceous hyphomycetes on Pandanaceae. 5. Sporidesmium sensu lato. Mycotaxon 56: 9-29.
Santa Izabel TS, Cruz ACR, Gusmao LFP. 2013. Conidial fungi from the semi-arid Caatinga biome of Brazil. Ellisembiopsis gen. nov., new variety of Sporidesmiella and some notes on Sporidesmium complex. Mycosphere 4: 156-163. http://dx.doi.org/10.5943/mycosphere/4/2/1
Shenoy BD, Jeewon R, Wu WP, Bhat DJ, Hyde KD. 2006. Ribosomal and RPB2 DNA sequence analyses suggest that Sporidesmium and morphologically similar genera are polyphyletic. Mycol. Res. 110: 916-928. http://dx.doi.org/10.1016/j.mycres.2006.06.004
Shenoy BD, Jeewon R, Hyde KD. 2007. Impact of DNA sequence-data on the taxonomy of anamorphic fungi. Fungal Diversity 26: 1-54.
Subramanian CV. 1992. A reassessment of Sporidesmium (hyphomycetes) and some related taxa. Proc. Nat. Acad. Sci. India B58: 179-190.
Summerbell RC, Gueidan C, Schroers HJ, de Hoog GS, Starink M, Arocha Rosete Y, Guarro J, Scott JA. 2011. Acremonium phylogenetic overview and revision of Gliomastix, Sarocladium, and Trichothecium. Stud. Mycol. 68: 139-162. http://dx.doi.org/10.3114/sim.2011.68.06
Sutton BC, Pascoe IG. 1988. Some dematiaceous hyphomycetes from branches and phyllodes of Acacia in Australia. Aust. Syst. Bot. 1: 127—138. http://dx.doi.org/10.1071/SB9880127
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ISSN (print) 0093-4666 © 2014. Mycotaxon, Ltd. ISSN (online) 2154-8889
MYCOTAXON
http://dx.doi.org/10.5248/129.47 Volume 129(1), pp. 47-56 July-September 2014
Taxonomy and phylogeny of Heterobasidion in South Korea
YEONGSEON JANG’, SEOKYOON JANG’, YOUNG WOON LIM’, CHANGMU KIM? & JAE-JIN Kim’*
‘Division of Environmental Science & Ecological Engineering, College of Life Sciences & Biotechnology, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 136-701, Korea School of Biological Sciences, Seoul National University, Seoul, 151-747, Korea National Institute of Biological Resources, Environmental Research Complex, Incheon, 404-708, Korea
*CORRESPONDENCE TO: jae-jinkim@korea.ac.kr
ABSTRACT — ITS and TEF gene sequences from eleven basidiome collections from South Korea and one mycelial culture were analyzed to infer the phylogeny of Korean Heterobasidion species. Comparison of morphological characters with phylogenetic analyses revealed that specimens previously recorded as ‘H. annosum’ and ‘H. araucariae’ in fact represent H. ecrustosum, while those recorded as “H. insulare’ represent H. orientale. The two species currently recognized in South Korea are described and illustrated.
Key worps — Basidiomycota, Bondarzewiaceae, polypore
Introduction
Heterobasidion Bref. is a well-known polypore genus that includes the destructive tree pathogen, H. annosum (Fr.) Bref. The genus is characterized by resupinate to pileate basidiocarps, a dimitic hyphal system with mostly simple septate generative hyphae and dextrinoid skeletal hyphae, and finely asperulate inamyloid basidiospores (Gilbertson & Ryvarden 1986). In addition, its skeletal hyphae are cyanophilous in Cotton Blue (Dai & Korhonen 2009). Until recently, five species — H. annosum, H. araucariae P.K. Buchanan, H. insulare (Murrill) Ryvarden, H. pahangense Corner, and H. rutilantiforme (Murrill) Stalpers — were accepted, but both H. annosum and H. insulare have been found to represent species complexes (Korhonen 1978, Dai et al. 2002), so that now eleven Heterobasidion species are recognized (Dai & Korhonen 1999,
48 ... Jang & al.
Tokuda et al. 2009, Dai & Korhonen 2009, Otrosina & Garbelotto 2010). Dai et al. (2003), Tokuda et al. (2009), and Dai & Korhonen (2009) accept H. australe Y.C. Dai & Korhonen, H. ecrustosum, H. linzhiense Y.C. Dai & Korhonen, H. orientale, and H. parviporum Niemela & Korhonen in northeast Asia (China and Japan).
In Korea, Lee (1988), Lee & Jung (2006), and Paul et al. (2012) have reported three Heterobasidion species: H. annosum, H. araucariae, and H. insulare. Heterobasidion insulare was reported (as Fomitopsis insularis) from a conifer trunk (Lee 1988), H. annosum was found at the base of Pinus rigida (Lee & Jung 2006), and H. araucariae was found as an endophyte from roots of red pepper, Capsicum annuum (Paul et al. 2012). However, as none of these three species had previously been reported for northeast Asia, re-examination of those materials was required.
In this study, we list the Heterobasidion species in South Korea and describe the morphological characteristics for specimens collected in the country and, when possible, for voucher specimens from previous studies. In addition, we analyzed sequences from the internal transcribed spacer rDNA (ITS) and translation elongation factor 1-alpha (TEF). The ITS was selected because it is used for barcoding fungal species (Schoch et al. 2012), and the TEF has been used to infer species relationships within Heterobasidion (Ota et al. 2006, Paul et al. 2012). We describe and illustrate the two accepted species and compare their key characters with the Heterobasidion species in northeast Asia.
Materials & methods
Collection and morphological examination
The basidiocarps of Heterobasidion spp. identified by Lee & Jung (2006) and materials deposited in Seoul National University Fungal Collection (SFC) and the Korea University Culture Collection (KUC) were used in this study (TABLE 1). The macro- and microscopic characteristics of the specimens were examined according to Jang et al. (2013). Color codes in the descriptions follow Munsell (2000).
Phylogenetic analysis
Genomic DNAs were extracted according to Jang et al. (2013) from the materials in TaBLe 1. PCR reactions for ITS regions were performed using Accupower PCR Premix Kit (Bioneer, Korea) with the primers ITSIF/ITS4 according to Jang et al. (2013). TEF region was amplified using the same kit with the primers, EF1-728F (5’-cATCGAGAAGTTCGAGAAGG-3’) /EF1-1567R (5 ’-ACHGTRCCRATACCACC- RATCTT-3’) and the PCR conditions: an initial denaturation step of 95°C for 7 min, followed by 35 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 1 min, with an elongation step of 72°C for 7 min at the end. The DNA was sequenced by the Macrogen sequencing service (Seoul, Korea). Sequences obtained in this study were deposited in GenBank, NCBI; the accession numbers are shown in TABLE 1. The ITS sequences
Heterobasidion in South Korea... 49
TABLE 1. The Korean Heterobasidion specimens and their ITS and TEF sequences used
in this study.
Cbnerne ORIGINAL SPECIMEN NO." GENBANK ITS GENBANK TEF DETERMINATION No. No.
H. ecrustosum H. annosum KUC20080904-43 _ — H. annosum KUC20110916-44 KF218829 — H. annosum KUC20111001-14 KEF218830 KF154267 H. annosum KUC20120810-11 KF218831 KF154268 H. annosum KUC20120810-14 KF218832 KF154269 H. araucariae CNU081069 JQ691621> JQ691622> H. annosum SFC 20020927-11 — — H. annosum SFC 20120820-01 KF218834 KF154270
H. orientale H. insulare KUC20081030C-04 KF218828 — H. insulare KUC20121019-01 — KF154271 H. insulare KUC20121019-30 KF218833 KF154272 H. insulare KUC20121123-14 — KF154273
“Examined basidiocarps now deposited in National Institute of Biological Resources (KB); H. ecrustosum CNU081069 is a mycelial isolate from Paul et al (2012).
> Sequences retrieved from GenBank, NCBI.
were aligned with the Heterobasidion sequences from Dalman et al. (2010). TEF sequences were aligned with the sequences from Ota et al. (2006) and Paul et al. (2012). The sequence alignment, model selection, and Bayesian phylogenetic analyses were performed according to Jang et al. (2013).
Results & discussion
Morphological comparisons
Although the voucher specimens of “Heterobasidion insulare’ in Lee (1988) were not available, we feel that his fungus represents H. orientale based on his description and colored illustration of the basidiocarps. He described the basidiocarps as sessile and 2.5-5 x 4-8 x 1-1.5 cm; the pileus had a brick-red surface and white to cream margin. The 1.5-3 mm context was white to cream, the tubes were 1 cm with round to labyrinthiform pores, and the spores were subglobose and 4-5 um long. In contrast, H. insulare has a mostly light brown pileus surface and round to angular (not labyrinthiform) pores (Tokuda et al. 2009). In addition, other specimens formerly known as ‘H. insulare’ (TABLE 1) are actually H. orientale: Tokuda et al. (2009) note that specimens previously recognized as H. insulare in China and Japan represent H. orientale and that H. insulare is known only from the type specimen found in the Philippines.
Re-examination ‘Heterobasidion annosum’ specimen SFC 20020927-11 from Lee & Jung (2006) revealed that it represents instead H. ecrustosum,
50 ... Jang & al.
and the species description in Lee & Jung (2006) is quite similar to that of H. ecrustosum by Tokuda et al. (2009). Furthermore, all specimens previously identified as ‘H. annosum’ have been since identified as H. ecrustosum (TABLE 1). Dai et al. (2003) showed that H. annosum has not been found from eastern Asia and Altai region and that Russia represents its easternmost distribution. ‘Heterobasidion araucariae from Paul et al. (2012) was found as an endophyte. The cultural characteristics of H. araucariae and H. ecrustosum are similar. The data of Paul et al. (2012) for their ‘H. araucariae’ (conidiophores <300 x 5.5-10.5 um; conidia 4-11.5 x 2-7 um) lie within the data range of Tokuda et al. (2009) for H. ecrustosum (conidiophores <300 x 4-10 um; conidia 3.2-17.1 x 2.9-12 um), but only partially overlap the data range of Buchanan (1988) for authentic H. araucariae (conidiophores <400 x 5.5-13.5 um; conidia 5-16 x 3-13.5 um). Authentic H. araucariae has been found only in Australia, New Zealand, and Fiji (Buchanan 1988; Ota et al. 2006; Tokuda et al. 2009), and
Tokuda et al. (2009) consider it a species restricted to the southern hemisphere.
Phylogeny
Among the 11 specimens examined in this study, ITS gene region sequences were obtained from seven specimens (594-627 bp) and TEF gene region sequences from seven specimens (718-774 bp) (TABLE 1). No sequence was amplified from KUC20080904-43 and SFC 20020927-11. The phylogenetic analyses supported our morphological conclusions that the examined ‘H. annosum and ‘H. araucariae’ specimens represent H. ecrustosum and that the examined ‘H. insulare’ specimens represent H. orientale.
Bayesian analysis of ITS sequences (not presented) was congruent with the tree from Dalman et al. (2010). No intraspecific ITS sequence variation was found in Korean H. ecrustosum and H. orientale. Heterobasidion ecrustosum was monophyletic with H. araucariae and our H. ecrustosum ITS sequences were 99% similar (5 positions different) to H. araucariae from Australia and New Zealand (GenBank FJ627521, FJ627522, FJ627526, and FJ627527). Heterobasidion orientale was monophyletic and sister to the H. araucariae/ H. ecrustosum clade.
The TEF gene region tree (Fic. 1) showed a topology similar to that of Ota et al. (2006) except that Japanese isolates of H. parviporum did not form a separate clade but were positioned on the basal part of the clade comprising H. occidentale, H. abietinum, and H. parviporum. Korean H. orientale collections produced two different TEF sequences although they were collected from the same location; their basal position within the H. orientale clade was supported with high posterior probability (1.0 p.p.). Heterobasidion orientale
Heterobasidion in South Korea... 51
‘North American S-group’ B1142, Mexico (AY273391)
‘North American S-group’ Tc122.11, USA (AY273387) ‘North American S-group’ Bc3.3, Canada (AY273388) 06 ‘North American S-group’ Faf10.2, USA (AY273386)
Heterobasidion abietinum Faf7.1, Italy (AY273385)
1 Heterobasidion abietinum Faf4.6, Italy (AY273383) Heterobasidion abietinum Faf8.5, Italy (AY273382) Heterobasidion abietinum OH2.8.c6, Switzerland (AY273380)
Heterobasidion parviporum B1314, China (AY273393)
1 Heterobasidion occidentale
Heterobasidion abietinum
0.78 Heterobasidion parviporum FSE.3, Finland (AY273368)
0.54 L_ Heterobasidion parviporum B1295, China (AY273392) ‘Heterobasidion annosum’ \WD1212, Japan (AB255540) ‘Heterobasidion annosum’ HF419, Japan (AB255571) ‘Heterobasidion annosum’ HF594S1, Japan (AB255575) ‘Heterobasidion insulare’ \WD142, Japan (AB255547) ‘Heterobasidion insulare’ WD651, Japan (AB255548) ‘Heterobasidion insulare’ WWD825, Japan (AB255549) ‘Heterobasidion insulare’ WD1981, Japan (AB255570) ‘Heterobasidion insulare’ \WWD2154, Japan (AB255550) ‘Heterobasidion insulare’ HF 448, Japan (AB255551) ‘Heterobasidion insulare’ HF449, Japan (AB255552) 4 Heterobasidion orientale KUC20121019-30, Korea (KF154272)
Heterobasidion orientale KUC20121123-14, Korea (KF154273)
Heterobasidion orientale KUC20121019-01, Korea (KF154271)
‘Heterobasidion sp.’ WD1945, Japan (AB255553)
0.961 _ ‘Heterobasidion sp.’ WD2107, Japan (AB255554)
‘Heterobasidion sp.’ WD2220, Japan (AB255555) Heterobasidion ecrustosum KUC20120810-14, Korea (KF154269) Heterobasidion ecrustosum KUC20120810-11, Korea (KF154268)
085! _ Heterobasidion ecrustosum KUC20111001-14, Korea (KF154267) 0.96. ‘Heterobasidion araucariae’ CNU081069, Korea (JQ691622) Heterobasidion ecrustosum SFC20120820-01, Korea (KF154270)
Heterobasidion araucariae ICMP9529, New Zealand (AB255556) Heterobasidion araucariae ICMP9533, New Zealand (AB255557)
0.96
Heterobasidion parviporum
0.9 Heterobasidion orientale
0.82
0.91
0.8
Heterobasidion ecrustosum
a Heterobasidion araucariae
Heterobasidion annosum P16.4, Sweden (AY273397) I Heterobasidion annosum ‘North American P-group’ P32.1, USA (AY273396) I Heterobasidion irregulare
0.1
Fic. 1. 50% major-rule consensus tree of Korean Heterobasidion spp. and allies using TEF region sequences. The tree, containing 36 taxa and 258 characters, was constructed from 15,000 trees produced by Bayesian analysis and was mid-point rooted. Posterior probabilities >50% are given. Korean specimens are shown in bold type. For each node, the former names, isolate numbers, and localities are given, and the species names accepted by recent studies (Tokuda et al. 2009, Otrosina & Garbelotto 2010) are presented on the right side of the tree. GenBank accession numbers are provided in parentheses.
KUC20121019-30 and KUC20121123-14 were 98% similar to Japanese isolates (254 out of 258 sequences matched), and KUC20121019-01 was 96% similar (247 out of 258 sequences matched). Korean H. ecrustosum collections (including ‘H. araucariae CNU081069 from Paul et al. 2012) had 100% identical TEF sequences and were sister to Japanese H. ecrustosum isolates with moderate support (0.8 p.p.). The H. ecrustosum clade again clustered with authentic H. araucariae with high support (1.0 p.p.). Korean H. ecrustosum collections were 98% similar to Japanese H. ecrustosum (245 out of 251 sequences matched) and 97% similar to H. araucariae (242 out of 249 sequences matched).
52 ... Jang & al.
Taxonomy
Fic. 2. Heterobasidion orientale (KUC20121019-01). A, B. Basidiocarp. Scale bar = 1 cm. C. Microscopic features. a, basidiospores; b, basidia; c, generative hyphae from trama; d, skeletal hyphae from trama; e, generative hyphae from subiculum; f, skeletal hyphae from subiculum.
Heterobasidion orientale Tokuda, T. Hatt. & Y.C. Dai, Mycoscience 50: 193. 2009. Fic. 2
BASIDIOCARPS annual, sessile, solitary to imbricate, <7 x 5 x 2 cm. Pileus broadly attached to the substrate, dimidiate, applanate to elongated. Pileus surface glabrous, sometimes radially rugose when dry, zonate with dusky red (10R3/3) to dark red (7.5R3/6) in the middle, yellow (10YR7/6) to very pale brown (10YR8/2-3) near the margin. Pileus margin acute. Pore surface very pale brown (10YR8/4) to yellow (10YR7/6-8, 10YR8/6-8). Pores round to angular or labyrinthiform, 2-4/mm, dissepiments eroded. Sterile margin 0.5 mm wide or absent. Context corky, very pale brown (10YR8/2), 1-2 mm thick. Tubes 3-6 mm deep, concolorous with context.
HYPHAL SysTEM dimitic; generative hyphae without clamp connection and skeletal hyphae.
CONTEXT generative hyphae without clamp connections, hyaline, thin to slightly thick-walled, IKI-, 2.0-3.5 um in diameter; skeletal hyphae abundant, hyaline, thick-walled, dextrinoid, 4.5-6.5 um in diameter.
TUBES generative hyphae without clamp connections, hyaline, thin-walled, IKI-, 2-2.5 um in diameter; skeletal hyphae abundant, hyaline, thick-walled, weakly dextrinoid, 3-5 um in diameter; cystidia none; basidia barrel-shaped, 4-sterigmate, slightly constricted in the middle, 17-29 x 5-7 um.
BASIDIOSPORES globose to subglobose, hyaline, finely asperulate, IKI-, (4.6-)4.8-6.7 x 3.9-5.4(-5.8) um, L = 5.6 um, W = 4.5 um (n = 88/3).
DISTRIBUTION — China, Japan, and Korea.
Heterobasidion in South Korea... 53
SPECIMENS EXAMINED: KOREA, KYEONGGI-DO, Mt. Bori, 37°39’06’N 127°32’02’E, on Pinus densiflora Siebold & Zucc., 30 October 2008, Yeongseon Jang (KB, KUC20081030C-04). GANGWON-DO, Mt. Odae, 37°43’59”N 128°35’23’E, on unknown wood, 19 October 2012, Yeongseon Jang (KB, KUC20121019-01); 37°44’13”N 128°35’16”E, Abies holophylla Maxim., 19 October 2012, Yeongseon Jang (KB, KUC20121019-30); 37°44’30”N 128°35’03”E, on unknown wood, 23 November 2012, Yeongseon Jang (KB, KUC20121123-14).
REMARKS — The basidia of Korean H. orientale specimens are larger than those given by Tokuda et al. (2009).
Fic. 3. Heterobasidion ecrustosum (KUC20110916-44). A, B. Basidiocarp. Scale bar = 1 cm. C. Microscopic features. a, basidiospores; b, basidia; c, generative hyphae from trama; d, skeletal hyphae from trama; e, generative hyphae from subiculum; f, skeletal hyphae from subiculum.
Heterobasidion ecrustosum Tokuda, T. Hatt. & Y.C. Dai, Mycoscience 50: 196. 2009. FIG. 3
BASIDIOCARPS annual, sessile, solitary to imbricate, <6 x 3.5 x 2 cm. Pileus broadly attached to the substrate, dimidiate, applanate to convex, somewhat inrolled when dry. Pileus surface glabrous, usually rough, subzonate to azonate, radially sulcate in one specimen (KUC20110916-44), very pale brown (10YR8/3-4) to yellow (10YR8/6) or reddish yellow (SYR6/8), with crust at the base or without crust, crust dark yellowish brown (10YR3/4) to very dark brown (10YR2/2) or dark reddish brown (5YR3/4). Pileus margin rounded. Pore surface very pale brown (10YR8/2-4) or pale to light yellowish brown (10YR6/4-6/6), pores round to angular, sometimes elongated, 2-3/mm, dissepiments entire. Sterile margin narrow, concolorous with the pore surface, 0.5-1.5 mm wide, or absent. Context corky, very pale brown (5YR8/2-3), 2-5 mm thick. Tubes concolorous with the context, 1-7 mm deep.
HYPHAL SYSTEM dimitic; generative hyphae without clamp connections and skeletal hyphae.
54 ... Jang & al.
CONTEXT generative hyphae without clamp connections, hyaline, thin to thick-walled, IKI-, 2.5-5 um in diameter; skeletal hyphae abundant, hyaline, dextrinoid, thick-walled, 3.5-5.5 um in diameter.
TUBES generative hyphae without clamp connections, hyaline, thin walled, IKI-, 2.0-4.5 um in diameter; skeletal hyphae abundant, hyaline, dextrinoid, thick-walled, 3-5.5 um in diameter; cystidia none; basidia barrel-shaped, 4-sterigmate, 17-26 x 4.5—7 um.
BASIDIOSPORES globose to subglobose, hyaline, finely asperulate, IKI-, 4,.9-6.9(-7.7) x (3.7-)4.1-5.9(-6.5) um, L = 5.9 um, W = 5.0 um (n = 99/3).
DISTRIBUTION — China, Japan, and Korea.
SPECIMEN EXAMINED: KOREA, SEOUL, Mt. Cheonggye, 37°25’47”N 127°02’51”E, on the branch of wood, 4 September 2008, Yeongseon Jang (KB, KUC20080904-43). Heonilleung, 37°28’03”N 127°05’00’E, on the stump of Pinus densiflora, 16 September 2011, Yeongseon Jang (KB, KUC20110916-44). JEOLLABUK-DO, Gochang, 35°27’35”N 126°30’51”E, on unknown wood, 1 October 2011, Yeongseon Jang (KB, KUC20111001- 14). JEOLLANAM-DO, Wando arboretum, 34°23’05”N 126°39’07”E, on unknown wood, 10 August 2012, Yeongseon Jang (KB, KUC20120810-11); on unknown wood, 10 August 2012, Yeongseon Jang (KB, KUC20120810-14). CHUNGCHEONGBUK-DO, Mt. Songni, on Pinus rigida Mill., 27 September 2002, Jin Sung Lee (KB, SFC 20020927-11). CHUNGCHEONGNAM-DO, Mt. Gaya, on Pinus densiflora, 20 August 2012, Young Woon Lim (KB, SFC 20120820-01). REMARKS — The ranges of basidia and basidiospore sizes of Korean H. ecrustosum specimens overlap with those given by Tokuda et al. (2009), but Korean specimens tend to have larger basidia and basidiospores.
We confirm that there are only two Heterobasidion species — H. ecrustosum and H. orientale — in South Korea and compare their characteristics with other northeastern Asian Heterobasidion species in TABLE 2. Because we analyzed
TABLE 2. Key characteristics of Heterobasidion species in northeast Asia.
BASIDIOCARPS PORES BASIDIOSPORES SUBSTRATES
SPECIES
(tum)
H. australe Perennial Mostly round, 4.1-6.2 x Keteleeria, Picea, (Dai & Korhonen occasionally 3.3-5.5 Pinus, Pseudolarix, 2009) angular, 4-5/mm Tsuga
H. ecrustosum Annual Angular to round, 4,.9-7.7 x Pinus (this study) 2-3/mm 3.7-6.5
H. linzhiense Annual Angular, 2-4/mm 5-10 x Abies, Picea (Dai et al. 2007) 4-10
H. orientale Annual Angular to round 4.6-6.7 x Abies, Pinus (this study) or labyrinthiform, 3.9-5.8
2-4/mm H. parviporum Mostly Round, regular, 3.3-5.6 x Abies, Larix, Picea,
(Tokuda et al. 2009) perennial 4-7/mm 2.9-4.8 Pinus, Populus, Tsuga
Heterobasidion in South Korea... 55
only a limited number of specimens, there might be other species present in this region. Thus, further studies are needed to determine the complete diversity of Heterobasidion in South Korea.
Acknowledgments
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF- 2013R1A1A2A10011390) and was funded by the project on survey and excavation of Korean indigenous species of NIBR under the Ministry of Environment, Republic of Korea. We are much obliged to Dr. Yu-Cheng Dai and Dr. Hayato Masuya for their valuable suggestions on the manuscript.
Literature cited
Buchanan PK. 1988. A new species of Heterobasidion (Polyporaceae) from Australasia. Mycotaxon a2) 3257337.
Dai YC, Korhonen K. 2009. Heterobasidion australe, a new polypore derived from the Heterobasidion insulare complex. Mycoscience 50: 353-356. http://dx.doi.org/10.1007/s10267-009-0491-3 Dai YC, Korhonen K. 1999. Heterobasidion annosum group S identified in north-eastern China. European Journal of Forest Pathology 29: 273-279. http://dx.doi.org/10.1046/j.1439-0329.1999.00153.x
Dai YC, Vainio EJ, Hantula J, Niemela T, Korhonen K. 2002. Sexuality and intersterility within the Heterobasidion insulare complex. Mycological Research 106: 1435-1448. http://dx.doi.org/10.1017/S0953756202006950
Dai YC, Vainio EJ, Hantula J, Niemela T, Korhonen K. 2003. Investigations on Heterobasidion annosum s. lat. in central and eastern Asia with the aid of mating tests and DNA fingerprinting. Forest Pathology 33: 269-286. http://dx.doi.org/10.1046/j.1439-0329.2003.00328.x
Dalman K, Olson A, Stenlid J. 2010. Evolutionary history of the conifer root rot fungus Heterobasidion annosum sensu lato. Molecular Ecology 19: 4979-4993. http://dx.doi.org/10.1111/j.1365-294X.2010.04873.x
Gilbertson RL, Ryvarden L. 1986. North American polypores I. Fungiflora, Oslo.
Jang Y, Lee SW, Lim YW, Lee JS, Hattori T, Kim J-J. 2013. The genus Wrightoporia in Korea. Mycotaxon 123: 335-341. http://dx.doi.org/10.5248/123.335
Korhonen K. 1978. Intersterility groups of Heterobasidion annosum. Communicationes Instituti Forestalis Fenniae 94: 1-25.
Lee JS, Jung HS. 2006. Taxonomic study on Korean Aphyllophorales (5) - on some unrecorded genera and species. Mycobiology 34: 166-175. http://dx.doi.org/10.4489/MYCO.2006.34.4.166
Lee JY. 1988. Coloured Korean mushrooms. Vol. 1. Academy Press. Seoul, Korea.
Munsell Color. 2000. Munsell soil color charts. Gretag Macbeth, New Windsor, NY.
Ota Y, Tokuda S, Buchanan PK, Hattori T. 2006. Phylogenetic relationships of Japanese species of Heterobasidion—H. annosum sensu lato and an undetermined Heterobasidion sp. Mycologia 98: 717-725. http://dx.doi.org/10.3852/mycologia.98.5.717
Otrosina WJ, Garbelotto M. 2010. Heterobasidion occidentale sp. nov. and Heterobasidion irregulare nom. nov.: A disposition of North American Heterobasidion biological species. Fungal Biology 114: 16-25. http://dx.doi.org/10.1016/j.mycres.2009.09.001
Paul NC, Deng JX, Shin KS, Yu SH. 2012. Molecular and morphological characterization of endophytic Heterobasidion araucariae from roots of Capsicum annuum L. in Korea. Mycobiology 40: 85-90. http://dx.doi.org/10.5941/MYCO.2012.40.2.85
56 ... Jang & al.
Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA, Chen W, Fungal Barcoding Consortium. 2012. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences 109: 6241-6246. http://dx.doi.org/10.1073/pnas.1117018109
Tokuda S, Hattori T, Dai YC, Ota Y, Buchanan PK. 2009. Three species of Heterobasidion (Basidiomycota, Hericiales), H. parviporum, H. orientale sp. nov. and H. ecrustosum sp. nov. from East Asia. Mycoscience 50: 190-202. http://dx.doi.org/10.1007/s10267-008-0476-7
ISSN (print) 0093-4666 © 2014. Mycotaxon, Ltd. ISSN (online) 2154-8889
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http://dx.doi.org/10.5248/129.57 Volume 129(1), pp. 57-61 July-September 2014
Two new combinations and a new record of Zasmidium from China
FENGYAN ZHAI, W.H. Hsien’, YINGJIE Liu’, & YINGLAN Guo?
' Henan Institute of Science and Technology, Xinxiang 453003, China ? Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan, Republic of China ° Institute of Microbiology, Chinese Academy of Science, Beijing 100101, China
* CORRESPONDENCE TO: guoyl@im.ac.cn
ABSTRACT — Stenella gynurae is recombined as Zasmidium gynurae, and S. bougainvilleae as Z. bougainvilleae; these species are described, illustrated and discussed. Zasmidium salicis is also reported as new to China.
Key worps —morphology, Mycosphaerellaceae, reallocation, taxonomy
Introduction
The type species of Stenella Syd., S. araguata Syd., which is characterized by pileate condiogenous loci and hila, is placed by molecular sequence analyses in Teratosphaeriaceae. In contrast, most other previous Stenella species have planate Cercospora-like scars and hila and belong in Mycosphaerellaceae, where they agree morphologically and cluster phylogenetically with the type of Zasmidium Fr.; they have therefore been transferred to Zasmidium (Braun et al. 2010a,b). Here we transfer two additional Stenella species, S. gynurae and S. bougainvilleae, which coincide morphologically with the current concept of Zasmidium. Another Zasmidium species, Z. salicis, is reported as new to China.
Taxonomy
Zasmidium gynurae (Sawada & Katsuki) W.H. Hsieh, Y.L. Guo & EY. Zhai, comb. nov. FIG. 1 MycoBank MB 808069 = Cercospora gynurae Sawada & Katsuki, Spec. Publ. Coll. Agric., Nat. Taiwan Univ. 8: 218, 1959. = Stenella gynurae (Sawada & Katsuki) Goh & W.H. Hsieh, Cercospora Similar Fungi Taiwan: 88, 1990.
58 ... Zhai & al.
Fic. 1. Zasmidium gynurae (NYU-PPE, holotype). Conidiophores and conidia. Scale bar = 10 um.
Leaf spots circular, greyish, without distinct margin, 3.0-5.0 mm diam. Fruiting hypophyllous. Primary mycelium immersed. Secondary mycelium superficial; hyphae subhyaline to pale olivaceous, septate, branched, finely verruculose, 1.0-2.0 um diam. Secondary conidiophores arising as lateral branches from external hyphae. Stromata none. Primary conidiophores emerging through stomata, 1-6-fasciculate, or arising as solitary branches from superficial hyphae, pale olivaceous to pale brown, paler towards the apex, irregular in width, straight to 1-2-geniculate, rarely branched, round to conical at the apex, 1-6-septate, 20.0-70.0(-230.0) x 2.5-3.5 um. Conidial scars conspicuously thickened, 0.5-1.0 um wide. Conidia cylindrical, catenate, later verruculose, subhyaline to pale olivaceous, straight to slightly curved, round to conical at the apex, obconical to obconically truncate at the base, 2-6-septate, 18.0-60.0 x 2.5-3.5 um; hila small but conspicuously thickened.
COLLECTION EXAMINED: CHINA. TaIwAN PROVINCE, Changhua County, on leaves
of Gynura bicolor (Willd.) DC. (Compositae), 14 XII 1909, coll. K. Sawada (NYU-PPE, holotype).
DISTRIBUTION: China.
Zasmidium bougainvilleae (J.M. Yen & Lim) Y.L. Guo, W.H. Hsieh & EY. Zhai, comb. nov. FIG.2 MycoBank MB 808070 = Stenella bougainvilleae J.M. Yen & Lim, Mycotaxon 16: 96, 1982. Leaf spots amphigenous, circular to subcircular, 1.0-4.0 mm diam., sometimes confluent, olivaceous-brown to pale brown, surrounded by a pale
Zasmidium combs. nov. (China) ... 59
Fic. 2. Zasmidium bougainvilleae (HMAS 242906). Conidiophores and conidia. Scale bar = 20 um.
brown to grayish brown margin, with 1-2 times zonate and pale yellow to pale grayish brown halo on the upper surface, paler on the lower surface. Fruiting hypophyllous. Primary mycelium immersed. Secondary mycelium superficial; hyphae emerging through stomata or from the base of primary conidiophores, or directly from the apex of conidiophores, pale olivaceous to olivaceous, branched, septate, verruculose, with lateral secondary conidiophores, 1.5-3.0 um diam. Stromata none or small, formed only by several brown cells. Primary conidiophores emerging through stomata, 2—8-fasciculate, or up to 25-fasciculate on small stromata, moderately olivaceous-brown to pale brown, paler towards the apex, irregular in width, narrower towards the apex, branched, straight to strongly curved, 0-3-geniculate, conical at the apex, 1-6-septate, sometimes constricted at the septa, 25.0-68.0 x 3.0-4.0 um. Secondary conidiophores olivaceous-brown to moderately olivaceous-brown, unbranched, erect or curved, 0-3-septate, 15.0-48.0 x 2.5-4.0 um. Conidial scars small but conspicuously thickened, 0.8-1.3 um wide. Conidia cylindrical to slightly obclavate-cylindrical, pale olivaceous, catenate, straight to slightly curved, finely verruculose, indistinctly 2-14-septate, obtuse to conical at the apex, obconical to subtruncate at the base, with small but conspicuously thickened hila, 15.0-68.0(-110.0) x 2.0-3.5 um. COLLECTIONS EXAMINED: CHINA. HAINAN PROVINCE, Lingshui, on leaves of Bougainvillea glabra Choisy (Nyctaginaceae), 11 IV 2011, coll. Y.L. Guo, no. HN345
(HMAS 242906). TArwAN PROVINCE, Changhua County, on leaves of Bougainvillea spectabilis Willd., 23 X 1985, coll. T.K. Goh (NCHUPP-184).
60 ... Zhai & al.
DISTRIBUTION: China, Singapore.
Notes: Hsieh & Goh (1990: 248) described Z. bougainvilleae on Bougainvillea spectabilis from Taiwan as leaf spots absent or merely indefinite yellowish discoloration, with shorter conidiophores (primary conidiophores 20.0-40.0 x 3.0-5.0 um, secondary conidiophores 5.0-15.0 x 2.0-4.0 um), and longer conidia (20.0-110.0 x 2.5-4.0 um). The morphological characters of our collection on B. glabra from Hainan are fairly similar to those described by Yen & Lim (1982) on B. spectabilis from Singapore (primary conidiophores 30.0-90.0 x 3.0-4.0 um, conidia 20.0-65.0 x 2.5-4.0 um).
Zasmidium salicis (Chupp & H.C. Greene) Kamal & U. Braun, Cercosporoid Fungi of India: 248, 2010. = Cercospora salicis Chupp & H.C. Greene, Am. Midland Naturalist 41: 757, 1949. = Stenella salicis (Chupp & H.C. Greene) Crous & U. Braun, Mycotaxon 78: 342, 2001. Leaf spots amphigenous, punctiform to irregular, 0.5-3.0 mm diam., often angular, limited by veins, brown to blackish brown on the upper surface, blackish brown on the lower surface. Fruiting amphigenous. Mycelium internal. Stromata substomatal, subglobose, brown, 10.0-26.0 um diam. Conidiophores arising from the upper cells of stromata, emerging through stomata, 3-25 per fascicle, pale olivaceous-brown, uniform in color, irregular in width, verruculose, straight to geniculate-sinuous, 0-4-geniculate, mostly unbranched, 0-2-septate, 8.0-28.0 x 2.5-5.0 um. Conidial scars darkened and thickened, 2.0-2.5 um wide. Conidia cylindrical, cylindrical-obclavate, solidary, pale olivaceous to olivaceous-brown, finely verruculose with age, straight to moderate curved, obtuse at the apex, subtruncate at the base, 1-7-septate, 15.5-96.0 x 2.5-5.0 um, hila thickened and darkened. COLLECTION EXAMINED: CHINA. JILIN PROVINCE, Changchun, on leaves of Salix
matsudana Koidz. (Salicaceae), 7 July 2006, coll. YJ. Liu & EY.Zhai 6178 (HMJAU 30005).
DISTRIBUTION: China, North America, Brazil.
Notes: This is the first report of Zasmidium salicis from China.
Chupp & Greene described Cercospora salicis from North America on Salix alba L. (Greene 1949; Chupp 1954). The holotype was re-examined by Crous & Braun (2001), who reallocated it to Stenella based on verruculose superficial hyphae and conidia. Kamal and Braun subsequently transferred the species to Zasmidium (Kamal 2010).
The conidiophores and conidia of our Chinese specimen are fairly similar to those of Z. salicis on Salix sp. in North America in shape and size but differ in lacking verruculose external mycelium, with the North American material having larger stromata (<50 um wide), 1-3-septate and somewhat
Zasmidium combs. nov. (China) ... 61
larger conidiophores (15-45 x 4-6 um), and 1-6-septate and slightly smaller conidia (20-60 x 3-4.5 um). The development of verruculose superficial hyphae is a basic feature of Zasmidium and well developed in most species, but may occasionally be absent (Crous & Braun 2003). A few species without any superficial mycelium have been assigned to this genus, e.g., Zasmidium hymenocallidis (U. Braun & Crous) U. Braun & Crous and Z. macluricola R.G. Shivas et al. (Shivas et al. 2009, Braun et al. 2010a).
Mycovellosiella salicis Deighton et al. (Deighton 1974) parasitic on Salix tetrasperma Roxb. is morphologically similar to Zasmidium salicis but differs in lacking obvious leaf spots and stromata and forming hypophyllous and diffusely fruiting, paler (pale olivaceous), and longer (<80 um) conidiophores, and paler (pale olivaceous) catenate conidia. Mycovellosiella salicis has been reallocated to Passalora salicis (Deighton et al.) U. Braun & Crous (Crous & Braun 2003).
Acknowledgments
The study was financed by the “Knowledge Innovation Program” of the Chinese Academy of Sciences - Flora Cryptogamarum Sinicarum Compilation and Research Foundation (Grant No. KSCXZ-EW-Z-9) and the National Natural Science Foundation of China (Grant No. 31100013). We express our deep appreciation to Dr. Uwe Braun and Shu-Yan Liu for their valuable suggestions, kind help, and their earnest assistance in the course of submission of this manuscript.
Literature cited
Braun U, Crous PW, Schubert, K, Shin, H-D. 2010a. Some reallocations of Stenella species to Zasmidium. Schlechtendalia 20: 99-104.
Braun U, Freire F, Urtiaga R. 2010b. New species and new records of cercosporoid hyphomycetes from Brazil, New Zealand and Venezuela. Polish Botanical Journal 55: 281-291.
Crous PW, Braun U. 2001. A reassessment of the Cercospora species described by C. Chupp: specimens deposited at BPI, Maryland, U.S.A. Mycotaxon 78: 327-343.
Crous PW, Braun U. 2003. Mycosphaerella and its anamorphs: 1. Names published in Cercospora and Passalora. CBS Biodiversity Series No. 1. 569 p.
Deighton FC. 1974. Studies on Cercospora and allied genera. V. Mycovellosiella Rangel, and a new species of Ramulariopsis. Mycological Papers 137. 76 p.
Greene HC. 1949. Notes on Wisconsin parasitic fungi XIII. The American Midland Naturalist 41: 740-758. http://dx.doi.org/10.2307/2421783
Hsieh WH, Goh TK. 1990. Cercospora and similar fungi from Taiwan. Maw Chang Book Company. Taichung Taiwan R.O.C. 367 p.
Kamal. 2010. Cercosporoid fungi of India. Dehra Dun: Bishen Singh Mahendra Pal Singh. 351 p.
Shivas RG, Young AJ, Braun U. 2009. Zasmidium macluricola. Fungal Planet 39. Persoonia 23: 190-191.
Yen JM, Lim G. 1982. Studies on parasitic fungi from South East Asia, 45. Parasitic fungi from Malaysia, 22. Mycotaxon 16(1): 96-98.
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http://dx.doi.org/10.5248/129.63 Volume 129(1), pp. 63-72 July-September 2014
New reports of Gymnopus from Pakistan based on ITS sequences
M. SABA* & A.N. KHALID
Department of Botany, University of the Punjab Quaid-e-Azam Campus, Lahore, 54590, Pakistan
* CORRESPONDENCE TO: rustflora@gmail.com
ABSTRACT — Specimens of Gymnopus luxurians and G. menehune from Pakistan are characterized morphologically and using nrDNA internal transcribed spacer sequences. These fungal species represent new records for South Asia. A key to the Gymnopus species known from Pakistan is provided.
Key worps — Abbottabad, Collybia, Khyber Pakhtoonkhaw, Marasmiaceae, saprobic fungi
Introduction
Gymnopus (Pers.) Roussel was monographed by Antonin & Noordeloos (1997) and many mushrooms from Collybia sections Vestipedes and Levipedes have now been transferred into this genus (Antonin & Noordeloos 1997). Gymnopus consists of delicate to semi-fleshy mushrooms commonly found on leaf and woody litter in tropical to temperate ecosystems, and Gymnopus sect. Vestipedes is characterised by a hairy or tomentose stipe surface and a simple pileus epicutis without a Rameales- or Dryophila-structure (Antonin & Noordeloos 1997). Ecologically, Gymnopus species are considered important in recycling nutrients (Singer 1986; Mata & Ovrebo 2009).
Five Gymnopus species have previously been reported from Pakistan (under their Collybia synonyms): Gymnopus confluens (Pers.) Antonin et al., G. dryophilus (Bull.) Murrill, G. fusipes (Bull.) Gray, G. erythropus (Pers.) Antonin et al. (as C. kuehneriana), and G. peronatus (Bolt.) Gray (Ahmad 1980; Iqbal & Khalid 1996; Shibata 1992; Sultana et al. 2011). During a collecting trip for macrofungi in Khyber Pakhtoonkhaw in 2012, many mushroom species were collected. Amongst them, three specimens belonging to Gymnopus sect. Vestipedes were identified as G. luxurians and G. menehune, which are new records from subtropical pine forests of Pakistan.
64 ... Saba & Khalid
TABLE 1. ITS-rDNA sequences of Gymnopus and Omphalotus spp.
used in the phylogenetic analysis
TAXON
G. biformis
G. confluens
G. cylindricus
G. dryophilus
G. erythropus
G. fibrosipes
G. fusipes
G. gibbosus
G. luxurians
G. menehune
G. mesoamericanus
G. polygrammus
G. readiae
G. subcyathiformis
G. subpruinosus
O. illudens
O. olearius
O. olivascens
COUNTRY
Costa Rica Costa Rica Russia Costa Rica Costa Rica Italy Czech Republic Slovakia USA Costa Rica France
Austria
Pakistan Pakistan USA Switzerland
Pakistan
Costa Rica Costa Rica Puerto Rico Not given New Zealand New Zealand Puerto Rico Dominican Republic USA
USA
USA
France
USA
GENBANK #
DQ450064 DQ450063 AY256697 HM240527 AY256696 DQ450057 JX536157 JX536158 DQ449996 DQ449998 AF505763 AY842953 AY256710 AF505777 AY263437 AY263438 KF803760 KF803761 AY256709 DQ450022 KF803762 JN182864 AY263426 DQ450036 AF505768 DQ450028 AY842954 DQ450034 HQ533036 DQ450041 DQ450042 DQ450026 DQ450027 AY313271 AY313277 AY313281
COLLECTION/ VOUCHER #
TFB7843 TFB7820 TENN58242 UBC F19677 TENN58024 TENN58024 BRNM707149 BRNM712600 SAV X12002 WTU JFA12910 TENN56660 PR23TN TENN59217 TENN59300 AWW66 AWW95 MSM#001 MSM#002 TENN57910 TENN50619 MSM#003
Not given DED5866 TENN58106 NYBG REH7379 TENN56589 PR 2542TN TENN53687 PDD95844 TENN58130 TENN59550 TENN56242 TENN59477 TENN54507 culture 9061b TENN55337
Gymnopus in Pakistan ... 65
Materials & methods
Basidiomata were collected, photographed, vouchered, dried under fan heater, and characterized morphologically. Specimen sections were mounted in 5% KOH for observation under a biological microscope (MX4300H, Meiji Techno Co., Ltd., Japan). Phloxine was used to increase contrast of structures, and Melzer’s reagent was used to test for dextrinoidity of basidiospores.
Measurements of anatomical features (basidiospores, basidia, cystidia, pileus hyphae, and stipe hyphae) are presented from at least 20 measurements made with an ocular micrometer and 100x oil-immersion objective; basidiospore abbreviations include x = arithmetic mean of length and width for all spores measured and Q = length divided by width. Line drawings were made with a camera lucida. Color designations are from Munsell (1975) colour system.
Genomic DNA was extracted from a small piece of pileus by a modified CTAB method (Bruns 1995). The internal transcribed spacers (ITS1 and ITS2) and 5.8S region of the nuclear ribosomal RNA gene were amplified with the primer pairs ITS1F/ ITS4 (White et al. 1990; Gardes & Bruns 1993) using the Extract-N-Amp plant DNA extraction Kit (Sigma-Aldrich, St. Louis, MO, USA). PCR was carried out under the following cycling parameters: initial denaturation (94 °C for 1 min), 35 cycles (94 °C for 1 min, 53 °C for 1 min, and 72 °C for 1 min), and final extension 72 °C (8 min). Amplified PCR products were sent for purification and bidirectional sequencing to Macrogen (Korea). Two sequences each of Gymnopus luxurians and G. menehune and other related sequences were retrieved from the Genbank and aligned by Muscle using the default setting in Molecular Evolutionary Genetics Analysis (MEGA) software (Tamura et al. 2011). Sequences were manually edited and assembled using BioEdit (www.mbio.ncsu.edu/bioedit/bioedit.html). Following Dentinger et al. (2011) for complete ITS sequences, all sequences were trimmed with the conserved motifs 5’-(... GAT) CATTA- and -GACCT (CAAA...)-3’ and the alignment portion between them was included in the analysis. All positions containing gaps and missing data were eliminated. A phylogenetic tree was constructed with the Maximum Likelihood (ML) algorithm and Jukes & Cantor (1969) model of nrITS sequences and nearest-neighbor- interchange (NNI) as ML heuristic search method using MEGAS5 software (Tamura et al. 2011). Phylogeny was tested by bootstrap value of 1000 replicates, and corresponding bootstrap values >50 % are cited in the tree. Sequences of Omphalotus spp. were used as outgroup based on results reported by Moncalvo et al. (2002) and Mata et al. (2004).
Sequences generated for this study were submitted to Genbank. A complete list of taxa used in this phylogenetic is shown in TaBLE 1. Percent identities (PID) and DNA divergence were calculated by DNAStar.
Taxonomy
Gymnopus luxurians (Peck) Murrill, N. Amer. Fl. 9(5): 362 (1916). Fic. 1
PILEUS 19-29 mm diam, convex when young, expanding with age to plane, flat, thin (collybioid), slightly umbonate; margin recurved or uplifted, flexuous, striate; surface dull, fibrillose; brown, dark brown or reddish brown (10R5/10), margin fading to pale brown to brown or buff (10R6/12). LAMELLAE adnexed,
66 ... Saba & Khalid
Fic. 1. Gymnopus luxurians (MSM#001; MSM #002). (A-C) Basidiomes. (D) Basidiospores. (E) Basidia and basidioles. (F) Cheilocystidia. (G) Caulocystidia. (H) Pileipellis. Scale bars: A-C = 2 cm; D-F = 10 um; G-H = 20 um.
indented or notched, close to subdistant, white at first (SYR9/2), pale grayish (S5YR8/6) at maturity. STIPE 13-25 mm, central, subequal, often with subbulbous
Gymnopus in Pakistan ... 67
base, hollow, striate, buff (2.5YR6/8) when young, base dark brown (10R2/6). RHIZOMORPHS white, numerous, branched. Odor and taste not recorded. BASIDIOSPORES 4.2-5 X 5.3-9.4 um [x = 4.6 x 7.6 um, Q = 1.65], oblong to ellipsoid in profile, apiculus present, smooth, thin-walled, hyaline in KOH, inamyloid. Basip1a 5.6-8 x 19-28.3 um, clavate, 2-spored basidia abundant, 4-spored also present, thin-walled, hyaline in KOH; sterigmata 2-4 um. BASIDIOLES subclavate, abundant. PLEUROCYSTIDIA absent. CHEILOCYSTIDIA 4-8 x 18-32 um, clavate or cylindrical, hyaline, thin-walled. PILEIPELLIS a cutis, hyphae cylindrical, 6-14 um, thin-walled, weakly to heavily encrusted with annular brown pigments, brown in KOH. Stipe hyphae cylindrical, 5-10 um, non-encrusted, hyaline to pale yellow in KOH. CauLocystip1a cylindrical, hyaline, thin-walled 9-14 x 53-124 um. CLAMP CONNECTIONS present in all tissues.
MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTOONKHAW, Abbottabad, Shimla,
under Pinus roxburghii Sarg., 14 September 2012, Malka Saba & Abdul Nasir Khalid,
MSM #001 (LAH; GenBank KF803760); MSM #002 (LAH; GenBank KF803761). COMMENTS: Gymnopus luxurians is a widely distributed species. It is characterized by brown or reddish brown convex pileus, slightly umbonate with striate margin, oblong to ellipsoid basidiospores, and cylindrical caulocystidia. It is frequently collected on woody debris across the continental United States and has been reported from Europe (Breitenbach & Kranzlin 1991), Hawaii (Desjardin et al. 1999), Dominican Republic (Mata et al. 2006), Costa Rica, and Panama (Mata & Ovrebo 2009). Gymnopus luxurians represents a new record for Pakistan.
Gymnopus luxurians falls in G. subsect. Vestipedes (Antonin & Noordeloos 1997). Other Gymnopus species from this subsection reported from Pakistan include G. confluens. (Ahmad 1980; Shibata 1992; Sultana et al. 2011), G. peronatus (Sultana et al. 2011), and G. menehune (see below). Gymnopus confluens is similar to G. luxurians in being reported from pine forests during the same season of the year but can be separated by the presence of thin and crowded lamellae and a whitish fuzz on the stipe that becomes noticeable as the fungus matures (Kuo 2013). Gymnopus peronatus can be distinguished by its remarkably distant gills and densely woolly-strigose stipe with white to yellow hairs (Antonin & Noordeloos 1997).
Gymnopus menehune Desjardin, Halling & Hemmes, Mycologia 91(1): 173 (1999). Fic. 2 PILEus 10-14 mm diam., convex to plano-convex and slightly umbilicate when young, infundibuliform, umbilicate at maturity, reddish brown (10R3/8) or brown fading near margin to grey or pale brown (5YR9/2); margin decurved, straight or flaring, uplifted, eroded, striate; surface dull, glabrous. LAMELLAE adnate to decurrent, ascending, close, with 3-6 series of lamellulae,
68 ... Saba & Khalid
off-white to pale orange white (10R8/4). Stipe 23-32 mm, central, equal, hollow, pubescent to tomentose, buff color near proximal end, brown to dark brown (10R2/6) at distal end. RHIZOMORPHS numerous, white, branched. Odor and taste not recorded. BAsip1osporeEs elongate to ellipsoid in profile, 3-4,2(-5.4) x (-5.5)7-9 um [x = 4.6 x 8.1 um, Q = 1.76], smooth, thin-walled, hyaline in KOH with cytoplasmic contents, inamyloid. Basrpra clavate, 6.3- 7(-8) x 18.8-24 um, commonly 2-spored, occasionally 4-spored; Sterigmata up to 4.9 um. BASIDIOLEs clavate or fusoid, abundant. PLEUROCysTIDIA absent. CHEILOCYSTIDIA broadly clavate or irregular in outline, sometimes lobed, thin-walled, 5-7 x 20-30 um, rarely observed. PILEIPELLIS a cutis, hyphae 3.7-6.3 diam., cylindrical with outgrowths or a few diverticula, thin-walled, weakly encrusted with brown pigments, terminal cells non-encrusted, lobed, pale brown in KOH, inamyloid. Strpz hyphae 5.9-6.4 um, cylindrical, parallel, thin-walled, hyaline to pale brown in KOH, inamyloid. CAuLocysTIDIA 5-6 x 31-54 um, cylindrical to sinuous, hyaline, thin-walled. Clamp CONNECTIONS present in all tissues.
MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTOONKHAW, Mansehra, Dadar,
under Pinus roxburghii, 15 September, 2012, Malka Saba & Abdul Nasir Khalid, MSM
#003 (LAH; GenBank KF803762). COMMENTS: Gymnopus menehune is diagnosed by its convex to plano-convex, umbilicate pileus, pubescent to tomentose stipe, and elongate to ellipsoid basidiospores. According to Desjardin et al. (1999) there are voluminous lobed cheilocystidia in G. menehune, but cheilocystidia were rarely observed in our Pakistan collection. The species was described first from Hawaii (Desjardin et al. 1999) and later from Indonesia (Wilson et al. 2004). Gymnopus menehune represents a new record for Pakistan.
Gymnopus menehune falls in G. subsect. Vestipedes (Antonin & Noordeloos
199%):
Phylogenetic analysis
Sequencing of the nrITS region of Gymnopus luxurians yielded fragments of 800 base pairs while G. menehune yielded 830 base pairs. Initial BLAST analysis of nucleotide sequences showed collections MSM#001 and MSM#002 with 100% and 99% maximum identity with Gymnopus luxurians (GenBank AY256709, DQ480106, DQ450022), while collection MSM#003 showed 97% maximum identity with G. menehune (GenBank JN182864, AY263426, AY263444, DQ450043, AY263443).
Sequences of closely related taxa were retrieved from Genbank for phylogenetic analysis. In addition to the three new Pakistani sequences of G. luxurians and G. menehune, the analysis included 30 other Gymnopus sequences from GenBank, and three Omphalotus sequences as outgroup (TABLE 1).
Gymnopus in Pakistan ... 69
pe NE
Fic. 2. Gymnopus menehune (MSM #003). (A-B) Basidiomes. (C) Basidiospores. (D) Basidia and basidioles. (E) Cheilocystidia. (F) Stipe elements and caulocystidia. (G) Pileipellis. Scale bars: A-B = 2 cm; C-E = 10 um; F-G = 20 um.
F ee
After removing and editing ambiguities from the aligned sequences, a total of 948 positions were in the final dataset. Of these, 508 characters were conserved, 418 were variable, 380 were parsimony informative, and 32 were singletons.
Clade I, and II (Fic. 3) included species of Gymnopus subsect. Vestipedes, Clade III species characteristic of G. sect. Gymnopus, and clade IV species of G. subsect. Levipedes. Pakistani sequences (=) generated in this study clustered with their respective morphological species, each one with strong bootstrap value. Gymnopus luxurians appears in a clade distinct from G. menehune.
70 ... Saba & Khalid
66 pM Gymnopus_menehune_KF803762 36 JL. Gymnopus_menehune_JN182864 92 Gymnopus_menehune_AY263426 Gymnopus_subcyathiformis_DQ450041 99! Gymnopus_subcyathiformis_DQ450042 Gymnopus_mesoamericanus_DQ450036 100 ! Gymnopus_mesoamericanus_AF505768 74 Gymnopus_confluens_AY256697 Clade | 100 & Gymnopus_confluens_HM240527 Gymnopus_readii_DQ450034 99 100 § Gymnopus_readiae_HQ533036 100 , Gymnopus_cylindricus_AY256696 Gymnopus_cylindricus_DQ450057 Gymnopus_biformis_DQ450064 100 Gymnopus_biformis_DQ450063
= 100; Gymnopus_subpruinosus_DQ450026 84 Gymnopus_subpruinosus_DQ450027 Gymnopus_polygrammus_DQ450028 98 !Gymnopus_polygrammus_AY842954 5 100) Gymnopus_fibrosipes_AF505763 Gysnnopas -Nprosipes NV hese Clade II 100 = Gymnopus_gibbosus_AY263437 31 Gymnopus_gibbosus_AY263438 i Gymnopus_luxurians_KF803760 Gymnopus_luxurians_DQ450022 97 | Hl Gymnopus_luxurians_KF803761 Gymnopus_luxurians_AY256709 100 y Gymnopus_fusipes_AY256710 Clade III Gymnopus_fusipes_AF505777 ain 100 yGymnopus_dryophilus_JX536158 Gymnopus_dryophilus_JX536157 Clade IV 98 Gymnopus_erythropus_DQ449998 100 ke Gymnopus_erythropus_DQ449996 Omphalotus_illudens_AY313271 100 Omphalotus_olearius_AY313277 Outgroup Omphalotus_olivascens_AY313281 oo.
Fic. 3. Molecular phylogenetic analysis of Gymnopus sequences by the Maximum Likelihood method. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history. The percentage of trees in which the associated taxa clustered together is shown next to the branches. New sequences reported in this study (#).
The percentage similarity, calculated by DNAStar, of Gymnopus luxurians showed 100% identity and 0% genetic divergence with G. luxurians sequences AY256709 and DQ450022, and G. menehune showed 99.9% identity and
Gymnopus in Pakistan ... 71
0.1% divergence with G. menehune sequences JN182864 and AY263426; this supports the results of the BLAST analysis and the phylogenetic tree.
Key to the Gymnopus spp. in Pakistan
1. Pileipellis a transition between cutis and trichoderm and comprising lobed
(often coralloid) elements, similar to a Dryophila-structure.......... G. fusipes 1. Pileipellis usually a simple cutis with some weakly to distinctly coralloid or
diverticulate terminal elements, without Dryophila-structure ............... Z Ze SUPE STO OU te, Mace bee ace be ees eae Le aes comes oats omic: Ciao, mes, Lee, Lome, 3 Da ADE AEN OF COMTER LOSE rn tp | Mecwuty eiecwartyl teawn ee stan see tara ee eenk Wee Hora Gears weit ees 4
3. Pileus dark red brown at centre, much paler yellow brown to yellow red LOWAPOSHNIQESIN 0!) asses oeasre 2 sete earings tease apogee eat ee G. erythropus 3. Pileus with ochre brown tinges, especially at centre, translucently striate uujaveo Hialitiie Palins es.. er ocne Red ees weeetntenGd times Miateres MAN esuce Ste G. dryophilus
4a. Pileus convex when young, expanding with age to plane, flat, thin (collybioid), slightly umbonate; lamellae adnexed, close to subdistant; basidiospores oblongite ellipsdid:4=5: 659s SUI ag hike a ete at ae oe ae we Oe G. luxurians 4b. Pileus convex to plano-convex and slightly umbilicate when young, infundibuliform, umbilicate at maturity; lamellae adnate to decurrent, close; basidiospore elongate to ellipsoid, 3-5.5 x 5.5-9 um........ G. menehune 4c. Pileus convex with an incurved margin when young, becoming broadly convex, bell shaped or nearly flat; lamellae narrowly attached to the stem or free, crowded or close; basidiospores lacrymoid to elliptical or nearly fusoid, SSDS OA ROSMAN 4 1a, ese Sei ya et dai dal oh lr da ey oak G. confluens 4d. Pileus convex then flattened, often with a low, broad umbo, tan to darker brown; lamellae adnexed to free, crowded; basidiospores elongate, 3-4 x 7-9 um LANs ste dently, etYiNi, seein, aelysMsy aetpaha 4 Uelwshs 4 oekvibe!y Witwahe 6 signe. oth aiba shee G. peronatus
Acknowledgments
We are cordially grateful for Higher Education Commission (HEC) for funding this project under Phase II, Batch I, Indigenous PhD fellowships program for 5000 scholars. We are highly indebted to Dr. Juan Luis Mata (Department of Biology, University of South Alabama, USA) and Dr. Amy Rossman (Systematic Mycology and Microbiology Laboratory, USDA-ARS, Beltsville, USA) for critically reviewing the manuscript. We are thankful to all lab fellows for accompanying us on the field trip. I am cordially thankful to Dr. Samina Sarwar (Lahore College for Women University) for her help and guidance preparing the phylogeny.
Literature cited
Ahmad S. 1980. A contribution to the Agaricales of Pakistan. Bulletin of Mycology 1: 35-89.
Antonin V, Noordeloos M. 1997. A monograph of Marasmius, Collybia and related genera in Europe. Part 2: Collybia, Gymnopus, Rhodocollybia, Crinipellis, Chaetocalathus and additions to Marasmiellus. Libri Botanici 17. 256 p.
72 ... Saba & Khalid
Breitenbach J, Kranzlin F. 1991. Fungi of Switzerland. Vol. 3. Boletes and agarics, first part. Verlag Mykologia, Luzern. 361 p.
Bruns TD. 1995. Thoughts on the processes that maintain local species diversity of ectomycorrhizal fungi. Plant and Soil 170: 63-73. http://dx.doi.org/10.1007/BF02183055
Dentinger BTM, Didukh MY, Moncalvo JM. 2011. Comparing COI and ITS barcode markers for mushrooms and allies (Agaricomycotina). PLoS One 6(9): e25081. http://dx.doi.org/10.1371/journal.pone.0025081
Desjardin DE, Halling RE, Hemmes DE. 1999. Agaricales of the Hawaiian Islands. 5. The genera Rhodocollybia and Gymnopus. Mycologia 91: 166-176. http://dx.doi.org/10.2307/3761206
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. http://dx.doi.org/10.1111/j.1365-294X.1993.tb00005.x
Iqbal SH, Khalid AN. 1996. Materials for the fungus flora of Pakistan. I. Checklist of agarics, their distribution and association with the surrounding vegetation. Science International (Lahore) 8(1): 51-64.
Jukes TH, Cantor CR. 1969. Evolution of protein molecules. 21-132, in: HN Munro (ed.). Mammalian Protein Metabolism. Academic Press, New York.
Kuo M. 2013. Gymnopus confluens. Retrieved from the MushroomExpert.com. http://www.mushroomexpert.com/gymnopus_confluens.html
Mata JL, Ovrebo CL. 2009. New reports and illustrations of Gymnopus for Costa Rica and Panama. Fungal Diversity 38: 125-131.
Mata JL, Hughes KW, Petersen RH. 2006. An investigation of Omphalotaceae (Fungi: Euagarics) with emphasis on the genus Gymnopus. Sydowia 58: 191-289.
Moncalvo JM, Vilgalys R, Redhead SA, Johnson JE, James TY, Aime MC, Hofstetter V, Verduin SJW, Larsson E, Baroni TJ, Thorn RG, Jacobsson S, Clémencon H, Miller OK. 2002. One hundred and seventeen clades of Euagarics. Molecular Phylogenetics and Evolution 23: 357-400.
Munsell ™. 1975. Soil color charts. Baltimore.
Shibata H. 1992. Higher basidiomycetes from Pakistan. Cryptogamic Flora of Pakistan 1: 145-164.
Singer R. 1986. The Agaricales in modern taxonomy. 4th edn. Koeltz, Koenigstein. 980 p.
Sultana K, Rauf CA, Riaz A, Naz F, Irshad G, Haque MI. 2011. Checklist of agarics of Kaghan valley - 1. Pakistan Journal of Botany 43(3): 1777-1787.
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28(10): 2731-2739. http://dx.doi.org/10.1093/molbev/msr121
White TJ, Bruns T, Lee S, Taylor JW. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. 315-322, in: MA Innis et al. (eds). PCR Protocols: a guide to methods and applications. Academic, New York.
Wilson AW, Desjardin DE, Horak E. 2004. Agaricales of Indonesia. 5. The genus Gymnopus from Java and Bali. Sydowia 56(1): 137-210.
ISSN (print) 0093-4666 © 2014. Mycotaxon, Ltd. ISSN (online) 2154-8889
MY COTAXON
http://dx.doi.org/10.5248/129.73 Volume 129(1), pp. 73-77 July-September 2014
First report of Callistosporium luteoolivaceum from Western Himalaya, Pakistan
M. SABA* & A.N. KHALID
Department of Botany, University of the Punjab Quaid-e-Azam Campus, Lahore, 54590, Pakistan
* CORRESPONDENCE TO: rustflora@gmail.com
ABSTRACT — Callistosporium luteoolivaceum was collected from pure pine forests of Western Himalaya, Pakistan, and identified based on morphological characters and nrDNA ITS sequences. It is characterized by dull yellow or olive yellow pilei, lamellae, and stipes that turn reddish brown with age and basidiospores that turn purplish in KOH. This species is new to the mycobiota of Pakistan.
Key worps — Abbottabad, Basidiomycota, Khyber Pakhtoonkhaw, saprobic fungi, Tricholomataceae
Introduction
Geographically, the areas of Pakistan and Indian-held Kashmir fall within the Western Himalaya. The climate ranges from tropical at the base of the mountains to permanent ice and snow at the highest elevations. The Himalayan forests have been reliably identified as a ‘biodiversity hotspot; a global priority for the conservation of biodiversity (Wikramanayake et al. 2002; Qamar et al. 2011).
During a 2012 mushroom collecting trip in pure pine forests inthe Himalayan range, many species of saprobic macrofungi were collected. Amongst them, Callistosporium luteoolivaceum was identified morphologically and molecularly as a new record from Pakistan. This very variable species occurs worldwide in temperate or tropical forests mostly outside of boreal and subalpine regions (Redhead 1982). According to Kuo (2006) this little mushroom is a decomposer found primarily on dead conifer wood. No Callistosporium species has previously been reported from Pakistan.
Materials & methods Basidiomata were collected, photographed, vouchered, dried under fan heater and characterized morphologically. Specimen sections were mounted in 5% KOH for
7A ... Saba & Khalid
observation under a MX4300H biological microscope (Meiji Techno Co., Ltd., Japan). Phloxine was used to increase contrast of structures, and Melzer’s reagent was used to test for dextrinoidity of basidiospores.
Measurements of morphological features (basidiospores, basidia, cystidia, and pileus and stipe hyphae) were taken from at least 20 measurements made with an ocular micrometer and 100x oil-immersion objective, where x = arithmetic mean of spore length and width for all spores measured, and Q = spore length divided by spore width. Line drawings were made with camera lucida. Color designations are from Munsell (1975). The collection was conserved in the Herbarium of the Department of Botany, University of the Punjab, Lahore, Pakistan (LAH).
Genomic DNA was extracted from a small piece of pileus by a modified CTAB method (Bruns 1995). The internal transcribed spacers (ITS1 and ITS2) and the 5.8S nuclear ribosomal gene were amplified with primer pairs ITS1F/ITS4 (White et al. 1990; Gardes and Bruns 1993) using the Extract-N-Amp plant DNA extraction Kit (Sigma-Aldrich, St. Louis, MO, USA). PCR cycling parameters were as follows: initial denaturation (94 °C for 1 min), 35 cycles (94 °C for 1 min, 53 °C for 1 min, and 72 °C for 1 min), and final extension 72 °C (8 min). Amplified PCR products were sent for purification and bidirectional sequencing to Macrogen (Republic of Korea). One C. luteoolivaceum sequence and other related sequences were retrieved from GenBank and aligned by Muscle using default setting in Molecular Evolutionary Genetics Analysis (MEGA) software (Tamura et al. 2011). Sequences were manually edited and assembled using BioEdit (www.mbio.ncsu.edu/bioedit/bioedit.html). Following Dentinger et al. (2011) for complete ITS sequences, all sequences were trimmed with the conserved motifs 5’-(...GAT) CATTA- and -GACCT (CAAA...)-3’ and the alignment portion between them were included in analysis. A sequence generated for this study was submitted to GenBank. Percent Identities (PID) and DNA divergence were calculated by Megalign (DNA Star Inc.).
Taxonomy
Callistosporium luteoolivaceum (Berk. & M.A. Curtis) Singer, Lloydia 9: 117 (1946). Fics 1, 2 PILEus 27-31 mm diam., convex to plano-convex, flat, thin (collybioid); margin straight or flaring, smooth; surface dull, smooth, olive yellow near margin (5YR7/10); disc moderately indented, dark brown (5YR1/4) at the center. LAMELLAE regular, adnate, close to crowded, pale yellow or olive yellow (SYR7/8); margin wavy. STIPE 55-58 mm, central, equal, hollow, striate, pale yellow to olive yellow (S5YR7/8); annulus absent; volva absent. RHIZOMORPHS white, few. ODOR AND TASTE not recorded. BASIDIOSPORES 4.6-6.6 x 3-4.5 um [x = 5.2 x 3.8 um, Q = 1.36], ellipsoid in profile, smooth, thin-walled, purplish in KOH, apiculus present; BAstp1A 16.9-24.5 x 5.4—7.8 um, clavate, two to four spored, thin-walled, some turning deep purple in KOH; sterigmata 2.8-4.2 um. Trama hyphae, parallel, thin-walled, hyaline in KOH. CHEILOCYSTIDIA 12-26.6 x 4.7-8 um, clavate or cylindrical, hyaline, thin-walled. PLEURO-
Callistosporium luteoolivaceum in Pakistan ... 75
Callistosporium luteoolivaceum (MSM #004, LAH). Basidiomes. Scale bars = 7 mm.
CYSTIDIA absent. PILEIPELLIS a cutis; hyphae cylindrical, 4-6 um, thin-walled, hyaline in KOH, some with brown pigments; pileal cystidia clavate, 34-61 x 5.1-8.2 um, thin walled, hyaline in KOH. Stipe hyphae cylindrical, 3.5-17 um, hyaline in KOH. All structures inamyloid. CLamp CONNECTIONS absent. MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTOONKHAW, Abbottabad, Shimla, under Pinus roxburghii Sarg., 14 September 2012, MSM #004 (LAH; GenBank KJ101607). Discussion: Callistosporium luteoolivaceum was first described from eastern North America. It is taxonomically complicated, and its variable characters and wide world distribution has resulted in its being described many times as a new taxon. Redhead (1982) critically examined C. elaeodes Bon, C. favrei Singer, C. graminicolor Lennox, C. luteofuscum Singer, C. luteofuscum var. major Singer, C. majus Singer, and C. xanthophyllum (Malencon & Bertault) Bon and reduced them to synonymy under C. luteoolivaceum because their distinguishing features intergrade. Despite the intergradations of critical characters, there remains a great diversity of forms within the broad species concept which when viewed individually appear quite distinct from one another (Redhead 1982).
76 ... Saba & Khalid
—
D Rass Callistosporium luteoolivaceum (MSM #004, LAH).
(A) Basidia. (B) Basidiospores. (C) Stipe elements. (D) Pileipellis and pileal cystidia. Scale bars: A = 10 um; B = 4.5 um; C, D = 17.5 um.
Our collection is in remarkable agreement with the description provided by Redhead (1982). This is the first report of C. luteoolivaceum from Pakistan. It has been reported from North and South America, Europe, and Asia (Hongo 1981; Horak 1987; Manimohan & Leelavathy 1989; Redhead 1982; Singer 1970). This species is listed among the rare and notable macrofungi of British Columbia (Redhead 1997: 6-8).
Sequencing of the nrITS region of C. luteoolivaceum yielded fragments of 690 base pairs when its PCR product was sequenced. Initial blast analysis showed our Pakistani sequence (GenBank KJ101607) with 97% maximum identity with C. xanthophyllum (GenBank JF907781; AF325667) and C. luteoolivaceum (GenBank AF325666). Further analysis reinforced the blast results, showing
Callistosporium luteoolivaceum in Pakistan ... 77
97.5% identity and 0.5% genetic divergence between our Pakistani sequence and the C. luteoolivaceum sequence (GenBank AF325666).
Acknowledgments
We are highly indebted to Higher Education Commission (HEC), Pakistan, for funding this project under Phase II, Batch I, Indigenous PhD fellowships program for 5000 scholars. We are cordially grateful to Dr. Juan Luis Mata (Dept. of Biology, University of South Alabama, USA) and Dr. Vladimir Antonin (Moravian Museum, Czech Republic) for critically reviewing the manuscript and their valuable comments. We are thankful to all lab fellows for accompanying us on field trip.
Literature cited
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Dentinger BTM, Didukh MY, Moncalvo JM. 2011. Comparing COI and ITS barcode markers for mushrooms and allies (Agaricomycotina). PLoS One 6(9): e25081. http://dx.doi.org/10.1371/journal.pone.0025081
Gardes M, Bruns TD. 1993. ITS primers with enhanced specificity for Basidiomycetes: application to the identification of mycorrhizae and rusts. Mol. Ecol. 2: 113-118.
Hongo T. 1981. Notulae mycologicae (17). Mem. Fac. Educ. Shiga Univ. Nat. Sci. 31: 33-36.
Horak E. 1987. Agaricales from Yunnan, China, I. Trans. Myc. Soc. Japan 28: 171-188.
Kuo M. 2006. Callistosporium luteo-olivaceum. Retrieved from the MushroomExpert.Com website: http://www.mushroomexpert.com/callistosporium_luteo-olivaceum.html.
Manimohan P, Leelavathy KM. 1989. Some agarics new to India. Sydowia 41: 200-208.
Munsell ™. 1975. Soil color charts. Baltimore.
Qamar FM, Ali H, Ashraf S, Daud A, Gillani H, Mirza H, Rehman HU. 2011. Distribution and habitat mapping of key fauna species in selected areas of Western Himalaya, Pakistan. Journal of Animal and Plant Sciences 21(2S): 396-399.
Redhead SA. 1982. The systematics of Callistosporium luteo-olivaceum. Sydowia 35: 223-235.
Redhead SA. 1997. Macrofungi of British Columbia: requirements for inventory. Res. Br., B.C. Min. For., and Wildl. Br., B.C. Min. Environ., Lands & Parks, Victoria, B.C. Working Paper 28/1997. 119 p.
Singer R. 1970. Omphalinae (Clitocybeae - Tricholomataceae, Basidiomycetes). Fl. Neotrop. Monogr. 3: 3-84.
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28(10): 2731-2739.
Wikramanayake E, Dinerstein E, Loucks CJ, Olson DM, Morrison J, Lamoureux J, McKnight M, Hedao P. 2002. Terrestrial ecoregions of the Indo-Pacific: a conservation assessment. Island Press, Washington DC. 643 p.
White TJ, Bruns T, Lee S, Taylor JW. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. 315-322, in: MA Innis et al. (eds). PCR Protocols: a guide to methods and applications. Academic, New York.
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MY COTAXON
http://dx.doi.org/10.5248/129.79 Volume 129(1), pp. 79-83 July-September 2014
Gastroboletus thibetanus: a new species from China
SHU-RONG WANG", Qi WANG’, DE-LI WANG’, & Yu L1’?*
' Institute of Grassland Science, Northeast Normal University, and Key Laboratory of Vegetation Ecology, Ministry of Education, Changchun, 130024, China
? Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi, Jilin Agricultural University, 2888 Xincheng Street, Changchun, 130118, China
* CORRESPONDENCE TO: “wzlj2005@163.com; *yuli966@126.com
ABSTRACT— Gastroboletus thibetanus sp. nov. is reported from Tibet, China. It was collected under Abies forrestii var. smithii and is characterized by pale green-yellow to coral red basidiomata, a percurrent columella, and relatively large basidiospores.
Key worps— Basidiomycota, Boletaceae, Boletales, macrofungi, taxonomy
Introduction
Gastroboletus Lohwag (Boletaceae, Boletales, Basidiomycota) is a small genus that is widely distributed in temperate regions (Kirk et al. 2008). It is characterized by hypogeous, sequestrate Boletus-like basidiomata that have a poorly developed or absent stipe with irregularly arranged tubes (usually covered by persistent membranes) and elliptical to spindle-shaped, smooth, brown to golden brown spores. Spore dispersal depends primarily on animal mycophagy (Nouhra et al. 2002). Gastroboletus has been reported from North and South America, Asia, South Africa, Western Europe, and eastern Australia (Cazares & Trappe 1991; Horak 1977; Lohwag 1926; Nouhra & Castellano 1995; Nouhra et al. 2002; Singer & Smith 1964; Smith & Singer 1959; Trappe & Castellano 2000; Thiers & Trappe 1969; Thiers 1979, 1989; Zang 1995). Thiers (1989) divided Gastroboletus sensu lato into several genera to reflect their phylogeny more correctly. Nouhra et al. (2002) provided a comprehensive key to all known Gastroboletus species.
Mt. Sejila, which is located in Tibet, China, is geographically isolated due to its high altitude and poor road access. Its fungal flora has been poorly studied, with only a few Cortinarius species previously reported (Teng et al. 2011). In an attempt to help rectify this, macrofungal field surveys were carried out in 2012
80 ... Wang & al.
FiGuRE 1. Gastroboletus thibetanus (HMJAU 30001, holotype). A. basidioma; B. vertical section of basidioma, showing gleba and columella. Scale bars = 2 cm.
and 2013. Here we describe a particularly interesting hypogeous sequestrate species, which we propose as G. thibetanus sp. nov.
Materials & methods
Specimens were collected at Mt Sejila, Tibet, under Abies forrestii var. smithii, the dominant species in the coniferous forest with Larix and Picea and with Rhododendron occasionally found in the understory (Xu 1995). The acidic brown soil has a pH of 4.9. All specimens are deposited in the Herbarium of Mycology of Jilin Agricultural University (HMJAU), China. Macroscopic characters were described based on fresh and dried material. Color names follow Kornerup & Wanscher (1978). For light microscopic observations, free-hand sections of the specimens were mounted in 5% KOH solution on glass slides. Forty randomly selected basidiospores were measured under 1000x magnification. The basidiospore surfaces were observed by scanning electron microscope after coating with gold (Bozzolla & Russell 1999).
Taxonomic description
Gastroboletus thibetanus Shu R. Wang & Yu Li, sp. nov. FIGS 1, 2 MycoBank MB 807041 Differs from Gastroboletus ruber and G. molinae by its larger basidiospores and the absence of cystidia. Type: China. Tibet, Lenzhi, Mount Sejila, 29°40’22.54”N 94°43’11.20’E, on soil under Abies forrestii var. smithii Viguié & Gaussen, 26 July 2012, Wang Shu-Rong T10020 (Holotype, HMJAU 30001).
Gastroboletus thibetanus sp. nov. (China) ... 81
FIGURE 2. Gastroboletus thibetanus. A. vertical section through the peridium and gleba of a basidioma; B. peridial structure; C. parallel hyphae in trama; D. basidia and basidiospores; E. basidiospores, showing yellowish color and thick walls; F SEM micrograph of basidiospores, showing smooth surface. Scale bars: A = 100 um; B, C = 50 um; D, E = 20 um; F = 5 um.
ErymMo_ocey: Referring to Tibet Autonomous Region, where the holotype was collected.
BASIDIOMATA depressed-globose to reniform, 6-40 mm across when dried, 10-48 mm across when fresh, surface smooth, sometimes cracked-areolate on top, “light green-yellow” to “coral red” (1A7—1A8, 9A7—9B7), becoming somewhat grayish yellow with age. GLEBA 5—23 mm broad, olive brown (4E5), with empty chambers and a prominent columella, changing bluish on bruising or exposure. COLUMELLA 7-15 mm broad, yellow to reddish-brown (2A8, 4A8) when fresh, sordid yellowish when dried, truncate to dendroid, with narrow, radiating branches, extended below as a reddish-brown sterile base, usually arising from yellowish rhizomorphs, changing bluish when bruised or exposed. Odor fruity to mushroomy.
PERIDIUM 60-150 um thick, yellow-brown. PERIDIAL HYPHAE 3-18 um diam., surface smooth, thin-walled, yellowish, more or less appressed, sometimes interwoven, slightly gelatinous to floccose in KOH. HYPHAE BELOW PERIDIUM 2.0-4.5 um diam., surface smooth, yellow-brown, thin-walled, loose and subparallel with the peridium. TRAMA olive brown, consisting of loose, subparallel hyphae (2.0—5.0 um thick). COLUMELLAR HYPHAE 3.0-6.0 um diam., thick-walled, pale yellow to pale brown, loose and rarely dichotomously branched. Basrp1a cylindrical, 2-4-spored, 25-36 x 10-14 um; sterigmata
82 ... Wang & al.
3.7—5 um long and 1.5—3 um thicker at base. Bastp1osporEs ellipsoid, smooth, thick-walled, yellowish in KOH, 11-23.5 x 7.5-12 um, L/W ratio = 1.64-1.94, Average = 1.80 + 0.02, symmetrical, basal apiculus prominent. CystTrp1a and CLAMP CONNECTIONS not seen.
Hasirat: Epigeous or subhypogeous on soil under Abies forrestii var. smithii. ADDITIONAL SPECIMENS EXAMINED: CHINA. TiBET, LENzHI, Mount Sejila, 29°40'22.54”N 94°43’11.20’E, 11 August 2013, coll. Wang Shu-Rong T50457 (HMJAU 30002); 29°40’22.64’N 94°43’10.22”E, 11 August 2013, coll. Wang Shu-Rong 150467 (HMJAU 30005); 29°40’22.50”N 94°43’10.44”E, 4 October 2013, coll. Li Shu T18269 (HMJAU 30006).
Discussion
The combined characters of the pale green-yellow to coral red basidiomata that become bluish on bruising or exposure, percurrent columella, relatively large (11-23.5 x 7.5-12 um) thick-walled basidiospores, and the absence of cystidia are diagnostic for G. thibetanus. Gastroboletus thibetanus belongs to G. sect. Gastrosubtomentosi based on the red overtones of the basidiomata, dry peridial surface, and structure of the peridium (Thiers 1989).
The color and size of G. thibetanus basidiomata are somewhat similar to those of G. ruber and G. molinae reported from U.S.A. (Cazares & Trappe 1991; Nouhra et al. 2002). The two American species differ from G. thibetanus by smaller basidiospores (8—20 x 4—6 um and 11-20 x 5—8 um, respectively) and the presence of cystidia. In addition, G. ruber always occurs in coniferous forests dominated by Tsuga spp. (Zeller 1939, Molina et al. 1992) while G. molinae is associated with Quercus chrysolepis (Nouhra et al. 2002).
Gastroboletus thibetanus also resembles G. citrinobrunneus, G. dinoffii, and G. doii by tissues that turn blue on bruising or exposure. Both G. citrinobrunneus and G. dinoffii, reported from California, U.S.A, are readily distinguished by their larger basidiomata and smaller basidiospores (Nouhra & Castellano 1995; Thiers 1979). Gastroboletus doii (from New Caledonia) can be differentiated by pale dull brown to ochre basidiomata, presence of cystidia, and a volva (Zang 1995). The type species of Gastroboletus, G. boedijnii from China (Lohwag 1926), is easily separated from G. thibetanus by the presence of cystidia and smaller (11-14 x 4—5.5 um) basidiospores (Smith & Singer 1959).
Acknowledgments
We express our deep appreciation to Dr. M. A. Castellano (USDA, Forest Service, Northern Research Station, Corvallis, Oregon, U.S.A) and Dr. Ian R. Hall (Truffles & Mushrooms (Consulting) Ltd., Dunedin, New Zealand), who critically reviewed the manuscript and provided invaluable suggestions. The first author is very grateful to Dr. M. Kakishima (Jilin Agricultural University, China) and Dr. E. Nagasawa (The Tottori Mycological Institute, Japan) for their improvement of the manuscript. This work was
Gastroboletus thibetanus sp. nov. (China) ... 83
supported by the Fungal Diversity Investigation and Key Technology in Sustainable Use of Rare Fungi in Tibet fund (No. 2012BAC01B04).
Literature cited
Bozzolla JJ, Russell LD. 1999. Electron microscopy: principles and techniques for biologists. 2nd ed. Jones & Bartlett Learning.
Cazares E, Trappe JM. 1991. Alpine and subalpine fungi of the Cascade Mountains. 3. Gastroboletus ruber comb. nov. Mycotaxon 42: 339-345.
Horak E. 1977. New and rare boletes from Chile. Bol. Soc. Argentina Bot. 18: 97-109.
Kirk PM, Cannon PF, Minter DW, Stalpers JA. 2008. Ainsworth & Bisby’s dictionary of the fungi, 10th edn. Wallingford, CAB International.
Kornerup A, Wanscher JH. 1978. Methuen handbook of colour. Eyre Methuen, London.
Lohwag H. 1926. Zur Entwicklungsgeschichte und Morphologie der Gastromyceten. Beihefte zum Botanischen Centralblatt, 2. Abt., 42: 117-334.
Molina R, Massicotte H, Trappe JM. 1992. Specificity phenomena in mycorrhizal symbioses: community-ecological consequences and practical implications. 357-423, in: MF Allen (ed.). Mycorrhizal functioning: an integrative plant-fungal process. Chapman & Hall, New York.
Nouhra E, Castellano MA. 1995. NATS truffle and truffle-like fungi 3: Gastroboletus dinoffii sp. nov. Mycotaxon 55: 179-183.
Nouhra E, Castellano MA, Trappe JM. 2002. NATS truffle and truffle-like fungi 9: Gastroboletus molinai [sic] sp. nov. (Boletaceae, Basidiomycota), with a revised key to the species of Gastroboletus. Mycotaxon 83: 409-414.
Singer R, Smith AH. 1964. Studies on secotiaceous fungi X. Additional data on Gastroboletus. Mycologia 56: 310-313. http://dx.doi.org/10.2307/3756548
Smith AH, Singer R. 1959. Studies on secotiaceous fungi. IV. Gastroboletus, Truncocolumella, and Chamonixia. Brittonia 11: 205-223. http://dx.doi.org/10.2307/2805006
Teng LJ, Xu AS, Liu XJ. 2011. The resources of Cortinarius in Sejila Mountain of Mountain of Tibet. Edible Fungi of China 5: 002.
Thiers HD. 1979. New and interesting hypogeous and secotioid fungi from California. Beihefte zur Sydowia 8: 381-390.
Thiers HD. 1989. Gastroboletus revisited. Memoirs of the New York Botanical Garden 49: 355-359.
Thiers HD, Trappe JM. 1969. Studies in the genus Gastroboletus. Brittonia 21: 244-254. http://dx.doi.org/10.2307/2805576
Trappe JM, Castellano MA. 2000. New sequestrate Ascomycota and Basidiomycota covered by the Northwest Forest Plan. Mycotaxon 75: 153-179.
Xu FX. 1995. Study on the forest ecosystem in Tibet. Shenyang. Liaoning University Press. [in Chinese].
Zang M. 1995. A new species of the genus Gastroboletus from New Caledonia. Mycotaxon 54: 407-412.
Zeller SM. 1939. New and noteworthy gasteromycetes. Mycologia 31: 1-32. http://dx.doi.org/10.2307/3754429
ISSN (print) 0093-4666 © 2014. Mycotaxon, Ltd. ISSN (online) 2154-8889
MY COTAXON
http://dx.doi.org/10.5248/129.85 Volume 129(1), pp. 85-95 July-September 2014
Biogeographical patterns in pyrenomycetous fungi and their taxonomy. 4. Hypoxylon and the southern United States
LARISSA N. VASILYEVA! & STEVEN L. STEPHENSON?
‘Institute of Biology and Soil Science, Far East Branch of the Russian Academy of Sciences, Vladivostok 690022, Russia *Department of Biological Sciences, University of Arkansas, Fayetteville, Arkansas 72701, USA
* CORRESPONDENCE TO: vasilyeva@biosoil.ru
ABSTRACT — A biogeographic region with a peculiar species composition and located in the southern states of the United States is discussed. It is proposed that this region is a part of a possible Caribbean center of fungal biodiversity. Hypoxylon confertisilvae, H. ilicinum, H. meridionale, H. minicroceum, and H. rolingii are described as species new to science.
Key worps — Ascomycota, distribution, Xylariaceae
Introduction
A group of the southern states of the United States (Arkansas, Alabama, Florida, Louisiana, Mississippi, Missouri, and Texas) represent a region of considerable interest with respect to the biogeography of pyrenomycetous fungi. First, one can observe typical species that are known only from southeastern North America, including such examples as Biscogniauxia atropunctata (Schwein.) Pouzar and Diatrype atlantica Lar.N. Vassiljeva on Quercus spp., Diatrype tremellophora Ellis on Magnolia spp., and Diatrype virescens (Schwein.) M.A. Curtis on Fagus grandifolia. Second, there are species that occur mainly in the southern states listed above, with one example being Camillea signata (S.C. Jong & C.R. Benj.) Lzessge et al. The same preference for this region is observed in Biscogniauxia schweinitzii Y.M. Ju & J.D. Rogers, Rosellinia glandiformis Ellis & Everh., and R. langloisii Ellis & Everh.
Over a period of several years (2006-11) surveys for pyrenomycetous fungi were carried out in a number of localities in this region, mostly in Arkansas (the Buffalo National River Park, the Ouachita Mountains Biological Station, and the Ozark National Science Center) and Texas (the Big Thicket National Preserve). The survey results combined with literature data suggest a very specific center of fungal biodiversity.
86 ... Vasilyeva & Stephenson
The southern United States as part of a possible Caribbean center
of fungal biodiversity
Some species reported for Mexican border regions—Biscogniauxia arima FE San Martin et al., Hypoxylon lividipigmentum F. San Martin et al., and Annulohypoxylon thouarsianum var. macrosporum (F. San Martin et al.) Y.M. Ju et al. also have been found in Texas (Big Thicket National Preserve). Several species have distributions that extend from the southern United States to northern South America. Thus, Hypoxylon venezuelense Y.M. Ju & J.D. Rogers, described originally from Venezuela (Ju & Rogers 1996), also has been found in Texas (Fic. 1). Hypoxylon rickii Y.M. Ju & J.D. Rogers is known from French Guiana, Mexico, and St. John (U.S. Virgin Islands) as well as from Louisiana (Fic. 2). Other Caribbean islands seem to share species of pyrenomycetous fungi with adjacent continental areas, such as Vivantia guadalupensis J.D. Rogers et al., described originally from Guadeloupe (Rogers et al. 1996) and later found in Texas (Fie. 3).
There are additional data from some other biogeographic comparisons that support an independent Caribbean center of fungal biodiversity. We have previously discussed the “Asa Gray disjunction,’ a well-known distribution pattern that exists for many organisms, including fungi (Vasilyeva & Stephenson 2010). This pattern, which is characteristic of species occurring both in northeastern North America and northeastern Asia, is represented by ‘sibling’ species that replace each other in these two widely separated world regions.
Unexpectedly, a southern parallel to the “Asa Gray disjunction” was found to exist between southeastern Asia (tentatively consisting of the Indochina Peninsula, southern provinces of China, and the Malay Archipelago) and the region in and around the Caribbean Sea and the Gulf of Mexico (Fic. 5). When efforts are made to identify species collected in the Indo-Malayan center of biodiversity (Vasilyeva et al. 2012), the other species they most closely resemble often appear to be those reported from Mexico, the southern United States, or the Caribbean coast of South America. Sometimes, the morphological resemblance is so high that the species from the eastern and western hemispheres differ in only one or two features. (The same phenomenon is observed for the species involved in the northern “Asa Gray disjunction.”) For example, Biscogniauxia schweinitzii from the southern United States and B. lithocarpi Lar.N. Vassiljeva et al. from northern Thailand have strikingly similar asci and ascospores but differ in their stromata. Hypoxylon rubroargillaceum Lar.N. Vassiljeva et al. is similar to H. flavoargillaceum J.H. Mill. (known from Colombia and Venezuela) but differs in ascal tips that do not turn blue in Melzer’s reagent and the presence of granules immediately beneath the surface and among the perithecia that are not yellowish-brown but bright-red.
Hypoxylon spp. nov. (U.S.A.) ... 87
Fics. 1-5. 1. Hypoxylon venezuelense: A. Stroma (VLA P-2605), B. Collection localities; 2. Hypoxylon rickii: A. Stroma (VLA P-2607), B. Collection localities; 3. Vivantia guadalupensis: A. Stroma (VLA P-2448), B. Collection localities; 4A-B. Stromata of Hypoxylon subchlorinum (VLA P-2604); 5. Tentative location of the Caribbean and Indo- Malayan centers of fungal diversity. Scale bars: 1A = 1.5 mm; 2A = 9 mm; 3A = 4.5 mm; 4A-B = 2.5 mm. [The maps modified to show our data are taken from the web sites <http://www.biomedcentral.com> (Fics 1B, 2B, 3B) and <http://www.designus.com/vector-world-map> (Fic. 5).]
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Such morphological similarities shown by species associated with eastern and western centers of biodiversity is not surprising, given that molecular studies also pull together exactly such species from those areas. Thus, the most interesting and relevant xylariaceous phylogenetic tree (Hsieh et al. 2005) clusters together several species (Hypoxylon crocopeplum Berk. & M.A. Curtis, H. dieckmannii Theiss., H. erythrostroma J.H. Mill., H. fendleri Cooke, and H. haematostroma Mont.) from both Mexico (the Caribbean center) and Taiwan (the Indo-Malayan center). While this might support conspecificity of specimens from the western and eastern hemispheres, the specimens could just as easily be assigned to different species, as has been done for Hypoxylon lividicolor Y.M. Ju & J.D. Rogers from Taiwan and H. lividipigmentum from Mexico, two taxa that also cluster together with the same level of support (Hsieh et al. 2005) and which we have already (Vasilyeva & Stephenson 2010) suggested might represent a vicarious pair of species. The pantropical distribution of a number of species as well as the species concepts themselves might have to be reconsidered in the light of the existence of more restricted biogeographic centers of biodiversity.
Supporting data from non-pyrenomycetous fungi
The peculiar species composition of the fungal assemblages found in the southern United States and the occurrence of many species in only some portions of the Caribbean have been emphasized by basidiomycete specialists. For some of these, Wu & Mueller (1997) described a “Florida-Texas distribution pattern.” The extensive literature on fleshy basidiomycetes described from Texas (Lewis & Ovrebo 2009) lists a number of species known only from Texas [e.g., Boletus lewisii (Both) Bessette et al., Clitocybe texensis H.E. Bigelow, Cortinarius lewisii O.K. Mill., Megacollybia texensis R.H. Petersen & D.P. Lewis, Russula lewisii Buyck, R. texensis Buyck et al.] or species more widely distributed in the southern United States bordering the Caribbean Sea. Thus, Phylloporus boletinoides A.H. Sm. & Thiers is known from Alabama, Florida, and Texas (Singer et al. 1990), whereas Amanita westii (Murrill) Murrill has been reported from Florida, Mississippi and Texas (Tulloss & Lewis 1994).
More recent information on basidiomycetes restricted to countries adjacent to the Caribbean Sea has been published. For example, Ganoderma ravenelii Steyaert is known from the southeastern states and Mexico (Torres-Torres & Guzman-Davalos 2008). Guzman-Davalos (2002) indicated that only 13% of Mexican Gymnopilus species are distributed worldwide, whereas the remaining 87% occur in only Mexico, in the United States and Mexico, in the United States, Mexico, and Cuba, or in Mexico and Central America. A number of agarics and boletes occur primarily in the montane oak forests of Costa Rica and Colombia (Halling & Mueller 2002). Lodge et al. (2002) note that “49 species of Hygrocybe,
Hypoxylon spp. nov. (U.S.A.) ... 89
Camarophyllopsis and Cuphophyllus are found in southeastern United States, excluding species in common with the Lesser Antilles, Trinidad and Venezuela” and that “58.8% (10/17) of the 38 southern Caribbean species would eventually be found in the Greater Antilles.” Here the reference to “Caribbean species” surely indicates the existence of a special center of fungal biodiversity.
There are even some data on vicarious species in the Caribbean and Indo- Malayan centers of diversity. As such, Trogia venenata Zhu L. Yang et al. was described recently from southern China counterbalanced by a closely related Neotropical species [Trogia buccinalis (Mont.) Pat.], described originally from Guyana (Yang et al. 2012).
Possible resurrection of some species
At present, the names of some species reported from the southeastern United States have been reduced to synonyms, but their biogeographic distributional pattern suggests a separate taxonomic identity. For example, Hypoxylon epiphaeum Berk. & M.A. Curtis, which is restricted to Magnolia spp., was reported [as a variety of H. investiens (Schwein.) M.A. Curtis] for Alabama, Florida, Georgia, Louisiana, New Jersey, New York, North Carolina, South Carolina, and West Virginia (Miller 1961). The species also has been found twice in Texas (both on Magnolia virginiana L. from the David Lewis property near Bleakwood in Newton Co. and the Turkey Creek Unit of the Big Thicket National Preserve), but H. epiphaeum is treated by Ju & Rogers (1996) as a synonym of H. monticulosum Mont., a fungus commonly reported from the tropics and subtropics on different kinds of wood. Granted, the two species share some similarities (e.g., papillate ostioles, ascospore size, perispore dehiscent in 10% KOH). One Big Thicket specimen lacked KOH-extractable pigments and so resembled H. monticulosum, while another (seemingly younger) specimen displayed dark-livid to dark-vinaceous pigments. We differentiate H. epiphaeum from H. monticulosum by the possession of glomerate stromata with an underlying orange-red subiculum (Fic. 6) and purple pigments in the young stages and its association with Magnolia species in the southeastern United States. Unfortunately, the holotype of “H. epiphaeum” in Kew has stromata characteristic of H. monticulosum, leaving the fungus associated with Magnolia without a proper name so that a new name should be proposed for a common species.
Another example is Hypoxylon subchlorinum Ellis & Calk. (Fic. 4), described originally from Florida and also found in Georgia and Texas. Ju & Rogers (1996, p. 124) cite the name H. subchlorinum under H. fuscum (Pers.) Fr., although they admit, “additional study might further subdivide this species.” They accept a rather wide ascospore length range (8-20 um) for H. fuscum, whereas northern temperate specimens usually have ascospores 12-15 um long
90 ... Vasilyeva & Stephenson
(Miller 1961). Hypoxylon subchlorinum has smaller ascospores and differently shaped stromata. There is also a discrepancy between the original description of H. subchlorinum and its type specimens. Ellis & Everhart (1888) described the stromata as yellow and the ascospores as 7-8 um long. However, the ascospores are 8-10.5 um long in both holotype and one isotype of H. subchlorinum (both parts of exsiccate North American Fungi 2115: Florida, Jacksonville, 1886, Calkins, W.W., corticated wood). A piece of the same exsiccate studied in Herbarium of Michigan University was characterized by the flattened and rounded stromata also observed in the material from the Big Thicket. The Texas material was primarily from Carpinus virginiana Mill., but very similar stromata were found in a specimen of H. subchlorinum on Celtis from Louisiana (Farlow Herbarium: St. Martinville, 28 Jan 1889, A.B. Langlois). This species, which seems to be a characteristic representative of Florida, Louisiana, and Texas should be segregated from the “H. fuscum” complex.
A third candidate in need of resurrection is Hypoxylon mulleri J.H. Mill., described originally from Puerto Rico (Miller 1933) and later found in Florida (Miller 1961) and Texas (Big Thicket National Preserve). Its name has been suggested as synonymous with H. placentiforme Berk. & M.A. Curtis, but based on material collected in Texas, the shiny black stromata with conspicuous perithecial mounds of H. mulleri differ from the brown vinaceous and very smooth stromata of H. placentiforme. The H. placentiforme concept of Ju & Rogers (1996), which accepts 8.5-18.5 um long ascospores, seems rather broad; such a range is very unusual, given the nature of repetitive variability within the Hypoxylon (cf. Vasilyeva & Stephenson 2010: Table 2). It would appear that the range has been ‘synthesized’ from several different taxonomic entities, and indeed, the synonyms cited under H. placentiforme have been applied to taxa with narrower ascospore ranges (Miller 1961, Whalley & Taligoola 1978). Prominent examples are H. sclerophaeum var. macrosporum (ascospores 14-20 um long) and H. sclerophaeum var. microsporum (ascospores 7.5-12 um long), whereas the lectotype of H. placentiforme [K(M)125651] from Cuba has ascospores 11-15 um long.
Taxonomy
Hypoxylon confertisilvae Lar.N. Vassiljeva & S.L. Stephenson, sp. nov. Fic. 6C MycoBank MB 808385 Differs from Hypoxylon fuscum by its finely papillate stromatal surface and KOH- extractable pigments between cinnamon and umber. TypE—USA, Texas, Big Thicket National Preserve, Turkey Creek Unit, Kirby Nature Trail, on bark of Acer sp., 4 Apr 2011, L. Vasilyeva (VLA P-2601).
EryMoLocy—refers to the “thick forest” implying the place of collection (Big Thicket National Preserve)
Hypoxylon spp. nov. (U.S.A.) ... 91
ee yr
Fic. 6. Stromata: A-B. “Hypoxylon epiphaeum” (VLA P-2454); C. Hypoxylon confertisilvae; D. Hypoxylon ilicinum; E. Hypoxylon meridionale; F. Hypoxylon croceum from the Great Smoky Mountains National Park (VLA P-1617); G. Hypoxylon minicroceum; H. Hypoxylon rolingii. Scale bars: A-B = 1.5mm; C = 1.7 mm; D = 2.4mm; E= 4.5 mm; F=1mm;G=1.7 mm;H =2.4mm.
STROMATA hemispherical to pulvinate, discrete 2-4 mm diam., with finely conspicuous perithecial mounds; surface dark-vinaceous; tissue black immediately beneath surface and among the perithecia, with KOH-extractable pigments between cinnamon (62) and umber (9). Perithecia 100-150 um diam., ostioles black and finely papillate. Ascr in spore-bearing part 85-90 x 6-7 um, stalks 30-40 um, with apical ring bluing in Melzer’s iodine reagent, discoid, 0.3-0.5 um high, 2.5-3 um broad. Ascosporss light brown, unicellular, elongated-ellipsoid, almost equilateral, 12-15 x 4.5-5 um, germ slit not observed, perispore indehiscent in 10% KOH.
ComMMENTS— This species is similar to Hypoxylon fuscum in stromatal shape and color but differs with respect to the stromatal surface and KOH-extractable pigments. It also is similar to H. minicroceum (see below) in ascospore shape and color and the KOH-extractable pigments but differs in the stromatal shape and the larger ascospores.
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Hypoxylon ilicinum Lar.N. Vassiljeva & S.L. Stephenson, sp. nov. Fig. 6D MycoBank MB 808386
Differs from Hypoxylon fuscum by its smaller ascospores and luteous KOH-extractable pigments.
TypE—USA, Texas, Big Thicket National Preserve, Jack Gore Baygall Unit, Blue Hole Trail, on branches of Ilex sp., 11 Apr 2011, L. Vasilyeva (VLA P-2593).
EryMoLoGy—refers to the association with branches of Ilex sp.
STROMATA hemispherical to pulvinate, discrete 2-4 mm diam. or confluent, with inconspicuous or slightly conspicuous perithecial mounds; surface livid- vinaceous to dark-vinaceous; light brownish granules immediately beneath surface and among the perithecia, with KOH-extractable pigments luteous. PERITHECIA spherical, 200-300 um diam., ostioles umbilicate. Asc in the spore-bearing part 75-80 x 6-7 um, stalks 35-40 um, with an apical ring bluing in Melzer’s iodine reagent, discoid, 0.3-0.5 um high, 2.5-3 um broad. Ascosporss brown, unicellular, ellipsoid-inequilateral, 8-12.5 x 4.5-5.5 um, with a straight germ slit extending the length of the spore on the concave side; perispore dehiscent in 10% KOH.
CoMMENTS—This species resembles H. fuscum in shape and color of the stromata but differs in having smaller ascospores and KOH-extractable pigments. It also is similar to H. porphyreum Granmo in general appearance (Granmo 1999: Fig. 31) and ascospore size, but pigments of H. porphyreum are said to be “brown with a greenish tone” in 10% KOH. Moreover, H. porphyreum is found only on Quercus in Norway and Sweden. Another similar species is H. vinosopulvinatum Y.M. Ju et al. from Taiwan (Ju et al. 2004), which has pulvinate stromata of the same color, similar KOH-extractable pigments, comparably sized ascospores, and a perispore dehiscent in 10% KOH. However, H. vinosopulvinatum has rather conspicuous perithecial mounds and a germ slit on the convex side of the ascospore. The two species—H. ilicinum and H. vinosopulvinatum—could be considered vicarious, replacing each other in the Caribbean and Indo- Malayan centers of fungal biodiversity.
Hypoxylon meridionale Lar.N. Vassiljeva & S.L. Stephenson, sp. nov. Fic. 6E MycoBank MB 808387
Differs from Hypoxylon dieckmannii by its larger and darker, as well as strongly inequilateral, ascospores.
TypE—USA, Texas, Big Thicket National Preserve, Big Sandy Creek Unit, Woodlands Trail, on wood, 3 Apr 2011, L. Vasilyeva (VLA P-2602).
EryMoLoGcy—refers to the south (of the United States). STROMATA widely effused, with slightly conspicuous perithecial mounds, surface brown vinaceous; cream-whitish granules immediately beneath surface, with grayish sepia (108) pigments. PERITHECIA spherical, 200-300 um
Hypoxylon spp. nov. (U.S.A.) ... 93
diam., ostioles umbilicate. Asc1 not observed. Ascospores brown, ellipsoid- inequilateral, 8-12(12.5) x 4-4.5 um, with a slightly oblique germ slit less than the length of the spore, perispore indehiscent in 10% KOH.
ComMMENTS—The species is similar to H. dieckmannii, which can also possess brown vinaceous stromata with grayish sepia KOH-extractable pigments, 6.5-10(11) um long ascospores, and a perispore indehiscent in 10% KOH. Hypoxylon dieckmannii has been reported from Brazil, French Guiana, Mexico, and Louisiana (New Orleans) in the United States, so its occurrence in Texas could be expected. However, the specimen from the Big Sandy Creek Unit— with a similar stromatal surface, pigments, and indehiscent perispore—has longer (although overlapping) ascospores (10-12 um) that do not fall into the characteristic ascospore range for H. dieckmannii. In addition, Miller (1961: 33, Fig. 48) described H. dieckmannii as having 6-8 um long ascospores and illustrated these as small and light brown; their shape (almost equilateral and ellipsoid) also differs from the ascospores of Texas specimens, which are darker and strongly inequilateral. It also should be noted that the fresh stromata produced an olivaceous pigment in NaOH, a feature that suggests H. anthochroum Berk. & Broome, but H. meridionale differs from H. anthochroum in having an indehiscent perispore.
Hypoxylon minicroceum Lat.N. Vassiljeva & S.L. Stephenson, sp. nov. Fic. 6G MycoBank MB 808388 Differs from Hypoxylon croceum by its smaller stromata and KOH-extractable pigments. TypE—USA, Texas, Big Thicket National Preserve, Jack Gore Baygall Unit, Blue Hole Trail, rotten wood, 11 Apr 2011, L. Vasilyeva (VLA P-2591).
EryMoLoGcy—refers to the similarity with Hypoxylon croceum.
STROMATA glomerate to effuse-pulvinate, with conspicuous to almost naked perithecial mounds; surface fuscous; dark brown woody tissue immediately beneath surface and among the perithecia, with KOH-extractable pigments between umber (9) and cinnamon (62). PERITHECIA spherical, (100-)120-150 um diam., ostioles papillate. Asci in spore-bearing part 80-95 x 4.5-5 um, stalks 17-20 um, with apical ring bluing in Melzer’s iodine reagent, discoid, 0.5-0.7 um high, 1.5-2 um broad. Ascosporss light brown, unicellular, narrow-ellipsoid, almost equilateral, 10-12.5 x 4-4.5 um, with a straight germ slit the length of the spore; perispore indehiscent in 10% KOH.
ComMMENTS— The stromata of H. minicroceum resemble those of H. croceum J.H. Mill. (Fic. 6F) but are much smaller. Both species have comparable ascospore size and a perispore indehiscent in 10% KOH, but they differ in their KOH-extractable pigments. Hypoxylon croceum has been reported from a restricted area in the southeastern United States (Georgia, North
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Carolina, Ohio, and Tennessee) and also has been reported from Venezuela (Ju & Rogers 1996). When the perithecial mounds are not very prominent, H. minicroceum is similar to H. submonticulosum Y.M. Ju & J.D. Rogers but differs in the cinnamon KOH-extractable pigments and ascospore shape. With respect to its small and inconspicuous stromata, H. minicroceum is similar to H. inconspicuum J.D. Rogers & Y.M. Ju, described originally from Costa Rica (Ju et al. 2005), which differs in a number of features (smaller ascospores, dark vinaceous KOH -extractable pigments and umbilicate ostioles).
Hypoxylon rolingii Lar.N. Vassiljeva & S.L. Stephenson, sp. nov. Fic. 6H MycoBAnk MB 808389
Differs from Hypoxylon crocopeplum by its very thin stromata of a different color and the
KOH-extractable pigments.
TypE—USA, Texas, Big Thicket National Preserve, large palmetto area along Little Pine
Island Bayou, on dead branches, 8 Apr 2011, L. Vasilyeva (VLA P-2590).
EryMoLoGcy—after the volunteer Paul Roling, who provided assistance to researchers in
the Big Thicket National Preserve. STROMATA effused-pulvinate, with conspicuous perithecial mounds, very thin (about 0.15-0.2 mm); surface bay (6) or dark-vinaceous (82), with dark tissue immediately beneath surface and among perithecia, with fresh KOH-extractable pigments chestnut (40) or sepia (63), but umber (9) or cinnamon (62) after the slides dry up. PERITHECIA spherical, 150-200 um diam., ostioles black and very faintly papillate. Asci in the spore-bearing part 70-75 x 5-6 um, stalks 25-30 um long, with an apical ring bluing in Melzer’s iodine reagent, discoid, 0.5-0.7 um high, 1.5-2 um broad. Ascosporss light brown, unicellular, ellipsoid, almost equilateral, 10-13.5 x 4-5 um, with a straight germ slit extending the length of the spore; perispore indehiscent in 10% KOH.
ComMMENTS— The stromata are very thin but are somewhat reminiscent of those in H. crocopeplum in having rather conspicuous perithecia mounds. In many other respects (e.g., color of surface and KOH-extractable pigments, indehiscent perispore), H. rolingii differs from H. crocopeplum.
Acknowledgments
We express our thanks to the personnel of the Big Thicket National Preserve, Buffalo National River Park, Ouachita Mountains Biological Station, and the Ozark National Science Center for providing an opportunity to collect at these localities. We also thank Dr. Santiago Chacon (Instituto de Ecologia, México) and David Lewis (Newton, TX) for serving as presubmission reviewers.
Literature cited Ellis JB, Everhart BM. 1888. Synopsis of the North American species of Hypoxylon and Nummularia. Journal of Mycology 4: 85-93. http://dx.doi.org/10.2307/3752760
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Granmo A. 1999. Morphotaxonomy and chorology of the genus Hypoxylon (Xylariaceae) in Norway. Sommerfeltia 26: 1-81.
Guzman-Davalos L. 2002. Tropical brown- and black-spored Mexican agarics with particular reference to Gymnopilus. 61-71, in: R Watling et al. (eds). Tropical Mycology: Vol. 1. Macromycetes. CABI Publishing, Wallingford.
Halling RE, Mueller GM. 2002. Agarics and boletes of Neotropical oakwoods. 1-10, in: R Watling et al. (eds). Tropical Mycology: Vol. 1. Macromycetes. CABI Publishing, Wallingford.
Hsieh JM, Ju YM, Rogers JD. 2005. Molecular phylogeny of Hypoxylon and closely related genera. Mycologia 97: 844-865. http://dx.doi.org/10.3852/mycologia.97.4.844
Ju YM, Rogers JD. 1996. A revision of the genus Hypoxylon. Mycologia Memoir 20: 1-365.
Ju YM, Rogers JD, Hsieh HM. 2004. New Hypoxylon species and notes on some names associated with or related to Hypoxylon. Mycologia 96: 154-161. http://dx.doi.org/10.2307/3761997
Ju YM, Rogers JD, Hsieh HM. 2005. New Hypoxylon and Nemania species from Costa Rica and Taiwan. Mycologia 97: 562-567. http://dx.doi.org/10.3852/mycologia.97.2.562
Lewis DP, Ovrebo CL. 2009. Mycological literature on Texas fleshy Basidiomycota, two new combinations, and new fungal records for Texas. Journal of the Botanical Research Institute of Texas 3: 257-271.
Lodge DJ, Baroni TJ, Cantrell SA. 2002. Basidiomycetes of the Greater Antilles project. 45-60, in: R Watling et al. (eds). Tropical Mycology: Vol. 1. Macromycetes. CABI Publishing, Wallingford.
Miller JH. 1933. Some new species of Hypoxylon. Mycologia 25: 321-329. http://dx.doi.org/10.2307/3754100
Miller JH. 1961. A monograph of the world species of Hypoxylon. University of Georgia Press, Athens. 158 p.
Rogers JD, Ju YM, Candoussau F. 1996. Biscogniauxia anceps comb. nov. and Vivantia guadalupensis gen. et sp. nov. Mycological Research 100: 669-674. http://dx.doi.org/10.1016/S0953-7562(96)80196-1
Singer R, Ovrebo CL, Halling RE. 1990. New Species of Phylloporus and Tricholomopsis from Colombia, with notes on Phylloporus boletinoides. Mycologia 82: 452-459. http://dx.doi.org/10.2307/3760016
Torres-Torres MG, Guzman-Davalos L. 2008. Taxonomic status and new localities for Ganoderma ravenelii. Mycotaxon 103: 33-40.
Tulloss RE, Lewis DP. 1994. Amanita westii - taxonomy and distribution. A rare species from states bordering the Gulf of Mexico. Mycotaxon 50: 131-138.
Vasilyeva LN, Stephenson SL. 2010. Biogeographical patterns in pyrenomycetous fungi and their taxonomy. 1. The Grayan disjunction. Mycotaxon 114: 281-303. http://dx.doi.org/10.5248/114.281
Vasilyeva LN, Stephenson SL, Hyde KD, Bahkali AH. 2012. Some stromatic pyrenomycetous fungi from northern Thailand - 1. Biscogniauxia, Camillea and Hypoxylon (Xylariaceae). Fungal Diversity 55: 65-76. http://dx.doi.org/10.1007/s13225-011-0150-9
Whalley AJS, Taligoola HK. 1978. Species of Hypoxylon from Uganda. Transactions of the Botanical Society of Edinburgh 42: 93-98. http://dx.doi.org/10.1080/03746607808685328
Wu QX, Mueller GM. 1997. Biogeographic relationships between the macrofungi of temperate eastern Asia and eastern North America. Canadian Journal of Botany 75: 2108-2116. http://dx.doi.org/10.1139/b97-922
Yang ZL, Li YC, Shi GQ, Zeng G. 2012. Trogia venenata (Agaricales), a novel poisonous species which has caused hundreds of deaths in southwestern China. Mycological Progress 11: 937-945. http://dx.doi.org/10.1007/s11557-012-0809-y
ISSN (print) 0093-4666 © 2014. Mycotaxon, Ltd. ISSN (online) 2154-8889
MY COTAXON
http://dx.doi.org/10.5248/129.97 Volume 129(1), pp. 97-108 July-September 2014
Rhizophagus natalensis, a new species in the Glomeromycota
JANUSZ BLASZKOWSKI’ , GERARD CHWAT’, ANNA GORALSKA’ & BRUNO T. GOTO?
‘Department of Ecology and Protection of Environment, West Pomeranian University of Technology in Szczecin, Szczecin, Stowackiego 17, PL-71434 Szczecin, Poland
’Departamento de Botanica, Ecologia e Zoologia, CB, Universidade Federal do Rio Grande do Norte, Campus Universitario, 59072-970, Natal, RN, Brazil
‘CORRESPONDENCE TO: janusz. blaszkowski@zut.edu.pl
AsstRract — Morphological and histochemical studies of spores and phylogenetic analyses of LSU and SSU nrDNA sequences have indicated that a fungal species found in maritime dunes of the Parque Estadual das Dunas de Natal “Journalista Luiz Maria Alves” located in Natal, Brazil, represents an undescribed species of Rhizophagus. The new species, R. natalensis, forms spores singly or (sometimes) in loose clusters in soil. Its spores are pastel yellow to light yellow and globose to subglobose [(75-)101(-133) um diam] or (rarely) ovoid (50-70 x 63-79 um) and have one subtending hypha with a pore that is open or occluded by a septum. Its spore wall comprises four layers: (1) a semi-permanent hyaline layer forming the spore surface, (2) a permanent hyaline unit layer, (3) a laminate pastel yellow to light yellow layer, and (4) a flexible hyaline layer. Layers 1 and 3 stain in Melzer’s reagent.
Key worps — arbuscular mycorrhizal fungi, molecular phylogeny, tropical ecosystem
Introduction
Arbuscular mycorrhizal fungi (AMF) of the phylum Glomeromycota live in symbiosis with ca. 70-90% of land plants growing in different habitats (Smith & Read 2008, Brundrett 2009). Sand dunes are especially favorable sites for development of AMF, mainly due to the sparse populations of other microorganisms competing for nutrients produced by living plants and the rare occurrence of AMF microparasites (Blaszkowski 1994, Dalpé 1989, Koske 1987, Lee & Koske 1994, Tadych & Blaszkowski 2000). On the other hand, the existence of plants growing in extremely poor and harsh dune sites may well depend highly on the establishment of such symbioses, in that AMF frequently increase the supply of nutrients to plants and decrease their sensitivity to
98 ... Blaszkowski & al.
different abiotic and biotic stresses (Bothe et al. 2010, Sch6nbeck 1978, Dehn & Schiiepp 1989, Grifhoen & Ernst 1989, Smith & Read 2008).
The approximately 250 AMF species that have been described (Schiifler & Walker 2010) probably represent less than 5% of world’s AMF species (Kriiger et al. 2009). Of the described species ca. 62% produce glomoid spores, i.e., spores similar in mode of formation, spore wall structure, and subtending hyphal characters to those of Glomus macrocarpum Tul. & C. Tul. (type species of the genus Glomus; Clements & Shear 1931).
Glomoid spores are produced by AMF of the genus Rhizophagus P.A. Dang. in the family Glomeraceae Piroz. & Dalpé (Redecker et al. 2013; Schiifler & Walker 2010). Long after Dangeard (1896) described Rhizophagus, Gerdemann & Trappe (1974) synonymized the genus with Glomus. Recently, however, Schiifler & Walker (2010) resurrected Rhizophagus based on both the original description of its type species, R. populinus P.A. Dang., stating that the fungus produced spores abundantly inside roots, and the phylogenetic sequence analyses of Rhizophagus and other AMF that tend to form intraradical spores.
We studied the morphological and histochemical characters of glomoid spores extracted from a pot trap culture inoculated with rhizosphere soil and root fragments of an unrecognized grass colonizing maritime sand dunes of the Parque Estadual das Dunas de Natal “Journalista Luiz Maria Alves” located in Natal, Brazil. Our research suggested that we had found an undescribed species similar to R. fasciculatus (Thaxt.) C. Walker & A. Schiissler. Later phylogenies inferred from analyses of sequences of the large (LSU) and small (SSU) subunit genes support our original hypothesis that the two fungi represent different species. The Brazilian fungus is described below as R. natalensis.
Materials & methods
Establishment and growth of trap and single-species cultures, extraction of spores, and staining of mycorrhiza
Spores examined in this study derived from a pot trap culture. The trap culture was established to obtain living spores and to initiate sporulation of specimens that may not have sporulated in the field collections (Stutz & Morton 1996). Methods used to establish the trap culture, growth conditions, and spore extraction follow Blaszkowski et al. (2012). The growing substrate of the trap culture was the field-collected rhizosphere soil and roots of the plant species sampled mixed with autoclaved coarse-grained sand (1:1 v/v).
Eight single-species cultures were also established and grown as given in Blaszkowski et al. (2012). The single cultures were set up with ca. 10 spores and small clusters of spores (5-12) attached by a common mycelium. Unfortunately, all cultures failed.
Microscopy and nomenclature Morphological features of spores and their wall structure were determined after examining at least 100 spores mounted in water, lactic acid, polyvinyl alcohol/lactic
Rhizophagus natalensis sp. nov. (Poland) ... 99
acid/glycerol (PVLG; Omar et al. 1979), and a mixture of PVLG and Melzer’s reagent (1:1, v/v). Glomerospores at all developmental stages were mounted in PVLG and PVLG+Melzer’s reagent, then covered with a cover slip, crushed to varying degrees by applying pressure to the cover slip, stored at 65°C for 24 h to clear their contents from oil droplets, and examined under an Olympus BX 50 compound microscope equipped with Nomarski differential interference contrast optics. Microphotographs were recorded on a Sony 3CCD color video camera coupled to the microscope.
Spore structure terminology follows Stiirmer & Morton (1997) and Walker (1983). Spore color was examined under a dissecting microscope on fresh specimens immersed in water. Color names are from Kornerup & Wanscher (1983). Nomenclature of fungi and the authors of fungal names are from Index Fungorum (http://www.indexfungorum. org/AuthorsOfFungalNames.htm). Voucher specimens were mounted in PVLG and a mixture of PVLG and Melzer’s reagent (1:1, v/v) on slides and deposited in the Department of Ecology and Protection of Environment (DEPE), West Pomeranian University of Technology in Szczecin, Szczecin, Poland, and in the herbarium at Universidade Federal do Rio Grande do Norte (UFRN) in Natal, Rio Grande do Norte, Brazil.
DNA extraction, polymerase chain reaction and DNA sequencing
Crude DNA was isolated from 3-8 single spores crushed with a needle in ultra clean water on sterile microscope slides under a dissecting microscope. Amplification, cloning, and sequencing procedures followed Btaszkowski et al. (2012). The large subunit nrDNA gene (partial) of the fungus newly described here was amplified using the primers LR1 (Tuinen et al. 1998) and FLR2 (Trouvelot et al. 1999) and then the primers 28G]1 and 28G2 (Silva et al. 2006), and SSU nrDNA gene (partial) was amplified with the primers AML1 and AML2 (Lee et al. 2008) as described in Blaszkowski et al. (2012). The segment spanning SSU (partial), internal transcribed region (ITS1, 5.8S and ITS2, full), and LSU (partial) nrDNA of Septoglomus africanum (Blaszk. & Kovacs) Sieverd. et al., S. furcatum Blaszk. et al., S. fuscum Blaszk. et al., and S. xanthium (Blaszk. et al.) G.A. Silva et al. were amplified using a nested procedure and the SsUmAf-LSUmAr primer pair for the first nested PCR and the SSUmCf-LSUmBr primer pair for the second nested PCR, as suggested by Kriiger et al. (2009). Representatives of sequences have been deposited in GenBank (KJ210823-KJ210828).
Sequence alignment and phylogenetic analyses
The glomeromycotan origin of the sequences was initially tested by BLAST (Zhang et al. 2000) search. To determine the generic affiliation of our new species we performed pilot phylogenetic analyses separately with all LSU and SSU sequences and those representing all recognized glomeromycotan genera with glomoid spores available in GenBank and published by Kriiger et al. (2012). The results of the SSU sequence analyses are not presented here. The final data set used to generate the LSU tree in Fic. 9 comprised six sequences from our new species, three sequences each from known Rhizophagus spp. and one to three sequences, published or obtained by us, from 12 other species in the Glomeraceae. Sequences representing Claroideoglomus claroideum (N.C. Schenck & G.S. Sm.) C. Walker & A. Schiissler served as outgroup in all analyses. The LSU and SSU sequences were aligned with PRANK, with the ,, option invoked
100 ... Btaszkowski & al.
to fix already inferred indels at their place and avoid another indel being inferred in an overlapping position during the second recursion of the algorithm (Léytynoja & Goldman 2008). Overhanging sequence fragments of both termini were trimmed. Bayesian (BI) analyses were performed with MrBayes 3.1 (Huelsenbeck & Ronquist 2001, Ronquist & Huelsenbeck 2003), and maximum likelihood (ML) analyses with PHYML (Guindon & Gascuel 2003). Before the analyses the best-fit substitution models for the alignments were estimated by the Akaike information criterion (AIC) using TOPALI v. 2.5 (Milne et al. 2004). In the BI analyses of both LSU and SSU sequences the model employed was GTR + G, and TrN + G was applied in the ML analyses of both types of sequences. In the BI analyses the Markov chain was run for 5,000,000 generations, sampling in every 500 steps with a burn-in at 3000. In the ML analyses the transition/transversion ratio for DNA models and the gamma distribution parameter were estimated. Six substitution rate categories were set. Topology and branch lengths and rate parameters were optimized. Support of branches in the ML analyses was estimated in bootstrap analyses with 1000 replicates. The details of the analyses are available on request. Phylogenetic trees were visualized and edited in MEGA5 (Tamura et al. 2011).
Taxonomy
Rhizophagus natalensis Btaszk., Chwat & B.T. Goto, sp. nov. Fics 1-8 MycoBAank MB 808282
Differs from Rhizophagus irregularis in spore color and shape and the number and phenotypic and histochemical characters of the spore wall layers.
Type: Brazil, Rio Grande do Norte, Natal, Parque Estadual das Dunas de Natal “Journalista Luiz Maria Alves’, under an unrecognized grass colonizing maritime sand dunes, 12 Sept. 2012, J. Blaszkowski 3381 (Holotype, DEPE; GenBank KJ210823), J. Blaszkowski 3382-3384 (Isotypes, DEPE; GenBank KJ210824-KJ210828).
ErymMo_oey. Latin, natalensis, referring to the ‘Natal’ dunes from which the species was collected. SPOROCARPS unknown.
GLOMEROSPORES formed singly, sometimes in loose clusters, in soil; developing blastically at the tip of sporogenous hyphae either directly from mycorrhizal extraradical hyphae (single spores) or branched from a parent hypha continuous with a mycorrhizal extraradical hypha (spores in clusters). Clusters 160-989 x 180-1200 um with 2-14 spores. Spores pastel yellow (3A4) to light yellow (3A5); globose to subglobose; (75-)101(-133) um diam; rarely ovoid; 50-70 x 63-79 um; with one subtending hypha.
Fics. 1-8. Rhizophagus natalensis spores (differential interference microscopy). 1. Spores in loose cluster. 2-5. Spore wall layers (swl) 1-4; note the unstained layer 4 in Fic. 4 and the swollen layer 1 in Fic. 5. 6. Spore wall layers (swl) 1-4 continuous with subtending hyphal wall layers (shwl) 1-4; note shwl4 extending along the inner surface of shw]3. 7. Spore wall layers (swl) 1-4, of which layer 4 forms a curved septum (s) in the subtending hyphal (sh) lumen. 8. Subtending hyphal wall layers (shwl) 1-3 continuous with spore wall layers 1-3; shwll and swll are not visible. Fics 1, 2, 6, 7, in PVLG; Fics 3-5, 8, in PVLG+Melzer’s reagent.
Rhizophagus natalensis sp. nov. (Poland) ... 101
/
20 pm Attn 10 ym
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SPORE WALL consists of four layers: layer 1, forming the spore surface, semi- permanent, hyaline, (1.0-)2.4(-5.3) um thick, slowly deteriorating with age, usually present as a more or less decomposed structure even in older spores; sometimes swelling and becoming up to 8.3 um thick; layer 2, a unit layer, permanent, smooth, hyaline, (0.8-)1.1(-1.5) um thick; layer 3 laminate, smooth, pastel yellow (3A4) to light yellow (3A5), (6.3-)8.7(-14.0) um thick, consisting of laminae up to 0.8-1.0 um thick, frequently easily separating from each other in crushed spores; and layer 4 flexible, smooth, hyaline, (0.8-)1.3(-2.0) um thick, usually separating from the lower surface of layer 3 in crushed spores. Layers 1 and 3 stain reddish white (9A2) to greyish red (10C5) and brownish violet (11D8) to violet brown (11E8) in Melzer’s reagent, respectively.
SUBTENDING HYPHA pastel yellow (3A4) to light yellow (3A5); straight or recurved, cylindrical to funnel-shaped, sometimes slightly constricted at the spore base; (16.0-)20.8(-29.8) tum wide at the spore base. Wall of subtending hypha pale pastel yellow (3A4) to light yellow (3A5); (7.3-)9.4(-14.3) um thick at the spore base; continuous with spore wall layers 1-4. Pore (1.5-)2.4(-3.3) um diam, open when spore wall layer 4 develops from subtending hyphal wall layer 4 arising far below the spore base and extending along the inner surface of subtending hyphal wall layer 3 or occluded by a curved septum continuous with spore wall layer 4 and open or occluded from the reasons mentioned above and gradually narrowing due to thickening of wall layer 3 of the subtending hypha towards the centre of its lumen.
GERMINATION unknown.
MYCORRHIZAL ASSOCIATIONS — In the field R. natalensis was associated with roots of an unrecognized plant species of Poaceae that colonized the Atlantic Ocean sand dunes in the Parque Estadual das Dunas de Natal “Journalista Luiz Maria Alves” (5°46’S, 35°12’W), one of the largest urban conservation areas with dune vegetation in Brazil. In a trap culture inoculated with the rhizosphere soil and root fragments of the plant, the fungus lived in symbiosis with Plantago lanceolata and sporulated abundantly. However, R. natalensis stopped producing spores after ca. two years of growing of this culture. All attempts to establish single-species cultures from spores extracted from the trap culture failed.
PHYLOGENETIC POSITION — In BI and NJ trees with LSU sequences the R. natalensis clade obtained high values of statistical support and was sister to
Fic. 9. 50% majority rule consensus phylogram of AMF species inferred from a Bayesian analysis of LSU nrDNA sequences of our new species and 33 other species, including Claroideoglomus claroideum as outgroup. The new species is shown in boldface. The fungal names are followed by GenBank accession numbers. The Bayesian posterior probabilities >0.50 and ML and NJ bootstrap values =50% are shown near the branches, respectively. Bar indicates 0.5 expected change per site per branch.
Rhizophagus natalensis sp. nov. (Poland)
0.98/100 reeptogiomus xanthium KF 154775 S. xanthium KF 154774 0.99/91 ILS. xanthium KF154772 S. fuscum KF060316 0.83/59 S. fuscum KF060314 4/100 Al S. fuscum KF060315 S. furcatum KFO60310 0.92/77| +S. furcatum KF060312 S. furcatum KFO60311 1/100] | |S. africanum KF060307 1/99 Rey S. africanum KF060306 0.55/5 NS S. africanum KF060304 os S. constrictum FJ461827 0.66/5 1S. constrictum JF439167 1/99 S. constrictum JF439176 1/96 S. deserticola JQ048859 4/99 S. deserticola JQ048857 S. deserticola JQ048925 ee S. viscosum KC182038
0.54/- S. vscosum KC182036 S. viscosum KC182037 1/99 Funneliformis caledonius FN547496 1/99 F. caledonius FN547499
F. caledonius FN547494 PASE F. coronatus FM876797 _|LF. coronatus FM876794 1/99-F| LF. coronatus FM876796 0.74/64 F. mosseae FN547476 F. mosseae FN547486 1/96 LF. mosseae FN547474 Glomus macrocarpum FR750526 1/10G G. macrocarpum FR750532 G. macrocarpum FR750535 Sclerocystis sinuosa FJ461846 0.97/99. Rhizophagus intraradices FM865577 R. intraradices FM865606 R. intraradices FM865559 R. clarus FM865538 R. clarus FM865541
0.58/100
1/9
11007 | «OLR. clarus FM865543 R. proliferus FM992390 0.99/8 R. proliferus FM992398
4/100 R. fasciculatus FR750071 Fig R. fasciculatus FR750072 R. fasciculatus FR750073 R. arabicus KF 154766 R. arabicus KF 154767
0.75/6 i R. proliferus FM992401
1/6 R. arabicus KF 154765 0.98/79 R. irregularis FR750112 naaie R. irregularis FM865550
R. irregularis FM992377 R. natalensis KJ210823 holotype R. natalensis KJ210824 R. natalensis KJ210825 R. natalensis KJ210826 R. natalensis KJ210827 R. natalensis KJ210828
1/99
Claroideoglomus claroideum FR750058 | ee |
FkQ3
104 ... Blaszkowski & al.
a clade with R. irregularis (Blaszk. et al.) C. Walker & A. Schiissler (Fic. 9). In BI and ML trees with SSU sequences R. natalensis was strongly supported as monophyletic and sister to a clade comprising R. fasciculatus, R. intraradices (N.C. Schenck & G.S. Sm.) C. Walker & A. Schissler, R. irregularis, and R. vesiculiferus (Thaxt.) C. Walker & A. Schissler distributed into two subclades (data not shown).
DISTRIBUTION & HABITAT — Spores of R. natalensis were found only in one pot trap culture inoculated with a mixture of the rhizosphere soil and root fragments of an unrecognized plant species of Poaceae growing in maritime sand dunes of the Parque Estadual das Dunas de Natal “Journalista Luiz Maria Alves” located in Natal, Brazil. Data on the climate, vegetation and soil chemical properties of the dunes in which R. natalensis occurred were described previously (Goto et al. 2012).
Spores of R. natalensis were not found in either ca. 3000 field-collected soils and ca. 3000 pot trap cultures representing different regions of Europe, Africa, Asia, Cuba, and the U.S.A. (Blaszkowski, pers. observ.).
However, BLAST queries indicated that the R. natalensis LSU sequences we obtained were 99% similar to: (1) five LSU sequences (FR871319, FR871318, FR871325, FR871323, FR871320) of AMF colonizing roots and rhizosphere soils of Brachypodium retusum (Pers.) P. Beauv. and Bromus rubens L. growing in semiarid degraded area located in the natural ecological park “Vicente Blanes” in Molina de Segura, Province of Murcia, southern Spain (Torrecillas et al. 2012); (2) an LSU sequence (EF066656) of an AMF coming from soil collected under Medicago spp. cultivated in a Mediterranean fallow land located at the Mas d’Imbert, France (Pivato et al. 2007); and (3) 55 SSU sequences of uncultured AMF from, e.g., Spain (Alguacil et al. 2012; Torrecillas et al. 2012). This suggests that R. natalensis has a wide world distribution.
Discussion
The distinguishing morphological and histochemical characters of R. natalensis are its light yellow, relatively large spores with a four-layered spore wall, of which layers 1 and 3 stain intensively in Melzer’s reagent. In addition, based on DNA sequence analyses, the species is phylogenetically unique.
Our LSU sequence analyses indicate that R. natalensis is most closely related to R. irregularis (see “Phylogenetic position” above and Fic. 9). Mature R. irregularis spores are frequently hyaline (vs. no mature hyaline spores in R. natalensis) and ovoid to oblong or irregular with deep local depressions (Blaszkowski 2012; Blaszkowski et al. 2008; vs. usually globose to subglobose without depressions). The spore wall of R. irregularis comprises three (vs. four) layers, of which layer 1 frequently is highly thickened at the spore top and forming a cap-like swell (vs. no such thickening), a laminate innermost layer
Rhizophagus natalensis sp. nov. (Poland) ... 105
(vs. a flexible innermost layer 4), and the only spore wall component (vs. layers 1 and 3) that stains in Melzer’s reagent. The subtending hypha of R. irregularis spores is 0.6-2.5-fold narrower and has a 1.7-4.9-fold thinner wall at the spore base and its pore may be open or closed by a septum, but the septum forms some innermost laminae of the laminate spore wall layer 3 (vs. spore wall layer 4 in R. natalensis). In addition, R. irregularis tends to form spores in oblong clusters inside roots and in soil, because the spores arise either terminally from or intercalary inside sporogenous hyphae developing from spore wall layer 1 of an earlier fully differentiated parent spore or/and a branch of the parent hypha continuous with a mycorrhizal extraradical hypha (Blaszkowski 2012; Blaszkowski et al. 2008; vs. no such mode of spore formation in R. natalensis).
SSU sequence analyses also support a close relationship of R. natalensis with R. fasciculatus, R. intraradices, and R. vesiculiferus (data not shown). Intact R. natalensis spores observed under low and high microscope magnifications are almost morphologically and histochemically identical to those of R. fasciculatus. Both species form spores singly and in clusters and the spores are similar in color and size and stain intensively in Melzer’s reagent (Blaszkowski 2012; Morton, http://invam.wvu.edu/). Examination of spores crushed in PVLG and PVLG+Melzer’s reagent readily separates both species. The spore wall of R. fasciculatus is 3-layered (lacking layer 2 of the 4-layered wall of R. natalensis). Spore wall layer 1 of R. natalensis, forming the spore surface, is semi-permanent (vs. permanent in R. fasciculatus), may be 2.0-2.9-fold thicker, sometimes swells up to 8.3 um thick, thereby showing its plasticity (vs. no such behaviour), and may stain more intensively in Melzer’s reagent (vs. reddish white; Blaszkowski 2012). In addition, the laminae of the laminate structural spore wall layer 3 of R. natalensis are ca. 2-fold thicker than those of the laminate structural spore wall layer 2 of R. fasciculatus (<0.5 um thick) and the R. natalensis laminae frequently separate from each other in crushed spores (vs. usually remaining adherent in R. fasciculatus). Finally, the subtending hypha of R. natalensis spores is less regular in shape (cylindrical to funnel-shaped vs. cylindrical in R. fasciculatus), has a 5.2-6.5-fold thicker wall at the spore base, and its pore may be open or occluded by a septum continuous with spore wall layer 4 (vs. closed by a septum continuous with spore wall layer 3).
Freshly mature spores of R. intraradices usually are light yellow with a greenish tint (Blaszkowski 2012, Morton http://invam.wvu.edu/, Stirmer & Morton 1997) lacking in R. natalensis spores. The spore wall of R. intraradices is 1.3-1.8-fold thinner, comprises only three (vs. four) layers of which only the laminate layer 3 (vs. layers 2-4) is permanent, and only spore wall layer 1 (vs. layers 1 and 3) stains in Melzer’s reagent. In addition, the subtending hypha of R. intraradices spores is less regular in shape (cylindrical to slightly flared vs. cylindrical to funnel-shaped) and 1.2-1.6-fold narrower at the spore base; its
106 ... Btaszkowski & al.
wall is 1.8-2.2-fold thinner at the spore base and its pore is open (vs. open or closed by a septum).
Rhizophagus vesiculiferus is most clearly separated from R. natalensis by the formation of sporocarps with a peridium-like layer of thin-walled vesicles (Berch & Fortin 1984, Gerdemann & Trappe 1974). In addition, R. vesiculiferus spores are ca. 1.6-fold smaller, their spore wall is 2-layered (vs. 4-layered) and 2.2-2.9-fold thinner, and their subtending hypha is ca. 1.7-fold narrower with an open pore (vs. open or occluded by a septum).
Acknowledgements
The authors acknowledge Dr. Fritz Oehl (Agroscope, Federal Research Institute for Sustainability Sciences, Plant-Soil Interactions, Ziirich, Switzerland) and Dr. Gladstone Alves da Silva (Departamento de Micologia, CCB, Universidade Federal de Pernambuco, Av. Prof. Nelson Chaves s/n, Recife, PE, Brazil) for reviewing the manuscript and making helpful suggestions. We also appreciate the corrections and suggestions by Dr. Shaun Pennycook (Nomenclature Editor) and Dr. Lorelei Norvell (Editor-in-Chief). This study was supported in part by Polish National Centre of Science, grants no. 05/B/NZ8/00498 and 07/N/NZ8/02363 and the Fundacao de Amparo a Ciéncia e Tecnologia do Estado do Rio Grande do Norte (FAPERN-PPP) and CASADINHO UFRN/FIOCRUZ (Process N® 552577/2011-1), which provided a financial support to J. Blaszkowski as “visiting professor”.
Literature cited
Alguacil MM, Torrecillas E, Roldan A, Diaz G, Torres MP. 2012.