Iv ... MYCOTAXON 124

MY COTAXON

VOLUME ONE HUNDRED TWENTY-FOUR — TABLE OF CONTENTS

COVER SECTION

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a chy Fea gee The ha a Micah ES I Sea eee Mal SET ed, WAN to Slay rey PU Sry RAS ib

SUDMESSION PIO CCHUTES 3 25 vs Pearich Youbet vss Red i 2. cae aps Mayme mechanic ok a gs Cah x

OP EPEIOCE LOT Roots B Fhe Ayo. tons hE Se eS Se a oy SY 3 eon ay Re xi

RESEARCH ARTICLES

A new Myrmecridium species from Guizhou, China

CHUN- YU JIE, QING-XIN ZHOU, WEN-SHENG ZHAO, YU-LAN JIANG,

KEVIN D. Hype, Eric H.C. MCKENZIE & YONG WANG

The occurrence of Rutstroemia coracina on Quercus ilex leaves in Spain

RICARDO GALAN, FRANCISCO PRIETO-GARCIA,

ANGELES GONZALEZ & CARLOS ENRIQUE HERMOSILLA

Two species of Cladosporium associated with wood discoloration in Korea

YEONGSEON JANG, YOUNG MIN LEE, GyU-HYEOK KIM & JAE-JIN KIM

Phialophora avicenniae sp. nov., a new endophytic fungus

in Avicennia marina in China

YUE-LIAN Liu, PING-GEN XI, XIAO-LAN HE & ZI-DE JIANG

Ramichloridium strelitziae associated with sooty blotch and flyspeck on

Ravenala madagascariensis in China

BAOJUN Hao, WENHUAN LI, CHEN CHEN, Liu GAo, RONG ZHANG,

BAOTONG WANG, GUANGYU SUN & MARK L. GLEASON

Polycoccum anatolicum sp. nov. on Lepraria incana and a key to

Polycoccum species known from Turkey

MEHMET GOKHAN HALICI, HATICE EsRA AKGUL,

CELALEDDIN OzTURK & Emre KILIG¢

Further additions to the macrolichen mycota of Vietnam

UDENI JAYALAL, ANDRE APTROOT, THI THUY NGUYEN,

NGUYEN ANH DZUNG, SANTOSH JOSHI, SOON-OK OH & JAE-SEOUN HuR

Inonotus niveomarginatus and I. tenuissimus spp. nov.

(Hymenochaetales), resupinate species from tropical China

Hat-You Yu, CHANG-LIN ZHAO & YU-CHENG Dal

Arenariomyces truncatellus sp. nov., an ascomycete on driftwood

from the north coast of Zealand, Denmark J. Kocu

New ascomycete records from Guatemala

RosARIO MEDEL, OSBERTH MORALES,

RANULFO CASTILLO DEL MORAL & ROBERTO CACERES

Coleosporium in Europe STEPHAN HELFER

APRIL-JUNE 2013... V

Septoglomus titan, a new fungus in the Glomeraceae (Glomeromycetes)

from Bahia, Brazil BRUNO ToMIO GoTo,

ADRIANE FREIRE ARAUJO, ANA CRISTINA FERMINO SOARES,

ARAESKA CARENNA DE ALMEIDA FERREIRA, LEONOR COSTA MaIA,

CARLA DA SILVA SOUSA & GLADSTONE ALVES DA SILVA 101

Geoglossaceous fungi in Slovakia 5. Geoglossum uliginosum:

taxonomy and nomenclature

VIKTOR KUCERA, JOHAN NITARE, PAVEL LIZON & JAN GAISLER 111

A new endophytic species of Chaetomium from Jatropha podagrica

ROHIT SHARMA, GIRISH KULKARNI,

MAHESH S. SONAWANE & YOGESH S. SHOUCHE 117

A contribution to the taxonomy of Lyromma (Lyrommataceae,

lichenized Ascomycota) with a species key

ADAM FLAKUS & EDIT FARKAS 127

Scolecobeltrania, an interesting new microfungus from Venezuela

TERESA ITURRIAGA, ROBERTO FERNANDEZ,RAFAEL F. CASTANEDA-RUIZ,

Davip W. MINTER & ALISSON CARDOSO RODRIGUES DA CRUZ 143

A new species of Pisolithus from Spain

Maria P. Martin, FATIMA DURAN, CHERDCHAI PHOSRI & Roy WATLING 149

A reassessment of excavated Tuber species from China based on

morphology and ITS rDNA sequence data

Li FAN, JIN-ZHONG CAo & Yu Li 155

A new species and a new record of the genus Entoloma from China

JiaN-RuI WANG & TOLGOR Bau 165

Russula atroaeruginea and R. sichuanensis spp. nov. from southwest China

Guo-JiE Li, Qt ZHAO, DONG ZHAO, SHUANG-FEN YUE,

SAI-FE1 L1, HUA-AN WEN & XING-ZHONG Liu 173

Five new Terfezia species from the Iberian Peninsula JuaN-JULIAN BoRDALLO,

ANTONIO RODRIGUEZ, JUSTO M. MUNoz-MOHEDANO,

LaAuRA M. Suz, MARIO HONRUBIA & ASUNCION MorTE 189

Hypochnicium pini, a new corticioid basidiomycete in East Asia

YEONGSEON JANG, SUNG WOOK LEE, YOUNG WOON LIM,

Jin SUNG LEE, NILS HALLENBERG & JAE-JIN Kim 209

Taxonomic studies on Mucor inaequisporus, isolated for the first time

in South America

ANDRE Luiz C.M. DE A. SANTIAGO, ANDRE RODRIGUES,

ENzO M. CANEDO & EDSON R. FILHO 219

Molecular phylogeny reveals Megacollybia virosa is a Cantharocybe

T.K. ARUN KUMAR & P. MANIMOHAN 231

New record of Scedosporium dehoogii from India

ROHIT SHARMA, GIRISH KULKARNI,

MAHESH S. SONAWANE & YOGESH S. SHOUCHE 239

vI ... MYCOTAXON 124

Inocybe nitidiuscula and its ectomycorrhizae associated with

Alnus nitida from Galyat, Pakistan S. ILyas, A. RAZAQ & A.N. KHALID

Xerocomus porophyllus sp. nov., morphologically intermediate between

Phylloporus and Xerocomus

WEN-JUAN YAN, Tai-Hutr Li, MING ZHANG & TING LI

Glomus mume and Kuklospora spinosa: two new species of

Glomeromycota from China

BANG-PING CAI, LIANG-DONG GUO, JUN-YU CHEN & QI-XIANG ZHANG

Russula changbaiensis sp. nov. from northeast China

Guo-J1E L1, DONG ZHAO, SAI-FEI LI,

Hual-JUN YANG, Hua-AN WEN & XING-ZHONG LIU

Studies on three rare coprophilous plectomycetes from Italy

FRANCESCO DOVERI, SABRINA SARROCCO & GIOVANNI VANNACCI

Morphological and genetic characterisation of Beauveria sinensis sp. nov.

from China MING-JUN CHEN, Bo Huana,

ZENG-ZHI LI & JOSEPH W. SPATAFORA

The lichen genus Fissurina (Graphidaceae) in Vietnam

SANTOSH JOSHI, THI , THUY NGUYEN, NGUYEN ANH DzuUNG,

UDENI JAYALAL, SOON-OK OH & JAE-SEOUN Hur

Studies of North American macrofungi, 1. Validation of Lactarius rubidus

comb. nov. and Leccinellum quercophilum sp. nov.

MICHAEL Kuo, ANDREW S. METHVEN,

ANDREW M. MINNIS, & Roy E. HALLING

Cladonia dunensis sp. nov. from southern Brazil, with notes on

the genus in beach dune environments EMERSON LuIz GUMBOSKI,

FLAVIO BEILKE & SIONARA ELIASARO

Coniolepiota spongodes (Agaricaceae, Basidiomycota) in

Bangladesh and China Mp. IqBaL HosEN & ZHU L. YANG

Pertusaria albiglobosa, a new lichen from China QIANG REN

Lichenological notes 6: nomenclatural acts

KERRY KNUDSEN & JANA KOCOURKOVA

Phylloporia tiliae sp. nov. from China Li-We!I ZHou

NOMENCLATURE

Nomenclatural novelties proposed in volume 124

247

255

263

269

2%9

301

309

320

333

341

349

353

361

367

APRIL-JUNE 2013... VII

ERRATA FROM PREVIOUS VOLUMES

VOLUME 122

p-30, table, after Lambertella sp.3 For: Leaf of Pinus sp. | READ: Leaf of Pyrola sp.

p-30, table, after Lambertella sp.8 For: Leaf of Pinus sp. READ: Leaf of Pyrola sp.

VOLUME 123

p. ii, lines 12-13 FOR: SEPPO HUHTINEN (2006-2012) Turku, Finland

READ: KAREN HANSEN (2013-2017) Stockholm, Sweden

p. iv, line 12: FOR: Lichenochora tertia

READ: Lichenochora tertia (Phyllochorales):

p. iv, line 22: FOR: Jatropha podarica READ: Jatropha podagrica

p. vi, line 8: FOR: TEODOR T. DENCHEV, HABIB AHMAD & ABDUL NASIR KHALID

READ: TEODOR T. DENCHEV, ABDUL NASIR KHALID & HABIB AHMAD

vill ... MYCOTAXON 124

PUBLICATION DATE FOR VOLUME ONE HUNDRED TWENTY-THREE

MYCOTAXON for JANUARY-MARCH, VOLUME 123 (I-v1 + 1-495)

was issued on August 1, 2013

APRIL-JUNE 2013 ... IX

REVIEWERS — VOLUME ONE HUNDRED TWENTY-FOUR

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 volume.

M. Catherine Aime

A. Aptroot

Bita Asgari

Timothy J. Baroni

Reinhard Berndt

Janusz Blaszkowski

Wolfgang von Brackel

Matias J. Cafaro

Paul EF Cannon

Cecilia Cristina Carmaran

R.E Castafieda-Ruiz

Pedro Crous

Yu-Cheng Dai

Kanad Das

FR.M. Dugan

Walter Gams

Genevieve M. Gates

Matteo Gelardi

Sergio P. Gorjon

Josep Guarro

Cécile Gueidan

Shouyu Guo

Ian R. Hall

Samuel Hammer

David L. Hawksworth

G. Sybren de Hoog

Vit Hubka

Seppo Huhtinen

K.D. Hyde

Mikael Jeppson

Evan Benjamin

Gareth Jones

Kerry Knudsen

Patrick R. Leacock

Teresa Lebel

James C. Lendemer

De-Wei Li

Tai-Hui Li

Zongqi Liang

Runjin Liu

Laszl6 Lékés

Guo-zhong Lit

Robert Licking

Marcelo P. Marcelli

P. Brandon Matheny

Patrick McCarthy

Eric H.C. McKenzie

Gabriel Moreno

Salvatore Moricca

Sanjeeva Nayaka

Abdul Rehman Niazi

Lorelei L. Norvell

Fritz Oehl

Esteri Ohenoja

Takamichi Orihara

Beatriz Ortiz-Santana

Todd W. Osmundson

Ka-Lai Pang

Shaun R. Pennycook

Evangelina Pérez-Silva

Leif Ryvarden

Jaya Seelan Sathiya Seelan

Ewald Sieverding

José Ivanildo de Souza

Viacheslav Spirin

Iben Margrete Thomsen

Giuseppe Venturella

Long Wang

A.J.S. Whalley

Pat Wolseley

Jian-Ping Xu

Yi-Jian Yao

Xiu-Guo Zhang

Zhongyi Zhang

Li-Wei Zhou

Wen-Ying Zhuang

X ... MYCOTAXON 124

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Mycotaxon publishes four 350-500-page volumes a year. Both open access and

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APRIL-JUNE 2013... XI

FROM THE EDITOR-IN-CHIEF

WELCOME KAREN HANSEN! — We wish to announce - somewhat belatedly

- that Sweden's Karen Hansen (Senior Curator, Kryptogambotanik,

Naturhistoriska Riksmuseet, Stockholm) has been elected to MycoTaxon’s

Editorial Advisory Board. She joins Chair Wen-Ying Zhuang (Beijing), Past

Chair Henning Knudsen (Copenhagen), Scott Redhead (Ottawa), Sabine

Huhndorf (Chicago), and Peter Buchanan (Auckland) on the 2013 Board.

Each member serves a six-year term, standing as Chair in the fifth year and

as Past Chair in his or her final year. We apologize profusely for the editorial

(!) lapse leading to the omission of Karen’s name from the masthead (on p. ii)

of Mycotaxon 123, the first volume for 2013.

MycoTaxon 124 — After some deliberation and in an effort to bring the

journal back on schedule this year, we have closed the delayed April-June

2013 volume at 380 pages. The current volume contains 38 papers by 151

authors (representing 26 countries) and revised by 74 expert reviewers.

Within its pages are one new genus and species (Scolecobeltrania from

Venezuela) and 33 other taxa new to science representing Arenariomyces

from Denmark; Beauveria, Entoloma, Glomus, Inonotus, Kuklospora,

Myrmecridium, Pertusaria, Phialophora, Phylloporia, Russula, Tuber, and

Xerocomus from China; Chaetomium from India; Cladonia and Septoglomus

from Brazil; Coleosporium from Europe; Hypochnicium from East Asia;

Leccinellum from the U.S.A.; Lyromma from Bolivia and Brazil; Pisolithus and

Terfezia from Portugal and/or Spain; and Polycoccum from Turkey.

In addition to range extensions and/or new hosts for previously named

taxa, we also offer new combinations in Aspicilia, Cantharocybe, Lactarius,

and Rutstroemia, newly established synonymies and typifications, and

conclusions from recent mycorrhizal (Inocybe) and monographic (Fissurina

in Vietnam, plectomycetes in Italy, ascomycetes in Guatemala) studies.

The next two volumes will also close ‘early’ to ensure that the four 2013

volumes will not spill over into next year. We shall begin 2014 with a much

shorter turn-around time between final submission and publication, and

hope eventually to return to publishing each volume at the beginning of a

quarter rather than well after!

Warm regards,

Lorelei L. Norvell (Editor-in-Chief)

29 September 2013

MY COTAXON

http://dx.doi.org/10.5248/124.1

Volume 124, pp. 1-8 April-June 2013

A new Myrmecridium species from Guizhou, China

CHUN- YU JIE“, QING-XIN ZHOU™, WEN-SHENG ZHAO’, YU-LAN JIANG’,

KEVIN D. Hype**, Ertc H.C. MCKENZIE° & YONG WANG'*

"Department of Plant Pathology, College of Agriculture, Guizhou University,

Guiyang 550025, China

? Institute of Agro-Food Science & Technology, Shandong Academy of Agricultural Sciences,

Jinan, Shandong 250100, China

* Department of Plant Pathology, China Agricultural University,

Beijing 100193, Peoples Republic of China

* Institute of Excellence in Fungal Research, and School of Science, Mae Fah Luang University,

Chiang Rai, Thailand

° School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand

° Landcare Research, Private Bag 92170, Auckland, New Zealand

* CORRESPONDENCE TO: yongwangbis@aliyun.com OR mppzhaws@cau.edu.cn

ABSTRACT — A strain of Myrmecridium was isolated from farmland soil in Guizhou

Province, China. The strain produced distinctive obovoid or fusoid conidia that taper to

a subtruncate darkened hilum at the base that distinguished it from other Myrmecridium

species. Phylogenetic analysis of combined ITS1-5.8S-ITS2 and large subunit rDNA

sequences supported this morphological separation. We describe Myrmecridium obovoideum

as new and provide a key to Myrmecridium species.

KEY worps — asexual fungi, hyphomycetes, molecular phylogeny

Introduction

Arzanlou et al. (2007) established Myrmecridium Arzanlou et al. based on its

hyaline mycelium with pale to unpigmented, pimple-like denticles that produce

obovoid or fusiform conidia, tapering towards a narrowly truncate base with a

slightly prominent unpigmented hilum and schizolytic conidial secession. The

genus belongs in Sordariomycetes and comprises three species and two varieties

(Arzanlou et al. 2007; Crous et al. 2011; Crous et al. 2012).

During a survey of anamorphic ascomycetes, we isolated a strain with

characters of Myrmecridium (Arzanlou et al. 2007) from farmland soil in China.

“C.-Y. Jie and Q.-X. Zhou contributed equally to the manuscript and should be considered as

joint first authors

2 ... Jie & al.

Combined ITS and LSU rDNA sequences were generated from the strain

and analyzed to evaluate the morphological findings. Based on morphology

and DNA sequence comparison, the strain is proposed as Myrmecridium

obovoideum. In the present paper, we provide detailed illustrations and a

description of this new species.

Materials & methods

Morphological and cultural studies

The Myrmecridium specimen — from a farmland soil sample collected from Huaxi

District, Guizhou, China, in 2005 —is deposited in the herbarium of the Department

of Plant Pathology, Shandong Agricultural University (HSAUP), and ex-type strains

are conserved in HSAUP and the Herbarium of Plant Pathology, Guizhou University

(HGUP). The specimen was examined in the laboratory and the taxon is described from

cultures grown at 25°C on potato dextrose agar (PDA). Conidia and conidiophores were

placed in a drop of 85% lactic acid and examined and photographed using a Nikon

80i microscope (Nikon Corporation, Japan) at 400x and 1000x magnification. The

taxonomic determinations were made by comparing with descriptions in the literature.

DNA extraction, amplification and DNA sequencing

Genomic DNA was extracted from colonies grown on potato-dextrose agar (PDA),

using the Fungal gDNA Kit GD2416 (Biomiga, CA, USA) following the manufacturer's

instructions. The universal primers ITS1/ITS4 (White et al. 1990) were used for the

ITS region (ITS1-5.8S-ITS2) amplification, and LROR/LR5 were used for a segment

of the large subunit rDNA (Vilgalys and Hester 1990). Amplification reactions were

performed in a Bio-RAD PTC-200 thermocycler in a 25 ul reaction mixture using the

following final concentrations or total amounts: 5 ng DNA, 1 x PCR buffer (20 mM

Tris/HCl pH 8.4, 50 mM KCl), 1 uM of each primer, 2.5 mM MgCl, 0.25 mM of each

dNTP, 0.5 U of Taq polymerase.

The PCR-amplified DNA fragments were fractionated in 1% agarose gels in 0.5x TBE

buffer and visualized by ethidium bromide staining and UV illumination. Sequencing

was performed with an ABI PRISM 3730 DNA autosequencer using either dRhodamine

terminator or Big Dye Terminator chemistry (Life Technologies™, USA). Sequence data

of the isolates used in the study were deposited in GenBank (TaBLE 1). Alignments are

available in TreeBASE (www.treebase.org/treebase-web/home.html) under the study ID

T3733.

Phylogenetic analyses

Preliminary nucleotide sequence alignments were constructed using Clustal X

1.81 (Thompson et al. 1997). A partition homogeneity test (Farris et al. 1994) was

applied to evaluate the feasibility of combining the datasets. Phylogenetic analyses of

LSU sequences and combined ITS and LSU rDNA sequence were computed using

MP analysis in PAUP* (Swofford 2002). In the MP analyses, trees were inferred using

heuristic search option with tree bisection reconnection (TBR) branch swapping and

1000 random sequence additions, maxtrees were 5000, branches of zero length were

collapsed and all parsimonious trees were saved. Measures calculated for parsimony

Myrmecridium obovoideum sp. nov. (China) ... 3

included tree length (TL), consistency index (CI), retention index (RI), and rescaled

consistence index (RC). Bootstrap analyses (Hillis & Bull 1993) were conducted with

1000 replications.

Results

Phylogenetic analysis

Partition homogeneity tests for combining the two (ITS and LSU) gene

regions yielded a P-value of 0.104, and the two gene/regions were combined

based on the tree topologies and P-value (Cunningham 1997, Dettman et

Myrmecridium phragmitis JQ044425

Myrmecridium schulzeri EU041770

Myrmecridium banksiae JX069871

Myrmecridium flexuosum EU041768

Myrmecridium obvoideum sp. nov KC136140

Rhodoveronaea varioseptata EU041813

Gondwanamyces wingfieldi JQ844903

Pseudoidriella syzygii JQ044421

Seiridium banksiae JQ044422

Diaporthe musigena JF951138

100

Phomopsis subordinaria GQ922519

Pleurothecium obovoideum EU041784

Carpoligna pleurothecii JQ429149

Ophiostoma stenoceras AF 484476

Veronaeopsis simplex EU041820

— 10

Fic. 1. One of the two equally most parsimonious trees of the analyzed ITS and LSU rDNA region/

genes (284 of 1485 characters were parsimony informative). Bootstrap support values of less than

50% are not shown. The tree is rooted with Pleurothecium obovoideum.

A ... Jie & al.

; Myrmecridium flexuosum EU041825

Myrmecridium obvoideum sp. nov KC136139

100 |_| Myrmecridium schulzeri EU041827

50 Myrmecridium phragmitis JQ044444

Myrmecridium banksiae YX069855

Rhodoveronaea varioseptata EU041870

Ophiostoma stenoceras DQ836904

Diaporthe musigena JF951158

99 Phomopsis asparagi AF 439634

75 Seiridium banksiae JQ044442

100 Polyscytalum algarvense GQ303318

100

Pseudophloeospora eucalypti HQ599593

Gondwanamyces wingfieldii JQ844902

tat Pleurothecium obovoideum EU041841

Carpoligna pleurothecii JQ429237

Veronaeopsis simplex EU041877

— 10

Fic. 2. The most parsimonious tree of the analyzed LSU rDNA gene (206 of 844 characters were

parsimony informative). Bootstrap support values of less than 50% are not shown. The tree is

rooted with Veronaeopsis simplex.

al. 2003). The aligned sequence data matrix contained five taxa (including

the outgroup Pleurothecium obovoideum) and 1485 (546 ITS and 839 LSU)

characters, of which 284 were parsimony informative. Two most parsimonious

trees were obtained, with one chosen to represent the topology of the strict

Myrmecridium obovoideum sp. nov. (China) ... 5

consensus tree (Fic. 1; Tree Length (TL) = 380, CI = 0.956, RI = 0.467, HI

= 0.044, and RC = 0.446). In this tree, five Myrmecridium species clustered

together supported by a 100% bootstrap value. Myrmecridium obovoideum

formed a single branch as the sister clade to the other four species with only

56% bootstrap support.

In order to evaluate the relationship of Myrmecridium with other

ascomycetes, we also generated a parsimonious tree (Fic. 2) based on LSU

rDNA gene region. The alignment with 16 taxa (Veronaeopsis simplex as

outgroup) comprised 844 characters, of which 206 were parsimony-informative

[Tree Length (TL) = 628, CI = 0.67, RI = 0.68, HI = 0.33, and RC = 0.45]. Only

one most parsimonious tree was obtained to represent the topology of the strict

consensus tree selected for presentation (Fic. 2). In this tree, the Myrmecridium

group had a close relationship with Rhodoveronaea and Ophiostoma supported

by a credible bootstrap value (74%), which was consistent with Arzanlou et

al. (2007). However, the connection between Rhodoveronaea and Ophiostoma

received weak support (<50%). Meanwhile, the placement of M. obovoideum

was movable, because it showed a closer relationship with M. flexuosum (de

Hoog) Arzanlou et al. supported by 82% bootstrap value, which differed from

the analysis combined ITS and LSU rDNA gene regions.

Taxonomy

Myrmecridium obovoideum Jie, Y.L. Jiang, McKenzie & Yong Wang bis,

sp. nov. Fig. 3

MycoBank MB 804082

Differs from other Myrmecridium species by its conidia with darkened hilum.

Type: China, Guizhou Province, Guiyang, Huaxi District, isolated from farmland soil,

5 November 2005, Y.L. Jiang (Holotype, HSAUP051001; isotype, HGUP0314; GenBank,

KC136140, KC136139).

EryMo_oey: in reference to the conidial shape of this new taxon.

Colonies on PDA at 25°C for 2 weeks reaching 30 mm diam. ‘The surface

effuse, grayish. Mycelium mostly superficial, partly immersed is contradictory

with lacking aerial mycelium. Conidiophores straight to geniculate-

sinuous, unbranched, subhyaline, 1-8-septate, <280 um tall, 3-3.6 um wide.

Conidiogenous cells terminal, integrated, cylindrical, pale or reddish brown

near the base, gradually becoming paler towards the apex, 50-210 um, often

with 1-6 thin septa, forming a rachis with scattered pimple-shaped denticles

less than 1 um long, sometimes fertile part slightly inflated. Conidia solitary,

aseptate, subhyaline, pale brown, thin-walled, smooth to finely verrucose,

surrounded by a wing-like gelatinous sheath, <1.5 um thick, obovoid or fusoid,

(7-)8-9(-10) x (3-)3.5(-4.2) um, tapering to a subtruncate, slightly thickened

hilum; darkened at rounded end.

6 ... Jie & al.

Fic. 3. Myrmecridium obovoideum (HSAUP051001, holotype) on PDA. a-b. Conidiophores.

c. Rachis with pimple-shaped denticles and slightly inflated fertile part. d. Conidia surrounded by

wing-like gelatinous sheaths. e-f. The thin septa of conidiogenous cells. Scale bars: a-c, e-f = 35

um; d= 10 um.

Myrmecridium obovoideum sp. nov. (China) ... 7

Discussion

Our LSU rDNA sequence analysis clustered Myrmecridium with

Rhodoveronaea (sexual state = Annulatascaceae) and Ophiostoma

(Ophiostomataceae) in the Sordariomycetes. Arzanlou et al. (2007) classified

Myrmecridium as “Incertae sedis (Sordariomycetes)”, and our results confirm

this placement, without any indication of a family affinity.

Only M. schulzeri var. tritici (M.B. Ellis) Arzanlou et al. and M. phragmitis

Crous produce septate conidia. Myrmecridium banksiae Crous differs from

M. obovoideum by its larger conidia (9-14 x 2.5-3.5 um); and M. flexuosum

differs by its smaller conidia (5-9 x 3-4 um). The conidia of M. schulzeri (Sacc.)

Arzanlou et al. var. schulzeri (6-12 x 3-4 um) are also larger than those of our

species. In Myrmecridium only M. obovoideum has conidia with a darkened

hilum, which enlarges the taxonomic criteria of Myrmecridium (Arzanlou et

al. 2007). Our ITS and LSU rDNA sequence analyses also confirmed with high

bootstrap value that our species differs from other Myrmecridium species. The

combined morphological and phylogenetic analysis supports M. obovoideum

as a new species.

Key to Myrmecridium species

Las Pile ofconidia ne td atkeneds5 Act, 0 Aeh, chit lead ubai dn Heald tn tnald a aluonttah sal 2

Ibe Hiltom-ofcomidia darketeden 5 fc. 6.5 242.5 nce pale yeh laine Melee Sale ims M. obovoideum

2a; Comidiawithouy septate, .2 Lin. + iden eg dh sees Pieter a Saber a Atnaes garners Abeta Ss Nees 3

2b. Septate conidia frequently-Observed ... guiicieclaicenglaneetlenes M. schulzeri var. tritici

ba. Conidia mostly lessthani 10. pint lone: sti. cea cshee Gta sda negate Gea co Bet wed ale 4

Sb, Conidiarip to P4afimislon es. isptea ras atls tay nll len cette tes tres steele M. banksiae

dace onidia rounded at the bases) fut. fit.ne PO! Wo hal Wet Weer 1 M. flexuosum

4b. Conidia slightly acuminate at the base......................00 0000. M. phragmitis

4c. Conidia tapering to a subtruncate base................ M. schulzeri var. schulzeri

Acknowledgments

The authors are grateful for pre-submission comments and suggestions provided

by Drs. R.E Castafieda-Ruiz and De-Wei Li. This project was supported by the

National Science Foundation of China (No. 31060005) and the Doctors’ Funding of

Guizhou University (no. 2010014). We are indebted to Prof. Tian-Yu Zhang (Shandong

Agricultural University, China) for supplying the culture of this new species.

Literature cited

Arzanlou M, Groenewald JZ, Gams W, Braun U, Shin HD, Crous PW. 2007. Phylogenetic and

morphotaxonomic revision of Ramichloridium and allied genera. Studies in Mycology 58:

57-93. http://dx.doi.org/10.3114/sim.2007.58.03

8 ... Jie & al.

Crous PW, Summerell BA, Shivas RG, Romberg M, Mel'nik VA, Verkley GJM, Groenewald JZ.

2011. Fungal Planet description sheets: 92-106. Persoonia 27: 130-162.

http://dx.doi.org/10.3767/003158511X617561

Crous PW, Summerell BA, Shivas RG, Burgess TI, Decock CA, Dreyer LL, Granke LL, Guest DI,

Hardy GEStJ, Hausbeck MK, Hiiberli D, Jung T, Koukol O, Lennox CL, Liew ECY, Lombard L,

McTaggart AR, Pryke JS, Roets E Saude C, Shuttleworth LA, Stukely MJC, Vanky K, Webster

BJ, Windstam ST, Groenewald JZ. 2012. Fungal Planet description sheets: 107-127. Persoonia

28: 138-182. http://dx.doi.org/10.3767/003158512X652633

Cunningham CW. 1997. Can three incongruency tests predict when data should be combined?

Molecular Biology and Evolution 14: 733-740. http://dx.doi.org/10.1093/oxfordjournals.

molbev.a025813

Dettman JR, Jacobson DJ, Taylor JW. 2003. A multilocus genealogical approach to phylogenetic

species recognition in the model eukaryote Neurospora. Evolution 57: 2703-2720.

http://dx.doi.org/10.1554/03-073

Farris JS, Kallersj6 M, Kluge AG, Bult C. 1994. Testing significance of incongruence. Cladistics 10:

315-320. http://dx.doi.org/10.1111/j.1096-0031.1994.tb00181.x

Hillis DM, Bull JJ. 1993. An empirical test of bootstrapping as a method for assessing confidence in

phylogenetic analysis. Systematic Biology 42: 182-192.

http://dx.doi.org/10.1093/sysbio/42.2.182

Swofford DL. 2002. PAUP*: Phylogenetic analysis using parsimony (*and other methods), version

4.0b10. Sinauer Associates, Sunderland, Massachusetts.

Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997. The Clustal X windows

interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.

Nucleic Acids Research 24: 4876-4882. http://dx.doi.org/10.1093/nar/25.24.4876

White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal

RNA genes for phylogenetics. 315-322, in: MA Innis et al. (eds). PCR protocols: a guide to

methods and applications. Academic Press, New York, U.S.A.

Vilgalys R, Hester M. 1990. Rapid genetic identification and mapping of enzymatically amplified

ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: 4239-4246.

MY COTAXON

http://dx.doi.org/10.5248/124.9

Volume 124, pp. 9-20 April-June 2013

The occurrence of Rutstroemia coracina on Quercus ilex leaves

in Spain

RICARDO GALAN!*, FRANCISCO PRIETO-GARCIA?’,

ANGELES GONZALEZ? & CARLOS ENRIQUE HERMOSILLA‘

” Department of Life Sciences, University of Alcala, Alcala de Henares, 28871 Madrid, Spain

? Marqués de la Valdavia 13 A, 28400, Collado Villalba, Madrid, Spain

° Prado Ibarra 29, 28270, Colmenarejo, Madrid, Spain

* Francisco Cantera 11, 1° izda., 09200 Miranda de Ebro, Burgos, Spain

* CORRESPONDENCE TO: ricardo.galan@uah.es

AxBsTRACT— ‘The authors report new findings of an apparently rare fungus, Rutstroemia

coracina (Rutstroemiaceae). The species grew on old fallen leaves of Quercus ilex subsp.

ballota at different localities in Guadalajara and Madrid provinces (center of Spain) where it

was first collected in 1983 and subsequent years. The apothecia are described and illustrated

in detail. Microscopic studies, including microtome sections, show a clear gel layer in the

ectal excipulum, which allows assignment in the genus Rutstroemia. Since the type material

deposited in PC is so scanty and deteriorated that any future revision will be impossible, an

epitype is selected here.

Key worps— Ascomycota, Helotiales, foliicolous fungi, taxonomy

Introduction

Palmer (1994) was the first to report the presence of Rutstroemia coracina

from the European continent. This species was initially recorded in Algeria and

was documented only by a coloured plate as “Peziza coracina DR. et Lév. in

Durieu (1848: pl. 28 fig. 4) reproduced here as Fic. 1, which depicts apothecia

(on a Quercus ilex leaf) with egg-shaped spores, paraphyses, and asci. Although

no text accompanied the 1848 protologue, the figure caption published in Durieu

(1869: 10) identified the substrate as the undersurface of a dried Q. ilex leaf and

detailed the individual elements included in the figure. Later Saccardo (1889:

237) provided a brief description as Helotium coracinum, apparently based on

the original figure since no microscopic measurements are given and enquiries

by J.T. Palmer with herb. PAD (Universita degli Studi di Padova) ascertained

that there is no exsiccatum under this epithet in Herb. P.A. Saccardo.

10 ... Galan & al.

The first authentic description seems to be by Dennis (1964: 39-40, fig. 12),

who studied the type collection preserved in PC. Reporting ascospores “11-13

x 5-5.5 um’ and an “[e]xcipulum composed of parallel hyphae with glassy

walls,” he transferred the species to Rutstroemia.

Spooner (1981: 283, 285, fig. 22) redescribed the species from a few apothecia

ona Quercus ilex leaf collected by M.C. Clark in 1977 near Somerset, southwest

England, and recombined it in Lanzia, as he failed to find the “glassy walls”

found by Dennis. He reported similar spore measurements to those mentioned

by Dennis for the type.

In 1992, Palmer visited the Allerford plantation between Bossington and

Porlock, Somerset, where Mr. Clark had collected his leaf, and collected

large samples of dead Q. ilex leaves from different parts of the wood, which

subsequently produced apothecia in natural culture. His measurements for the

living spores (in tap water) were 12.4-16.8 (18.4) x 4.8-7.6 (8) um.

Palmer (1994) also restudied the type remnants in PC and found a minute

fragment (approximately haft an apothecium) among the stipes of immature

apothecia on one of the two spiny leaves of the holotype; although it was not

possible to make a detailed study, he managed to draw ovoid spores without

any sign of guttulation (measuring 10.6-13.9 x 4.5-6.5 (7.3) um), one ascus

(measuring 106 x 12 um), and scarce hyphae suspected to be from the gelatinous

layer in the ectal excipulum protruding as obtuse “hairs” (3.3-6.0 um wide) -

reproduced here as Fic. 2. In addition, Palmer (1994: 226-228, figs 1-5) made

good collections in northern Greece in March 1988, mainly on old, fallen leaves

of Quercus coccifera and Q. ilex on Mount Athos and Mount Olympus but also

on a leaf of Quercus pubescens (= Q. humilis) on Mt Athos.

This species was collected and identified for the first time in Spain by

R.G. in 1983 on old, fallen leaves of Quercus ilex subsp. ballota at Tamajon

(Guadalajara province). The identification was later confirmed by Palmer,

who had the opportunity to record the species in subsequent years, including

collections by Dr. A. Raitviir (Palmer & Galan 1999, Galan 2001). The area is

open with scattered trees (intermixed with Juniperus thurifera), and leaf litter is

sparse. More recently, during a field trip through Guadalajara as part of the XIII

Congress of European Mycologists in Alcala de Henares (September, 1999),

several attendees had the opportunity to collect the species in abundance,

including Drs. B. Spooner, M.M. Nauta and H.O. Baral, who kindly provided

us with herbarium samples. More recent findings of the species by two of us

(E.P.G. & A.G.) on old fallen leaves of Quercus ilex subsp. ballota at Colmenarejo

(Madrid province) allow us not only to widen the distribution of Rutstroemia

coracina in Spain but also to complete its description. In addition, Palmer (pers.

comm.) also recorded and identified this species in 1990 on Minorca (Balearic

Islands, Spain) growing on Quercus ilex leaves.

Rutstroemia coracina on Quercus (Spain) ... 11

Fic. 1. Illustration of the type of Peziza coracina Durieu & Léveille,

Expl. Sci. Algérie, Bot. 1: t. 28, f. 4 (1848).

12 ... Galan & al.

From the beginning, Palmer (1994) found it noteworthy that the greyish

green tinges of apothecia in both freshly collected material or that developed

in damp chambers were overlooked in Spanish material, which more closely

resembled the reddish brown color in the original Algerian plate. That might

suggest the existence of two different taxa, but otherwise all specimens appeared

to be microscopically identical.

Since the type material preserved in PC allows no further revision, we

here select as epitype a very rich Spanish collection (about 30 fallen leaves of

Quercus ilex subsp. ballota, bearing ca. 60-70 apothecia) whose habit, colour,

and microscopic features (mainly ascospore shape and size) match the holotype

very well.

There has been continued controversy since Dennis (1964) reported an

“excipulum composed of parallel hyphae with glassy walls.” This was why the

taxon was first combined in Rutstroemia and why Spooner (1981) transferred

the species to Lanzia, remarking, “I have not been able to find glassy walls.”

Spooner later had opportunity to re examine one of the Greek collections (J.T.P.

4418) identified as R. coracina and agreed entirely with Palmer’s identification.

He noted the presence of gelatinized tissue, which made him review the British

material where he found “a gelatinous matrix to the ectal hyphae, something

which I overlooked when referring the species to Lanzia” (B.M. Spooner, in

letter). The microtome sections (Fics 6, 7) of English, Greek, and Spanish

material show a clear gel layer in the ectal excipulum, although the thickness

is not as prominent as in other Rutstroemia spp. and also is somewhat variable

in width from collection to collection. Nonetheless, we feel that the species was

correctly placed in Rutstroemia by Dennis (1964).

Rutstroemia coracina appears to be known only from Algeria (where the

actual type locality is unknown), Britain (Spooner 1981), Greece (Palmer 1994),

France (Palmer, pers. comm.), Turkey [K(M) 49709 Spooner, pers. comm.],

and Spain.

Materials & methods

Measurements of living cells (marked *) were made in tap water or IKI (1% Lugol’s

solution), with dead cells (marked f) in tap water, Melzer’s reagent (MLZ), KOH

2%, and aqueous cresyl blue (CRB) 0.5%. Line drawings were made using a NIKON

Labophot-2 either by hand without the aid of a camera lucida (C.E.H.) or with a drawing

tube incorporated into the light microscope (R.G.) with magnifications up to 1250x.

Photomicrographs were made with using a NIKON Labophot-2 equipped with phase

contrast and camera. Macroscopic pictures of fructifications in fresh state showing the

general habit were taken in situ (by EP.G.) or at the lab (by C.E.H.). Apothecial colours

are cited according to Locquin (1985). Specimens have been deposited in AH and J.T.P.

(personal herbarium of the late Mr. J. Terence Palmer, currently deposited in CUP)

with additional specimens in H.O.B. (personal herbarium of Hans O. Baral, Tubingen,

Germany) and K.

Rutstroemia coracina on Quercus (Spain) ... 13

NATIONAL HERBARIA

J.T.P. Refis WX institute: Museum D'Histo res NaTa@cwe - abbreviation: PC

De Paris:-LReorato (ae he Cevetoeamis, PARIS

ORTOINAL DETERMINATION: PEZIZA CoRAciN« revid.: = RYTIRRORMA CORACIN'A

Leg. Date: Devi

Locality: ALG &RIA Sle eon A RE erst

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feziza coraciha DAR er Liv. eet Coops ota ice

ker } ae” Deen cee ! 140

, Neletyous in PC - gy ee 3o 4 Ho $0 yo 100 uo Ps : UK

106 ¥l2 prot, : ¥ y .

Ifolety pus comb nites 2 leaves wired se ff |

typical of We Soiny fon of Puenens ile ,

[luce to ao sheet Untlh one Sepavarted. v

a (e\ 2B Kw. we 4 iN

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Fic. 2. Rutstroemia coracina (holotype, PC): Annotations and drawings by J.T. Palmer showing

excipular hyphae and an ascus (up), ascospores, and “hyphoid” hairs (down).

14 ... Galan & al.

Fic. 3. Rutstroemia coracina (AH 7641, epitype) growing in natural habitat.

Photo EPG. in situ (bar = 10 mm).

=a Pe. see. ee et” a Ora}

Fic. 4. Apothecia of Rutstroemia coracina (C.E.H. 08468) under hi

Photo C.E.H. ex situ (bar = 1 mm).

Rutstroemia coracina on Quercus (Spain) ... 15

Taxonomy

Rutstroemia coracina (Dur. & Lév.) Dennis, Persoonia 3: 39 (1964) Fics. 1-7

= Peziza coracina Dur. & Lév., Expl. Sci. Algérie, Bot. 1: t. 28, f. 4 (1848)

= Helotium coracinum (Dur. & Lév.) Sacc., Syll. Fung. 8: 237 (1889)

= Calycina coracina (Dur. & Lév.) Kuntze, Revis. Gen. Pl. 3(3): 448 (1898)

= Ciboria coracina (Dur. & Lév.) Boud., Hist. Class. Discom. Eur.: 106 (1907)

= Lanzia coracina (Dur. & Lév.) Spooner, Trans. Br. Mycol. Soc. 76: 283 (1981)

Type: Algeria (Holotype, PC). Spain, Madrid, Colmenarejo, UTM 30TVK1489, 885

m a.s.l., on petioles, lower (more exceptionally upper) faces and veins of fallen leaves

of Quercus ilex subsp. ballota, 27.X1.2011, Prieto & Gonzalez, det. R.G. , rev. C.E.H.

(Epitype designated here, AH 7641).

APOTHECIA superficial, scattered, cupulate, stipitate, castaneus to reddish-

brown, merging from darkened areas of the substrate. pisc slightly concave

becoming plane, flesh colour (ie., russus B4f) to yellowish-olivaceous (i.e.,

venetus S2d), tending to darken with age, 0.5-2.0 mm in diameter. RECEPTACLE

cupulate, covered with a sparse and irregular net of brown fibrils, vertically

arranged with adpressed ends, sometimes protruding and giving a fringed

appearance, colour similar to or concolorous with disc and stipe, sometimes

chestnut (with varying reddish brown shades). MARGIN ochre, chestnut to

reddish-brown, crenulate. sTIPE equal in length to or longer than disc diameter,

reddish-brown often almost black at the base, 0.3-3.0(-6.0) mm long. ECTAL

EXCIPULUM composed of three layers: outer zone, 12-38 um thick, formed of

some sparse rows of loosely branched, flexuous, brown hyphae (3.7-5.5 um

in diam. +) arranged parallel to the surface, with thin walls encrusted with

pigment giving a typically banded aspect and protruding as agglutinated

“hairs” on flank and stipe or in groups as “clavate hairs” at the margin to form

teeth; the medial layer of textura oblita, 15-60 um thick, formed of flexuose,

subhyaline to light pale ochre hyphae, arranged parallel or at a low angle to

the surface, with very narrow lumina and strongly gelatinized walls; the inner

zone of imprecise thickness, formed of prismatic to doliform cells (textura

prismatica) and not well delimited from the medullary excipulum, hyphae 5-

10 um (ft) in diameter, with pale chestnut content and thin walls. MEDULLARY

EXCIPULUM well developed, of textura intricata and subhyaline cells with thin,

smooth walls (3-5 um in diam. ft). SUBHYMENIUM compact, poorly developed

and indistinguishable from the medullary area, apart from the brownish colour

and highly intricate texture. asci 8-spored, cylindric-clavate, tapering at base

to form a slender stalk, emerging from croziers, a round, truncate to fissured

apex, with a long pore outlined J+ (deep blue) in both IKI and Melzer’s reagent,

125-152 x11-13 um (*), 110-130(-140) x9-11 um (+). spores biseriate to

uniseriate or irregularly biseriate (in dead state), hyaline, ellipsoid to elliptic-

16 ... Galan & al.

fusoid, slightly inequilateral, regularly unicellular, rarely one to three-septate

when over-mature, exceptionally producing subglobose microconidia (2-2.5

um * diam.) at one or both apices, with high number of globose lipid bodies

(1-2 um) occupying most of the cytoplasm in the living spores (multiguttulate),

forming two large distinct apical drops in dead state by coalescence, measuring

14-17(-18) x 6-7.5 um (*), 12-15(-16.8) x (5—)5.5-6.8(—7.2) um (+). PARAPHYSES

straight, filiform to subclavate ca. 2-2.5 um diam. (*), enlarged to 4—-5.5 um (*)

at the apex, sparsely septate, simple or branched below, equaling or scarcely

exceeding the asci in length, bearing brown to reddish-brown vacuolar contents

(“refractive vacuolar bodies” sensu Baral 1985) in the living state.

ADDITIONAL SPECIMENS EXAMINED: SPAIN, GUADALAJARA, Tamajon, surroundings

of Ermita del Humilladero, on a fallen leaf of Quercus ilex subsp. ballota in an open

mixed wood (with Juniperus thurifera) with scattered trees and leaf litter sparse on

the ground, 18.XI.1983, Galan, Moreno & Checa, det. R.G., rev. Palmer (AH 6235;

duplicate, J.T.P. as permanent slides); on stromatized petioles and veins of fallen old

Quercus ilex subsp. ballota leaves, 3.X1I.1984, Galan, Moreno & Palmer, det. J.T.P. (J.T.P.

4125); 3.XII.1991, Galan & Raitviir, det. R.G. & J.T.P. (AH 6720; duplicate, J.T.P. 4695).

Cifuentes, between Oter and Carrascosa de Tajo, on fallen leaves of Quercus ilex subsp.

ballota, 25.1X.1999, Nauta & Spooner, det. J.T.P. (J.T.P. 4978; duplicates, AH 7334, K).

Mirabueno, surroundings, 1000 m a.s.l., on side and veins of fallen leaves of Quercus ilex

subsp. ballota lying on ground, 24.1X.1999, Baral & Marson, det. Baral, rev. J.T.P. (J. T-P.

4977; duplicates, AH 7335, H.O.B. 6477E). MAprRID, Colmenarejo, UTM 30TVK1489,

885 ma.s.l., on petioles, lower (more exceptionally upper) faces and veins of fallen leaves

of Quercus ilex subsp. ballota, 18.X1.2011, det. R.G. (AH 7640); 11-XII.2011, det. EP, rev.

C.E.H. (C.E.H. 08468). ENGLAND, SoMERSET, between Allerford and Bossington,

on old, fallen leaves of Quercus ilex sampled, (apothecia developed in damp chamber

from 26.[V—30.V.1992), 20.11.1992, Palmer, det. J.T.P., rev. R.G. (J.T.P. 4713; duplicate,

AH 6813); (J. T-P. 4714; duplicate, AH 6812). GREECE, Maceponlia, Athos Mountain,

surroundings of Monastery Iveron, on stromatized fallen leaves of Quercus ilex subsp.

ilex, 25.11.1988, Palmer, det. J.T.P., rev. R.G. (J.T.P. 4415; duplicate, AH 6604).

CoMMENTS—There are at least three additional Rutstroemia species usually

or occasionally reported from decaying Quercus leaves: R. sydowiana (Rehm)

W.L. White (widely distributed at the Mediterranean basin) with “allantoid”

spores (nearly ovoid, with basally bent beaks); R. petiolorum (Roberge ex Desm.)

W.L. White (typically reported on Fagus leaves in Europe but occasionally also

appearing on Quercus leaves) with allantoid to reniform spores with small polar

gel caps; and R. latispora K.S. Thind & Saini, later transferred to Lambertella

(Spooner 1987), found on Quercus incana leaves in the north-west Himalayas,

having asymmetrical and much broader ascospores and longer asci than the two

previous species (Thind & Saini 1968). In addition Rutstroemia “kalevi” Baral

(ined.), which that could be misidentified as R. coracina (despite its Mollisia-

like vacuoles in the paraphyses and narrower spores) grows on stromatized

veins and petioles of Acer spp. in Central Europe (Baral & Marson 2005).

Rutstroemia coracina on Quercus (Spain) ...

Fic. 5. Rutstroemia coracina (AH 7641, epitype): A. Apothecia in their natural habitat (bar =

10 mm); B. Enlarged view of fresh apothecia (bar = 1 mm); C. Cross section of an apothecium

(marginal region) (bar = 50 um); D. Encrusted wall of hyphae of the outer ectal excipulum

(bar = 5 um); E. Hymenium showing asci arising from croziers and paraphyses (bar = 50 um);

F, apex of a mature ascus with an euamyloid ring (bar = 10 um); G. Paraphyses containing

many refractive vacuolar bodies in apical cells (bar = 5 um); H. Free 0-3-septate ascospores,

exceptionally forming small conidia on germ tubes (bar = 20 um). Mountants: tap water,

except fig. F (IKI). Watercolour by C.E.H.

Rutstroemia coracina on Quercus (Spain) ... 19

Fic. 6 (left). Rutstroemia coracina: A. Radial section of an apothecium (AH 6812; bar = 200 um);

B. Frontal view of the outer ectal excipulum forming marginal teeth (AH 6604; bar = 50 um);

C. Radial section of an apothecium at middle flanks showing a three layered ectal excipulum

(AH 6812; bar = 50 um); D, E, G, H. Asci with ascospores (AH 6235; bar = 20 um for D, G, H;

25 um for E); F. Mature asci, free spores and tips of paraphyses (AH 7641; bar = 25 um). Mountants:

2% KOH (figs. A, B & C); CRB (figs. D & H); MLZ (figs. E & G); tap water (fig. F). Dead state except

fig. F. Photographs by R.G. & C.E.H. (fig. F). Abbreviations: outer (= oee), medial(= mee) and inner

(= iee) layers of the ectal excipulum; medullary excipulum (= me).

Fic. 7 (above). Rutstroemia coracina (AH 6812): A. Radial section of an apothecium (bar = 200 tm);

B. Enlarged view of an apothecium (bar = 100 um), at flank and margin, showing: the outer

(= oee), medial(= mee) and inner (= iee) layers of the ectal excipulum; medullary excipulum

(= me); hymenium (= h). Dead state (KOH 2%). Drawings by R.G. & C.E.H.

20 ... Galan & al.

Acknowledgements

We would like to thank Dr. Richard P. Korf for suggestions and improvement in

English grammar. Also to A. Pueblas of the “Gabinete de Dibujo y Fotografia Cientifica”

at the Alcala University for their invaluable help in the digital preparation of the

photographs. We are grateful to Dr. Scott LaGreca (CUP), Dr. J. Rejos (AH), Dr. B.M.

Spooner & Dr. B. Aguirre-Hudson (K), Mme J. Perreau (PC) for providing institutional

specimens data, and Dr. M.N. Nauta and H.O. Baral for information on Rutstroemia

coracina in their personal herbaria. One author (R.G.) extends his gratitude to the late

Mr. J.T. Palmer for invaluable help with sclerotiniaceous fungi and a lifetime friendship.

Finally, we wish to express our gratitude to Dr. S. Huhtinen and Dr. W.-Y. Zhuang for

reviewing the manuscript and for their useful comments.

Literature cited

Baral HO. 1985. Vital versus herbarium taxonomy: morphological differences between living and

dead cells of Ascomycetes, and their taxonomic implications. Mycotaxon 44: 333-390.

Baral HO, Marson G. 2005. In vivo veritas. Over 10000 images of fungi and plants (microscopical

drawings, water colour plates, photo macro- & micrographs), with materials on vital taxonomy

and xerotolerance. DVD, 3rd edition.

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species referred by Saccardo and others to the genera Helotium, Pezizella or Phialea. Persoonia

3: 29-80.

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1841, 1842, Botanique vol. 1(livr. 8): 281-320, pl. 28, 38, 41, 54, 59, 83. Paris: Imprimerie

Impériale.

Durieu de Maisonneuve MC. 1869. Exploration Scientifique de [Algérie pendant les années 1840,

1841, 1842, Botanique, Atlas: 1-39. Paris: Imprimerie Impériale.

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Peninsula Ibérica e Islas Baleares. Notas a los numeros 1624-1676. 13-14, 101-139, in: F Pando,

JC Hernandez (eds). Bases Coroldgicas de Flora Micoldégica Ibérica. Adiciones y numeros

1572-1765. Madrid (CSIC).

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Palmer JT. 1994. Sclerotiniaceae aus Nordgriechenland. Z. Mykol. 60(1): 225-230.

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basin and beyond. XIII Congress of European Mycologists. Book of Abstracts. Alcala de

Henares: 101.

Saccardo PA. 1889. Discomyceteae et Phymatosphaeriaceae. Sylloge Fungorum 8: 3-859.

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265-301.

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Hyaloscyphaceae. Bibliotheca Mycologica 116: 1-711.

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Sciences — Plant Sciences 67(4): 141-147.

MYCOTAXON

http://dx.doi.org/10.5248/124.21

Volume 124, pp. 21-29 April-June 2013

Two species of Cladosporium associated with

wood discoloration in Korea

YEONGSEON JANG, YOUNG MIN LEE, GYU-HYEOK KIM & JAE-JIN K1m*

Division of Environmental Science & Ecological Engineering,

College of Life Sciences & Biotechnology, Korea University

5-1 Anam-dong, Seongbuk-gu, Seoul, 136-701, Korea

*CORRESPONDENCE TO: jae-jinkim@korea.ac.kr

AxBstTRACT — During our previous study on the diversity of molds inhabiting wood, many

Cladosporium isolates were collected and phylogenetically identified. Among them were

isolates of two species previously unrecorded from Korea. Here, we confirm the isolates as

Cladosporium perangustum and C. ramotenellum and provide descriptions and illustrations

of cultural and microscopic characters on standard media. We draw attention to a broadening

of the range of several characters for C. ramotenellum and to possible alternative phylogenetic

relationships of C. perangustum when comparing our multi-gene analysis with previous

analyses.

KEY worDs — ascomycetes, phylogeny, taxonomy

Introduction

Cladosporium Link is one of the most widely known (ifunder-studied) molds.

It is a cosmopolitan genus with more than 772 names (Dugan et al. 2004) and

recently Bensch et al. (2012) recognized a total of 169 Cladosporium species.

It has been isolated from air, soil, and many other substrates (Schubert et al.

2007, Bensch et al. 2010, 2012). Members are variously known to be saprobic,

endophytic, or plant pathogenic (Schubert et al. 2007, Bensch et al. 2010, 2012).

In Korea, about 20 Cladosporium species have been reported (Min 1985, Shin

& Braun 1995, Shin & Im 1999, Kwon et al. 2000, 2001, Paul & Yu 2008, Korean

Society of Plant Pathology 2009, Bensch et al. 2010). Recently Lee et al. (2012)

phylogenetically examined Cladosporium representatives inhabiting wood

products in Korea and recognized nine Cladosporium species. Among them

were C. perangustum and C. ramotenellum, not previously recorded in Korea.

Here, we confirm their identities by morphology and phylogenetic re-analysis

and provide detailed descriptions and figures for these two Cladosporium

species.

22 ... Jang & al.

Materials & methods

Morphological examination

Four Cladosporium isolates, KUC1462, KUC1767, KUC3027, and KUC5085 studied

by Lee et al. (2012) were used in this study. They were inoculated onto SNA (Synthetic

nutrient-poor agar: KH,PO, 1 g, KNO, 1 g; MgSO,°7H,O 0.5 g; KCI 0.5 g; glucose 0.2

g; sucrose 0.2 g; agar 20 g; distilled water 1000 ml) and cultivated at 25°C for 7 days.

Preparations were mounted in Shear’s solution (CH,COOK 3 g; distilled water 150

ml; glycerin 60 ml; 95% ethanol 90 ml) according to Crous et al. (2009). Microscopic

observations were made using an Olympus BX51 light microscope (Olympus, Tokyo,

Japan) and photos were taken using the same microscope and an Axio Imager Al

microscope (Zeiss, Jena, Germany). For determination of cultural characteristics,

colonies were cultivated on PDA (Potato dextrose agar, Difco), MEA (Malt extract agar:

malt extract 30 g; agar 15 g; distilled water 1000 ml), and OA (Oatmeal Agar, Difco)

at 25°C for 14 days in the dark. The isolates were deposited at the National Institute of

Biological Resources, Incheon, South Korea (KB) with the acronym KUC, which refers

to the Korea University Culture Collections, Korea University, Seoul, South Korea.

Phylogenetic analysis

DNA sequences of four Cladosporium isolates (KUC1462, KUC1767, KUC3027,

KUC5085) from the internal transcribed spacer (ITS), actin (ACT), and translation

elongation factor-la (TEF) regions were obtained from Lee et al. (2012). ITS, ACT,

TEF sequences were aligned separately with the reference sequences from Bensch et

al. (2010) using MAFFT 6.885 (Katoh & Toh 2008). The L-INS-I alignment option was

used for all datasets. Each dataset was manually edited with MacClade 4.08 (Maddison

& Maddison 2005). Gaps were treated as missing. The best-fit model was applied for

each dataset using AIC in MrModeltest 2.3 (Nylander 2004) and the three datasets were

combined. Bayesian analysis was performed with the combined dataset using MrBayes

3.2.1 (Ronquist et al. 2012). Two independent runs (four chains each) of 705,000

generations were performed and every 100th tree was sampled. The first 25% of sampled

trees were discarded and the remaining 75% were used for construction of a 50% major-

rule consensus tree. The graphic representation of the likelihood scores of the sampled

trees was checked. The standard deviation of split frequencies is below 0.01 and the

potential scale reduction factor (PSRF) was close to 1.0. The tree was viewed via FigTree

1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/).

Taxonomy

Cladosporium perangustum Bensch, Crous & U. Braun, Stud. Mycol. 67: 65. 2010.

Fics 1-2

Mycelium internal and superficial; 1.5-4 um wide, septate, sometimes slightly

constricted at septa, sometimes intercalary swellings and constrictions present,

subhyaline or pale olivaceous-brown, smooth or verruculose. Conidiophores

macronematous or micronematous arising terminally and laterally from hyphae,

straight or slightly curved, filiform to narrowly cylindrical-oblong, unbranched,

occasionally branched, conidiophores 14-142 x 2-3.5 um, pale olivaceous-

Cladosporium species discoloring wood (Korea) ... 23

Fic. 1. Cladosporium perangustum (KUC1767) grown in 9 cm diam Petri dishes for 14 d at 25°C.

A: PDA. B: MEA. C: OA.

]

Fic. 2. Cladosporium perangustum. A, B. Macro- and micronematous conidiophores (KUC1462).

C. Ramoconidia and conidia (KUC1767). Scale bar = 10 um.

brown, asperulate-verruculose towards the base of conidiophores, at the apex

smooth, sometimes slightly attenuated towards the apex. Conidiogenous cells

integrated, mainly terminal, sometimes also intercalary, narrowly cylindrical-

24 ... Jang & al.

oblong, in intercalary cells loci situated on small peg-like lateral prolongations

or just below the septum, 13-36 um long, with 1-4 apically crowded loci.

Ramoconidia cylindrical-oblong, up to 44 um long, 2.5-3.5 um wide, rarely

septate, base truncate, secondary ramoconidia narrowly ellipsoid to cylindrical-

oblong, 8-21 x 2-3 um (av. + SD: 13.7 + 4.1 x 2.5 + 0.2), with 2-4 distal hila, pale

olivaceous-brown. Conidia numerous, catenate, in branched chains, branching

in all directions, 1-4 conidia in the terminal unbranched part of the chain,

small terminal conidia globose, subglobose or ovoid to obovoid, 2-3.5 x 1.5-2

um (av. + SD: 2.9 + 0.4 x 1.8 + 0.2), intercalary conidia ovoid, limoniform to

ellipsoid, somewhat fusiform, 4-8.5 x 2-3 um (av. + SD: 5.9 + 1.4 x 2.2 + 0.2),

attenuated towards apex and base, with 1-3 distal hila, pale olivaceous-brown.

CULTURE CHARACTERISTICS Colonies on PDA attaining 32-38 mm diam

after 14 d at 25°C, greenish grey to dull green, reverse olive or dark green, fluffy,

floccose or powdery, margins glabrous, whitish, olive grey or pale regular or

somewhat undulate, aerial mycelium loosely floccose or felty, occasionally

numerous small to large prominent exudates formed, sporulation abundant.

Colonies on MEA reaching 20-29 mm diam after 14 d at 25°C, dark green or

brown, reverse olive brown, velvety to floccose, margins white to light yellow,

narrow to broad, regular to undulate, glabrous, aerial mycelium abundantly

formed, sometimes covering most parts of colony surface, loosely to densely

floccose or felty, growth habit plane to sometimes elevated, sporulation

abundant. Colonies on OA 35-49 mm diam after 14 d at 25°C, grey to dark

green, fluffy to felty-floccose, margins colorless or white, up to 2 mm diam,

aerial mycelium abundant, covering large parts of the colony surface, dense,

low to high, white, sporulating profusely.

SPECIMENS EXAMINED: KOREA, GYEONGSANGBUK-DO, Bonghwa-gun, on the surface

of Pinus densiflora log, 05 June 2001 (KB, KUC1462; GenBank JN033481, JN033508,

JN033536); GYEONGSANGNAM-DO, Pusan, on the surface of Pinus radiata log, August

2000 (KB, KUC1767; GenBank JN033468, JN033495, JN033523); INCHEON, on the

log surface of Picea abies, 16 June 2007 (KUC5085; GenBank JN033460, JN033487,

JN033515).

REMARKS — The colony morphology and microscopic features of the isolates

agreed well with the description of C. perangustum (Bensch et al. 2010). The

fungus has been reported from Africa, Asia, Australasia, Europe, and North

America. According to Bensch et al. (2010), Cladosporium exile Bensch et

al. and C. scabrellum Bensch et al. are similar to C. perangustum but C. exile

has longer and wider conidiophores, wider ramoconidia and conidia, and

shorter intercalary conidia. Cladosporium scabrellum differs in having mainly

macronematous, wider conidiophores, and wider secondary ramoconidia.

Compared to the other species studied by Lee et al. (2012), C. perangustum

showed medium discoloration on Pinus densiflora and Pinus radiata.

Cladosporium species discoloring wood (Korea) ... 25

Fic. 3. Cladosporium ramotenellum (KUC3027) grown in 9 cm diam Petri dishes for 14 d at 25°C.

A: PDA. B: MEA. C: OA.

Cladosporium ramotenellum K. Schub., Zalar, Crous & U. Braun, Stud. Mycol. 58:

137. 2007. FIGS 3-4

Mycelium internal and superficial, 1.5-4 um wide, septate, without

swellings and constrictions, hyaline or subhyaline, smooth. Conidiophores

macronematous and micronematous, arising terminally or laterally from

branches of plagiotropous hyphae, straight or slightly flexuous, oblong or

cylindrical, unbranched, sometimes branches often only as short lateral

prolongations, 15-117 x 2-3.5 um, septate, pale olivaceous-brown or brown,

smooth to verruculose. Conidiogenous cells integrated, terminal, sometimes

also intercalary, cylindrical, 15-49 um long, proliferation sympodial, with

few conidiogenous loci, mostly 1-3, loci sometimes situated on small lateral

prolongations. Ramoconidia cylindrical-oblong, up to 46.5 um long, 2-3.5

um wide, usually 0-1-septate, pale olivaceous, smooth or verruculose, with a

broadly truncate base, secondary ramoconidia subcylindrical to cylindrical-

oblong, 12.5-22 x 2-4 um (av. + SD: 18 + 4 x 3.1 + 0.5), pale olivaceous,

apex broadly rounded or slightly attenuated towards apex and base. Conidia

numerous, catenate, in branched chains, branching in all directions, small

terminal conidia numerous, 1-3 conidia in the terminal unbranched part of

Fic. 4. Cladosporium ramotenellum (KUC3027). A, B. Macro- and micronematous conidiophore.

C. Ramoconidia and conidia. Scale bars = 10 um.

26 ... Jang & al.

the chain, globose, subglobose or ovoid, obovoid, 3.5-7 x 2-3 um (av. + SD: 4.3

+ 0.7 x 2.5 + 0.3), intercalary conidia ellipsoid to subcylindrical, or limoniform,

4-11 x 2.5-4(-4.5) um (av. + SD: 6.6 + 1.6 x 3 + 0.3).

CULTURE CHARACTERISTICS Colonies on PDA reaching 41-47 mm diam

after 14 d at 25°C, olive due to abundant sporulation, margins entire, white,

glabrous, aerial mycelium absent or sparse, growth flat with a somewhat folded

and wrinkled colony center, no exudates, sporulation profuse. Colonieson MEA

reaching 46-49 mm diam after 14 d at 25°C, olive, velvety, greyish green to dark

green in reverse, margins entire, colorless, glabrous to feathery, aerial mycelium

sparse, diffuse, growth flat with slightly elevated colony center, prominent

exudates not formed, abundantly sporulating. Colonies on OA attaining 39-41

mm diam after 14 d at 25°C, olivaceous, margin entire, colorless or white, aerial

mycelium sparse, growth flat, without exudates, sporulation profuse.

SPECIMEN EXAMINED: KOREA, on the surface of Chromated Copper Arsenate (CCA)-

treated wood product (Pinus radiata), 2003 (KUC3027; GenBank JN033464, JN033491,

JN033519).

REMARKS — The colony morphology and microscopic features of our isolate

agreed well with the description of C. ramotenellum (Schubert et al. 2007),

although it has smaller intercalary conidia and secondary ramoconidia.

The fungus previously has been reported only from Slovenia. According

to Schubert et al. (2007), C. ramotenellum resembles C. cladosporioides

(Fresen.) G.A. de Vries and C. tenellum K. Schub. et al. but differs from C.

cladosporioides by having narrower conidiophores and conidia. Cladosporium

tenellum possesses conidiophores with numerous conidiogenous loci, and

shorter and wider conidia. Also, C. ramotenellum grows faster in culture than

C. tenellum. Cladosporium ramotenellum showed a discoloration rate similar

to C. perangustum on Pinus densiflora, but a little bit more prominent than

C. perangustum on Pinus radiata (Lee et al. 2012).

Phylogeny

The resulting alignment contained 36 taxa (including the outgroup taxon)

and 481, 139 and 201 characters (including alignment gaps) were used in

the ITS, ACT and TEF partitions, respectively. The model used for ITS was

SYM+G model. For ACT and TEE, HKY+I+G model and GTR+G model were

used, respectively. For the Bayesian analysis, 10,705 trees were obtained from

which the consensus tree and posterior probabilities were calculated (Fic. 5).

The species relationships in the tree were sometimes not as well resolved as

in previous studies, or resolved differently (Bensch et al. 2010, Bensch et al.

2012). The phylogenetic analysis revealed that Cladosporium perangustum

isolates, KUC1462, KUC1767 and KUC5085 were clustered within the

C. perangustum species clade with a high posterior probability value (1.0

Cladosporium species discoloring wood (Korea) ... 27

Cladosporium perangustum KUC1767 (JN033468)

Cladosporium perangustum KUC1462 (JN033481)

Cladosporium perangustum CBS 125996 (HM148121)

Cladosporium perangustum CPC 13870 (HM148142)

1p Cladosporium perangustum CPC 11046 (HM148125)

Cladosporium perangustum KUC5085 (JN033460)

Cladosporium exasperatum CBS 125986 (HM148090)

1f Cladosporium chalastosporoides CBS 125985 (HM148001)

Cladosporium hillianum CBS 125988 (HM148097)

Cladosporium scabrellum CBS 126358 (HM148195)

Cladosporium phyllophilum CPC 13873 (HM148155)

Cladosporium licheniphilum CBS 125990 (HM148111)

Cladosporium varians CBS 126362 (HM148224)

4 Cladosporium chubutense CBS 124457 (FJ936158)

Cladosporium colombiae CBS 274.80B (FJ936159)

Cladosporium pini-ponderosae CBS 124456 (FJ936160)

Cladosporium asperulatum CBS 126340 (HM147998)

Cladosporium myrtacearum CBS 126349 (HM148116)

Cladosporium funiculosum CBS 122128 (HM148093)

Cladosporium pseudocladosporioides CPC 14992 (HM148192)

Cladosporium australiense CBS 125984 (HM147999)

ip Cladosporium cucumerinum CBS 176.54 (HM148078)

Cladosporium subuliforme CBS 126500 (HM148196)

Cladosporium xylophilum CPC 14364 (HM148234)

Cladosporium delicatulum CPC 14363 (HM148088)

Cladosporium inversicolor CPC 14368 (HM148109)

Cladosporium lycoperdinum CBS 274.80C (HM148114)

Cladosporium basiinflatum CBS 822.84 (HM148000)

0.81

0.67,

0.59

0.61., | Cladosporium ramotenellum CPC 12047 (EF679385)

Cladosporium ramotenellum KUC3027 (JN033464)

Cladosporium ramotenellum CPC 12043 (EF679384)

Cladosporium spinulosum CBS 102044 (EF679387)

Cladosporium iridis CBS 138.40 (EF679370)

Cladosporium ossifragi CBS 843.91 (EF679382)

Cercospora beticola CPC 11557 (AY840527)

Fic. 5. 50% majority-rule consensus tree of 10,575 trees resulting from a bayesian analysis of 36

sequences in a combined ITS, ACT, and TEF alignment. Bayesian posterior probabilities 250%

are shown. The tree was rooted to sequences of Cercospora beticola strain CPC 11557. GenBank

Accession numbers of ITS sequences are shown in parentheses.

28 ... Jang & al.

p-p.). In our analysis, C. perangustum was sister to C. exasperatum Bensch et

al., a result differing substantially from the analysis of Bensch et al. (2010). In

our analysis, Cladosporium ramotenellum KUC3027 was monophyletic with

C. ramotenellum CPC 12047, but not with C. ramotenellum CPC 12043 and

CBS 170.54. Also the monophyly of C. ramotenellum KUC3027 and CPC

12047 was weakly supported (61% posterior probability value). Nevertheless,

the cultural and microscopic observations of our C. ramotenellum KUC3027

were consistent with the descriptions of C. ramotenellum (Schubert et al. 2007;

Bensch et al. 2010).

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 grateful to Dr. Pedro Crous and Dr. FM. Dugan for their valuable

suggestions on the manuscript.

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H-D, Dugan FM, Schroers H-J, Braun U, Crous PW. 2010. Species and ecological diversity

within the Cladosporium cladosporioides complex (Davidiellaceae, Capnodiales). Stud. Mycol.

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1-401. http://dx.doi.org/10.3114/sim0003

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1-103.

Katoh K, Toh H. 2008. Recent developments in the MAFFT multiple sequence alignment program.

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herbarum in Korea. Mycobiology 29: 110-112.

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of major molds inhabiting woods. Part 3. Genus Cladosporium. Holzforschung 66: 537-541.

http://dx.doi.org/10.1515/hf.2011.184

Maddison D, Maddison W. 2005. MacClade 4: Analysis of phylogeny and character evolution.

Version 4.08. Sinauer Associates, Sunderland, MA, USA.

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Korea. Kor. J. Mycol. 14: 1-8.

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Paul NC, Yu SH. 2008. Two species of endophytic Cladosporium in pine trees in Korea. Mycobiology

36: 211-216. http://dx.doi.org/10.4489/MYCO.2008.36.4.211

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PW. 2007. Biodiversity in the Cladosporium herbarum complex (Davidiellaceae, Capnodiales),

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MYCOTAXON

http://dx.doi.org/10.5248/124.31

Volume 124, pp. 31-37 April-June 2013

Phialophora avicenniae sp. nov., anew endophytic fungus

in Avicennia marina in China

YUE-LIAN Liu*?, PING-GEN XI, XIAO-LAN HE! & ZI-DE JIANG"

‘College of Natural Resources and Environment, South China Agricultural University,

Guangzhou 510642, China

Agricultural College, Guangdong Ocean University, Zhanjiang 524088, China

*CORRESPONDING AUTHOR: Zdjiang@scau.edu.cn

ABSTRACT — Three isolates of an endophytic fungus were obtained from Avicennia marina.

Morphological and molecular evidence indicates that they are identical and represent a new

species of Phialophora, described here as P. avicenniae. The new species is characterized by

brown colonies, branched conidiophores, and catenulate conidia.

Key worps — Herpotrichiellaceae, ITS sequence, taxonomy

Introduction

The morphologically vaguely delimited hyphomycete genus Phialophora

Medlar is characterized by dark and slow-growing colonies, pigmented

hyphae, solitary or aggregated conidiophores, and flask-shaped phialides

usually with a flaring collarette. The type of Phialophora is P. verrucosa Medlar,

diagnosed by its slow-growing dark olivaceous colonies and usually pigmented

phialides bearing an even darker collarette (Medlar 1915). Phialophora has

been regarded as highly polyphyletic, comprising anamorphs of discomycetes,

pyrenomycetes, and loculoascomycetes (Gams 2000). Gams & Holubova-

Jechova (1976) distinguished sect. Catenulatae with catenate conidia having a

truncate base. Some members of this section and other phialophora-like species

with catenulate conidia, such as Brachyalara straminea Réblova & W. Gams,

Exochalara longissima (Grove) W. Gams & Hol.-Jech., and Infundichalara

microchona (W. Gams) Réblova & W. Gams, have been segregated from

Phialophora by molecular phylogeny analyses and morphological characters

(Bogale et al. 2010; Réblova et al. 2011). Phialophora s. str. is recognized as a

member of the Herpotrichiellaceae (Untereiner et al. 1995).

32 ... Liu & al.

We obtained three isolates of an endophytic fungus from twigs of Avicennia

marina (Forssk.) Vierh. (Acanthaceae) in Zhanjiang, Guangdong Province,

China, in 2007. The taxonomic status of these isolates was determined using

morphological and molecular methods, showing them to represent a new

Phialophora species. The isolates are stored in the laboratory of the Agricultural

College, Guangdong Ocean University, and subcultures at CBS in Utrecht, the

Netherlands.

Materials & methods

Isolates

Healthy twigs (0.4-0.8 cm diam.) of Avicennia marina were thoroughly washed in

running tap water and then sterilized by washing in 75% ethanol for 2 min and 0.1%

mercuric chloride (vw) for 2 min. The twigs were rinsed 3 times in sterile distilled water

and then cut into pieces (0.5 cm in length). The small pieces were evenly spaced in

Petri dishes (9 cm diam) containing a medium (Arnold et al. 2000) consisting of (per

litre distilled water) dextrose 20 g, peptone 10 g and agar 20 g, with addition of 60 mg

streptomycin before pouring the plates. The Petri dishes were incubated at 27°C. Hyphal

tips were transferred to potato dextrose agar (PDA) dishes.

Morphological examination

Three cultures of every isolate grown on PDA plates were investigated for colony

characteristics. Thirty conidia were measured under an OLtympus-BX51 microscope.

Scanning electron microscopy (SEM) was used to observe phialide morphology.

Specimens were flash-frozen (—196°C) in liquid nitrogen under vacuum for cryo-SEM,

transferred to the preparation chamber, and then to the SEM chamber where ice particles

were sublimated (—80°C). Samples were sputter-coated with E-1010 in the preparation

chamber for 75 sec under 2.0 KV at -170°C. Specimens were viewed under 5 KV at

-188°C with a Puitips SL30 scanning electron microscope.

DNA extraction, PCR amplification, DNA sequencing, and phylogenetic analyses

After the isolates were grown and incubated on PDA plates at 27°C for 10 days, the

mycelia were scraped off. Genomic DNA was extracted from the mycelia using SDS

procedure (Lee & Taylor 1990) with a few modifications. The quality and quantity

of DNA were visually assessed by staining with ethidium bromide in 1% agarose gel

electrophoresis. The partial ITS sequences were amplified according to Geiser et al.

(2005) using primers ITS1 and ITS4 (White et al. 1990) in TP600 (TaKaRa PCR Thermal

Cycler Dice). Shanghai Invitrogen Biotechnology Co., Ltd. sequenced the DNA. The ITS

sequences of the isolates were aligned using MEGA version 4.1 (Tamura et al. 2007)

and manually optimized to ensure positional homology. Gaps were considered as

missing data. Seventeen sequences of Phialophora and one sequence of an unidentified

ascomycotan species were downloaded from GenBank, and Capronia semiimmersa

(Herpotrichiellaceae) was chosen as outgroup. A phylogenetic tree was constructed using

the neighbor-joining method of MEGA version 4.1 (bootstrapped 1000 replicates).

Phialophora avicenniae sp. nov. (China) ... 33

Taxonomy

Phialophora avicenniae Yue L. Liu & Z.D. Jiang, sp. nov. Fras. 1-2

MycoBank MB561173

Differs from Phialophora clavispora and P. olivacea by its branching conidiophores and

endophytic habit and from P oxyspora by its ovoid to globose conidia and endophytic

habit.

Type: China, Guangdong Province, Zhanjiang, endophytic in twig of Avicennia marina,

18 April 2007, Liu Yuelian Q37 (Holotype, CBS 130286 [lyophilized culture]; GenBank,

GQ500118).

EryMo.ocy: named after the host genus, Avicennia.

COLONIES initially yeast-like, gradually becoming cottony, brown, reaching 2-3

cm diam. after 5 days at 27°C on PDA plates; reaching 9 cm diam. after 14

Figure 1. Phialophora avicenniae (CBS 1302876). a. Colony; b-f. Conidiophores; g. Phialides;

h-l. Conidial chains. Scale bars: b-f, h-] = 10 um; g = 2 um.

34 ... Liu & al.

FiGuRE 2. Phialophora avicenniae. Conidiophore and conidia. Bar = 10 um.

days and changing from brown to black-brown. HyPHAE hyaline to subhyaline,

smooth; occasionally brown, verrucose, 1.5-3.0 um wide. CONIDIOPHORES

hyaline or brown, branched, rarely simple, septate, smooth, and variable in size.

PHIALIDES usually aggregated on conidiophores, flask-shaped or cylindrical,

hyaline or brown, smooth and thick-walled, 6.5-11 x 1.8-3.5 um. Collarette

somewhat darker than the phialide body, funnel-shaped or flaring, 0.5-1.0 x

0.3-0.5 um. Conip14 catenulate, hyaline or brown, 1-celled, smooth, ovoid to

globose, slightly apiculate at the base, 1.6-2.5 x 1.8-3.5 um. CHLAMYDOSPORES

absent.

TELEOMORPH: unknown

Hasirat: endophytic in twigs of Avicennia marina.

ADDITIONAL CULTURES EXAMINED: CHINA. GUANGDONG PROVINCE, Zhanjiang,

endophytic in twigs of Avicennia marina, 10 August 2007, Liu Yuelian Q44 (CBS 130287;

GenBank, HM055753); Liu Yuelian Q48 (CBS 130288; GenBank, HM055754).

Phialophora avicenniae sp. nov. (China) ... 35

Notes: Phialophora verrucosa, the type of Phialophora, differs from the new

species by olivaceous-black colonies and broadly ellipsoidal conidia adhering

in slimy heads. The new species resembles P. clavispora W. Gams, P. olivacea

W. Gams, and P. oxyspora W. Gams by its catenulate conidia. However,

P. clavispora and P. olivacea have simple conidiophores, and P oxyspora has

fusiform conidia (Gams & Holubova-Jechova 1976). In addition, the new species

is endophytic, while the other three species are so far only known as saprobes.

Phylogenetic analyses

ITS rDNA sequences obtained from the isolates of CBS 130286, CBS

130287, and CBS 130288 were submitted to GenBank and received the

P. olivacea AB190379

P. reptans EUS14699

P. europaea EF540756

P. sessilis EUS14700

54 P. oxyspora AB190870 clade |

@ CBS 130286 GQ500118.1

@ CBS 130287 HM055753.2

98 | @ CBS 130288 HM055754.1

54 Ascomycota sp. FJ037728

P. japonica AB190378

@ P. verrucosa DQ404353

P. clavispora AB190868

P. chrysanthemi AB190387

8 | en P. intermedia AB190399

P. cyclaminis AB190390

so P. geniculata AB190395

1004 P. alba AB190382 ciate I

53 P. atrovirens AB190383

100 P. foetens AB190394

57 P. cinerescens AB190388

91®© P. mustea JN1I23359

Capronia semiimmersa FJ225726

0.1

FiGuRE 3. Phylogenetic tree based on neighbor-joining of ITS sequences, showing the relationships

among Phialophora avicenniae and related species.

36 ... Liu & al.

accession numbers GQ500118, HM055753, and HM055754. BLast (Basic

Local Alignment Search Tool) analysis showed that the ITS sequences are 99%

similar to an unidentified strain labeled “Ascomycota sp. GX6-1C” (GenBank

FJ037728; isolated as an endophyte from an unknown mangrove host in

China) and 79-87% similar to various GenBank isolates of Phialophora spp.

A neighbor-joining analysis shows the relationships of the three strains with

17 other species of Phialophora (Fic. 3). The phylogram also shows that the

unidentified strain forms a monophyletic clade (bootstrap 98%) with the three

P. avicenniae strains, and is probably conspecific.

The Phialophora species shown here form two distinct clades (clade I and

clade II) where P. avicenniae is part of clade I (bootstrap 54%), showing a sister

relationship with P olivacea, P. reptans, P. sessilis, P europaea, and P. oxyspora.

Acknowledgements

We express our sincere thanks to Prof. W. Gams who revised the manuscript and

deposited the strains in CBS. We also thank Prof. Yi-Jian Yao (Key Laboratory of

Systematic Mycology and Lichenology, Institute of Microbiology, Chinese Academy of

Sciences) for reviewing the manuscript. The authors are grateful to Dr. Martina Réblova

(Department of Taxonomy, Institute of Botany, Academy of Sciences, Prihonice, Czech

Republic) for providing valuable suggestions. This study was supported by the National

Science Foundation of China (No. 30870015).

Literature cited

Arnold E, Maynard Z, Gilbert G, Coley PD, Kursar TA. 2000. Are tropical fungal endophytes

hyperdiverse. Ecology Letters 3: 267-274. http://dx.doi.org/10.1046/j.1461-0248.2000.00159.x

Bogale M, Orr MJ, O'Hara MJ, Untereiner WA. 2010. Systematics of Catenulifera (anamorphic

Hyaloscyphaceae) with an assessment of the phylogenetic position of Phialophora hyalina.

Fungal Biology 114: 396-409. http://dx.doi.org/10.1016/j.funbio.2010.02.006

Gams W. 2000. Phialophora and some similar morphologically little-differentiated anamorphs of

divergent ascomycetes. Studies in Mycology 45: 187-199.

Gams W, Holubova-Jechova V. 1976. Chloridium and some other dematiaceous hyphomycetes

growing on decaying wood. Studies in Mycology 13: 62-99.

Geiser DM, Lewis-Ivey ML, Hakiza G, Juba JH, Miller SA. 2005. Gibberella xylarioides

(anamorph Fusarium xylarioides), a causative agent of coffee wilt disease in Africa, is a

previously unrecognized member of the G. fujikuroi species complex. Mycologia 97: 191-201.

http://dx.doi.org/10.3852/mycologia.97.1.191

Lee SB, Taylor JW. 1990. Isolation of DNA from fungal mycelia and single spores. 283-287, in: MA

Innis et al. (eds), PCR protocols: a guide to methods and applications. Academic Press, New

York.

Medlar EM. 1915. A new fungus, Phialophora verrucosa, pathogenic for man. Mycologia 7:

200-203. http://dx.doi.org/10.2307/3753363

Réblova M, Gams W, Stépanek V. 2011. Thenewhyphomycete genera Brachyalara and Infundichalara,

the similar Exochalara and species of Phialophora sect. Catenulatae (Leotiomycetes). Fungal

Diversity 46: 67-86. http://dx.doi.org/10.1007/s13225-010-0077-6

Phialophora avicenniae sp. nov. (China) ... 37

Tamura K, Dudley J, Nei M, Kumar S. 2007. MEGA4: Molecular Evolutionary Genetics

Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24: 1596-1599.

http://dx.doi.org/10.1093/molbev/msm092

Untereiner WA, Straus NA, Malloch D. 1995. A molecular-morphotaxonomic approach to

the systematics of the Herpotrichiellaceae and allied black yeasts. Mycological Research 99:

897-913.

White TJ, Bruns T, Lee S, Taylor JW. 1990. Amplification and direct sequencing of fungal ribosomal

RNA genes for phylogenetics. 315-322, in: MA Innis et al. (eds). PCR protocols; a guide to

methods and applications. Academic Press, New York, USA.

MY COTAXON

http://dx.doi.org/10.5248/124.39

Volume 124, pp. 39-45 April-June 2013

Ramichloridium strelitziae associated with sooty blotch and

flyspeck on Ravenala madagascariensis in China

BAojJUN HAo*?, WENHUAN LI**?, CHEN CHEN’, Liu GAo’, RONG ZHANG’,

BAOTONG WANG', GUANGYU SUN" & MARK L. GLEASON?

'State Key Laboratory of Crop Stress Biology in Arid Areas and College of Plant Protection,

Northwest A&F University, Yangling, Shaanxi, 712100, P. R. China

*Department of Plant Pathology, Iowa State University, Ames, Iowa 50011, USA

College of Life Science, Northwest A&F University, Yangling, Shaanxi, 712100, P. R. China

“CORRESPONDENCE TO: sgy@nwsuaf.edu.cn

ABSTRACT — ‘The first report of Ramichloridium strelitziae from China is documented. In

a survey of host plants for SBFS fungi, we isolated the fungus from colonies on stems of

Ravenala madagascariensis collected from Haikou, Hainan, China. It is distinguished from

the other species in the genus by morphological characters and phylogenetic analysis based

on ITS sequences.

KEY worps — microfungi, Capnodiales, taxonomy, phylogeny, traveller's palm

Introduction

Sooty blotch and flyspeck (SBFS) are epiphytes that colonize the waxy

cuticle of a wide range of plants in humid regions worldwide. SBFS fungi can

result in a black or sooty appearance leading to cosmetic damage that can cause

significant economic losses (Batzer et al. 2005).

For approximately 70 years, SBFS was viewed as two distinct diseases, each

caused by a single species of fungus. Recently, this concept was revised by

combining morphological characterization with genetic analysis. Presently,

it is evident that SBFS includes a spectrum of intergraded mycelial types and

comprises more than 80 species (Batzer et al. 2008, Frank et al. 2010, Gleason

et al. 2011, Liet al. 2012, Yang et al. 2010).

In this study, we identified one isolate that was described as the first record

of R. strelitziae on stems of Ravenala madagascariensis Sonn. (Strelitziaceae;

traveller's palm) from China based on morphological comparison and

phylogenetic relationships.

# = Joint first authors

AO ... Hao &al.

Materials & methods

Isolates

Ravenala madagascariensis stems with flyspeck signs (Gleason et al. 2011) were

found in Jinniuling Park in Haikou city, Hainan Province (20°00'26"N 110°20'31"E),

China, in October 2011. Thalli on the stem were transferred directly from colonies to

potato dextrose agar (PDA) slants in a sterile environment and cultured at 25°C for 1

month in darkness (Sun et al. 2003). Hyphal tips were then transferred to malt extract

agar (MEA) plates. The isolates were allowed to grow onto an adjacent, sterile cover slip

that had been partially inserted into the agar surface at a 60° angle in order to measure

and observe fungal structures (Li et al. 2011). Microscopic examination was made

after 14 d of incubation. Thirty measurements per relevant microscopic structure were

gathered where possible. Colony descriptions (surface and reverse) were made after 2

weeks of growth on MEA plates at 25+1°C in the dark. The isolate acronym HKLRJ-4

was a temporary laboratory number. Representative dried culture and plant specimens

were deposited in the Fungal Herbarium of Northwest A&F University (HMUABO),

Yangling, Shaanxi Province, China.

DNA extraction, PCR, and phylogenetic analysis

Genomic DNA for polymerase chain reaction (PCR) was obtained according to the

protocol of Li et al. (2011). The primer pair ITS1-F and ITS4 was used to amplify the

internal transcribed spacer (ITS) region of nuclear ribosomal DNA. The PCR reactions

were carried out with Taq polymerase, 1x PCR buffer, 2 mM MgCl, 0.2 mM of each

dNTP, 0.4 uM of each primer, and 2 ul of template DNA, and was made up to a total

volume of 25 ul with sterile water. Reactions were performed on a Bio-Rad PCR System

S1000TM Thermal Cycler. The amplification conditions were: initial denaturation at

94°C for 90 min followed by 35 cycles of denaturation at 94°C for 35 s, annealing at 52°C

for 60 s, extension at 72°C for 1 min, and a final extension step at 72°C for 10 min. The

PCR products were sequenced by Sangon Biotech Co., Shanghai, China.

The ITS nucleotide sequence generated in this study was added to other sequences

with high homology as the result of a BLAST search. Cladosporium bruhnei was used

as the outgroup taxon. Sequences were imported into BioEdit 5.0.9.1 (Hall 1999).

Preliminary alignments were performed using CLUSTAL-X (Thompson et al. 1997),

then manually adjusted. Phylogenetic analysis of aligned DNA sequences was carried

out with PAUP version 4.0b 10 for 32-bit Microsoft Windows (Swofford 2003). Heuristic

searches were performed with 1,000 random sequence additions. Clade stability was

assessed by 1,000 bootstrap replications. Other measures calculated for parsimony

analysis included tree length, consistency index (CI), retention index (RI), rescaled

consistency index (RC) and homoplasy index (HI).

The ITS sequence described in this study is deposited in GenBank as JX502176,

and the alignment and representative tree were deposited in TreeBase (http://purl.org/

phylo/treebase/phylows/study/TB2:S13803).

Results

Phylogenetic analyses

The ITS alignment contained 29 taxa (including the outgroup) and 520

characters including alignment gaps. Of these characters, 266 were constant,

Ramichloridium strelitziae, new for China... 41

62 were variable and parsimony-uninformative, and 192 were parsimony-

informative. One of the 10 equally most parsimonious trees saved from the

maximum parsimony analysis is shown in Fic. 1. From the most parsimonious

tree, two major clades were resolved. One clade, with 100% bootstrap support,

contained fifteen species in Ramichloridium sensu stricto, Uwebraunia,

Dissoconium, and Pseudoveronaea (all in Dissoconiaceae). The other major clade

55 | Ramichloridium apiculatum EU041794

99 | R. mali EF627452

541 'R cucurbitae JQ622087

100

Ramichloridium

R. luteum CPC 18962

R. punctatum CPC 18974

Uwebraunia musae CBS 122454

U. australiensis EF394854

100 Uwebraunia

U. commune DQ302948

U. dekkeri EU851919

Dissoconium aciculare CPC 10805

68 F- D. eucalypti CBS 120039

100 Dissoconium

100) LD. aciculare AF 173308

D. proteae CBS 122900

100 [~ _Pseudoveronaea ellipsoidea CPC 18970

Pseudoveronaea

Pseudoveronaea obclavata CPC 18972

gf Zasmidium angulare JQ622088

Z. nocoxi CBS 125009

541 __ Ramichloridium cerophilum AF050286

Z. xenoparkii CBS 111089

PA Z. lonicericola CPC 11671

go] 2 citri CPC 10522

95} © Z. anthuriicola CBS 118742 Mycosphaerellaceae

100 Z. scaevolicola HM122240

1007 Ramichloridium streliziae CBS 121711

$3 HKLRJ-4

R. musae EU041801

R. biverticillatum EU041796

40 R. australiense EU041795

Cladosporium bruhnei DQ780401

Fic. 1. One of 10 equally parsimonious trees determined from ITS sequences. Bootstrap support

values (>50%) based on 1000 replicates are shown at the node. (CI = 0.6611, RI = 0.8657,

RC = 0.5723, HI = 0.3389). The scale bar shows 10 changes. The tree is rooted to Cladosporium

bruhnei and the new sequence is presented in bold.

42 ... Hao &al.

had a bootstrap value of 100% including twelve species in Ramichloridium sensu

lato and Zasmidium (all in Mycosphaerellaceae). Our isolate (HKLRJ-4) grouped

with Ramichloridium strelitziae (Mycosphaerellaceae) with 100% bootstrap

support, indicating that they might represent the same species.

Morphology and culture characteristics

Ramichloridium strelitziae Arzanlou, W. Gams & Crous, Stud. Mycol. 58: 74. 2007.

Fic. 2

MYcELIvM consisting of smooth, thin-walled, septate, verrucose, pale brown

hyphae, 2-3 um wide. CONIDIOPHORES erect, solitary, arising from creeping

aerial hyphae, subhyaline, later becoming pale brown, thick-walled, 1-3(-4)-

septate, smooth or verruculose, 2 um wide, up to 43 um long. CONIDIOGENOUS

cells terminally integrated, (8-)11-38 um long, subhyaline, later turning

pale brown, fertile part as wide as the basal part, sympodially proliferating,

forming a straight rachis; Scars thickened and darkened, approx. 0.5 um diam.

ConipiA (3.5-)4-5 x (1-)2(-3) um, solitary, aseptate, subhyaline, smooth or

verruculose, oblong, ellipsoidal to clavate, base truncate with unthickened,

non-pigmented hilum.

CULTURAL CHARACTERISTICS — CoLony diameter after 14 d on MEA at

24°C reached 6 mm diam with entire margin and compact, raised, dense aerial

mycelium, surface olivaceous-grey, reverse olivaceous-black.

Host CHARACTERISTICS — On stem surface, shiny, black, sclerotium-like

bodies, round to oval (120-220 um diam) with no visible mycelial mat, densely

arranged (3-5/mm_°). The flyspeck symptom on Ravenala madagascariensis did

not physically damage the plants, but greatly reduced their ornamental value.

Based on phylogenetic analysis and morphological characters of the anamorph,

we identified isolate HKLRJ-4 as Ramichloridium strelitziae.

SPECIMEN EXAMINED: CHINA. HAINAN PROVINCE, Haikou City, Jinniuling Park,

20°00'26"N 110°20'31”E, on stems of Ravenala madagascariensis, Oct 2011, Li WH

(HMUABO HKLRJ-4; GenBank, JX502176).

Discussion

The modern circumscription of Ramichloridium is still heterogenous,

containing species in two _ different families (Dissoconiaceae and

Mycosphaerellaceae), and only species clustering in Capnodiales were

considered to be true Ramichloridium (Arzanlou et al. 2007). According to

Li et al. (2012) and Arzanlou et al. (2007), R. apiculatum (the generic type),

R. cucurbitae, R. indicum, R. luteum, R. mali, and R. punctatum (Ramichloridium

s. str.) clustered with Dissoconium species (all now placed in Dissoconiaceae),

and other Ramichloridium s. lat. species grouped in Mycosphaerellaceae. Our

strain clustered among the mycosphaerellacean species with R. strelitziae.

Ramichloridium strelitziae, new for China... 43

Fic. 2. Ramichloridium strelitziae (HKLRJ-4). A. Signs on stem of Ravenala madagascariensis

with close-up view. B. Colony on MEA after 14 days. C-E Conidiophores. G-H. Conidia. Bars:

C-F = 10um; G-H = 5um.

Arzanlou et al. (2007) reported Ramichloridium strelitziae from leaves of

Strelitzia nicolai in South Africa. The species has also been found in bathrooms

and washing machines and is able to degrade surfactants, soap, and shampoo

(Hamada & Abe 2010). Our results show that R. strelitziae may also be involved

in the sooty blotch and flyspeck complex.

Acknowledgments

This work was supported by National Natural Science Foundation of China

(31170015, 31171797), the 111 Project from Education Ministry of China (B07049), Top

Talent Project of Northwest A&F University and the earmarked fund for Modern Agro-

industry Technology Research System (nycytx-08-04-04). The authors wish to thank

Dr Eric H.C. McKenzie (Landcare Research, Private Bag 92170, Auckland, New Zealand)

and Professor Zhongyi Zhang (College of Plant Protection, Yunnan Agricultural

University, Kunming, Yunnan, China) for reviewing the manuscript.

Literature cited

Arzanlou M, Groenewald JZ, Gams W, Braun U, Shin H-D, Crous PW. 2007. Phylogenetic and

morphotaxonomic revision of Ramichloridium and allied genera. Studies in Mycology 58:

57-93. http://dx.doi.org/10.3114/sim.2007.58.03

44 ... Hao &al.

Batzer JC, Gleason ML, Harrington TC, Tiffany LH. 2005. Expansion of the sooty blotch and flyspeck

complex on apples based on analysis of ribosomal DNA gene sequences and morphology.

Mycologia 97: 1268-1286. http://dx.doi.org/10.3852/mycologia.97.6.1268

Batzer JC, Diaz Arias MM, Harrington TC, Gleason ML, Groenewald JZ, Crous PW. 2008. Four

species of Zygophiala (Schizothyriaceae, Capnodiales) are associated with the sooty blotch and

flyspeck complex on apple. Mycologia 100: 246-258.

http://dx.doi.org/10.3852/mycologia.100.2.246

Frank J, Crous PW, Groenewald JZ, Oertel B, Hyde KD, Phengsintham P, Schroers H-J. 2010.

Microcyclospora and Microcyclosporella: novel genera accommodating epiphytic fungi causing

sooty blotch on apple. Persoonia 24: 93-105. http://dx.doi.org/10.3767/003158510X510560

Gleason ML, Batzer JC, Sun GY, Zhang R, Diaz Arias MM, Sutton TB, Crous PW, Ivanovic

M, McManus PS, Cooley DR, Mayr U, Weber RWS, Yoder KS, Del Ponte EM, Biggs AR,

Oertel B. 2011. A new view of sooty blotch and flyspeck. Plant Disease 95: 368-383.

http://dx.doi.org/10.1094/PDIS-08- 10-0590

Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program

for Windows 95/98/N'TT. Nucleic Acids Symposium Series 41: 95-98.

Hamada N, Abe N. 2010. Growth characteristics of four fungal species in bathrooms. Biocontrol

Science 15: 111-115. http://dx.doi.org/10.4265/bio.15.111

Li HY, Sun GY, Batzer JC, Crous PW, Groenewald JZ, Karakaya A, Gleason ML. 2011. Scleroramularia

gen. nov. associated with sooty blotch and flyspeck of apple and pawpaw from the Northern

Hemisphere. Fungal Diversity 46: 53-66. http://dx.doi.org/10.1007/s13225-010-0074-9

Li HY, Sun GY, Zhai XR, Batzer JC, Mayfield DA, Crous PW, Groenewald JZ, Gleason ML. 2012.

Dissoconiaceae associated with sooty blotch and flyspeck on fruits in China and the United

States. Persoonia 28: 113-125. http://dx.doi.org/10.3767/003158512X651157

Sun GY, Zhang R, Zhang Z, Zhang M. 2003. Isolation of sooty blotch and flyspeck fungi from apple

surface by picking up the thalli. Acta Phytopathologica Sinica 33: 479-480.

Swofford DL. 2003. PAUP*. Phylogenetic analysis using parsimony (* and other methods). Version

4.0. Sinauer Associates, Sunderland, Massachusetts, USA.

Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997. The Clustal_X windows

interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.

Nucleic Acids Research 25: 4876-4882. http://dx.doi.org/10.1093/nar/25.24.4876

Yang HL, Sun GY, Batzer JC, Crous PW, Groenewald JZ, Gleason ML. 2010. Novel fungal genera

and species associated with the sooty blotch and flyspeck complex on apple in China and the

USA. Persoonia 24: 29-37. http://dx.doi.org/10.3767/003158510X492101

MY COTAXON

http://dx.doi.org/10.5248/124.45

Volume 124, pp. 45-50 April-June 2013

Polycoccum anatolicum sp. nov. on Lepraria incana

and a key to Polycoccum species known from Turkey

MEHMET GOKHAN HAtici’, HATICE Esra AKGUL’,

CELALEDDIN OzTURK? & EMRE KILIc?

' University of Erciyes, Faculty of Science, Department of Biology 38039 Kayseri, Turkey

* University of Selcuk, Faculty of Science, Department of Biology 42075 Konya, Turkey

* CORRESPONDENCE TO: mghalici@gmail.com

ABSTRACT — Polycoccum anatolicum is described as new from the thallus of Lepraria incana

on the trunk of a Prunus sp. in western Turkey and represents the first Polycoccum known to

occur on Lepraria. The new species is typical with its relatively small subglobose ascomata

and verruculose relatively large ascospores. It is compared with similar Polycoccum species,

and a key to Polycoccum species known from Turkey is provided.

Key worps — Ascomycota, lichens, lichenicolous fungi, biodiversity

Introduction

Polycoccum Saut. ex Korb. comprises approximately 50 species of

lichenicolous fungi (Halici et al. 2007a, 2009; Brackel & Berger 2010; Zhurbenko

2010). The type species of the genus is P sauteri Kérb. with lichenicolous

habit on Stereocaulon condensatum Hoftm. Species are characterized by dark

perithecioid ascomata, pseudoparenchymatous exciple, fissitunicate asci with

brown one-septate ascospores, and a hamathecium of persistent, branched

and anastomosing interascal filaments. Following the genus synopsis by

Hawksworth & Diederich (1988), Atienza et al. (2003) recognized 37 species.

Brackel & Berger (2010) described three Polycoccum species occurring on the

lichenised fungus Placopsis.

Attention to the lichenicolous fungi of Turkey has increased since Halici

(2008) published the key to the lichenicolous Ascomycota. Of the 181

lichenicolous species known from Turkey (Halici et al. 2012), ten Polycoccum

species have been identified (Halic1 2008, Halici et al. 2009; Yazici et al. 2011). In

2012, the second author (EA) collected an interesting specimen of Polycoccum

on epiphytic Lepraria incana (L.) Ach. s. lat. from Turkey’s Mediterranean

46 ... Halici & al

Region. After comparison with the described species of the genus, we concluded

that the specimen represents a new species, which we describe here. We also

provide a key to the eleven Polycoccum species now reported from Turkey.

Material & methods

The type of the new species is deposited in Erciyes University Herbarium Kayseri,

Turkey (EUH). Sections were prepared by hand and examined in I [Lugol's iodine

(MERCK 9261) with (KI) and without (I) pre-treatment with 10% KOH], 10% KOH,

and water. Ascospores were measured in water. Ascospore measurements are given as:

(min -) X-sd - X - X+sd (-max.), where ‘min’ and ‘max’ are the extreme values, ‘X’

the arithmetic mean, and ‘sd’ the corresponding standard deviation. The length/breadth

ratio of ascospore is indicated as 1/b and given in the same way. The microphotographs

were taken with a Leica DFC 420 digital microscope camera with a c-mount interface

and with a 5 megapixel CCD. The descriptive notes provided in the key are based on

Turkish specimens examined by the first author (MGH), except for Polycoccum evae,

which is based on the description given by Calatayud & Rico (1995).

Taxonomy

Polycoccum anatolicum Halici & E. Akgiil, sp. nov. FIGURE 1

MycoBank MB804154

Differs from Polycoccum dzieduszyckii by its 8-spored asci and its Lepraria host.

Type: Turkey, Isparta, Siitciiler, Yazili Canyon, 41°48’N 36°31’E, alt. 290 m, on Lepraria

incana on Prunus sp., 18 July 2012, E.Akgiil (Holotype, EUH (MGH 0.3118)).

Erymo.oey: The epithet refers to Anatolia, the Asian part of Turkey, where the type

specimen was collected.

Vegetative hyphae hyaline, scant, very narrow, the infection causing no

visible necrosis in host, but eventually bleaching the infected parts. Ascomata

perithecioid, subglobose, arising singly, in the early stage immersed with only

the ostiole area visible, semi-immersed at maturity, 75-90 um. Ascomatal wall

dark reddish brown, darkest in the upper half, 10—15 um thick, and thickening

in the ostiole region; composed of several layers of compressed cells, 10-15

x 6-8 um, forming a textura angularis. Hamathecium consisting of septate,

branched and anastomosing pseudoparaphyses, 1-2 um thick. Hymenial

gelatin K/I-, I-. Asci arising from the lower part of the ascomatal cavity,

cylindrical to elongate-clavate, shortly stalked, bitunicate, the apex thickened, 8-

spored, (71—)83—91(—103) x (11—)14—18(—21) um (n = 10); wall I-, endoplasm

dextrinoid. Ascospores monostichously arranged in the asci, sole-shaped, apices

rounded, some tending to taper towards the rounded ends, 1-septate, not or

slightly constricted at the septum, upper cell slightly larger, brown, verruculose,

non-halonate, some ascospores multi-guttulate, (25—)26—28—29.5(-32) x

(8.5—)9-10-11(-13) um (n = 30), I/b = (2.2—)2.52—2.76-3(—3.3). Conidiomata

not seen.

Polycoccum anatolicum sp. nov. (Turkey) ... 47

ioe

00pm —|

L S00 pm

Fic. 1. Polycoccum anatolicum (Holotype): A, Habitus; B, Ascoma (in water); C, Ascospores

(in water); D, Asci showing monostichously arranged ascospores (in water); E, Anastomosed

pseudoparaphyses (in methylene blue).

A8 ... Halici & al

ECOLOGY & DISTRIBUTION: Polycoccum anatolicum is currently known

only from the type locality in the Western Mediterranean part of Turkey. The

new species was collected on Lepraria incana on the trunks of Prunus sp. The

species seems to be weakly pathogenic as moderate bleaching was observed in

the infected parts of the host thallus. As the host lichen has a wide distribution,

P. anatolicum may also have a wide distribution.

Notes: Most Polycoccum species are confined to one host species or genus,

and P. anatolicum is the only species known to occur on Lepraria. It is closest

to P. dzieduszyckii (Boberski) D. Hawksw., a species known only on Verrucaria

spp. (and probably other lichens on limestone). Polycoccum dzieduszyckii has

small ascomata (50-100 um) and large verruculose ascospores (25—35(—44) x

8—10(—14) um) (Hawksworth & Diederich 1988, Halici et al. 2007b), but clearly

differs from P. anatolicum in having consistently 2-spored asci and infecting a

different host genus.

Polycoccum opulentum (Th. Fr. & Almgq.) Arnold, a species known on

Polyblastia intercedens (Nyl.) Lonnr., Lecidea plana (J. Lahm) Nyl., and a

sterile lichen on calcareous rocks, has similar sized ascospores (25-28 x

11-14 um) but thicker pseudoparaphyses (3 um), larger ascomata (100-170

um), and hymenial gelatin turning blue in iodine (Hawksworth & Diederich

1988; Atienza et al. 2003). Polycoccum squamarioides (Mudd) Arnold, another

species known on Placopsis spp., has similar sized ascomata (75-100 um) but

narrower ((18—)19-—26 x (5.5—)6—7(—8) um) and smooth-walled ascospores

(Hawksworth & Diederich 1988, Brackel & Berger 2010). Polycoccum evae

Calat. & V.J. Rico is restricted to Dimelaena oreina (Ach.) Norman and has larger

ascomata (100-200 um) and shorter verruculose ascospores ((17—)18—23(—25)

x (6—-)7—10(-11) um) (Calatayud & Rico 1995). Polycoccum crassum Vézda,

a species known on Peltigera spp., has similar sized verruculose ascospores

((25—)30—32(—36) x 8—10(—11) um) but much larger ascomata (300-500 um)

and consistently 4-spored asci (Hawksworth & Diederich 1988; Atienza et al.

2003; Halici et al. 2012).

A key to the Polycoccum species known from Turkey

Mek ASCOSPOLES i OT LOMoee 8 Fhnns Bhat! utattul Mhnsyt Tek mR! etna Leas AL 2

lL. GAscospores S20 Atmos di... a dibete a disnteeg ding ed dintery dente dcnopea’ tin apes diggs Pe)

2 weAscospores'smioothiwalleds .. 034.002 44nctddhed adhe eadead ered den bes 3

2. Ascospores verruculose, 13-14 x 6 um, on Acarospora spp. P. microsticticum

3... “SNOLcatising swellinssion-thehostlichens %. 2.31 d tt het eed he el 4

3. Ascomata c. 200 um, causing swellings on the host lichens.

Ascospores 14—18 x 5—6 um, on Peltigera spp. ........... 0. eee ee P. peltigerae

Polycoccum anatolicum sp. nov. (Turkey) ... 49

4, | Ascomata 100—140 um. Interascal filaments very thin, c. 1.0 um.

Ascospores (11—)12—14.5(—15.5) x (4.5—)5-6 um,

On-Xanthoria sp: ON: COaStall VOCKS 0) ssiew-t teers oracuee ofS athe oa ate tw Bat P. teresum

4, | Ascomata 150-235 um. Interascal filaments thicker. Ascospores

(11—)13.5—15(—16) x 6.5—7 um, on Aspicilia cinerea ................ P. aksoyi

Bim EVSCIBSZSDOEGA Toe Se tee sok eee to eaten ren ee tay Ee eee Neen Neen ee ER 6

5. Asci 2-spored. Ascospores verruculose, 25—28 x 8-11 um,

on endolithic Verrucaria spp. on hard calcareous rocks ....... P. dzieduszyckii

Gimm, ASCISARPOTEd ss kg ea task tees i vleale tele ls + pels § Deals pte Fe OE OG Gt OH ys

6. Asci 4-spored. Ascomata c. 400 um. Hymenial gelatin I+ orange-red.

Ascospore cells + equal in size, verruculose, 24—30 x 8-10 um,

CP PEIISEPAS DIOL ses tod Nes elo ehioer sey hat hy Spotty When Dee Mon bey eede eittinals P. crassum

Tait eh SOON ALA cee LOG) MRT o,f eR ec erat Mel hey Me Me Ay bel aly et al 8

7. Ascomata 75-90 um. Hymenial gelatin I—. Ascospores verruculose,

(25—)26—29.5(—32) x (8.5—)9-11(—13) um, on Lepraria incana ... P. anatolicum

Sepa Vee al alle Fea eet Pattee ARR se, Je Th tcl Bled de lg teil Md eel Wi ssagelt Miacele 9

8. | Ascomata c. 220 um. Hymenial gelatin I+ blue. Ascospores verruculose,

24-27 x 10-13 um, on endolithic Verrucaria spp. ............ P. marmoratum

9. Ascospores verruculose and cells + equal in size ........... 0. ee eee eee eee 10

9. | Ascomata 170-200 um. Ascospores smooth walled and lower cell often

attenuate, 22-23 x 10 um, on Sporastatia testudinea............ P. sporastatiae

10. Ascomata 100-200 um. Ascospores (17—)18—23(—25) x (6—)7—10(—13) pm,

OD TDECLAC TID OP CUIA pseu sot srvtsnbes drstsnben dicts spben-d Wacken drhesekes driep.t-gedhtfebou'y hig boue P. evae

10. Ascomata 180-250 um. Ascospores (25—)28.5—31.5(—34) x (7—)8-9.5 um,

Ol, ACATOSPOTO:CETVING Strate, Bhivarees avs coe timed oe hea or uthhos argent P. acarosporicola

Acknowledgements

The manuscript was reviewed by Kerry Knudsen (USA) and Wolfgang von Brackel

(Germany). This study was financially supported by the Selcuk University Scientific

Research Project Coordinator with the project coded (BAP-12101025).

Literature cited

Atienza V, Calatayud V, Hawksworth DL. 2003. Notes on the genus Polycoccum (Ascomycota,

Dacampiaceae) in Spain, with a key to the species. Lichenologist 35: 125-135.

http://dx.doi.org/10.1016/S0024-2829(03)00014-8

Brackel W, Berger F. 2010. Gall-inducing species of Polycoccum (Ascomycota) on the lichen genus

Placopsis. Herzogia 23: 195-204.

Calatayud V, Rico VJ. 1995. Polycoccum evae (Dothideales), a new lichenicolous fungus on

Dimelaena oreina. Mycotaxon 53: 29-32.

Halici MG. 2008. A key to the lichenicolous Ascomycota (including mitosporic fungi) of Turkey.

Mycotaxon 104: 253-286.

Halici MG, Atienza V, Hawksworth DL. 2007a. Two new Polycoccum species from Turkey.

Mycotaxon 101: 157-163.

50 ... Halici & al

Halici MG, Candan M, Ozdemir Tiirk A. 2007b. New records of lichenicolous and lichenized fungi

from Turkey. Mycotaxon 100: 255-260.

Halic1 MG, Knudsen K, Candan M, Tiirk A. 2009. A new species of Polycoccum (Dothideales,

Dacampiaceae) from Turkey. Nova Hedwigia 89: 431-436.

http://dx.doi.org/10.1127/0029-5035/2009/0089-0431

Halici MG, Candan M, Ozdemir Tiirk A. 2012. A key to the peltigericolous fungi in Turkey.

Mycotaxon 119: 277-289. http://dx.doi.org/10.5248/119.277

Hawksworth DL, Diederich P. 1988. A synopsis of the genus Polycoccum (Dothideales), with a key

to accepted species. Transactions of the British Mycological Society 90: 293-312.

http://dx.doi.org/10.1016/S0007-1536(88)80101-3

Yazic1 K, Etayo J, Aslan A. 2011. A note about lichenicolous fungi from Ardahan (Turkey).

Cryptogamie, Mycologie 32: 429-437.

Zhurbenko MP. 2010. New and interesting lichenicolous fungi from Eurasia. I]. Mycosphere 1:

213-222.

MY COTAXON

http://dx.doi.org/10.5248/124.51

Volume 124, pp. 51-59 April-June 2013

Further additions to the macrolichen mycota of Vietnam

UDENI JAYALAL ', ANDRE APTROOT *, THI THUY NGUYEN ?, NGUYEN ANH

DZUNG 3, SANTOSH JOSHI ', SOON-OK OH ' & JAE-SEOUN Hur **

'Korean Lichen Research Institute, Sunchon National University, Suncheon-540 742, Korea

?ABL Herbarium, Gerrit van der Veenstraat 107, NL-3762 XK Soest, The Netherlands

Biotechnology Center, Tay Nguyen University, 567 Le Duan, Buon Ma Thuot City, Vietnam

“CORRESPONDENCE TO: jshur1@sunchon.ac.kr

ABSTRACT — Seven new records of foliose lichens are reported from Chu Yang Sin

National Park of Dak Lak province in Vietnam. Morphological, anatomical and chemical

characteristics are described for Heterodermia obscurata, Hypotrachyna flavida, Leptogium

ulvaceum, Parmotrema sancti-angelii, Parmeliella brisbanensis, Physma byrsaeum, and Sticta

marginifera.

Key worps — tropical, macrolichens, Parmeliaceae, Da Lat Plateau

Introduction

Although larger organisms are well known in the tropics, the fungi and their

lichenized counterparts in tropical forests are less known (Coppins & Wolseley

2002). Aptroot & Sipman (1997) observed that the tropical forest lichens

represent about one-third to one-half of the world’s lichen diversity. Vietnam,

being a moderately large country with the monsoon tropical climate (VARCC

2009), provides a range of conditions to support lichen growth (Nguyen et al.

2010, 2011).

Although the lichen mycota of Vietnam is underworked, recently many

species have been reported or described. Aptroot & Sparrius (2006) provided

the first checklist of Vietnam lichens, which has been followed by new records

of macrolichens by Giao (2009), some findings on foliicolous lichens by Nguyen

et al. (2010, 2011), and reports of some Graphidaceae species by Joshi et al.

(2012).

The Korean Lichen Research Institute (KoLRI) has initiated collaboration

with Tay Nguyen University in Vietnam, which has resulted in the collection

of many lichen specimens, which have been deposited in the herbarium of the

KoLRI. This paper reports seven taxa (in five lichen families) from Chu Yang

Sin National Park, Vietnam.

52 ... Jayalal & al.

Materials & methods

Chu Yang Sin National Park is located in the central highlands of Vietnam in Krong

Bong and Lak Districts of Dak Lak Province. This park is situated within the elevations

ranging from less than 600 m to 2442 m at the summit of Mount Chu Yang Sin with the

area of 58,947 ha. The Park is the largest protected area on the Da Lat Plateau, in the

central highlands (Hughes 2010).

The dominant vegetation type in the Park is broadleaved evergreen forest, covering

over 65% of the area. This forest is dominated by the vascular plant families Fagaceae,

Lauraceae, Meliaceae, and IIliciaceae with a canopy height often in excess of 35 m. The

high altitudinal range and varied topography gives rise to high vascular plant diversity

in different forest types. Montane and submontane forests are at >900 m and lowland

semi evergreen forests at <900 m (Hughes 2010).

Morphological and anatomical investigations were performed under a binocular

dissecting (NIKON SMZ645) and light microscope (Zeiss Scope. Al). Measurements of

all the microscopic features were made on hand-cut thallus and apothecial sections. The

sections were examined by mounting in water, 10% KOH, and Lugol’s iodine solutions.

Ascospore measurements were determined in water. Chemical constituents were identified

by spot tests and thin layer chromatography performed in solvent systems A (toluene:

dioxin:acetic acid 180:45:5) and C (toluene:acetic acid 85:15) (Orange et al. 2010).

Taxonomic descriptions

Heterodermia obscurata (Nyl.) Trevis., Nuovo Gior. Bot. Ital. 1: 114 (1869) Fic.14

Thallus foliose, suborbicular, adnate, 2-6 cm across; lobes narrow, up to 1-2

mm wide, flat; upper surface whitish gray, shiny, epruinose, sorediate; soredia

granular, labriform to capitate on lateral and terminal lobes; lower cortex

absent; lower surface (medulla) felted with yellowish-brown pigment, K+

purple, marginally rhizinate; rhizines black, simple to squarrosely branched;

apothecia not seen.

CHEMISTRY — Cortex K+ (yellow), C-, KC-, P+ (pale yellow); medulla K-,

C-, KC-, P-. TLC: atranorin, chloroatranorin, zeorin.

EcoLoGcy & DISTRIBUTION — Found on sandy rock in broad-leaved

evergreen forest in Chu Yang Sin National Park. According to Moberg & Nash

(2001), this species is growing on tree trunk and mossy rocks in open humid

conditions. Mostly cosmopolitan and has been reported from many countries

in Africa, Asia, Europe, North America, South America, and Oceania (Aptroot

& Feijen 2002, Calvelo & Liberatore 2002, Egea 1996, Eliasaro & Adler 1997,

Elix & McCarthy 1998, Fryday et al. 2001, Gowan & Brodo 1988, Hafellner &

Tiirk 2001, Knezevic & Mayrhofer 2009, Kurokawa 1960, Llimona & Hladun

2001, Lépez—Figueiras 1986, Osorio 1992, Singh & Sinha 2010, Sipman 1993,

Streimann 1986, Tenorio et al. 2002, Wei 1991).

SPECIMEN EXAMINED: VIETNAM. Dak LAK PROVINCE: Chu Yang Sin National Park,

on rock, 12°27'57.0"N 108°20'34.9"E, elev. c. 780 m, 20.04.2012, S.O. Oh, J.S. Hur

VN120185 (KoLRI).

Macrolichens newly recorded for Vietnam ... 53

REMARKS — Heterodermia obscurata (Physciaceae) is characterized by its

robust appearance, labiate soredia, lack of lower cortex, and the rusty-brown

pigmented (K+ purple) lower surface or medulla. According to Moberg & Nash

(2001), this species is closely related to H. speciosa (Wulfen) Trevis., which

differs in having a lower cortex and in lacking yellowish pigmentation. It is also

easily confused with H. japonica (M. Sat6) Swinscow & Krog, which has no

lower cortex and which is white to violet.

Hypotrachyna flavida (Zahlbr.) Hale, Smithson. Contr. Bot. 25: 37 (1975) Fic. 1B

Thallus adnate to loosely attached, coriaceous, green, 4-6 cm across; lobes

sublinear, separate, 2-4 mm wide, margins eciliate; upper surface greenish grey,

emaculate, plane to convex, continuous or irregularly cracked on older lobes;

medulla white; lower surface moderately rhizinate; rhizines long, sparsely

dichotomously branched, often projecting beyond the lobe margins; apothecia

adnate, 1-3 mm in diameter, disc brown, flat to concave; Asci clavate, 8 spored,

spores colour less, 6-8 um; pycnidia not seen.

CHEMISTRY — Cortex K-, C-, KC-, P-; medulla K+ (pale yellowish to

brown), C-, KC-, P+ (orange-red). TLC: usnic acid, protocetraric acid.

EcoLoGy & DISTRIBUTION — Found on a sandy rock in broad-leaved evergreen

forest in Chu Yang Sin National Park. Reported as saxicolous species at somewhat higher

elevations by Hale (1975). This species has previously been reported from Central and

South America (Calvelo & Liberatore 2002, Eliasaro & Adler 1997, Hale 1975, Riddle

1920, Tenorio et al. 2002).

SPECIMEN EXAMINED: VIETNAM. DAK LAK PROVINCE: Chu Yang Sin National Park,

on rock, 12°28'12.3"N 108°20'59.9”E, elev. c. 763 m, 20.04.2012, S.O. Oh, J.S. Hur

VN120120 (KoLRI).

REMARKS — Hypotrachyna flavida (Parmeliaceae) is characterized by a

saxicolous habit and a medulla containing usnic acid and protocetraric acid.

According to Hale (1975), H. flavida is fairly restricted to northern South

America. Externally, this species very closely resembles H. protoboliviana

(Hale) Hale, which differs in its corticolous habitat and medulla containing

barbatic acid.

Leptogium ulvaceum (Pers.) Vain., Ann. Acad. Sci. Fenn., Ser. A, 15(6): 38

(1921) FIG. 1C

Thallus foliose, loosely adnate, 4-8 cm across, bluish gray; lobes rotund,

2-8 mm wide, homiomerous, margins entire, surface smooth to somewhat

rough; isidia, phyllidia absent; lower surface smooth, etomentose, pale bluish

with sparse tufted rhizines; apothecia laminal, shortly pedicellate, 0.5-2

mm diam., disc flat to concave, red brown, exciple smooth to wrinkled, cells

paraplectenchymatous and continuous to below the hypothecium; ascospores

54 ... Jayalal & al.

ellipsoid, muriform, 30-40 x 12-18 um, apices acute to elongate; pycnidia

submarginal; conidia 2-3 um long.

CHEMISTRY — No chemical detected.

ECOLOGY & DiIsTRIBUTION — Found on a sandy rock in a shady place in

broad-leaved evergreen forest in Chu Yang Sin National Park. This species has

previously been reported from Asia, Europe, South America, and Oceania

(Awasthi 1988, Elix & McCarthy 1998, Hafellner 1995, Marcelli 1991).

SPECIMEN EXAMINED: VIETNAM. DAK LAK PROVINCE: Chu Yang Sin National Park,

on rock, 12°26'51.9"N 108°20'18.1"E, elev. c. 816 m, 21.04.2012, S.O. Oh, J.S. Hur

VN120170 (KoLRI).

REMARKS — Leptogium ulvaceum (Collemataceae) is characterized by smooth

upper surface lacking vegetative propagules. This species is morphologically

similar to L. cochleatum (Dicks.) P.M. Jorg. & P. James, which differs in its

periclinally wrinkled thalline exciple.

Parmeliella brisbanensis (C. Knight) P.M. Jorg. & D.J. Galloway, Flora of Australia,

54: 314 (1992) FIG. 1D

Thallus rosette-forming, orbicular, 5-7 cm across, ona thick, black prothallus

projecting 1-2 mm beyond lobe apices, closely appressed to substratum; lobes

narrow, 0.6-1 mm wide, discrete at margins, flat; margins entire, somewhat

thickened, occasionally ascending; upper surface, slightly maculate, smooth,

occasionally striate, grayish when wet, whitish gray when dry, isidiate; isidia

delicate, simple to finger-like, to 0.5 mm tall and 0.1 mm thick, laminal and

marginal; medulla white to blackish, photobiont Nostoc; apothecia, 0.5-2 mm

wide, rounded to contorted; exciples thick, thalline, persistent, crenulate-

striate, concolorous with thallus; disc flat to concave, red-brown; ascospores

simple, ellipsoidal, 10-15 x 8-10 um; pycnidia not seen

CHEMISTRY — No chemical detected.

ECOLOGY & DISTRIBUTION — Found on a sandy rock in a shady place in

broad-leaved evergreen forest in Chu Yang Sin National Park. According to

Jorgensen and Galloway (1992), Parmeliella brisbanensis also occurs on tree

bark in tropical to subtropical rain forests. This species has been recorded from

many countries in the tropics, in Asia, Australia, and Oceania (Elix & McCarthy

1998, Jorgensen & Galloway 1992, Singh & Sinha 2010, Sipman 1993, Wolseley

et al. 2002).

SPECIMEN EXAMINED: VIETNAM. DAK LAK PROVINCE: Chu Yang Sin National Park,

on rock, 12°28'04.4"N 108°20'39.0"E, elev. c. 622 m, 20.04.2012, S. O. Oh, J. S. Hur

VN120046 (KoLRI).

REMARKS: Parmeliella brisbanensis (Pannariaceae) is characterized by its rosette

form, black hypothallus, and finger-like isidiate upper surface. According to

Jorgensen & Galloway (1992), this species is the isidiate counterpart of the

P. mariana (Fr.) P.M. Jorg. & D.J. Galloway complex.

Macrolichens newly recorded for Vietnam ... 55

FiGurE 1: External morphology of Vietnamese lichen specimens (Scale bars = 1 cm). A. Heterodermia

obscurata VN120185 (KoLRI); B. Hypotrachyna flavida VN120120 (KoLRI); C. Leptogium ulvaceum

VN120170 (KoLRI); D. Parmeliella brisbanensis VN120046 (KoLRI); E. Parmotrema sancti-angelii

VN120288 (KoLRI); F. Physma byrsaeum VN120107 (KoLRI); G. Sticta marginifera VN120053

(KoLRI).

Parmotrema sancti-angelii (Lynge) Hale, Phytologia 28: 339 (1974) FIG. 1E

Thallus loosely attached to the substratum, up to 6 cm across; lobes rotund,

4-10 mm wide; margins crinate, ciliate; cilia numerous, simple, 2-4 mm long;

upper surface pale grey, emaculate, smooth, sorediate; soralia marginal, soredia

granular; medulla white, yellowish brown pigments below; lower surface, with

56 ... Jayalal & al.

relatively narrow, smooth, erhizinate marginal zone; rhizines sparse, simple;

apothecia and pycnidia not seen.

CHEMISTRY — Cortex K+ (yellow), C-, KC-, P-; medulla K- C+ (rose),

KC+ (red), P-. TLC: atranorin, chloroatranorin, gyrophoric acid and unknown

compound.

EcoLoGy & DISTRIBUTION — Found on a sandy rock in an open place in

broad-leaved evergreen forest in Chu Yang Sin National Park. A common and

widespread species in the pantropical region (Elix 1994), it has been reported

from Asia, Australia, South America, and Oceania (Calvelo & Liberatore 2002,

Divakar & Upreti 2005, Elix 1994, Elix & McCarthy 1998, Elix & Schumm 2001,

Hale 1974, Kurokawa 1993, Wei 1991, Wolseley et al 2002).

SPECIMEN EXAMINED: VIETNAM. DAK LAK PROVINCE: Chu Yang Sin National Park,

on rock, 12°28'04.4"N 108°20'39.0"E, elev. c. 622 m, 20.04.2012, S.O. Oh, J.S. Hur-

VN120288 (KoLRI).

REMARKS — Parmotrema sancti-angelii (Parmeliaceae) is characterized by an

emaculate upper surface, sorediate margins, yellowish lower medulla, and

brown to black lower surface. This species is very close to P indicum Hale,

which differs in having norlobaridone and in lacking pigmentation on the

lower medulla.

Physma byrsaeum (Ach.) Tuck., Syn. N. Amer. Lich. 1: 115 (1882) FIG. 1F

Thallus foliose, rosulate, adnate, 6-8 cm across, 200-350 um thick; lobes

radiating, oblong 1-5 mm wide; margins entire, thickened, re-curved at the

lobes end; upper surface ridged, somewhat wrinkled, brownish black, lacking

isidia; cortex thin, photobiont Nostoc; lower surface pale brown, rhizinate;

rhizines of interwoven hyphae forming a cushion-like indumentum, whitish

to black; ascomata apothecial, abundant, laminal, sessile, 1-4 mm wide; disc

concave to plane, reddish brown; thalline exciple thick, wrinkled, lobed,

concolorous with thallus; ascospores simple, ellipsoidal, 12-15 x 10-12 um;

episporium, 2-3 um thick; pycnidia laminal, conidia 2-3 um long.

CHEMISTRY — No chemicals detected.

EcoLoGy & DISTRIBUTION — Found on sandy rock in a shady place in

broad-leaved evergreen forest in Chu Yang Sin National Park. Physma byrsaeum

has been reported from many countries in the paleotropics, including Asia,

Australia, Central America, and Oceania (Aptroot & Seaward 1999, Aptroot &

Sipman 1991, Elix & McCarthy 1998, Sipman 1993, Tenorio et al. 2002, Verdon

1992, Wolseley et al. 2002).

SPECIMEN EXAMINED: VIETNAM. Dak LAK PROVINCE: Chu Yang Sin National Park,

on rock, 12°28'12.3"N 108°20'59.9"E, elev. c. 763 m, 20.04.2012, S.O. Oh, J.S. Hur

VN120107 (KoLRI).

REMARKS — Physma byrsaeum (Collemataceae) is characterized by a well-

developed cortical layer and wrinkled thalline apothecial exciple. This species

Macrolichens newly recorded for Vietnam ... 57

is morphologically similar to P pseudoisidiatum Aptroot & Sipman, which is

isidiate.

Sticta marginifera Mont., Annls Sci. Nat., Bot., Sér. 2 18: 265 (1842) FIG. 1G

Thallus palmate to irregularly branched in clumps, 1-2 cm across, arising

from a rooted holdfast; lobes flabellate to irregularly dichotomously branching,

1-3 mm wide, proliferating round to oblong lobules attached to primary lobes

by thin terete stalks; margins entire or irregularly notched, thickened below;

upper surface grayish glaucous when dry, dark blue-black when wet, thin,

papery, smooth, rather fragile, isidiate, phyllidiate and minutely maculate;

isidia marginal, sometimes on the upper surface, styliform to coralloid, to 0.5

mm tall; phyllidia marginal, developing from isidia, to 1 mm tall, irregularly

lobed; photobiont Nostoc; lower surface white to pale at margins, brown

centrally, minutely pubescent; cyphellae scattered, round, to 0.5 mm diam.; pit

membrane white; apothecia not seen.

CHEMISTRY — No chemicals detected.

EcoOLoGy & DisTRIBUTION — Found on sandy rock in a shady place in

broad-leaved evergreen forest in Chu Yang Sin National Park. According to

Galloway (2001), Sticta marginifera prefers humid, deeply shaded habitats in

montane rain forests on different substrates. This species has been recorded

from Asia, Australia, South America, and Oceania (Awasthi 2007, Calvelo &

Liberatore 2002, Elix & McCarthy 1998, Galloway 2001, Streimann 1986).

SPECIMEN EXAMINED: VIETNAM. Dak LAK PROVINCE: Chu Yang Sin National Park,

on rock, 12°28'04.4"N 108°20'39.0"E, elev. c. 622 m, 20.04.2012, S.O. Oh, J.S. Hur

VN120053 (KoLRI).

REMARKS — Sticta marginifera (Lobariaceae) is characterized by the presence

of secondary lobules and the coralloid isidia. According to Galloway (2001),

the similar S. brevipes (Mull. Arg.) Zahlbr. also has similar flabellate lobes with

thickened and revolute apices but does not produce secondary lobules.

Acknowledgements

This work was supported by a grant from the National Research Foundation of Korea

(#2011-0031494) and the Korea National Research Resource Center Program. Authors

are grateful to Ms. Pat Wolseley and Dr. Sanjeeva Nayaka for their valuable comments

on manuscript.

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MY COTAXON

http://dx.doi.org/10.5248/124.61

Volume 124, pp. 61-68 April-June 2013

Inonotus niveomarginatus and I. tenuissimus spp. nov.

(Hymenochaetales), resupinate species from tropical China

Hatr-You Yu, CHANG-LIN ZHAO & YU-CHENG Dat

Institute of Microbiology, P.O. Box 61, Beijing Forestry University, Beijing 100083, China

* CORRESPONDENCE TO: yuchengd@yahoo.com

ABSTRACT — Two new species are described from tropical Yunnan, southwestern China.

Inonotus niveomarginatus is characterized by resupinate basidiocarps with distinct white

margins, a monomitic hyphal structure, absence of hymenial and hyphoid setae, and

yellowish, thick-walled, ovoid to subglobose basidiospores. Inonotus tenuissimus can be

distinguished from other Inonotus species by the combination of resupinate habit, a mono- to

dimitic hyphal structure, absence of the hymenial and hyphoid setae, and yellowish thick-

walled ellipsoid basidiospores.

Key worps — Hymenochaetaceae, polypore, taxonomy, wood-inhabiting fungi

Introduction

Yunnan Province in southwestern China is one of the biodiversity “hotspots”

in the world and rich with many vascular plants and fungi. Many wood-

decaying fungi have been described from this province (Chen & Cui 2012; Cui

et al. 2009; Dai 2010, 2011; Dai et al. 2002, 2009; He & Dai 2012; Li & Cui

2010, 2013; Yuan & Dai 2008; Zhao et al. 2013). During a systematic survey

of Inonotus P. Karst. species in southwestern China, two resupinate species

were found that lacked hymenial and hyphoid setae and possessed yellowish

thick-walled ovoid to subglobose or ellipsoid basidiospores. These prominent

characters distinguish the two species from all other known Inonotus species,

and they are described here as new.

Materials & methods

The studied specimens were deposited at the herbarium of Institute of Microbiology,

Beijing Forestry University (BJFC). The microscopic procedures were follows as in Cui

& Decock (2013). Sections were studied at magnifications up to 1000x using a Nikon

Eclipse 80i microscope with phase contrast illumination. Drawings were made with the

aid of a drawing tube. Microscopic features, measurements, and illustrations were made

62 ... Yu, Zhao & Dai

from the slide preparations stained with Cotton Blue and Melzer’s reagents. Spores were

measured from sections cut from the tubes. To present the spore size variation, the 5%

of measurements excluded from each end of the range are given in the parentheses;

spine lengths are not included in the basidiospore measurements. Abbreviations include

IKI = Melzer’s reagent, IKI- = negative in Melzer’s reagent, 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, and n = number of spores

measured from the given number of specimens. Special color terms follow Petersen

(1996).

Taxonomy

Inonotus niveomarginatus H.Y. Yu, C.L. Zhao & Y.C. Dai, sp. nov. Fics 1-2

MycoBank MB805435

Differs from other Inonotus species by its resupinate growth habit, absence of hymenial

and hyphoid setae, and yellowish, thick-walled, ovoid to subglobose basidiospores.

Type: China, Yunnan Province, Xishuangbanna, Jinghong County, Sanchahe Nature

Reserve, on fallen angiosperm trunk, 7.VI.2011, Dai 12318 (holotype, BJFC).

ETYMOLOGY: niveomarginatus (Lat.): refers to the white margin of the basidiocarp.

FruitBopy — Basidiocarps annual, resupinate, cushion-shaped, adnate,

without odor or taste when fresh, becoming corky upon drying, <4 cm long, 2.5

cm wide, 4 mm thick at centre. Pore surface deep olive and margin white when

fresh, dark brown to yellowish brown upon drying; pores circular, 6-8 per mm;

dissepiments thin, entire. Subiculum brown, corky, <1 mm thick. Tubes dull

brown to blackish brown, corky, <3 mm long.

HyPHAL STRUCTURE — Hyphal system monomitic; generative hyphae

simple septate; tissues darkening in KOH.

SUBICULUM — Generative hyphae yellow, thin- to thick-walled with a

wide lumen, frequently septate, very rarely branched, more or less straight,

interwoven, 3-4.5 um in diam.

TuBEs — Generative hyphae yellowish, thin- to thick-walled, frequently

branched and septate, more or less straight, subparallel along the tubes, 2.5-3.5

um in diam. Basidia barrel-shaped, with four sterigmata and a basal simple

septum, 13-16 x 6-8.5 um; basidioles in shape similar to basidia, but slightly

smaller than basidia.

SpoRES — Basidiospores ovoid to subglobose, yellowish, thick-walled,

smooth, occasionally bearing a guttule, IKI-, CB-, (4.5-)4.9-5.7(-6) x (4.2-)

4.5-5.2(-5.5) um, L = 5.35 um, W = 4.95 um, Q = 1.06 (n = 30/1).

REMARKS — Four other Inonotus species — I. costaricensis Ryvarden, I. rigidus

B.K. Cui & Y.C. Dai, I. truncatisporus Corner, and I. venezuelicus Ryvarden —

also are characterized by resupinate basidiocarps, thick-walled colored spores,

Inonotus spp. nov. (China) ... 63

FiGure 1. Inonotus niveomarginatus (holotype). Basidiocarp.

and a lack of both hymenial and hyphoid setae (Corner 1991; Cui et al. 2011;

Ryvarden 1987, 2005). Inonotus costaricensis differs from I. niveomarginatus by

having globose and longer basidiospores (5.5-6.5 um long; Ryvarden 1987).

Inonotus rigidus differs from I. niveomarginatus by its ellipsoid and smaller

64 ... Yu, Zhao & Dai

———N

Lay)

said | \

10 um

10 pm

FIGURE 2. Inonotus niveomarginatus (holotype) microscopic structures.

a: Basidiospores. b: Basidia and basidioles. c: Hyphae from trama.

d: Hyphae from subiculum.

spores (3.9-4.5 x 2.9-3.7 um; Cui et al. 2011). Inonotus truncatisporus is

distinguished from I. niveomarginatus by its larger pores (4-6 per mm) and

truncate basidiospores (Corner 1991). Inonotus venezuelicus has larger pores

(3-4 per mm), ellipsoid basidiospores (5-6 x 4.5-5 um), and has been found

only in South America (Ryvarden 1987).

Inonotus tenuissimus H.Y. Yu, C.L. Zhao & Y.C. Dai, sp. nov. Figs 3-4

MycoBANnk MB805434

Differs from other Inonotus species by its resupinate basidiocarps, a mono- to dimitic

hyphal structure, absence of hymenial and hyphoid setae, and yellowish, thick-walled,

ellipsoid basidiospores.

Type: China, Yunnan Province, Puer County, Laiyanghe Forest Park, on fallen

angiosperm trunk, 6.V1.2011, Dai 12365 (holotype, BJFC).

ETyMOLOoGy: tenuissimus (Lat.): refers to the very thin subiculum.

Inonotus spp. nov. (China) ... 65

FiGuRE 3. Inonotus tenuissimus (holotype). Basidiocarp.

FRuITBODy — Basidiocarps annual, resupinate, adnate, becoming corky upon

drying, <15 cm long, 7 cm wide, 1.5 mm thick at centre. Pore surface vinaceous

grey to greyish brown when fresh, pale brown upon drying; pores angular, 3-4

per mm; dissepiments thin, entire. Subiculum brown, corky, up to 0.2 mm

thick. Tubes dull brown, corky, <1.3 mm long.

HyPHAL STRUCTURE — Hyphal system mono- to dimitic; generative hyphae,

simple septate; tissues darkening in KOH.

66 ... Yu, Zhao & Dai

<<‘

| mencieniiaael

10 ym

ane

oe)

uae,

Figure 4. Inonotus tenuissimus (holotype) microscopic structures.

a: Basidiospores. b: Basidia and basidioles. c: Hyphae from trama.

d: Hyphae from subiculum.

SUBICULUM — Generative hyphae yellow, thick-walled, frequently septate,

more or less straight, interwoven, 2.5-4.5 um in diam. Some skeletal-like

hyphae similar to generative hyphae, rarely septate.

TuBES — Generative hyphae yellowish, thick-walled, occasionally branched,

rarely septate, subparallel along the tubes, 3-4 um in diam. Some skeletal-like

hyphae similar to generative hyphae, rarely septate. Basidia clavate to barrel-

shaped, with four sterigmata and a basal simple septum, 12-18 x 4.5-6.5 um;

basidioles barrel-shaped, smaller than basidia.

Spores — Basidiospores ellipsoid, yellowish, thick-walled, smooth,

IKI-, CB-, (4-)4.3-5(-5.2) x (3-)3.2-4(-4.2) um, L = 4.8 um, W = 3.6 um,

Q = 1.31-1.38 (n = 90/3).

Inonotus spp. nov. (China) ... 67

ADDITIONAL SPECIMENS EXAMINED: CHINA. YUNNAN PROVINCE, PUER COUNTY,

Laiyanghe Forest Park, on fallen angiosperm branch, 6.VI.2011, Dai 12245 & Dai 12255

(BJFC); 9.VI.2011, Dai 12330 (BJFC).

REMARKS — Inonotus tenuissimus resembles I. venezuelicus in its resupinate

basidiocarps, similar pores, thick-walled and colored spores, and absence of

both hymenial and hyphoid setae. However, I. venezuelicus has greyish pores

when dry, a distinctly monomitic hyphal structure, larger and ellipsoid to ovoid

basidiospores (5-6 x 4.5-5 um), and has been found only in South America

(Ryvarden 2005).

Acknowledgments

We express our gratitude to Dr. Li- Wei Zhou (IFP, China) and Mr. Jaya Seelan Sathiya

Seelan (Clark University, USA) who reviewed the manuscript. The research was financed

by the National Natural Science Foundation of China (Nos. 31070022, 30910103907).

Literature cited

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1-175.

Chen JJ, Cui BK. 2012. Studies on Wrightoporia from China 2. A new species and three new records

from South China. Mycotaxon 121: 333-343. http://dx.doi.org/10.5248/121.333

Cui BK, Decock C. 2013. Phellinus castanopsidis sp. nov. (Hymenochaetaceae) from southern China,

with preliminary phylogeny based on rDNA sequences. Mycological Progress 12: 341-351.

http://dx.doi.org/10.1007/s11557-012-0839-5

Cui BK, Dai YC, Bao HY. 2009. Wood-inhabiting fungi in southern China 3. A new species of

Phellinus (Hymenochaetales) from tropical China. Mycotaxon 110: 125-130.

http://dx.doi.org/10.5248/110.125

Cui BK, Du P, Dai YC. 2011. Three new species of Inonotus (Basidiomycota, Hymenochaetaceae)

from China. Mycological Progress 10: 107-114. http://dx.doi.org/10.1007/s11557-010-0681-6

Dai YC. 2010. Hymenochaetaceae (Basidiomycota) in China. Fungal Diversity 45: 31-343.

http://dx.doi.org/10.1007/s13225-010-0066-9

Dai YC. 2011. A revised checklist of corticioid and hydnoid fungi in China for 2010. Mycoscience

52: 69-79. http://dx.doi.org/10.1007/s10267-010-0068-1

Dai YC, Niemela T, Kinnunen J. 2002. The polypore genera Abundisporus and Perenniporia

(Basidiomycota) in China, with notes on Haploporus. Annales Botanici Fennici 39: 169-182.

Dai YC, Cui BK, Yuan HS. 2009. Trichaptum (Basidiomycota, Hymenochaetales) from China with a

description of three new species. Mycological Progress 8: 281-287.

http://dx.doi.org/10.1007/s11557-009-0598-0

He SH, Dai YC. 2012. Taxonomy and phylogeny of Hymenochaete and allied genera of

Hymenochaetaceae (Basidiomycota) in China. Fungal Diversity 56: 77-93.

http://dx.doi.org/10.1007/s13225-012-0174-9

Li HJ, Cui BK. 2010. A new Trametes species from Southwest China. Mycotaxon 113: 263-267.

http://dx.doi.org/10.5248/113.263

Li HJ, Cui BK. 2013. Two new Daedalea species (Polyporales, Basidiomycota) from South China.

Mycoscience 54: 62-68. http://dx.doi.org/10.1016/j.myc.2012.07.005

Petersen JH. 1996. Farvekort. The Danish Mycological Society’s colour-chart. Foreningen til

Svampekundskabens Fremme, Greve. 6 p.

68 ... Yu, Zhao & Dai

Ryvarden L.1987. New and noteworthy polypores from tropical America. Mycotaxon 38:

525-541.

Ryvarden L. 2005. The genus Inonotus, a synopsis. Synopsis Fungorum 21: 1-149.

Yuan HS, Dai YC. 2008. Polypores from northern and central Yunnan Province, southwestern

China. Sydowia 60: 147-159.

Zhao CL, Cui BK, Dai YC. 2013. New species and phylogeny of Perenniporia based on morphological

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http://dx.doi.org/10.1007/s13225-012-0177-6

MYCOTAXON

http://dx.doi.org/10.5248/124.69

Volume 124, pp. 69-72 April-June 2013

Arenariomyces truncatellus sp. nov., an ascomycete on driftwood

from the north coast of Zealand, Denmark

J. KocuH

GI. Dronninggadrds Allé 12, DK-2840 Holte, Denmark

ABSTRACT — Incubated driftwood from the north coast of Zealand, Denmark, yielded a

new species, Arenariomyces truncatellus, belonging to the Halosphaeriaceae, Ascomycota. The

species is characterized by globose, spiny ascomata formed on wood with asci deliquescing

before spore maturity and 1-septate ascospores with three delicate appendages at each

spore end. The species is compared with A. majusculus, A. parvulus, A. trifurcatus, and

A. triseptatus. A key to the species is provided.

Key worps — higher marine fungi, Microascales, taxonomy

Introduction

Investigations into the incidence and distribution of higher marine fungi

colonizing wood along the Danish coasts were initiated by Héhnk (1955) and

Kohlmeyer (1968). Since then more data have been accumulated and published

in “A check list of higher marine fungi on wood from Danish coasts” (Koch

& Petersen 1996) and further in Petersen (1997), Petersen & Koch (1997a,b),

and Koch et al. (2007). Also added (Koch unpublished) are Corollospora

angusta Nakagiri & Tokura, C. californica Kohlm. & Volkm.-Kohlm., and

C. pseudopulchella Nakagiri & Tokura. In this article, a new species of

Arenariomyces Hohnk (Halosphaeriaceae) is described from the north coast of

Zealand, Denmark.

Material & methods

Driftwood entrapped among stones and wood from the intertidal zone was collected

at Tisvildeleje on the north coast of Zealand, Denmark, in January 2008. Salt content

was around 10-20%. The wood was subsequently incubated at 16-19°C and examined

periodically for fruiting bodies. From the first formed A. truncatellus ascomata,

ascospores were used for inoculation of sterilized pieces of driftwood, on which ascomata

developed over a 4-8 month incubation period at 16-19°C ascomata developed.

70 ... Koch

Arenariomyces truncatellus Jorg. Koch, sp. nov. FIGs 1-5

MycoBAnk 50644

Differs from Arenariomyces majusculus by its smaller ascospores with two cells often

conspicuously different in size and with short delicate appendages.

Type: Denmark, North Zealand, Tisvildeleje (UTM 691000E 6217000N), from

driftwood (Picea sp.), Jan. 2008, Jorg. Koch 998, incubated nine months in humid

chamber (Holotype, CP [piece of conifer wood + slides 1-4]; isotype, CP [remainder

of collection Jorg. Koch 998]). [The original material also contained Dictyosporium

pelagicum (Linder) G.C. Hughes ex E.B.G. Jones and Halosphaeriopsis mediosetigera

(Cribb & W. Cribb) T.W. Johnson. ]

EryMo oey: From the Latin truncatellus = somewhat truncate, referring to the slightly

flattened ends of the ascospores, when appendages are not unfolded.

ASCOMATA on wood (rarely seated with subicula on grains of sand) measured

in situ 78-131-156 um (n = 19) in diam. Globose-subglobose with the basal

part immersed in the outermost layer of the substrate and anchored with brown

tortuous hyphae, often in tufts. Ascomata gregarious, black, shiny when young,

carbonaceous, brittle, covered with scattered brownish black spines, slightly

curved, up to 16 um long and 1.5-2 um wide, but sometimes with a swollen

base 4-8 um wide and 4 um high. Immature ascomata from 20 um in diam. are

covered with spines up to 8 um long. Neither necks nor ostioles were observed.

PERIDIUM membranous, single layered, composed of one layer of flat, on the

outside domed, thick-walled cells forming a textura angularis with edges 3-4-6

um, brownish black in transparent light. SustcuLUM composed of hyaline,

epidermoid cells merging into the thin-walled pseudoparenchyma, globose

or ellipsoidal as free cells (10-20-26 um in diam.), with scattered cytoplasm

and small oil globules filling the centre of young ascomata. In some cells a few

cytoplasmic strands stretching towards the cell wall indicate pit connections to

neighbouring cells.

Asci eight-spored, pyriform, 36-56 x 20-32 um, thin-walled, unitunicate,

deliquescing before maturity of the ascospores, developing from the base of the

ascocarp. No catenophyses or paraphyses. ASCOSPORES 18-21.4—24 x 8-10.2-

12 um (n = 66) measured in seawater (12%), cylindrical, 1-septate, more or less

constricted at the septa, often one cell is shorter and narrower than the other,

hyaline, with three subterminal-terminal appendages at each end. APPENDAGES

attenuate, straight or curved, delicate, up to 12 um long, about 1 um wide at the

base, expanded at right angles to the axis, 120° between each appendage. When

not unfolded the spore ends seem slightly flattened.

SUBSTRATE: Driftwood (conifer wood) between stones (intertidal zone).

Discussion

Jones et al. (2009) recognized the four species A. majusculus Kohlm. &

Volkm.-Kohlm., A. parvulus Jorg. Koch, A. trifurcatus Hohnk, and A. triseptatus

Arenariomyces truncatellus sp. nov. (Denmark) ... 71

Tei ry -

| le

- J

~~ ,

Fic. 1. Arenariomyces truncatellus. Group of ascomata formed on sterilized driftwood

inoculated with ascospores. Scale bar = 160 um.

Fics 2-5. Arenariomyces truncatellus. 2. Young asci. 3. Different stages of immature ascospores

forming an eight-spored cluster indicating an early deliquescing of the ascus wall. 4. Three

ascospores, one germinated after 72 hours in sea water (12%). 5. Part of the peridium with spines

and underlying epidermoid flattened cells. Scale bars: 2-4 = 10 um; 5 = 14 um.

Kohlm. in the genus Arenariomyces. The latest, A. truncatellus, is different from

these with respect to ascospore dimensions, the uneven sized spore cells, and the

short and delicate structure of the appendages. A. truncatellus and A. majusculus

ascomata are formed in the outermost part of the wood substrate and bearing

spines in contrast to A. parvulus, A. trifurcatus, and A. triseptatus. However, the

stability of ascomatal characters in the genus is questioned (on wood / on hard

surfaces, spines / no spines). For example, A. trifurcatus ascomata are sometimes

seen with a few spines and also directly on wood (Koch unpublished). Further

observations of the rare Arenariomyces species could perhaps reveal a similar

variability. But general similarities such as the thin-walled pseudoparenchyma

of the young ascomata, unitunicate asci that deliquesce early before ascospore

maturation, together with the three polar appendages point to A. truncatellus

72 ... Koch

as morphologically well accommodated as a new species in Arenariomyces. It

is recognized that examination of A. truncatellus in the electron microscope,

especially in regard to appendage development, and at the molecular level is

needed to establish its relationship to A. trifurcatus, the type species.

Key to Arenariomyces species

ba-Ascomatamain ly On WOO, of Lie. ade. opal webs hd Ss old by wlth Ate ash talade itty 2

1b. Ascomata mainly.on hard stirfaces.¢< ota. sags sae has blame Os a od ee ee 3

2a ASCOSPOLES ASH MLAS «bt AV oikes dy Foweten dy brwcon dying cdog rs poten dri schon dying op A. majusculus

2bunscospores 214 LO 2a tav: ted aloes 2 ota teed seated See ts es A. truncatellus

Day -ASCOSPOLES" SED CALS lay. ots! cot! atest: shade diet ohale do aha late aha lah! aha linia A. triseptatus

SH AASCOsPOresel Se ptate ew peels eiucten Bbube 4 aude y Bouin y Mouide y Meeede natauele wa Ry wal 4

Aad, BSCOSPOLES Ox 29 D0 D= Goll trea oan fe weehanll oa eee ele Ree RHEL aul A. parvulus

Aby AS COSPOLES 2BASZ, 29 = VG UIT aee sg dives eogdhireseg drt scene eeadrh sea gre aes A. trifurcatus

Acknowledgments

Iam grateful to Iben M. Thomsen and Flemming Rune, both at Forest and Landscape

Denmark (Faculty of Life Sciences, University of Copenhagen) for photographic work,

and to Iben M. Thomsen for preparing the manuscript for printing. Also I am grateful

to Dr. E.B. Gareth Jones and Dr. K.L Pang for reviewing the manuscript and giving

valuable comments.

Literature cited

Hoéhnk W. 1955. Studien zir Brack- und Seewassermykologie V. Héhere Pilze des submersen

Holzes. Veréffentlichungen des Instituts fiir Meeresforschung in Bremerhaven 3: 199-227.

Jones EBG, Sakayaroj J, Suetrong S, Somrithipol S, Pang KL. 2009. Classification of marine

Ascomycota, anamorphic taxa and Basidiomycota. Fungal Diversity 35: 1-203.

Koch J, Petersen KLR. 1996. A checklist of higher marine fungi on wood from Danish coasts.

Mycotaxon 60: 397-414.

Koch J, Pang KL, Jones EBG. 2007. Rostrupiella danica gen. et sp. nov. a Lulworthia-like

marine lignicolous species from Denmark and the USA. Botanica Marina 50: 294-301.

http://dx.doi.org/10.1515/BOT.2007.034

Kohlmeyer J. 1968. Danische Meerespilze (Ascomycetes). Berichten der Deutschen Botanische

Gesellschaft 81: 53-61.

Petersen KLR. 1997. Ultrastructural studies of the marine ascomycete Groenhiella bivestia. Botanica

Marina 40: 71-75. http://dx.doi.org/10.1515/botm.1997.40.1-6.71

Petersen KLR, Koch J. 1997a. Substrate preference and vertical zonation of lignicolous marine fungi

on mooring posts of oak (Quercus sp.) and larch (Larix sp.) in Svanemollen harbour, Denmark.

Botanica Marina 40: 451-463. http://dx.doi.org/10.1515/botm.1997.40.1-6.451

Petersen KLR, Koch J. 1997b. Buxetroldia bisaccata gen. et sp. nov., a marine lignicolous

halosphaeriacean fungus from coastal waters, Denmark. Mycological Research 101: 1524-1528.

http://dx.doi.org/10.1017/S0953756297004383

MY COTAXON

http://dx.doi.org/10.5248/124.73

Volume 124, pp. 73-85 April-June 2013

New ascomycete records from Guatemala

ROSARIO MEDEL”*', OSBERTH MORALES’,

RANULFO CASTILLO DEL MORAL! & ROBERTO CACERES?

"Instituto de Investigaciones Forestales, Universidad Veracruzana,

Apartado Postal 551, 91070, Xalapa, Veracruz, México

*Departamento de Microbiologia, Facultad de Ciencias Quimicas y Farmacia,

Universidad de San Carlos de Guatemala, Guatemala

* CORRESPONDENCE TO: medel.rosario@gmail.com OR romedel@uv.mx

ABSTRACT — Thirty-two species of ascomycetes were previously recorded from Guatemala.

This paper describes 12 newly recorded species, representing five orders and twelve

genera. Three previously reported species are redescribed because there are few data in the

Guatemalan literature.

Key worps — Helotiales, Hypocreales, Orbiliales, Pezizales, Xylariales, Central America

Introduction

Guatemala is a country of >108,000 km*. The name Quauhtemallan

(Guatemala) means “land of trees,’ referring to the original wide extent and

diversity of forests. Guatemala has been categorized as biologically mega-

diverse (Tolisano & Lopez 2010), but little is known about the fungal diversity

of this country and adjacent areas. There have been few studies on fungal

diversity, and the knowledge of the ascomycetes is poor; only 33 ascomycetes

species have been reported in previous studies (TABLE 1). The present study is

part of a project that began in 2007 to document the ascomycete diversity of

Guatemala.

Materials & methods

The study was based on a bibliographic revision and additional material collected

mainly from oak forests and cloud forests of the west of Guatemala. The following

literature was used for identifications: Abbot & Currah (1997); Breitenbach & Kranzlin

(1984); Dennis (1954; 1963; 1970; 1978), Glawe & Rogers (1984), Haines & Dumont

(1984); Hsieh et al. (2005); Ju & Rogers (1996); Medel (2002) Medel & Calonge, (2004);

Méndez-Mayboca et al. (2007); Ramamurthi et al. (1957); San Martin et al. (1998), and

7A ... Medel & al.

TABLE 1. Ascomycete species recorded from Guatemala.

(New records in bold font.)

Helotiales

Bisporella citrina This paper

Chlorociboria aeruginascens

(Nyl.) Kanouse ex C.S. Ramamurthi et al. Flores et al. (2012)

C. aeruginosa This paper

Lachnum abnorme This paper

L. brasiliense Haines & Dumont (1984); this paper

L. cyphelloides This paper

L. virgineum This paper

Leotia lubrica (Scop.) Pers. Sommerkamp & Guzman 1990

Trichoglossum farlowii (Cooke) E.J.Durand Flores et al. (2002, 2012)

Hypocreales

Cordyceps melolonthae (Tul. & C. Tul.) Sacc. Flores et al. (2012)

C. militaris (L.) Fr. Flores et al. (2012)

Hypomyces hyalinus (Schwein.) Tul. & C. Tul. Sommerkamp & Guzman (1990)

H. lactifluorum (Schwein.) Tul. & C. Tul. Bran et al. (2003)

Ophiocordyceps gracilis This paper

Orbiliales

Hyalorbilia inflatula This paper

Orbilia juruensis This paper

Pezizales

Aleuria aurantia (Pers.) Fuckel

Cookeina sulcipes (Berk.) Kuntze

Gyromitra infula

Helvella acetabulum

H. crispa (Scop.) Fr.

H. elastica Bull.

H. lacunosa Afzel.

H. macropus (Pers.) P. Karst.

Melastiza chateri (W.G. Sm.) Boud.

Morchella costata (Vent.) Pers.

M. elata Fr.

M. esculenta (L.) Pers.

M. guatemalensis Guzman et al.

Otidea onotica (Pers.) Fuckel

Phillipsia guatemalensis Paden

Pithya cupressina (Batsch) Fuckel

Scutellinia scutellata (L.) Lambotte

Wynnea americana Thaxt.

Xylariales

Annulohypoxylon thouarsianum

Daldinia concentrica

D. fissa Lloyd

D. vernicosa Ces. & De Not.

Diatrypella pulvinata

Phylacia poculiformis (Mont.) Mont.

Poronia pileiformis

Xylaria cubensis (Mont.) Fr.

X. multiplex (Kunze) Fr.

X. polymorpha (Pers.) Grev.

Sommerkamp & Guzman (1990)

Bran et al. (2003): this paper

This paper

Bran et al. (2003)

Sommerkamp & Guzman (1990)

Bran et al. (2003); Sommerkamp & Guzman (1990)

Flores et al. (2002)

Sommerkamp & Guzman, (1990)

Bran et al. (2003)

Sommerkamp & Guzman (1990); Bran et al. (2003)

Guzman et al. (1985)

Flores et al. (2002)

Paden 1977

Sommerkamp & Guzman (1990)

Flores et al (2002; 2012)

Sharp (1948), Flores et al. (2012); this paper

This paper

Morales et al. (2006)

Bran et al. (2003)

This paper

Sommerkamp & Guzman (1990)

This paper

Sharp (1948)

Sommerkamp & Guzman (1990)

Flores et al. (2012)

Ascomycetes from Guatemala ... 75

Spooner (1987). All material studied was deposited in the mycological collection at XAL

herbarium (Instituto de Ecologia, A.C., Veracruz, Mexico) and duplicate specimens

were deposited in mycological collection MICG (Universidad de San Carlos, Facultad

de Ciencias Quimicas, Guatemala, Guatemala). Microscopic features were examined in

KOH 5% and Melzer’s iodine reagent, and at least 20 measurements of ascospores were

made per collection. Photographs were taken with a Sony Cyber Shot Camera, and some

photographs of spores were cleared of spots, stains and bubbles using Photoshop CS5

software.

Helotiales

Bisporella citrina (Batsch) Korf & S.E. Carp., Mycotaxon 1: 58 (1974) Fic. 1

Apothecia discoid to cupulate shaped, attached to the substrate by a short

stalk. Disc 1 mm diameter. Stalk 0.6 mm long, cylindrical. Hymenium bright

yellow and smooth outer surface. Asci cylindrical, 57-80 x 5-7 um, small pore

blue in Melzer’s reagent. Ascospores elliptical, smooth hyaline, with 2 guttules,

with a single septum when mature, 7-10 x 2-3 um. Paraphyses filiform with

rounded tips, 1-1.5 um thick, hyaline, thin walled.

HABITAT — Gregarious, lignicolous; in cloud forest at 1500 m.

MATERIAL EXAMINED — GUATEMALA. CHIMALTENANGO DEPARTMENT, San Andres

Itzapa Municipality, Aldea La Hierbabuena, 27 June 2007, Medel 1451 (XAL).

The bright yellow apothecia and ascospore size are diagnostic for this species.

Bisporella citrina is widely distributed (Dennis 1970, 1978; Breitenbach &

Kranzlin 1984; Medel & Calonge 2004; Méndez-Mayboca et al. 2007). This

represents a new record for Guatemala.

Chlorociboria aeruginosa (Oeder) Seaver ex C.S. Ramamurthi, Korf &

L.R. Batra, Mycologia 49: 859 (1958) [“1957”] Fic. 2

Apothecia globose to discoid shaped, irregularly distorted, 12 mm diameter,

attached to the substrate by a short central stipe; hymenium smooth, blue-

green. Stalk cylindrical, 0.5 mm long. Asci cylindrical, 8-spored, (57—) 70-80

x 5-6 um, small blue pore in Melzer’s reagent. Ascospores irregularly fusiform,

smooth, hyaline, with 2 guttules, 9-15 x 2-3 um. Paraphyses with rounded tips,

1-1.5 um thick.

HABITAT — Gregarious, lignicolous; in Cupressus forest at 1500 m.

MATERIAL EXAMINED — GUATEMALA. GUATEMALA DEPARTMENT, Universidad

de San Carlos de Guatemala, University Campus, Parque Ecoldgico Las Ardillas, 28

June 2007, Medel 1495 (XAL); CHIMALTENANGO DEPARTMENT, San Andrés Itzapa

Municipality, Aldea la Hierbabuena, 27 June 2007, Medel 1446 (XAL, MICG).

According to Dixon (1975) this species is close to C. aeruginascens, which also

has the same green color but which differs in smaller ascospores (6-10 x 1.5-2

um). Chlorociboria aeruginosa is distributed in North, South, and Central

76 ... Medel & al.

America, China, India, Japan, and the Philippines (Dixon 1975) and Venezuela

(Mardones-Hidalgo & Iturriaga 2011). Despite being a widely distributed

species, in Guatemala the species was not known.

Lachnum abnorme (Mont.) J.H. Haines & Dumont, Mycotaxon 19: 10 (1984)

Fics 3-4

Apothecia discoid, with white hairs, stipe very small and sometimes almost

sessile, 1 mm wide x 0.5 mm tall, hymenium light to dark yellow. Hairs sub-

cylindrical with blunt apex, with coarse granules, hyaline, with 2-3 septa, thin

walled, (40-)60-88 x (3-)3.5-4 um. Asci cylindrical, 8-spored (75-)87-94 x

7-8 um, small blue pore in Melzer’s reagent. Ascospores straight to s-shaped,

hyaline, (38-)40-57 x 2 um. Paraphyses lanceolate, (77-)94-102(-110) x 3-3.5

um, hyaline, 3-4-septate.

HaBITAT — Gregarious, lignicolous; in Cupressus forest at 1500 m.

MATERIAL EXAMINED — GUATEMALA. GUATEMALA DEPARTMENT, Universidad de

San Carlos de Guatemala, University City, Parque Ecolégico Las Ardillas, 28 June 2007,

Medel 1486 (XAL).

The studied specimen fitted well with the descriptions by Dennis (1963),

Haines & Dumont (1984), and Spooner (1987). Lachnum abnorme is widely

distributed in tropical regions of the world: Australia, New Zealand, Chile,

India, Central America, and Costa Rica (Haines & Dumont 1984); Panama

(Piepenbring 2006); and Venezuela (Mardones-Hidalgo & Iturriaga 2011). This

is the first record to Guatemala.

Lachnum brasiliense (Mont.) J.H. Haines & Dumont, Mycotaxon 19: 23 (1984)

Apothecia scattered on bark, light to dark yellow hymenium, 1.2 mm wide

disc, shallow receptacle, shaped cup with a hairy short stalk, white to pale

buff color and a blue-black base. Hairs cylindrical with obtusely rounded tips,

(52-)60-75(-80) x 3-4 um septate, thin walled, granulated. Asci cylindrical,

(66-—)78-82(-84) x 7-9 um, 8-spored, hyaline, small blue pore in Melzer’s

reagent. Ascospores narrowly fusiform, cylindrical, usually slightly curved,

occasionally becoming 3-septate, (30—)32-43 x 2.5-3 um. Paraphyses cylindrical,

(68-)72-91 x 2-3 um, 1-3-septate.

Hasitat — Gregarious, lignicolous; in cloud forest at 1800 m.

MATERIAL EXAMINED — GUATEMALA. CHIMALTENANGO DEPARTMENT, San Andrés

Itzapa Municipality, Aldea La Hierbabuena, 27 July 2007, Medel 1475 (XAL).

The dark to medium yellow ascomata clothed with white hairs and the blue-

black base of the stalk are distinctive characters. Lachnum brasiliense is a

common species in the tropics and has previously reported from Guatemala

(Haines & Dumont, 1984) in pine forest. The material examined was found in

cloud forest.

Ascomycetes from Guatemala ... 77

Figures 1-8. Bisporella citrina: 1, ascospores. Chlorociboria aeruginosa: 2, apothecia. Lachnum

abnorme: 3, ascospores; 4, apothecia. Lachnum cyphelloides: 5, apothecia. Lachnum virgineum:

6, paraphyses in Congo red. Hyalorbilia inflatula: 7, apothecia. Orbilia juruensis: 8, paraphyses.

Bars: 1, 8 = 10 um; 2 = 1 cm; 3, 6 = 20 um; 4, 7 = 2 mm, 5= 1 mm.

Lachnum cyphelloides (Pat.) J.H. Haines & Dumont, Mycotaxon 19: 30 (1984) Fic. 5

Apothecia yellow translucent, disc 1 mm wide x 1.2 mm of height, long

stipitate, small white hairs to almost smooth. Hairs cylindrical, with obtusely

rounded tips, 48-70 x 3-4 um septate, hyaline, thin wall, granulated. Asci

cylindrical 66-67 x 6-7 um, small blue pore in Melzer’s reagent. Ascospores

fusiform, 31-40 x 1.5-2 um hyaline. Paraphyses lanceolate, (68-) 72-91 x 2-3

um hyaline, 1-3-septate.

Hasitat — Gregarious, lignicolous; in cloud forest at 1800 m.

78 ... Medel & al.

MATERIAL EXAMINED — GUATEMALA. GUATEMALA DEPARTMENT, Universidad de

San Carlos de Guatemala, University Campus, Parque Ecoldégico Las Ardillas, 27 July

2005, Medel 1465-A (XAL); 28 July 2005, Medel 1446 (XAL, MICG).

According to Haines & Dumont (1984), L. cyphelloides is covered with white

hair, has a yellow pale disc, and its stipe lacks a blue-black base. The species

is distributed in Central America, northern South America, and the Lesser

Antilles (Haines & Dumont, 1984; Piepenbring 2006). ‘This is first record for

Guatemala.

Lachnum virgineum (Batsch) P. Karst., Bidr. Kann. Finl. Nat. Folk 19: 169 (1871)

Fic. 6

Apothecia concave smooth disc, 1-1.5 mm in diameter, white to creamy

white, and margin enrolled when it dries. Central stipe, covered with white hairs,

1-1.2 mm of height. Hairs cylindrical, with obtusely rounded tips, 75-100 x

4-5 um septate, thin walled, granulated hyaline. Asci cylindrical asci, 8-spored,

34-45 x 4-5um, thin walled, small blue pore in Melzer’s reagent. Ascospores

fusiform, 6-8 x 1.5-2 um, thin walled, hyaline. Paraphyses lanceolate, 70-100

x 5-6 um, septate, hyaline, longer than the asci.

HaBITaT — Gregarious, lignicolous; in tropical forest at 160 m.

MATERIAL EXAMINED — GUATEMALA. ALTA VERAPAZ DEPARTMENT, Coban

Municipality, Ecorregién Lachua, 17 September 2005, Lopez y Quezada 2473 (XAL).

Lachnum virgineum is characterized by apothecia covered by straight, white

granulate hairs. Spooner (1987) reported the species from North and South

America, Europe, North Africa, Central Asia, India, Japan, Papua New Guinea,

and Australia. This is a new record for Guatemala.

Hypocreales

Ophiocordyceps gracilis (Grev.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora,

Stud. Mycol. 57:43 (2007)

Stromata with long cylindrical apices terminating in 5 x 3 mm heads, red

ochraceous, with dark dotted perithecial ostioles. Stipe yellow-olive. Asci with

many individual spores, 250-280 x 6 um, pore not blue in Melzer’s reagent.

Ascospores cylindrical, smooth, 7-10 x1-2 um, hyaline, lined chainlike upon

each other within the ascus. Paraphyses not observed.

Hasitat — Solitary, on larvae of Lepidoptera; in cloud forest, 1800 m.

MATERIAL EXAMINED — GUATEMALA. CHIMALTENANGO DEPARTMENT, San Andrés

Itzapa Municipality, Aldea La Hierbabuena, 21 June 2007, Medel 1466 (XAL).

Our specimen was consistent with the descriptions by Mains (1958), Dennis

(1978), and Breitenbach & Kranzlin (1984). Ophiocordyceps gracilis is always

associated with lepidopteran larvae. This is the first report for Guatemala.

Ascomycetes from Guatemala ... 79

Orbiliales

Hyalorbilia inflatula (P. Karst.) Baral & G. Marson, Micologia 2000 (Trento): 44

(2000) Fic. 7

Apothecia less than 1.5 mm wide, orange to yellow color, with rolled margin.

Asci cylindric-clavate, rounded apex, 8-spored, 21-23(-29) x 3-3.5(-4) um,

pore not blue in Melzer’s reagent. Ascospores cylindrical, straight or slightly

curved, rounded ends, 4-8 x 0.8-1 um. Paraphyses cylindrical hyaline, 18-21 x

2(-2.5) um, septate, with apices encrusted to form a thin epithecial layer.

Hasitat — Gregarious, lignicolous; in cloud forest and Cupressus forest at 1800 m.

MATERIAL EXAMINED — GUATEMALA. CHIMALTENANGO DEPARTMENT, San Andrés

Itzapa Municipality, Aldea La Hierbabuena, 27 June 2007, Medel 1467, 1484 (XAL);

GUATEMALA DEPARTMENT, Universidad de San Carlos de Guatemala, University

Campus, Parque Ecoldgico Las Ardillas, 28 June 2007, Medel 1485-B (XAL, MICG).

Our specimen agreed with the description by Spooner (1987). Liu et al. (2006)

note that Hyalorbilia inflatula is widely distributed in China. The species has

also been recorded from Venezuela (Mardones-Hidalgo & Iturriaga 2011). This

is the first report for Guatemala.

Orbilia juruensis Henn., Hedwigia 43: 270 (1904) Fic. 8

Apothecia superficial, sessile or subsessile, disc convex or undulate,

with crenulate margin, translucent orange-yellow, smooth, 1 mm diameter.

Receptacle smooth, concolorous or slightly paler than the disc, thin at the

margin and thicker towards the centre. Asci cylindric-clavate , broadest at the

apex and tapered downwards to a narrow base which is sometimes forked, apex

truncate or obtusely rounded, 29-37 x 3.5-4 um, pore not blue in Melzer’s

reagent. Ascospores straight or slightly inequilateral to fusoid, hyaline, (6-)

7-9(-10) x 1-1.5 um, not septate. Paraphyses capitate, tips up to 3.5-4 um

diameter, 21-30 x 2-3 um, 1-2-septa in the lower part.

HaBITAT — Gregarious, lignicolous; in Cupressus forest at 1500 m.

MATERIAL EXAMINED — GUATEMALA. GUATEMALA DEPARTMENT, Universidad de

San Carlos de Guatemala, University Campus, Parque Ecolédgico Las Ardillas, 28 June

2007, Medel 1491 (XAL).

This species is known form South America, Australasia, and Solomon Island

(Spooner 1987), Argentina (Wright & Wright 2005), and Panama (Piepenbring

2006). This is the first record for Guatemala.

Pezizales

Gyromitra infula (Schaeff.) Quél., Enchir. Fung.: 272 (1886)

Ascoma bilobate or saddle-shaped, generally fused with the stipe. The

hymenium covers the whole surface, which is cinnamon to dark brown. The

80 ... Medel & al.

interior is hollow and whitish. The stipe is smooth, often somewhat furrowed

or pitted, and finely felty towards the base. Asci cylindrical, (140-)150-250 x

15-17 um. Ascospores elliptical, smooth, with 2 guttules, hyaline, 18-22 x 8-10

um. Paraphyses clavate, 80-100 x 7-10 um.

Hasitat — Solitary or gregarious, humicolous; in Pinus-Abies forest, 2900 m.

MATERIAL EXAMINED — GUATEMALA. ToTONICAPAN DEPARTMENT, Totonicapan

Municipality, Mercado municipal de Totonicapan, 18 September 2001, Morales 295

(MICG); 19 August 2006, Morales 561 (MICG); Totonicapan Municipality, Aldea

Panquix, 31 August 2003, Morales 485 (MICG).

The bilobate or saddle-shaped ascoma is characteristic of this species (Abbott

& Currah 1997). Bran et al. (2003) previously reported this species from

Guatemala, and it is also known from Mexico (Medel 2005).

Helvella acetabulum (L.) Quél., Enchir. Fung. 275 (1886)

Ascoma cup-shaped with an irregular margin, 25-50 mm broad; fertile

surface dull brown. Lower surface smooth to subglabrous, light brown with

conspicuous pallid to cream raised ribs extending from the stipe base to middle

of the cup; thick whitish deeply ridged. Asci cylindrical, 200-300 x 14-17 um.

Ascospores broadly elliptical, hyaline, 15-21(-22) x 11-15 um, smooth, with

one guttule. Paraphyses with rounded tips, 5-6 um thick.

Hasitat — Solitary, humicolous; in pine-oak forest at 2300 m.

MATERIAL EXAMINED — GUATEMALA. CHIMALTENANGO DEPARTMENT, Tecpan

Municipality, Mercado municipal de Totonicapan, 7 October 1999, Morales 47 (MICG)

The ridged pale brown apothecium is characteristic of this species (Calonge

& Arroyo 1990, Abbot & Currah 1997). The species is known from Canada

(Abbot & Currah 1997), China (Zhuang 2004), Mexico (Medel & Calonge

2004; Vite-Garin et al. 2006) and Spain (Calonge & Arroyo 1990). This is the

first record for Guatemala.

Xylariales

Annulohypoxylon thouarsianum (Lév.) Y.M. Ju, J.D. Rogers & H.M. Hsieh,

Mycologia 97: 861 (2005) Fics 9-10

Stromata sessile, globose, 12 mm wide x 7 mm high, with papillate ostioles

in the middle ofa deep ring, surface dark brown to black, carbonaceous, context

fibrous woody, radially lined to faintly concentrically zoned grey and brown.

Asci not observed. Ascospores inequilateral narrowly elliptical to slightly

fusoid, light to medium brown, smooth, 23-27 x 6-7 um, with a germ slit spore

length on the flattened side. Paraphyses not seen.

Hasitat — Gregarious, lignicolous; in oak forest.

MATERIAL EXAMINED — GUATEMALA. CHIMALTENANGO DEPARTMENT, Tecpan

Municipality, Astillero Municipal, 21 July 2007, Medel 1436 (XAL).

Ascomycetes from Guatemala ... 81

FiGurEs 9-14. Annulohypoxylon thouarsianum: 9, ascospores in KOH 5%; 10, stromata. Daldinia

concentrica: 11, ascospores in 5% KOH. Diatrypella pulvinata: 12, ascospores in 5% KOH. Poronia

pileiformis: 13, ascus with blue pore + in Melzer’s solution; 14, stromata (ostiole and perithecia).

Bars: 9 = 20 um; 10, 14 = 1 cm; 11 = 15 um; 12 = 10 um; 13 = 40 um.

The species was first recorded from Guatemala by Sharp (1948) as Hypoxylon

malleolus Berk. & Ravenel,, a synonym of A. thouarsianum (Ju & Rogers 1996,

Hsieh et al. 2005). This is a common xylariaceous species with greenish pigments

in KOH and papillate ostioles in the middle of a deep disc (Ju & Rogers 1996;

Medel 2002). The species is also known from Mexico (Pérez-Silva 1987; Medel

2002) and Panama (Piepenbring 2006).

Daldinia concentrica (Bolton) Ces. & De Not., Comm. Soc. Crittog. Ital. 1: 197

(1863) Fic. 11

Stromata, globose circular to oval shaped 20-30 mm wide, sessile and,

attached to the substrate, hard surface, reddish-brown to blackish young,

and finally black. Asci cylindrical, 100-130 x 7-8 um, uniseriate. Ascospores

broadly elliptical to bean shape, dark brown, germ line straight, 13-15 x (6-)

6.5-7 um. Paraphyses not seen.

Hasitat — Gregarious, lignicolous; in oak forest.

MATERIAL EXAMINED — GUATEMALA. CHIMALTENANGO DEPARTMENT, Tecpan

Municipality, Astillero Municipal, 21 July 2007, Medel 1423 (XAL).

82 ... Medel & al.

The important diagnostic characters of this species are the concentric lines in

the stromata, and purple stromatal pigment in 5% KOH (Ju et al. 1997; Rogers

et al. 1999). In Guatemala two species have been cited: D. vernicosa (Bran et al.

2003) and D. fissa (Morales et al. 2006). Daldinia concentrica, not previously

reported for Guatemala, is also known from Mexico (Pérez-Silva 1978) and

Europe, India, New Zealand, Taiwan, and the United States of America (Ju &

Rogers 1997).

Diatrypella pulvinata Nitschke, Pyrenomyc. Germ. 1: 72 (1867) Fic. 12

Stromata pulvinate to oval, ostioles 3-5 sulcate, with endostromatic yellow

whitish tissue. Asci multispored. Ascospores 7-9 x 1.5-2 um, slightly curved

(allantoid), hyaline, with 2 guttules. Paraphyses not seen.

HaBiTaT — Gregarious, lignicolous; in Cupressus forest at 1500 m.

MATERIAL EXAMINED — GUATEMALA. GUATEMALA DEPARTMENT, Universidad de

San Carlos de Guatemala, University Campus, Parque Ecoldégico Las Ardillas, 28 July

2007, Medel 1489 (XAL).

The black sessile and pulvinate stromata with papillate ostioles are typical.

Diatrypella pulvinata is known from Costa Rica, Europe, and the United States

of America (Glawe & Rogers 1984; Chacén & Humafia 2006). This is the first

record for Guatemala.

Poronia pileiformis (Berk.) Fr., Nova Acta R. Soc, Scient. Upsal., Ser. 3, 1: 129

(1851) Fics 13-14

Stromata simple to branched, at the top of the stalk it has a small umbelliform

4-6 mm broad head, beige with dark dots (perithecial ostioles), smooth and

leathery, later becoming hard and brittle. Ostioles papillate. Stipe dark brown to

black, 60-70 mm high x 1-6 mm wide, smooth or ribbed with a thickened base.

Asci cylindrical, 100-130 x 4-5 um, pore blue in Melzer’s reagent. Ascospores

ellipsoid, dark brown, 7-11 x 4-5 um, with a straight germ line as long as the

length of the spore. Paraphyses not seen.

Hasitat — Gregarious, fimicolous, in tropical forest at 160 m.

MATERIAL EXAMINED — GUATEMALA. ALTA VERAPAZ DEPARTMENT, Coban

Municipality, Ecorregién Lachua, 19 September 2005, Lopez y Quezada 2473 (XAL).

This species is distinguished by the simple or branched stromata with a beige

head with black papillate ostioles, growing on dung (Dennis 1957; San Martin

et al. 1998). It is known from Costa Rica, Mexico, Peru, and Philippines. This is

the first record for Guatemala

Discussion

With the new records, there are now 44 ascomycete species known from

Guatemala (TABLE 1). The species encompass 26 genera of which Helvella

Ascomycetes from Guatemala ... 83

(5) and Morchella (4) provided the most records. The Pezizales was the most

diverse order with 11 genera and 18 species. About 50% of the 200 specimens

collected during the last survey in 2007 might represent new records or even

new species.

The continued study of ascomycetes and other fungi from Guatemala

will improve the knowledge of these organisms. This is essential because

deforestation threatens to decrease the fungal biodiversity in this country. Recent

deforestation data from Guatemala indicate a net 2006-2010 deforestation rate

of over 38,000 ha / year (Regalado et al. 2012).

Acknowledgments

The authors thank to M.C. Bran (Universidad San Carlos de Guatemala) for

supporting the expedition to collected the studied material of this study. Thanks to Dr.

Gaston Guzman and Juan Lara (INECOL) for providing literature and facilities to consult

the herbarium, and to the reviewers Dra. Evangelina Pérez Silva (Universidad Nacional

Auténoma de México) and Dra. Cecilia Carmaran (Universidad de Buenos Aires), who

significantly improved this manuscript. Special thanks to Dr. Shaun Penycook for his

critical review.

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MY COTAXON

http://dx.doi.org/10.5248/124.87

Volume 124, pp. 87-99 April-June 2013

Coleosporium in Europe

STEPHAN HELFER

Royal Botanic Garden Edinburgh, 20A Inverleith Row, Edinburgh EH3 5LR, UK

CORRESPONDENCE TO S.helfer@rbge.ac.uk

ABSTRACT — The species of Coleosporium (Uredinales) occurring in Europe are not clearly

differentiated morphologically. Most taxa in Europe have been combined into formae speciales

(fE.spp.) of Coleosporium tussilaginis by the majority of researchers. This study includes the

taxa C. doronici, C. inulae, and C. telekiae as further ff.spp. in C. tussilaginis and redefines

the existing ffspp. campanulae-rapunculoidis and senecionis-silvatici. It is hoped that this

treatment will establish a pragmatic basis on which future taxonomic work can be built.

KEY worps — rust fungi, cryptic species, host specialism

Introduction

Coleosporium Lév. isa genus of Uredinales G. Winter (rust fungi) with the aecial

stage parasitic on two-needle pines (Pinus spp.) and the telial stage on members

of a wide range of angiosperm species. Its distribution is mostly limited to the

northern hemisphere, where it is widespread and common in Europe, Asia, and

North America (Farr & Rossman 2013, GBIF 2013). However, specimens have

also been reported from South America (Arthur 1918, Hennen 2005) and New

Zealand (McKenzie 1998). Their presence on two-needle pines has not been

confirmed in these southern areas and specimens from Chile were reported to

lack the teleomorph, possibly as a result (Arthur 1918). In New Zealand it is

likely that already infected hosts from the northern hemisphere were planted or

intercepted, and there is one reference of introduced Coleosporium rust on an

indigenous species but no infections on two-needle pines have been reported

(McKenzie 1998, Farr & Rossman 2013).

Léveillé established the genus in 1847. In a short note, he devised a system

to bring more clarity into the confusing number of identical names of clearly

distinct species and species with multiple names on account of their occurrence

on a number of host plant species or genera (Léveillé 1847).

The type species is Coleosporium campanulae (lectotype, Laundon 1975).

88 ... Helfer

TABLE 1. Distribution of Coleosporium in host plant families in Europe

PLANT FAMILY Host GENERA Host SPECIES PREDOMINANT HOST GENUS

Asteraceae 35 110 Senecio (34 host taxa)

Campanulaceae 10 82 Campanula (63 host taxa)

Orobanchaceae 8 38 Euphrasia (16 host taxa)

Ranunculaceae 2 9

Pinaceae 1 10

CASUAL HOSTS & MISDETERMINATIONS NOTES

Tropaeolaceae 1 3 non-native plants

Orchidaceae 1 1 border intercept plant

Solanaceae 1 1 South American plants

[Amaranthaceae] [1] [1] ? misidentified host

Amarldccd r Pia seperti

[Boraginaceae] [2] [2] = Melampsorella symphyti

[Caryophyllaceae] [1] [1] = Caeoma coronariae

[Rosaceae] [1] [1] = Pucciniastrum agrimoniae

Most authors agree that the European taxa of Coleosporium are

indistinguishable in their morphology (e.g. Sydow & Sydow 1915, Hylander

et al. 1953, Gdumann 1959, Wilson & Henderson 1966, Boerema & Verhoeven

1972). Therefore, many authors used the host plant identity to distinguish

species (Gdumann 1959, Minkevicius & Ignataviciité 1991, Brandenburger

1994). Many current inventories treat all Coleosporium taxa distinguished in

Europe as a single species and synonymise most of the original species into

Coleosporium tussilaginis (Encyclopedia of Life 2012, Index Fungorum 2012).

However, C. doronici, C. inulae, C. ligulariae, and C. telekiae have never been

formally included in C. tussilaginis, although Hylander et al. (1953) did

synonymise C. ligulariae with C. senecionis. The three remaining taxa are

transferred to C. tussilaginis in the present study.

While some authors do not name the taxa included under C. tussilaginis

(e.g. Majewski & Ruszkiewicz-Michalska 2008, Termorshuizen & Swertz 2011),

others distinguish and name them (e.g., Gdumann 1959, Boerema & Verhoeven

1972, Encyclopedia of Life 2012, Bahcecioglu & Kabaktepe 2012), most often

as formae speciales, i.e., biologically specialised forms (Eriksson 1894, Anikster

1984, Kirk et al 2008; see also the next paragraph below). The latter approach

reflects the host specificity of Coleosporium analysed by extensive inoculation

experiments (e.g., Klebahn 1904, 1924, Gaumann 1959, EURED names 2012).

I follow this approach and extend it to all the European taxa.

Coleosporium in Europe... 89

Formae speciales (ff.spp.) are defined as taxa below the species level which

are morphologically not or hardly distinguishable but are separated by host

plant specificity. Nomenclature at this rank is not covered by the provisions of

the International Code of Nomenclature for algae, fungi, and plants (McNeill

et al. 2012: Article 4 Note 4); therefore in this paper I present them as newly

named taxa, rather than as “comb. nov.’

This study covers the European taxa of Coleosporium on native plants. In

the absence of molecular data, a morphological species concept is applied,

extended by the use of discrete host plant ranges, in defining the sub-specific

taxa of formae speciales, as described above. The aim is to present a pragmatic

and effective basis for specimen sampling for molecular analysis, in order to

generate data for further studies.

Materials & methods

The herbaria of Berlin (B), Paris (PC), Copenhagen (CP), St Petersburg (LE), Beltsville

(BPI) and Edinburgh (E) were consulted in this research. Furthermore information was

acquired from Ziirich and Bern (Helfer et al. 2011) and from databases at Stockholm

(Krypto-S 2012), BPI (Farr & Rossman 2013) and National Herbarium Netherlands

(NHL 2012). Label information was extracted and compared with accepted host plant

taxonomy using the Plant List (2012) database. Geographical information was checked

compiled according to the areas in the Atlas Florae Europaeae delimitations of Europe

(Atlas Florae Europaeae 2012). Where reports were from non native or cultivated plants,

this is indicated by the use of square brackets [ ]. Type material for C. telekiae, C. doronici,

and C. ligulariae was obtained from Berlin (B).

Light microscopy: small portions of herbarium specimens were sectioned by hand

and mounted in lactophenol-cotton blue for examination under a Carl Zeiss Axiophot©

microscope with Carl Zeiss Axiocam© digital camera equipment. Measurements were

achieved using the Axiocam software. Urediniospore dimensions as measured by light

microscopy are reported in the following format: (min—) average +/-standard deviation

(-max) length x breadth.

Scanning electron microscopy (SEM): small portions of herbarium specimens were

removed and fastened to aluminium stubs with adhesive carbon tabs. Prior to sputter

sorus sizes were measured and images taken with Carl Zeiss Axiocam© digital camera

equipment as above. Specimens were coated with Palladium for 2min resulting in a

deposition of approx 12nm and examined in a Carl Zeiss Supra©55VP SEM at 1-5kV

and 5mm working distance. Measurements were achieved using the Axiocam software

as above. Morphometric data were subjected to statistical analysis using the statistics

package R (R Development Core Team 2011).

Details of the specimens examined are displayed with each taxon. Further information

is available from the EURED specimens (2013) list.

Herbarium label images for B, PC, CP, LE and E used in this study can be viewed at

the EURED website at E (Helfer et al. 2011). This website is continuously added to and

improved.

90 ... Helfer

Results

The herbarium data searches for European taxa of Coleosporium resulted in

249 host plant relationships (TABLE 1), represented in five families. A further

seven taxa in five additional host families had been found on intercepted plants

or as single occurrences and four taxa in three families were misinterpretations.

In the past more than 50 valid names have been used for these rust taxa (EURED

names 2012).

The microscopic examinations of the specimens were inconclusive with

respect to urediniospore dimensions and sorus characters: while statistically

significant differences could be found between most samples involving various

host species there were also significant differences between collections of

the same host/fungus relationship (EURED stats 2012). Furthermore, data

ranges overlapped widely, indicating that urediniospore dimensions and sorus

characters are unsuitable for identification purposes and for the characterisation

or delimitation of taxa in this genus (EURED spores 2012). Similarly, spore

surface characters observed by SEM revealed no distinctive features. It is

therefore concluded that, morphologically, the taxa are all part of the same

species.

Taxa present in Europe

Coleosporium tussilaginis (Pers.) Lév., in Orbigny, Dict. Univ. Hist. Nat.

12: 786 (1849) f.sp. tussilaginis PLaTE 1

= Uredo tussilaginis Pers., Syn. meth. fung. 1: 218 (1801).

= Ustilago tussilaginis Losa, Anales Jard. Bot. Madrid 5: 126 (1945).

ALTERNATE HOSTS: Pinus halepensis, P. mugo, P. nigra, P. pallasiana, P. pinaster,

P. sylvestris (PLATE 1), [PB echinata, P. ponderosa, P. rigida].

Primary Hosts: Tussilago farfara, [Emilia coccinea, Erechtites glomeratus], E. hieraciifolius,

[Euryops acraeus, E. evansii, Kleinia fulgens, K. grandiflora].

EUROPEAN DISTRIBUTION: pan-European.

SPECIMENS EXAMINED:

(as C. sp.) PC0022213.

(as C. tussilaginis) E00458054, E00458055, E00458056, E00458059, E00458060,

E00458061.

UREDINIOSPORE SIZE: (21.7—)28.3+3.2(-39.6) x (15.9-)20.8+ 2.4(-26.6) um.

Notes: Artificial infections were obtained on Tropaeolum minus

(Tropaeolaceae) and Schizanthus grahamii (Solanaceae) (Gaumann 1959);

weak infections were also obtained on plants of Senecio vulgaris (Klebahn

1924), calling into question the biological separation of this taxon from f.sp.

senecionis discussed below. New specimens in many herbaria are deposited as

C. tussilaginis, irrespective of host plant identity.

Coleosporium in Europe... 91

PLATE 1 Aeciospore of Coleosporium tussilaginis on Pinus sylvestris. SEM; bar = 2 um.

Coleosporium tussilaginis f.sp. campanulae-rapunculoidis Boerema & Verh.,

Netherlands J. Pl. Pathol. 78(Suppl. 1): 7 (1972).

= Coleosporium campanulae (Pers.) Fr., Summa Vegetabilium

Scandinaviae: 512 (1849), as “campanulacearum”.

= Coleosporium campanulae-rapunculoidis Kleb., Wirtswechselnde Rostpilze: 365 (1904).

= Coleosporium campanulae-rotundifoliae Kleb., Wirtswechselnde Rostpilze: 366 (1904).

= Coleosporium campanulae-trachelii Kleb., Wirtswechselnde Rostpilze: 366 (1904).

= Coleosporium tussilaginis f.sp. campanulae-rotundifoliae Boerema

& Verh., Neth. Jl Pl. Path. 78(Suppl. 1): 7 (1972).

= Coleosporium tussilaginis f.sp. campanulae-trachelii Boerema

& Verh., Neth. Jl Pl. Path. 78(Suppl. 1): 7 (1972).

ALTERNATE HOstTs: Pinus nigra, P. sylvestris.

Primary Hosts: Adenophora liliifolia, [A. triphylla], Asyneuma giganteum, A. limonifolium,

Campanula alliariifolia, C. alpina, [C. americana], C. barbata, C. baumgartenii,

C. bononiensis, [C. californica], C. carnica, C. carpatica, C. caucasica, C. cervicaria,

C. cespitosa, C. cochleariifolia, C. drabifolia, C. erinus, C. glomerata, [C. grandis],

C. grossekii, C. hagielia, C. hawkinsiana, C. hispanica, C. incurva, C. isophylla,

C. lactiflora, C. lanata, C. latifolia, C. lusitanica, C. lyrata, C. macrorhiza subsp. gypsicola,

92 ... Helfer

C. medium, C. moesiaca, C. patula, C. pelia, C. persicifolia, C. persicifolia subsp.

subpyrenaica, C. pulla, |C. punctata], C. pyramidalis, C. rapunculoides, C. rapunculus,

C. rhomboidalis, C. romanica, C. rotundifolia, C. rumeliana, C. rupestris, C. sarmatica,

C. saxatilis, C. scheuchzeri, C. serrata, C. sibirica, C. sparsa, C. sparsa subsp. sphaerothrix,

C. spathulata, C. speciosa subsp. affinis, C. speciosa, C. stenosiphon, C. stevenii, C. tatrae,

C. tomentosa, C. trachelium, C. transsilvanica, C. versicolor, C. wanneri, Jasione montana,

Legousia hybrida, L. speculum-veneris, [Lobelia cardinalis, L. xalapensis|, Michauxia

campanuloides, M. laevigata, Petromarula pinnata, Phyteuma betonicifolium, P. nigrum,

P. orbiculare, P. scheuchzeri, P. spicatum, [Triodanis perfoliata, Wahlenbergia denticulata].

EUROPEAN DISTRIBUTION: Au, Be, Bel, Br, Bu, Cg, Cs, Ct, Da, EU, Fe, Ga, Ge, Gr, He, Ho,

Hs, Hu, Is, It, La, Lu, Mo, No, Po, Rm, Rus, Se, Sk, Su, Tu, Uk.

SPECIMENS EXAMINED:

(as C. campanulae) E00159053, E00458064, PC0022266, PC0022293, PC0022305,

PC0022308, PC0022317, PC0022324, PC0022354.

UREDINIOSPORE SIZE: (12.0—)20.5+4.8(-30.2) x (9.0-)14.9+3.2(-26.4) um.

Notes: Boerema & Verhoeven (1972) treat this in three separate formae

speciales: f.sp. campanulae-rapunculoidis, f.sp. campanulae-rotundifoliae, and

fisp. campanulae-trachelii. Gaumann (1959) lists several overlapping host

ranges and infections on unrelated angiosperms, such as Tropaeolum minus,

and I see no justification to maintain these three special forms.

Coleosporium tussilaginis f.sp. doronici S. Helfer, f.sp. nov.

MycoBank MB804294

= Coleosporium doronici Namysl., Spraw. Kom. fizyogr.

Akad. Umijetn. Krakowie 45: 125 (1911).

ALTERNATE HOSTS: Pinus sp. (presumed).

PRIMARY Hosts: Doronicum austriacum, D. columnae.

EUROPEAN DISTRIBUTION: Au, Cs, Hu, Po, Rm.

SPECIMENS EXAMINED:

(as C. doronici) B700015048, B700015049, B700015050, B700015051, B700015052,

B700015053, PC0022411, PC0022412.

UREDINIOSPORE SIZE: (10.8-)27.8+3.8(-34.1) x (7.0-)19.143.6(-24.8) um.

Notes: This appears to be a geographically as well as biologically separate

special form on Doronicum sp. (Miller 2003), since its hosts are widely

distributed over Europe (Atlas Florae Europaeae 2012). The report in Gaumann

(1959) that this rust occurs in Spain is based on a misunderstanding of the

geographical name Galicia (“Galizien”), in fact referring to a region in the Tatra

mountain border region of Poland.

Coleosporium tussilaginis f.sp. inulae S. Helfer, f.sp. nov.

MycoBank MB804295

= Uredo erigerontis Req. ex Duby, Bot. Gall. Edn 2: 893 (1830), as “erigeronis”.

= Coleosporium inulae Rabenh., Bot. Zeitung 9: 455 (1851).

= Coleosporium carpesii Sacc., Rivista Period. Lav. Regia

Accad. Sci. Lett. Arti Padova 24: 208 (1874).

Coleosporium in Europe ... 93

= Coleosporium jasoniae Gonz. Frag., Trab. Mus. Nac.

Cienc. Nat. Madrid, é Bot. 9: 23 (1916).

= Coleosporium asterisci-aquatici (Sacc.) Syd. & P. Syd., Ann. mycol. 19: 249 (1921)

ALTERNATE Hosts: Pinus halepensis, P. sylvestris.

PRIMARY HOSTS: Asteriscus aquaticus, Carpesium cernuum, Centaurea hierapolitana,

Dittrichia graveolens, D. viscosa, Inula aschersoniana, I. aspera, I. britannica, I. candida,

I. conyza, I. ensifolia, I. germanica, I. helenioides, I. helenium, I. helvetica, I. heterolepis,

I. hirta, I. methanaea, I. oxylepis, I. parnassica, [I. royleana], I. salicina, I. spiraeifolia,

Jasonia tuberosa.

EUROPEAN DISTRIBUTION: Al, Au, Bl, Bu, Cg, Co, Cs, Ct, Da, Es, Fe, Ga, Ge, Gr, He, Hs,

Hu, It, La, Lu, Mk, No, Po, Rm, Rus, Su, Tu, Uk.

SPECIMENS EXAMINED:

(as C. inulae) E00458051, E00458052, E00458053, E00458063.

(as Uredo erigeronis) PC0022413, PC0022415.

UREDINIOSPORE SIZE: (21.2—)30.3+3.3(-39.5) x (15.7-)23.4+3.1(-30.7) um.

Notes: A special form on most of the genera of tribus Inuleae Cass. of

the Compositae. Significant damage caused by this rust on elecampane (Inula

helenium) grown for medicinal use has been reported in Serbia (Pavlovic et al.

2003).

Coleosporium tussilaginis f.sp. melampyri Boerema & Verh., Netherlands

J. Pl. Pathol. 78(Suppl. 1): 8 (1972).

= Coleosporium melampyri (Rebent.) Kleb., Annls Sci. Nat., Bot., sér. 4, 4: 136 (1854).

ALTERNATE HOstTs: Pinus mugo, P. sylvestris.

PRIMARY HOosTs: Melampyrum arvense, M. nemorosum, M. polonicum Soo, M. pratense,

[Schizanthus sp.].

EUROPEAN DISTRIBUTION: Au, Bel, Br, Cs, Da, Fe, Ga, Ge, He, Ho, Hs, Hu, It, No, Po,

Rm, Rus, Sl, Su.

SPECIMENS EXAMINED:

(as C. euphrasiae) PC0022164.

(as C. melampyri) E00458065, E00458066, E00458067, E00458068.

(as C. tussilaginis) E00458050.

(as C. rhinanthacearum) PC0022434.

UREDINIOSPORE SIZE: (20.6—)25.9+2.6(-32.4) x (13.3-)18.942.2(-24.2) um.

Notes: Heavy infections of Melampyrum have been reported in Finland

(Pohjakallio & Vaartaja 1948). This rust was found to infect cultivated

Schizanthus sp. (Solanaceae) in Sweden (Hylander et al. 1953) and Norway

(Gjaerum 1974); please also see notes to f.sp. rhinanthacearum below.

Coleosporium tussilaginis f.sp. petasitis Boerema & Verh., Netherlands

J. Pl. Pathol. 78(Suppl. 1): 8 (1972).

= Coleosporium petasitidis Lév., Annls Sci. Nat., Bot., sér. 3, 8: 373 (1847).

= Coleosporium petasitis de Bary, Microscopic fungi: 213 (1865).

ALTERNATE HOstTs: Pinus mugo, P. nigra, P. sylvestris.

94 ... Helfer

PRIMARY HOsTs: Petasites albus, P. frigidus, P. frigidus var. palmatus, P. hybridus,

[P japonicus, a naturalized alien in central and northern Europe], P kablikianus,

P. paradoxus, P. radiatus, P. spurius.

European distribution: Au, Br, Bu, Cs, Da, Fe, Ga, Ge, Gr, Hb, He, Ho, It, La, No, Po,

Rm, Rus, Sk, Su.

SPECIMENS EXAMINED:

(as C. petasitis) E00458069, E00458070, E00458071.

UREDINIOSPORE SIZES: (18.4—)26+2.3(-31.6) x (14.3-)20.742.2 (-26.6) um.

Notes: Together with f.sp. senecionis, f.sp. tussilaginis, and f.sp. doronici, this

rust develops on members of the tribus Senecioneae Cass. of the Compositae.

Coleosporium tussilaginis f.sp. pulsatillae Boerema & Verh., Netherlands

J. Pl. Pathol. 78(Suppl. 1): 8 (1972).

= Uredo tremellosa var. pulsatillae F. Strauss, Ann. Wetterauischen

Ges. Gesammte Naturk. 2: 89 (1811) [“1810”].

= Coleosporium pulsatillae (F. Strauss) Fr., Summa Vegetabilium

Scandinaviae: 512 (1849), as “pulsatillarum”.

ALTERNATE Hosts: Pinus sylvestris.

PRIMARY HOSTS: Anemone alpina, A. halleri, A. montana, [A. patens], A. pratensis,

A. pulsatilla, A. slavica, Pulsatilla grandis, P. nigricans.

EUROPEAN DISTRIBUTION: Au, Cs, Da, Fe, Ga, Ge, He, Ho, Hu, It, La, No, Po, Rm, Rus,

Sk, Su, Uk.

SPECIMENS EXAMINED:

(as C. pulsatillae) E00458057, E00458062.

UREDINIOSPORE SIZE: (20.8-)28.7+4.2(-40.3) x (15.5-)19.6+2.2(-24.4) um.

Notes: This special form occurs on members of the Ranunculaceae. Despite

its morphological likeness, many authors (Braun 1981, 1982, Su et al. 2012)

consider this as a separate species. I concur with Boerema & Verhoeven (1972)

in the treatment as a special form, as no reliable distinction can be made using

morphometric methods.

Coleosporium tussilaginis f.sp. rhinanthacearum Boerema & Verh.,

Netherlands J. Pl. Pathol. 78(Suppl. 1): 8 (1972).

= Coleosporium rhinanthacearum (DC) Lév., Annls Sci. Nat., Bot., sér. 3 8: 373 (1847).

= Coleosporium flavum Bonord., Abh. Naturf. Ges. Halle: 186 (1860).

= Coleosporium euphrasiae (Schumach.) G. Winter, Rabenh.

Krypt.-Fl., Edn 2, 1(1): 246 (1881) [“1884”].

ALTERNATE HOstTs: Pinus mugo, P. sylvestris.

Primary HOSTS: Bartsia alpina, |B. lutea, B. trixago], B. viscosa, Euphrasia x reuteri,

E. arctica, E. brevipila, E. hirtella, E. liburnica, E. micrantha, E. minima, E. nemorosa,

E. parviflora, E. pectinata, E. picta subsp. kerneri, E. rostkoviana, E. rostkoviana vat.

fennica, E. salisburgensis, [E. striata], E. tricuspidata, Odontites litoralis, O. purpureus,

O. vulgaris, Orthantha lutea, Parentucellia viscosa, Pedicularis palustris, Rhinanthus

Coleosporium in Europe ... 95

aestivalis, R. alectorolophus, [R. apterus], R. glaber, R. glacialis, R. pulcher subsp. alpinus,

R. riphaeus, R. serotinus.

EUROPEAN DISTRIBUTION: Au, Be, Bel, Br, Bu, Cg, Cs, Da, Fe, Ga, Ge, Hb, He, Ho, Hs,

Hu, It, La, Lu, No, Po, Rm, Rus, Sk, Su, Uk.

SPECIMENS EXAMINED:

(as C. euphrasiae) PC0022172, PC0022176, PC0022178, PC0022200, PC0022420.

(as C. flavum) PC0022202.

(as C. rhinanthacearum) E00458082, PC0022434, PC0022459.

UREDINIOSPORE SIZE: (19.2—)22.9+2.0(-28.4) x (14.1-)17.941.7(-22) um.

Notes - Besides f.sp. melampyri this is the second forma specialis on these

more advanced members of the Orobanchaceae (Tank et al. 2006). In many

herbaria the specimens of rusts on Rhinanthus, Euphrasia, Melampyrum and

related hosts are labelled or arranged under any of the names Coleosporium

euphrasiae, C. melampyri, C. rhinanthacearum, C. tussilaginis.

Coleosporium tussilaginis f.sp. senecionis-silvatici Boerema & Verh.,

Netherlands J. Pl. Pathol. 78(Suppl. 1): 9 (1972)

= Coleosporium senecionis f.sp. senecionis-silvatici Wagner ex

Gaum., in Beitr. KryptogFlora Schweiz 12: 124. 1959.

= Coleosporium senecionis (Pers.) Fr., Summa Vegetabilium Scandinaviae: 512 (1849).

= Coleosporium cacaliae G.H. Otth, Mitt. naturf. Ges. Bern: 179 (1865).

= Coleosporium ligulariae Thiim., Bull. Soc. Imp. Nat. Moscou 52: 140 (1877).

ALTERNATE Hosts: Pinus halepensis, P. mugo, P. nigra, P. pinaster, P. pinea, P. sylvestris.

PRIMARY HOSTS: Adenostyles alliariae, A. alpina, [Delairea odorata, Farfugium japonicum

var. giganteum, Hasteola suaveolens], Jacobaea alpina, J. arnautorum, J. erucifolia subsp.

praealta, J. paludosa, [Ligularia fischeri, L. macrophylla], L. sibirica, Pallenis maritima,

Parasenecio hastatus, Pericallis cruenta, [P. hybrida, Senecio candicans], S. carpetanus,

[S. congestus], S. doria, S. doronicum, S. duriaei, |S. elegans], S. gallicus, |S. glaberrimus],

S. hercynicus, [S. inaequidens], S. incrassatus, S. jacobaea, S. leucanthemifolius,

S. leucanthemifolius subsp. vernalis, [S. leucostachys], S. lividus, S. macrophyllus,

[S. moorei], S. nemorensis, S. ovatus, [S. pulcher], S. pyrenaicus, [S. scandens, S. smithii],

S. squalidus, S. subalpinus, S. sylvaticus, [S. tristis], S. umbrosus, [S. viravira, S. viscidulus],

S. viscosus, S. vulgaris, Tephroseris palustris.

EUROPEAN DISTRIBUTION: Au, Az, Be, BI, Br, Bu, Cg, Co, Cs, Da, Fe, Ga, Ge, Gr, Hb, He,

Ho, Hs, Hu, It, La, Lu, No, Po, Rm, Rus, Sk, Su, Uk.

SPECIMENS EXAMINED:

(as C. cacaliae) PC0022228.

(as C. ligulariae) B700015041, B700015042, B700015044, B700015045, B700015046,

B700015047.

(as C. senecionis) E00458072, E00458073, E00458074, E00458075, E00458076,

E00458077.

UREDINIOSPORE SIZE: (18.6—)25.7£3.2(-37.1) x (11.8-)18.742.2(-26.4) um.

Notes: This is probably a pan-European form. Its distinctness from f.sp.

tussilaginis needs to be confirmed by detailed host range studies or molecular

investigations (see notes there).

96 ... Helfer

Coleosporium tussilaginis f.sp. sonchi Boerema & Verh., Netherlands J. Pl. Pathol.

78(Suppl. 1): 9 (1972).

= Uredo tremellosa var. sonchi F. Strauss, Ann. Wetterauischen Ges. Gesammte

Naturk 2: 90 (1811) [“1810”]; see Hylander et al. (1953).

= Coleosporium sonchi Lév., Annls Sci. Nat., Bot., sér. 4 2: 190 (1854).

= Coleosporium sonchi-arvensis (Pers.) Lév., in Orbigny, Dict. Univ. Hist. Nat. 12: 786 (1849).

ALTERNATE HOstTs: Pinus sylvestris.

PRIMARY HOSTS: Aposeris foetida, Arnoglossum atriplicifolium, Crepis palaestina,

C. tectorum, Dendroseris litoralis, Lactuca muralis, Lapsana communis, Picris cupuligera,

Sonchus arvensis, S. asperl, S. brachyotus, S. oleraceus, S. palustris, S. tenerrimus,

S. uliginosus.

EUROPEAN DISTRIBUTION: Au, Bel, BH, Br, Bu, Cg, Cs, Da, Fe, Ga, Ge, Hb, He, Ho, Hs,

It, La, Lu, No, Po, Rm, Rus, Sk, Su, Uk.

SPECIMENS EXAMINED:

(as C. sonchi) E00458078, E00458079.

(as C. sonchi-arvensis) E00458080.

UREDINIOSPORE SIZE: (15.9—)21.8+2.5(-29.6) x (9.6-)17.2+3.0(-23.9) um.

Notes: This forma specialis is restricted to the tribus Cichorieae Lam. & DC. of the

Compositae.

Coleosporium tussilaginis f.sp. telekiae S. Helfer, f.sp. nov.

MycoBank MB804296

= Coleosporium telekiae Thim., Fungi austriaci exsiccati no. 850 (1873)

ALTERNATE HOsTs: Pinus sp. (presumed)

Primary Hosts: Telekia speciosa, Xerolekia speciosissima.

EUROPEAN DISTRIBUTION: Au, Bu, Cs, [Fe,] Ge, It, [No,] Po, Rm, Sk, Uk.

SPECIMENS EXAMINED:

(as C. telekiae) B700004933, B700010610, B700010611, B700015019, B700015020,

B700015021, B700015022, B700015023, B700015024, B700015026, B700015027,

B700015030, B700015033, B700015034, B700015035, B700015037, B700015038,

B700015039, B700015040, E00458058.

UREDINIOSPORE SIZE: (15.8—)22.6+3.0(-31.8) x (10.6-)17.2+2.4(-24.1) um.

Notes: As well as in its natural range in Eastern Central Europe (Kokes

2004, Dietrich 2009), this rust was recently found on cultivated Telekia speciosa

in Western Norway (Gjaerum et al. 2008).

Discussion

The present study used the host plant taxonomy of the Plant List (2012)

database and revised the host plant spread to at least 239 taxa in 55 genera

represented in 4 angiosperm families as well as ten Pinus species as alternate

hosts (TABLE 1). The most common host family is the Compositae, represented

by 96 genera and 706 taxa worldwide and 35 genera and 110 taxa in Europe (Farr

& Rossman 2013). Kenneth & Palti (1984), who published a detailed study of

Coleosporium in Europe ... 97

the fungus-plant interactions of this family, found that globally Coleosporium

was most widely distributed on the tribe Heliantheae with 14 genera affected

followed by Senecioneae with 11 genera. Members of Heliantheae are not native

in Europe, leaving Senecioneae as the most important host tribe, as is confirmed

in this study.

To date few molecular investigations into Coleosporium have been published.

A total of 32 sequences is currently deposited in GenBank (GeneBank 2013),

mostly of the Asian/North American species C. asterum (Dietel) Syd. & P. Syd.

Maier et al. (2003) compared four accessions of Coleosporium using the nuclear

large subunit ribosomal DNA. In their neighbor joining analysis C. asterum

(synonym Stichopsora asterum Dietel) was clearly separated from C. cacaliae,

C. campanulae, and C. tussilaginis, which were not resolved.

Recent molecular studies have revealed the presence of cryptic species

among morphologically indistinguishable specimens of Melampsora Castagne

(Bennett et al. 2011, Milne et al. 2012), a rust genus of comparable host diversity,

and it is probable that similar speciation is taking place in Coleosporium.

Conclusions

The genus Coleosporium is well defined within the Uredinales. Its taxa,

however, are seriously confused. While host specificity appears to restrict

the host range in some taxa, it does not seem to do so in others. Molecular

taxonomic investigation of Coleosporium taxa is only in its infancy, and new

initiatives are urgently needed to deal with the relationships in this genus.

Acknowlegements

The author acknowledges the support of the herbaria in B, CP, LE, PC, Z+ZT, BERN

for access to specimens. The study was financially supported by SYNTHESYS (for visits

to B [DE-TAF-924] and PC [FR-TAF-5178]) as well as a travel grant from the British

Society for Plant Pathology to visit CP. Thanks are also expressed to Dr Reinhard Berndt

and Prof. Salvatore Moricca for helpful comments during the peer review process and

to the Nomenclature Editor.

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MY COTAXON

http://dx.doi.org/10.5248/124.101

Volume 124, pp. 101-109 April-June 2013

Septoglomus titan, a new fungus in the Glomeraceae

(Glomeromycetes) from Bahia, Brazil

BRUNO ToMIO GOTO!" »,ADRIANE FREIRE ARAUJO?, ANA CRISTINA FERMINO SOARES’,

ARAESKA CARENNA DE ALMEIDA FERREIRA’, LEONOR Costa Malta},

CARLA DA SILVA SOUSA? & GLADSTONE ALVES DA SILVA;

‘Departamento de Botanica, Ecologia e Zoologia, CB, Universidade Federal do

Rio Grande do Norte, Campus Universitario, 59072-970, Natal, RN, Brazil

*Nucleo de estudo em Microbiologia Aplicada, CCA, Universidade Federal do Recéncavo da Bahia,

Centro, S/N, CEP 44380-000, Cruz das Almas, BA, Brazil

*Departamento de Micologia, CCB, Universidade Federal de Pernambuco,

Av. Prof. Nelson Chaves, S/N, CEP 50670-420, Cidade Universitaria, Recife, PE, Brazil

“CORRESPONDENCE TO: brunogoto@hotmail.com & adrianebiologa@yahoo.com. br

ABSTRACT — A new fungus of the Glomeraceae found in the rhizosphere of Agave sisalana

is described under the epithet Septoglomus titan. It forms large glomerospores, (243-)265 x

325(-400) um in diameter. They generally are subglobose and have three spore wall layers:

a semi-persistent short lived sub-hyaline to yellow-brown outer layer (0.5-1.4 um thick), an

adherent smooth light-yellow to orange-brown middle layer (2.5-5.1 um), and a laminate

smooth thick orange brown to dark red brown innermost layer (12.8-19.2 um). Spore size

and colors of the spore wall layers separate this species from other yellow-brown to dark-

brown species.

Key worps — Glomerales, Agavaceae, Glomus group A, crop system, rDNA

Introduction

Approximately 230 species of arbuscular mycorrhizal fungi (AMF;

Glomeromycota) have been described, of which 118 exhibit glomoid spore

development (sensu lato; Oehl et al. 2011a, Estrada et al. 2011). Historically,

identification of the glomoid species was considered extremely difficult because

of the limited morphological characters (Morton 1988), and the molecular

approach has been used to solve the hypothetical separation into different

genera, families, and orders (Walker & Schiifler 2004, 2007, Schiifler & Walker

2010). For this reason recent taxa from different glomeromycotan groups have

been poorly described morphologically (Walker & SchiiBler 2004, Schiifler &

Walker 2010), including groups with diversified morphological data sets such

as Acaulospora, Kuklospora, and Ambispora (Kaonongbua et al. 2010, Kriiger et

102 ... Goto & al.

al. 2011, SchiiBler & Walker 2010). Recently Oehl et al. (2011a,b,c) combined

significant morphological characters with concomitant molecular data to

reorganize the Glomeromycota, especially the groups with glomoid spore

development. This newly available morphological data set is being successfully

used to identify distinct groups with glomoid spores (Estrada et al. 2011,

Furrazola et al. 2011, Goto et al. 2012).

Glomoid species are very commonly found in Brazilian agrosystems (Silva

et al. 2007, Goto et al. 2010, Maia et al. 2010, Nobre et al. 2010) with reports

of many undescribed species (Carrenho et al. 2010) but with new descriptions

rarely available. Studies on AMF diversity in Agave sisalana (common name:

sisal) crop systems in Bahia State, Brazil, have revealed an undescribed species

forming large spores with glomoid development, which we describe here as

Septoglomus titan.

Material & methods

Study areas, soils sampling and soil parameters

Study areas were in the municipalities of Sao Domingos located at 12°48'S 41°37'W,

and Ourolandia located at 10°58'S 41°05'W, both in the semi-arid region of Bahia State,

in the Northeast Brazil. This semi-arid region is characterized by tropical dry climate

(type Am of K6ppen-Geiger; Kottek et al. 2006), with mean annual temperature of 30°C

and precipitation of 400-600 mm. The vegetation was agriculturally managed sisal, a

perennial culture of 40-year-old systems, in a Caatinga biome characterized by low tree

stratum without a continuous canopy, trees and shrubs with thin stems and small or

composite leaves, deciduous in the dry season (Queiroz 2006). The predominant local

geology is characterized by a granite-greenstone formed by the Santa Luz complex,

Archaean basement, and comprises an assemblage of migmatitic gneisses with

subordinate granitoids. The fungus was found in soil samples collected around Agave

sisalana roots in farms of both municipalities. Soil characteristics in Ourolandia were:

pH (CaCl) = 6.4, organic matter = 13 g dm’, available P = 18 mg dm’ and in Sao Domingos

were: pH (CaCL,) = 6.3, organic matter = 32 g dm’, and available P = 58 mg dm’.

AMF bait cultures

The native AMF communities were cultured for two cycles (5 months each) with

Sorghum bicolor, Brachiaria decumbens, and Panicum miliaceum in 500 mL pots

filled with autoclaved sand substrate (250 g per pot) mixed with the natural field soil

as AMF inoculum (250 g per pot), at the greenhouse of the Center for Agricultural,

Environmental and Biological Research, Universidade Federal do Recéncavo da Bahia,

Cruz das Almas, Brazil. Additionally, multiple glomerospores from the new species were

separated and used as infective propagules in single species cultures on S. bicolor. The

new fungus could successfully be propagated in bait cultures together with Acaulospora

scrobiculata, Ambispora appendicula, Claroideoglomus etunicatum, Entrophospora

infrequens, Funneliformis mosseae, Glomus intraradices, G. sinuosum, Paraglomus

brasilianum, and P. occultum. However, no single species cultures were obtained from

the fungus. Glomerospores isolated exclusively from the trap cultures were used for

morphological and molecular analyses.

Septoglomus titan sp. nov. (Brazil) ... 103

Morphological analyses

Spores of the new species were extracted as described in Sieverding (1991). Trap

cultures were established according to Goto et al. (2012) modified by using Hoagland’s

solution without phosphorus. The spores were thereafter mounted in polyvinyl alcohol-

lacto-glycerin (PVLG), in PVLG + Melzer’s reagent, and in water (Brundrett et al. 1994,

Spain 1990). The terminology used is based on recent papers (Furrazola et al. 2011,

Goto & Maia 2006, Goto et al. 2012, Estrada et al. 2011)

Molecular analyses

DNA EXTRACTION: DNA was extracted from three single spores of the new species.

Individual spores on a slide with a drop (5-10 ul) of ultrapure water were crushed with

a needle. The resulting material was used directly in the PCR reactions.

AMPLIFICATION AND SEQUENCING: DNA extract was used as template for a semi-

nested PCR using the primers ITS3 (White et al. 1990) - 28G2 (Silva et al. 2006) and

LR1 (van Tuinen et al. 1998) - 28G2 consecutively. PCR reactions were carried out in a

volume of 50 ul, 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); cycling parameters were 5 min at 95°C (1 cycle), 45 s at 94°C, 1

min at 55°C, 1 min at 72°C (40 cycles), and a final elongation of 7 min at 72°C following

the last cycle. The amplified products were purified with a PureLink PCR Purification

Kit (Invitrogen), following the manufacturer’s instruction and sequenced. Sequencing

was provided by the Human Genome Research Center (Sao Paulo, Brazil).

SEQUENCE ALIGNMENT: Querying the National Center for Biotechnology Information

databases with the BLASTn program, we verified that the sequences obtained from S.

titan were affiliated with the Glomerales (Glomeromycota) before phylogenetic analysis.

The AM fungal sequences (partial LSU rRNA) obtained in our laboratory were aligned

with other glomeromycotean sequences from the GenBank using the program ClustalX

(Larkin et al. 2007) and edited with the BioEdit program (Hall 1999) to obtain a final

alignment. The sequences were deposited at GenBank under the accession numbers

JQ312667-312669.

PHYLOGENETIC ANALYSES: Maximum parsimony (MP) analysis with 1000 bootstrap

replications was performed using the Phylogenetic Analysis Using Parsimony (PAUP)

program version 4 (Swofford 2003). Bayesian (two runs over 1 x 10° generations with a

burnin value of 2500) and maximum likelihood (1000 bootstrap) analyses were executed,

respectively, in MrBayes 3.1.2 (Ronquist & Huelsenbeck 2003) and PhyML (Guindon &

Gascuel 2003), launched from Topali 2.5. The model of nucleotide substitution (GTR +

G) was estimated using Topali 2.5 (Milne et al. 2004). Sequences from Claroideoglomus

claroideum and C. etunicatum were used as outgroup.

Results

Molecular analyses

Phylogenetic analyses of LSU rRNA gene sequences (Fic. 1) revealed that

the new fungus is a Septoglomus species (former Glomus group Aa3 sensu Oehl

et al. 2011a).

104 ... Goto & al.

Claroideoglomus etunicatum AJ623310

C. claroideum AJ271929

100

1.00

0.1

Glomus sinuosum FJ461846

G. intraradices AF396797

G. intraradices AY842577

G. proliferum FM992402

G. proliferum FM992398

G. manihotis AM158947

G. clarum AJ510243

G. clarum AJ510242

Septoglomus deserticola AJ746249

0.73 72 S. constrictum FJ461827

70 1.00

83 S. xanthium AJ849467

D.99)

S. titan JQ312667

100

1.00) S. titan JQ312669

3 S. titan JQ312668

1.00

Funneliformis coronatus AF 145739

F. mosseae AY639273

100 F. mosseae AF 145735

100

1.00

F. fragilistratus AF 145747

F. caledonius AJ628059

F. caledonius AJ510239

F. coronatus AF 145740

F, geosporus AF 145742

F. geosporus AF 145743

Fic. 1. Phylogenetic tree of the Glomeraceae based on LSU rDNA analysis and rooted by

Claroideoglomus claroideum and C. etunicatum. Sequences are labeled with database accession

numbers. Support values are from maximum parsimony (MP), maximum likelihood (ML) and

bayesian analyses, respectively. Septoglomus titan sequences are in bold. Only topologies with

250% bootstrap values are shown. (Consistency Index = 0.62; Retention Index = 0.79).

Septoglomus titan sp. nov. (Brazil) ... 105

Fics 2-10. Septoglomus titan. 2. Spore formed on subtending hypha (sh) with three spore wall layers

(SWL1, SWL2, SWL3) continuous with subtending hyphal wall layers. 3-6. Spore wall with three

layers in young and mature spore (SWL1, SWL2 and SWL3). [3, 4. Young spores with inner layer

(SWL3) less pigmented than outer layers (SWL1 and SWL2). 5, 6. Mature spores with inner layer

(SWL3) more pigmented than outer layers (SWL1 and SWL2)] 7, 8. Subtending hyphae slightly

funnel-shaped to slightly constricted and all three spore wall layers continuous with subtending

hyphal wall (SW). 9, 10. Detail of septum formed by sublaminae of laminated inner layer (SWL3)

typical of Septoglomus.

Taxonomic analyses

Septoglomus titan B.T. Goto & G.A. Silva, sp. nov. Fics 2-10

MycoBank MB 564321

Differs from Septoglomus africanum, S. deserticola, and S. xanthium by its larger spores

with 3-layered walls; and from S. constrictum by its thicker 3-layered spore walls.

TyPE: BRAZIL: Bahia, near Ourolandia, in soil cultivated with Agave sisalana, 20.Apr.2011,

B.T. Goto, permanent PVLG slides (Holotype, URM83528; GenBank JQ312667,

JQ312668, JQ312669. Isotypes, URM83529; URM83530; Z+ZT Myc 15119).

ETyMoLoey: titan = in reference to the large spores formed by the new species.

GLOMEROSPORES are yellow brown to orange brown when young to dark red

brown at maturity, formed terminally on hyphae, and globose to sub-globose

(243-)265-375(-400) um or (rarely) oblong to irregular, (225-)245-285 x

308-325(-400) um.

106 ... Goto & al.

SPORE WALL is (15.3—)17.9-23.0(-—25.6) um thick in total, comprising three

smooth layers (SWL1-3). SWL1 is sub-hyaline to light yellow, thin (0.5-1.4 um)

observed in young and mature spores or in the subtending hyphal wall. SWL2

is pigmented, yellow brown to orange brown, unit (2.5-5.1 um); darker than

SWL3 in young spores, SWL2 acquires pigmentation during maturation. The

SWLS3 is dark yellow to yellow brown in young spores becoming orange brown

to dark red brown in mature spores, laminated,12.8-19.2 um thick. Spore wall

layers are continuous with the subtending hyphal wall layers, and the SWL2

and SWL3 pigments continue into the subtending hyphal wall. The three layers

do not stain when exposed to Melzer’s reagent.

SUBTENDING HYPHA (sh) generally present, single, straight, cylindrical to

constricted, light yellow to orange brown, 17.9-33.2 um wide (mean = 25.4 um)

at the spore base. The subtending hyphal wall is 7.6-8.9(-10.2) um thick (mean

8.2 um) near the spore base, tapering to approximately 2.5-5.2 um about 100 um

distant from the spore base; occlusion formed by bridging septum arising from

sublaminae of SWL3, although the pore at the subtending hypha sometimes

appears partly open.

GERMINATION by regrowth of the subtending hypha.

GLOMEROSPORE DEVELOPMENT deduced from spores found in different

developmental stages. The sub-hyaline hyphal wall layer differentiates into

sub-hyaline to yellow, evanescent (SWL1) and unified (SWL2) spore wall

layers after which the laminate layer 3 differentiates with increasing number of

developing sublaminae and becomes pigmented. Finally, the pore is not closed

by introverted thickening of SWL3 but by a bridging septum arising from the

laminate wall layer at the spore base.

ARBUSCULAR MYCORRHIZA FORMATION associated with roots of Sorghum

bicolor (L.) Moench.

DISTRIBUTION — Currently known only from agricultural soils mainly

cultivated with Agave sisalana Perrine (Agavaceae) in Ourolandia and Sao

Domingos, Bahia State (Brazil).

ADDITIONAL COLLECTIONS EXAMINED: BRAZIL: Banta, near SAo Domingos, in soil

cultivated with Agave sisalana, UFRN1895 UFRN1896 UFRN1897.

Discussion

Sequence analyses of the partial LSU rRNA region confirm with high support

that the new species represents a Septoglomus that differs from the four other

described Septoglomus species (Fic. 1). The morphology of the subtending

hyphal structure (Fics 3-8) also supports the new species in Septoglomus as

circumscribed by Oehl et al (201 1a).

Septoglomus africanum (Btaszk. & Kovacs) Sieverd. et al. S. deserticola

(Trappe et al.) G.A. Silva et al., and S. xanthium (Blaszk. et al.) G.A. Silva et al.

Septoglomus titan sp. nov. (Brazil) ... 107

differ from S. titan by their smaller spores with 2-layered walls [S. africanum

(60-)87(—125) um (Blaszkowski et al. 2010); S. deserticola 47-54(-115) x

38-52(-102) um (Trappe et al. 1984); and S. xanthium 20-55 x 45-100 um

(Blaszkowski et al. 2004)]. Although Septoglomus constrictum (Trappe) Sieverd.

et al. also has large (150-330 um) spores, it differs from S. titan by its thinner,

2-layered spore walls with a total thickness of 7-15 um (Trappe 1977, Oehl et

al. 201:1a).

Septoglomus titan is easily distinguished from all other glomoid non-

Septoglomus species by spore wall structure. A few dark species such as Glomus

tenebrosum (‘Thaxt.) S.M. Berch and Funneliformis geosporus (T.H. Nicolson

& Gerd.) C. Walker & A. Schiiéler resemble S. titan. Glomus tenebrosum has

spores with only two spore wall layers (Thaxter 1922, Trappe 1977, Walker

1982). Funneliformis geosporus has spores with three wall layers, but the inner

layer is flexible and thin (<1.0 um), whereas in S. titan the SWL3 is laminated

and thick.

Goto et al. (2012) noted that the spore wall of G. trufemii B.T. Goto et al. was

one of the thickest (7.4-15.5 um) among the glomoid-spored glomeromycotan

species, but S. titan has even thicker spore walls.

Acknowledgements

The authors acknowledge Dr. Janusz Blaszkowski (Department of Plant Protection,

West Pomeranian University of Technology, Szczecin, Poland) and Dr. Fritz Oehl

(Agroscope Reckenholz-Tanikon ART, Zurich, Switzerland) for reviewing the

manuscript and making helpful comments and suggestions and appreciate the

corrections by Dr. Shaun Pennycook (Nomenclature Editor) and suggestions by Dr.

Lorelei Norvell (Editor-in-Chief). This work was supported by: Conselho Nacional

de Desenvolvimento Cientifico e Tecnolégico (CNPq) through Protax (Programa de

Capacitacao em Taxonomia); PPBio (Programa de Biodiversidade do Semiarido) and

INCT-HV (Herbario Virtual da Flora e dos Fungos); FACEPE (Fundacao de Amparo a

Ciéncia e Tecnologia do Estado de Pernambuco); UFRN (Programa de Pés-Graduacgao

em Sistematica e Evolugao); UFPE (Programa de Pés-Graduacao em Biologia de Fungos)

and CAPES-PNPD (Coordenacao de Aperfeigcoamento de Pessoal de Nivel Superior

- Programa Nacional de Pés-Doutorado). The Graduate Program in Microbiologia

Agricola da Universidade Federal do Recéncavo da Bahia is acknowledged for providing

technical support.

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MY COTAXON

http://dx.doi.org/10.5248/124.111

Volume 124, pp. 111-115 April-June 2013

Geoglossaceous fungi in Slovakia 5.

Geoglossum uliginosum: taxonomy and nomenclature

VIKTOR KUCGERA’ , JOHAN NITARE’, PAVEL LIZON! & JAN GAISLER?

‘Institute of Botany, Slovak Academy of Sciences,

Dubravskad cesta 9, SK-845 23, Bratislava, Slovakia

*Skogsstyrelsen, SE-551 83 Jonkdping, Sweden

>Crop Research Institute Prague, Grassland Research Station,

Rolnickd 6, CZ-460 01, Liberec, Czech Republic

*CORRESPONDENCE TO: viktor.kucera@savba.sk

ABSTRACT — Geoglossum uliginosum is a new record for Slovakia and second record for

Central Europe. Geoglossum glabrum var. uliginosum is a forgotten and disused name

representing a different fungus with unknown circumscription. Recent collections of

G. uliginosum from Slovakia are described and compared with the Swedish holotype and the

Czech collections, and the nomenclature and distribution of the species are discussed.

Key worps — Geoglossaceae, Scandinavia, conservation

Introduction

During field research of geoglossaceous fungi in non-forested stands in

Slovakia, an interesting species of Geoglossum was collected and identified as

Geoglossum uliginosum. Although collections from the Czech Republic have

already been published (Kucera & Gaisler 2012), the Slovak specimens were

collected and identified first. Geoglossum species are rare in Slovakia, and only

a few taxa had been reported before 2000 when we started our research (Kucera

et al. 2008, 2010, 2012; Kucera 2012). Geoglossaceous fungi, which have been

used in studies evaluating European grasslands (Rald 1985; Nitare 1988; Jordal

1997; McHugh et al. 2001; Adamcik & Kautmanova 2005), are of conservation

interest. In Sweden, Geoglossum uliginosum is treated in a special ‘action plan’

for nature conservation (Nitare 2007).

Materials & methods

Macromorphological characters were observed in fresh material. The

micromorphological structures were observed in dried material using a light microscope

112... Kuéera & al.

with an oil immersion lens. Fragments of fruit-bodies were examined in tap water, 5%

KOH, Melzer’s reagent, and a solution of Congo red in ammonia. Quantitative values

for micromorphological characters are presented as mean + 1 standard deviation of

30 measurements for each character (with minimum/maximum values in parentheses).

Herbarium acronyms are in accordance with Index Herbariorum (Thiers 2013). Voucher

specimens were deposited in herbaria PRM, BRNM, SAV, and UPS. Localities are geo-

referenced and the coordinates are given in the WGS 84 system. The description is based

on collections from Slovakia.

Description of studied Slovak collections

Geoglossum uliginosum Hakelier, Svensk Bot. Tidskr. 61: 422, 1967.

Ascocarps (35-)38-58(-66) mm high, clavate, stipitate, scattered, solitary.

Fertile part (10-—)14-22(-25) x (2-)3-6(-8) mm, usually flattened, lanceolate,

black, occasionally vertically grooved, glabrous. Sterile part 20-40(-50) x 1-3

mm, clearly delimited from the fertile part, cylindrical, black, smooth, viscid

when fresh, + shiny after drying [22 fruitbodies examined]. Asci (143-)

156-189(-214) x (11-)15-20(-24) um, clavate, apex rounded, narrowed, 8-

spored, pore blued in Melzer’s reagent. Spores (48-)59-75(-80) x 4-5(-6) um,

cylindrical, slightly curved, tapering towards one end, dark fuliginous, in one

cluster in the upper part of the ascus, 7-septate, occasionally 5- or 6-septate.

ParAPHYSES Slightly protruding above the asci, fragile, brownish in the apical

part, <9 um diam, remotely septate in the basal portion, closely septate in the

apical part, constricted at the septa. The constrictions occur at every second

septum. The cell pairs form chains and are easily broken off at the constrictions.

One of the cell pairs may be inflated to pyriform or globular form, <6-8 um

diam, especially in the apical part of the paraphyses. Instructive drawings

of paraphyses (and asci and spores) taken from the holotype specimen were

published by Nitare (2007).

HaBitTaT — Peat-bogs and wet meadows in association with Sphagnum

sp., Eriophorum sp., Deschampsia caespitosa, Nardus sp., Molinia caerulea, and

various species of Carex.

DISTRIBUTION — Czech Republic (Kucera & Gaisler 2012), Norway (Fadnes

2008; Nitare 2007), Slovakia (this paper), Sweden (Hakelier 1967; Nitare 1984,

2007; Ohenoja 2000, Turander 2012). Reports from Scotland and Northern

Ireland probably relate to a different species (Nitare 2007).

SPECIMENS STUDIED: SWEDEN, VASTMANLAND, Viker parish, Alvhyttan, Venen,

11.9.1965, N. Hakelier (holotype, UPS BOT: F-013939). SLOVAKIA, BaNnsKkA

Bystrica, Polana Mts., Hrinova, near mountain hotel “Polana’, along the trail to

the top of the Polana hill, 48°37'39.73"N 19°28'01"E, alt. 1304 m, wet meadow with

Deschampsia caespitosa, Eriophorum sp., Sphagnum sp. 27.9.2009, V. Kucera (SAV F-

10162); 6.10.2011, V. Kucera (SAV F-10529).

Geoglossum uliginosum (Slovakia) ... 113

Discussion

NOMENCLATURE — The name Geoglossum uliginosum could potentially

be confused with G. glabrum [var.] 6 uliginosum Pers. (Persoon 1800: 62,

tab. 3 fig. 4). As Persoon’s name was apparently never raised to specific rank,

however, Hakelier’s name is legitimate and not in conflict. G. glabrum var.

uliginosum certainly represents a different taxon because it was described as

having a squamulose stipe, while that of G. uliginosum is completely smooth.

We were unable to locate any specimen labelled either Geoglossum glabrum

var./f. uliginosum or Geoglossum uliginosum in Persoon’s herbarium (L), and

identification of Persoon’s fungus based only on the original (sparse) description

is impossible.

Taxonomy — Geoglossum uliginosum is characterised by having 1) ascocarps

with a slightly slimy-viscid stipe and 2) paraphyses with characteristically

long and easily broken chains of cells (or cell pairs) that are swollen at one or

sometimes both ends. The species grows only in slightly wet grasslands among

Sphagnum. ‘The material from Central Europe (both from Slovak and Czech

Rpublics) does not differ in any essential character from the holotype and other

Swedish collections; minor differences are presented in TABLE 1.

TABLE 1. Comparison of asci and ascospores in Geoglossum uliginosum from different

countries.

AscI (um) ASCOSPORES (ttm) No. oF

COUNTRY

LENGTH WIDTH LENGTH WIDTH SEPTA

(143-)156- (11-)15- (48-)59-

OAR 189(-214) 20(-24) 75(-80) AS(=8) C7

(151-)167- (13-)15- (50-)63-

CZECH REPUBLIC 190(-196) 19(-24) 78(90) 5-7 (5-)7(-9)

SWEDEN 140-175 14.5-17 60-80 4.5-6 7

Specimen data: Slovakia — SAV F-10162, SAV F-10529; Czech Republic — PRM 860478, PRM 860479,

PRM 860480, BRNM 737698, BRNM 737699, SAV F-10531, SAV F-10532 (see Kucera & Gaisler

2012); Sweden — Holotype UPS BOT F-013939 (Hakelier 1967).

CONSERVATION — Geoglossum uliginosum is well known from Scandinavia

(Sweden and Norway) and it was assumed that its distribution is restricted to

slightly wet and unfertilised semi-natural grasslands in the hemiboreal and

southern boreal vegetation zons in Scandinavia (Nitare 2007). Records from

Central Europe are therefore noteworthy.

We expect to find Geoglossum uliginosum in other locations in Central

Europe with habitats similar to those in Polana and Jizerské hory mountains.

The species is apparently rare and should be included in the next edition of the

Red list of Slovakia and Red list of Czech Republic. Currently the species is red-

listed in Sweden (Gardenfors 2010).

114... Kuéera & al.

Semi-natural grasslands are valuable landscape elements with high diversity

of plant and animal life and represent a reservoir of indigenous biodiversity.

Such habitats are critically threatened all over Europe, their number decreasing

mainly due to the fertilization, lack of grazing animals, and industrial pollution

(Newton et al. 2003).

Acknowledgements

We would like to thank Paul Cannon (CABI and Royal Botanic Gardens, Kew, UK)

and Esteri Ohenoja (University of Oulu, Finland), who read and commented on the

manuscript, and Shaun Pennycook (Manaaki Whenua Landcare Research, Auckland,

New Zealand) for his support. Vaclav Kautman, Stanislav Glejdura, and Nikola

Rybarikova are acknowledged for their assistance in the field, and Gerard Thijsse and

the staff of the Nationaal Herbarium Nederland in Leiden for the opportunity to study

Persoon’s herbarium. This study was supported by grant VEGA 2/0062/10 to VK and PL

and grant MZE 0002700604 to JG.

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rapport- 2/2008. [accessed 11-6-2012:

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country. Catathelasma 14: 11-14.

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Nitare J. 2007. Atgardsprogram for sumpjordtunga 2007-2011 Geoglossum uliginosum.

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MY COTAXON

http://dx.doi.org/10.5248/124.117

Volume 124, pp. 117-126 April-June 2013

A new endophytic species of Chaetomium

from Jatropha podagrica

ROHIT SHARMA’, GIRISH KULKARNI,

MAHESH S. SONAWANE’ & YOGESH S. SHOUCHE!’

"Microbial Culture Collection & *Molecular Biology Unit, National Centre for Cell Science,

NCCS Complex, Ganeshkhind, Pune- 411 007 (Maharashtra), India

* CORRESPONDENCE TO: rohit@nccs.res.in

ABSTRACT — A new endophytic species of Chaetomium from fruits of Jatropha podagrica is

described from Maharashtra State, India. A combined sequence dataset of the ITS region,

LSU rDNA, and $-tubulin genes supports recognition of this fungus as a new species that is

largely concordant with morphological characters. Chaetomium jatrophae is characterized by

spirally coiled, predominantly unbranched, and coarsely roughened terminal ascomatal hairs

and elongate-ellipsoidal ascospores with a subapical or lateral germ pore. It is a mesophilic

Chaetomium species with a temperature maximum of 40+0.2°C.

Key worps — Chaetomiaceae, Euphorbiaceae, phylogeny, taxonomy

Introduction

Chaetomium Kunze (Kunze 1817) (Chaetomiaceae) accommodates more

than a hundred species. Chaetomium species are widespread and found

on various substrates, most with remarkable cellulolytic activity. Some are

pathogenic for humans (Barron et al. 2003, Al-Aidaroos et al. 2007, de Hoog et

al. 2009, Hubka et al. 2011) and others are plant endophytes (Syed et al. 2009).

Chaetomium is a widely studied genus (Ames 1969; von Arx et al. 1986; Gené &

Guarro 1996; Decock & Hennebert 1997; Udagawa et al. 1997; Rodriguez et al.

2002; Wang & Zheng 2005a,b; Asgari & Zare 2011) that is mainly characterized

by superficial ostiolate ascomata covered with hairs. During our biodiversity

survey of endophytic fungi, we isolated 20 strains of fungi from Jatropha

podagrica belonging to 13 species. Two strains belonged to Chaetomium:

C. bostrychodes Zopf (MCC 1019), isolated from a leaf petiole, and a new species

isolated from the fruit of the same plant. This new species is described here as

Chaetomium jatrophae based on morphological and combined phylogenetic

data of three loci.

118 ... Sharma & al.

TABLE 1. Achaetomium and Chaetomium isolates used in phylogenetic analysis.

GENBANK ACCESSION NUMBERS

ie oyu ITS LSU B-TUBULIN

A. strumarium CBS 333.67 (T) AY681204 AY681170 AY681238

C. acropullum CBS 126783 HM365258 HM365258 HM365292

C. ancistrocladum CBS 126784 HM365241 HM365241 HM365290

CBS 126673 HM365242 HM365242 HM365291

C. atrobrunneum CBS 379.66 (T) HM365255 HM365255 HM365294

C. bostrychodes IMI 325809 HM365261 HM365261 HM365296

C. carinthiacum CBS 126669 HM365265 HM365265 HM365299

C. coarctatum CBS 162.62 (T) HM365260 HM365260 HM365281

C. crispatum CBS 152.49 HM365267 HM365267 HM365293

C. cruentum CBS 371.66 (T) HM365266 HM365266 HM365270

C. elatum CBS 126657 HM365236 HM365236 HM365282

CBS 126656 HM365238 HM365238 HM365284

Bet oan a HM365237 HM365237 HM365283

C. funicola IMI 300511 HM365262 HM365262 HM365300

C. globosum CBS 148.51 HM365254 HM365254 HM365269

C. grande CBS 126780 (T) HM365253 HM365253 HM365273

C. indicum CBS 126667 HM365248 HM365248 HM365301

CBS 126668 HM365249 HM365249 HM365302

C. interruptum CBS 126661 HM365245 HM365245 HM365276

CBS 126662 HM365244 HM365244 HM365275

CBS 126660 (T) HM365246 HM365246 HM365277

C. iranianum CBS 126670 (T) HM365257 HM365257 HM365297

C. jatrophae Beaman an 1Q246354 HE981193 - HE981190

C. jodhpurense CBS 280.79 HM365243 HM365243 HM365295

C. madrasense CBS 126663 HM365252 HM365252 HM365274

C. megalocarpum CBS 533.79 HM365259 HM365259 HM365271

CBS 126666 HM365264 HM365264 HM365272

C. murorum CBS 126785 HM365256 HM365256 HM365288

CBS 636.83 HM365268 HM365268 HM365289

C. rectangulare CBS 126659 HM365240 HM365240 HM365286

CBS 126778 (T) HM365239 HM365239 HM365285

C. subaffine CBS 126777 HM365247 HM365247 HM365280

C. truncatulum CBS 126782 (T) HM365263 HM365263 HM365298

C. undulatulum CBS 126776 HM365250 HM365250 HM365278

CBS 126775 (T) HM365251 HM365251 HM365279

Sequences in bold font were generated in this study. (T) = ex-type strain.

CBS = Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands.

IMI = CABI BioScience, Egham, UK.

Chaetomium jatrophae sp. nov. (India) ... 119

Materials & methods

Sampling, fungal isolation and growth

The fungus was isolated from a healthy fruit of Jatropha podagrica, an ornamental

plant, collected from Pune (Pimpri), Maharashtra, India (18°37'07.04"N 73°48'13.43"E).

Various plant parts (leaves, petioles, fruits, flowers, young stems) were used to isolate

various endophytic fungi upon Potato Dextrose Agar (PDA) and Czapek Dox Agar

(CDA). Healthy fruits were harvested with clean blades and immediately dipped in

beakers containing water. The fruits were subsequently washed with tap water (10

min), cut into fragments, washed twice in sterile distilled water (5 min), 70% ethanol

(2-3 min), and 100% ethanol (20-30 sec). The fungal strains were isolated on CDA at

room temperature after 3 d of incubation. Colony growth rate and characteristics were

recorded on 2% MEA (Hi-Media, India) at room temperature (28—30°C) after 7 d in

dark. Growth rate was also measured at different temperatures (20-45°C, at intervals

of 5+0.2°C) on five media including PDA, CDA (Hi-Media, India), PCA (Potato Carrot

Agar), MEA (Malt Extract Agar) and OA (Oatmeal Agar) after 7 d.

Morphological observations and scanning electron microscopy (SEM)

The ascomata and ascospores were studied on slides mounted in water or

lactophenol. The slides were observed under Nikon YS100 microscope (Nikon, Japan).

Microphotographs were taken on Olympus BX53 (Olympus Corporation, Japan)

fitted with ProgRes C5 camera (Jenoptik, USA). Measurements of morpho-taxonomic

characters were recorded and compared with type descriptions of known species (Ames

1969, Tiwari et al. 1977, Wang & Zheng 2005a,b; Asgari & Zare 2011). Fifty readings were

recorded for each measurement using Jenoptik software. Ascomatal hair ornamentation

was determined by scanning electron microscopy (SEM). Dry ascomata were placed on

black double-sided tape on a stub (1 cm diam.), coated with platinum in a Jeol sputter

coater (Jeol-JFC 1600), and then visualized in SEM (Jeol-JSM 6360A) at 10 kV.

DNA extraction

Genomic DNA for amplification of internal transcribed spacer (ITS) region (ITS1-

5.8S-ITS2) was extracted using QIAamp® DNA Mini Kit (Qiagen, Inc., Valencia CA).

Genomic DNA for D1, D2 region of 28S large subunit (LSU) of ribosome and $-tubulin

(BTUB) genes was extracted by crushing fungal mycelia in cetyl trimethylammonium

bromide (CTAB) extraction buffer, pH 8 (Hi-Media, India). Thereafter 20 ul of proteinase

K (20 mg/ml) was added and incubated at 65+0.2°C for 1 h (Baker & Mullin 1994),

followed by addition of 20 ul RNase (10 mg/ml) and further incubation at 65+0.2°C

for 15 min. Chloroform: isoamylalcohol (24:1) was added (500 ul) to the supernatant

collected after centrifugation (8000 rpm, 10 min). The mixture was shaken for 5 min

and centrifuged at 10,000 rpm, 4°C for 15 min. Two volumes of CTAB precipitation

buffer (1% CTAB, 50 mM Tris, pH 8.0, 10 mM EDTA) was added to the supernatant and

kept at room temperature for 1 h. The pellet collected after centrifugation was dissolved

in 500 ul of 1.2 M NaCl and then 500 ul of chloroform: isoamylalcohol (24:1) was added.

Two volumes of absolute alcohol were added to the aqueous phase to precipitate the

DNA (Murray & Thompson 1980, Brandfass & Karlovsky 2008). The quantity of DNA

was measured by the amount of absorbance of the sample at 260 nm on NanoDrop

(ND-1000, Thermo Scientific, USA) and purity was checked on 0.8% agarose gel

electrophoresis.

120 ... Sharma & al.

PCR amplification and DNA sequencing

The ITS-LSU rDNA region was amplified using the 5.88 (CGCTGCGTTCTTCATCG)

and LR7 (TACTACCACCAAGATCT) primers (http://www.botany.duke.edu/fungi/mycolab/

primers.htm). The reaction mixture (25 ul) containing genomic DNA (100 ng), 1X PCR

buffer, 0.2 mM dNTPs, 10-20 pmoles of each primer, and 1 unit of Taq DNA polymerase

was amplified with Gene Amplifier PCR System 9700 (Perkin Elmer, USA) using the

following protocol: initial denaturation at 95°C for 3 min, 35 cycles of 94°C for 1 min,

57°C for 30 sec, 72°C for 2 min, and final extension at 72°C for 10 min. The BTUB gene

was amplified using primers Tl (AACATGCGTGAGATTGTAAGT) and Bt2b (ACCCTCAGTG

TAGTGACCCTTGGC) (O'Donnell & Cigelnik 1997) using the same reaction mixture and

equipment as for the ITS-LSU region using the following protocol: initial denaturation

at 95°C for 3 min, 35 cycles of 94°C for 30 sec, 52°C for 30 sec, 72°C for 30 sec, and

final extension at 72°C for 10 min. Five uL of the PCR amplified product was run on

1.0% agarose gel in 1X TBE buffer (54 g Tris base, 27.5 g boric acid, 20 ml EDTA 0.5

M, pH 8). Electrophoresis was carried out for about 90 min at 80 V. The gel was stained

in 1% ethidium bromide for 15 min and observed under UV illumination. The PCR

products were purified according to Sambrook et al. (1989). The sequencing reactions

were carried out using the ABI PRISM BigDye Terminator Cycle Sequencing Ready

Reaction Kit’ (Perkin Elmer Applied Biosystems Division, Foster City, CA) according to

the manufacturer’s protocol. DNA sequencing was carried out on ABI 3730xl Automated

Sequencer.

Phylogenetic analysis

An NCBI and DDBJ BLASTn search for ITS region (555 bp) was conducted for

sequence similarity (Zhang et al. 2000). The phylogenetic relationships of C. jatrophae and

other Chaetomium species were examined using ITS, LSU, and BTUB gene sequences.

Thirty-three sequences corresponding to 23 Chaetomium species were retrieved from

GenBank along with the outgroup, Achaetomium strumarium (TaBLE 1). The sequences

generated in this study were manually edited using ChromasPro version 1.34. The ITS

region, rDNA LSU, and BTUB gene sequences were analyzed individually and together

in MEGA 5.0. All sequences were aligned by multiple alignments using MUSCLE

program in MEGA 5.0 (Edgar 2004, Tamura et al. 2011). Sequences were analyzed by

maximum parsimony (MP) statistical method using bootstrap method of phylogeny

test (1000 replicates and 10 initial trees) (Tamura et al. 2011). Gaps and missing data

were deleted. MP tree was obtained by close-neighbor-interchange (CNI) on random

trees algorithm (Nei & Kumar 2000). Bootstrap values below 50% were removed from

tree. Sequences were also analyzed by neighbour- joining (NJ) and maximum likelihood

(ML).

Results

Taxonomy

Chaetomium jatrophae Rohit Sharma, sp. nov. PLATES 1-2

MycoBAnk 563940

Differs from Chaetomium atrobrunneum by its larger ascomata with spiraling coarsely

roughened terminal hairs, its peridium of texture intricata, and its ascospores with a

sub-apical to lateral germ pore.

Chaetomium jatrophae sp. nov. (India) ... 121

PLATE 1. Chaetomium jatrophae (MCC 1025). Fic. 1: Mature ascoma, showing terminal coiled

terminal hairs. Fic. 2, 3: Ascomatal hairs (SEM). Fic. 4: Ascospores (SEM). Scale bars: 1 = 100

um; 2,3 = 10 um; 4=5 um.

Type: India, Maharashtra State, Pune, Pimpri, 18°37'07.04"N 73°48'13.43”E, endophytic

in fruit of Jatropha podagrica Hook. (Euphorbiaceae), 25.1V.2011, Rohit Sharma

(Holotype, AMH 9558; ex-type culture, MCC 1025, NFCCI 2596, CBS 134263).

EryMo_ocy: Refers to the host genus Jatropha.

Colonies reaching 60 mm diam. on MEA after 7 d at room temperature

(28-30°C), olivaceous-buff to grayish-sepia with sparse aerial mycelium;

reverse similar in color to colony surface. Mycelium composed of hyaline to

subhyaline, septate, smooth hyphae, 9.0—-9.4 um diam. Ascomata superficial

to immersed, scattered, ostiolate, globose to subglobose, 187.3 (161.1—193.8)

x 151.6 (129.2-161.1) um, brown to dark brown, maturing in 20 d, loosely

attached to mycelium with brown to olivaceous brown rhizoids. Peridium

brown-vinaceous to brown-olivaceous, membranaceous with textura intricata.

Terminal hairs of different length, 312.5 (250.0—375.0) um long, numerous, pale

yellowish-brown to dark brown, straight below and spirally coiled above, 8-11

coils (generally 7—8), 3.9 (3.2—4.8) um wide at the base gradually tapering and

paling towards the apex, hairs forming dense tuft around ostiole, unbranched,

rarely branched, thick walled, coarsely roughened, septate. Lateral hairs straight,

short, coarsely roughened, 56.0 (32.3—83.9) um in length and 3.5 (3.2—4.0) um

122 ... Sharma & al.

PLATE 2. Chaetomium jatrophae (MCC 1025). Fic. 5: Ascomatal hairs. Fic. 6: Ascospores.

Fic. 7: Mature ascomata. Scale bars: 5, 6 = 5 um, 7 = 150 um.

wide at the base. Paraphyses absent. Asci clavate, hyaline, evanescent. Ascospores

one-celled, brown when mature, fusiform or elongate-ellipsoidal, symmetrical

or sometimes asymmetrical, 12.3 (6.5-16.2) x 7.6 (6.5—9.7) um, thick-walled,

with a distinct sub-apical or lateral germ pore. Anamorph absent.

Cultural characteristics

The optimum growth of C. jatrophae was determined as 30+0.2°C (mesophilic

in nature) and its temperature maximum at 40+0.2°C (Fic. 8). The temperature

Le —-MEA

s -™-CDA

Z “ —*-OMA

> 30 =<—PCA

= —<PDA

0 T T T T T T 1

20°C 25°C 30°C 35°C 40°C 45°C

Temperature (°C)

Fic. 8. Temperature-growth relationship of three Chaetomium jatrophae stains on Malt Extract

Agar (MEA), Czapek Dox Agar (CDA), Oatmeal Agar (OA), Potato Dextrose Agar (PDA), and

Potato Carrot Agar (PCA) (mm diam. of 7 d old colonies).

Chaetomium jatrophae sp. nov. (India) ... 123

E C. elatum CBS 126657

— Ec. elatwm CBS 126656

71 'C. elatwm CBS 910.70

C. coarctatum CBS 162.627

C. subaffine CBS 126777

74| 96] (- C. undulatulwn CBS 126776

97° © undulatuhwn CBS 1267757

C. rectangulare CBS 126659

65| 99'C. rectangulare CBS 1267787

96|C. megalocarpum CBS 533.79

621 'C. megalocarpum CBS 126666

C. grande CBS 1267807

99} 8? C. madrasense CBS 126663

55| |C. interruptum CBS 126662

99|| C. interruptum CBS 126661

75 73'C. interruptum CBS 1266607

C. cruentum CBS 371.667

100! C. globosum CBS 148.51

C. carinthiacwn CBS 126669

64 C. iranianwn CBS 1266707

C. truncatulum CBS 1267827

jatrophae CBS 134263

99 C. atrobrunneum CBS 379.66"

9g|C. ancistrocladum CBS 126784

C. ancistrocladum CBS 126673

100] + C. murorwnCBS 126785

94'C. murorum CBS 636.83

C. bostrychodes IMI325809

C. acropullwn CBS 126783

99 C. crispatwn CBS 152.49

C. jodhpurense CBS 280.79

C. funicola IM1300511

100 C. indicum CBS 126667

99'C. indicum CBS 126668

Achaetomium strumarium CBS 333.677

Fic. 9. Maximum parsimony phylogram of Chaetomium jatrophae (MCC 1025) based on combined

dataset of ITS-LSU rDNA-BTUB gene sequences, with Achaetomium strumarium as outgroup.

Bootstrap values > 50% (1000 replicates) are shown; CI = 0.531860, RI = 0.705642, RCI = 0.375302;

gaps and missing data deleted. C. = Chaetomium, (T) = ex-type strain.

maximum is an important physiological character for species differentiation (Li

et al. 2012). The maximum fungal growth occurred primarily on MEA followed

by OA. Ascomata developed after 25-30 d of incubation on MEA.

Molecular analyses

The phylogenetic analysis of 24 taxa of Chaetomium was conducted using

ITS and LSU (D1, D2 region) of rDNA and BTUB gene and sequence data

124 ... Sharma & al.

analyzed individually (not shown) and in combination. The ITS alignment

consisted of 576 bases (438 conserved, 131 variable, 91 parsimony informative,

40 singleton). The maximum parsimony tree had CI = 0.687500 RI = 0.805654

and RCI = 0.553887 (for all sites). The LSU alignment consisted of 386 bases

(361 conserved, 25 variable, 22 parsimony informative, 3 singleton), and the

BTUB gene alignment consisted of 425 bases (209 were conserved, 215 variable,

193 parsimony informative, 22 singleton). The combined data set had 1392 sites

(1008 conserved, 371 variable, 306 parsimony informative, 65 singleton). One

most parsimonious tree is shown in Fic. 9. All phylogenetic analyses (NJ, MP,

ML) supported the novelty of this species.

Discussion

The newly described species, C. jatrophae, is morphologically and

molecularly differentiated from related species. Maximum parsimony

analysis of combined dataset of ITS-LSU rDNA-BTUB gene (Fic. 9) groups

C. jatrophae in a clade with C. atrobrunneum L.M. Ames with 97% bootstrap

support. Although the LSU and BTUB sequence data is unavailable for

C. jabalpurense D.P. Tiwarietal., the ITS sequence analysis (not shown) distinctly

separates C. jatrophae from the morphologically similar C. jabalpurense and

C. perlucidum Sergeeva. Chaetomium jatrophae resembles C. jabalpurense,

C. atrobrunneum, and C. perlucidum mainly by ascospore shape and germ pore.

Nevertheless, C. jabalpurense (Tiwari et al. 1977) can also be distinguished from

C. jatrophae by its larger ascomata (154.7—304.5 x 150.3-297.2 um), narrower

(3.2 um) terminal hairs, spirally coiled lateral hairs, and narrower (5.3—6.2

um diam.) ascospores. Chaetomium atrobrunneum (Ames 1949), also close to

C. jatrophae, is distinguished mainly by smaller ascomata (75—120 um), peridium

of textura angularis, straight seta-like mostly smooth-walled ascomatal hairs,

and fusiform or elongate pyriform ascospores with a slightly subapical germ

pore. Chaetomium perlucidum (Ames 1969) is differentiated from C. jatrophae

by its olive-yellow to pale-brown smaller (105-135 x 100-130 tm) ascomata

and narrower (5.6—6 um diam.) ascospores.

The fact that Chaetomium jatrophae was isolated from Jatropha podagrica,

a xerophytic latex-producing plant may help fungi to survive better than in

outside environment. The occurrence of this Chaetomium species in fruits

suggests that further research is needed to better understand its biology as well

as its obligatory or facultative endophytic nature, and its role as an endophyte in

J. podagrica. However, Syed et al. (2009) pointed that endophytic Chaetomium

species are similar to Chaetomium strains obtained from other substrates (e.g.,

scat, seed, soil) both physiologically (utilizing the same carbon source) and

genetically (similar ITS rDNA sequence data).

Chaetomium jatrophae sp. nov. (India) ... 125

Acknowledgments

The authors are grateful to DBT, India for funding, NCCS, India for laboratory

facilities and Dr. S.V. Shinde (University of Pune, India) for SEM microscopy. We are

also indebted to Dr. Xue-Wei Wang (State Key Laboratory of Mycology, China) and

Dr. Yei-Zeng Wang (National Museum of Natural Science, Taiwan) for their assistance

during preparation of manuscript. Dr. Bita Asgari (Iranian Research Institute of Plant

Protection, Iran) and Dr. Vit Hubka (Charles University, Czech Republic) are also

acknowledged for critical review of the manuscript.

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MY COTAXON

http://dx.doi.org/10.5248/124.127

Volume 124, pp. 127-141 April-June 2013

A contribution to the taxonomy of Lyromma

(Lyrommataceae, lichenized Ascomycota)

with a species key

ADAM FLAKUS! & EDIT FARKAS?

‘Laboratory of Lichenology, W. Szafer Institute of Botany, Polish Academy of Sciences,

Lubicz 46, PL-31-512 Krakow, Poland

?Institute of Ecology and Botany, Centre for Ecological Research, Hungarian Academy of Sciences,

H-2163 Vacratét, Alkotmany u. 2-4, Hungary

CORRESPONDENCE TO: 'a.flakus@botany. pl, *farkas.edit@okologia.mta.hu

ABSTRACT — The paper presents a new concept of conidial-ascosporic relationship in the

foliicolous lichenized genus Lyromma and a revised key to species determination. Lyromma

coronatum sp. nov. (Bolivia) is characterized by reduced perithecial appendages composed

of clavate hyphae that form a crown around the ostiole. Lyromma multisetulatum sp. nov.

(Bolivia, Brazil) is characterized by numerous large strongly recurved perithecial appendages

composed of individual acicular hyphae. Undescribed ascosporic states for Lyromma

dolicobelum and L. palmae and conidial states for L. ornatum and L. pilosum are described

based on observations of perithecia and pycnidia for the first time in natural conditions.

Confirmation of a conidial-ascosporic connection for L. nectandrae suggests L. confusum

as a synonym. Lyromma dolicobelum, L. nectandrae, and L. ornatum are reported as new to

Bolivia, and L. pilosum as new to Bolivia and Brazil.

Key worps —Neotropics, new species, South America

Introduction

This is a new contribution to the foliicolous lichens of Bolivia (Flakus &

Liicking 2008) within an inventory aimed to establish knowledge on the

diversity of lichens and allied fungi in the country (e.g. Flakus & Wilk 2006,

Flakus et al. 2008, Flakus 2009, Knudsen & Flakus 2009, Kukwa & Flakus 2009,

Flakus & Kukwa 2012, Flakus et al. 2012, Kukwa et al. 2012).

The foliicolous lichenized genus Lyromma Bat. & H. Maia (Lyrommataceae

Liicking, Chaetothyriales), containing six species accepted by Liicking (2008), is

characterized by globose to short barrel-shaped perithecia and elongate barrel-

shaped pycnidia, sessile on smooth ecorticate thalli of rounded patches formed

by symbiotic relationship with Phycopeltis. Perithecia are composed of a thin

128 ... Flakus & Farkas

layer of irregular dark brown hyphae, and persistent or evanescent paraphyses

comprise the hamathecium. The bitunicate obclavate to saccate asci (I-, KI-)

contain 8 transversely septate fusiform colorless ascospores. Pycnidia (formed

by a thin layer of distinctly parallel dark brown hyphae) produce multiseptate

filiform colourless macroconidia or non-septate bacilliform microconidia.

Mature perithecia and pycnidia both form appendages in the upper part near

ostiole, composed of either individual or agglutinated hyphae (Batista & Maia

1965, Cavalcante et al. 1972, Licking 1992, 2008, Liicking et al. 1998).

Lyromma was established as a monospecific pycnidial genus for L. nectandrae

(Batista & Maia 1965, Liicking et al. 1998). Perithecia were described originally

for L. nectandrae (Licking 1992, Aptroot et al. 1997) and later for L. ornatum

(Licking & Kalb 2000). For a long time, L. nectandrae was the only species

known to produce both perithecia and pycnidia (Liicking 1992, Aptroot et al.

1997). However, a re-examination of the known Lyromma material by Licking

(2008) showed that no single collection contained both perithecia and pycnidia

on the same thallus. Moreover, specimens assigned to L. nectandrae with

perithecia were shown to belong to different taxa described later as L. confusum

and L. pilosum (Liicking 2008). Since ascosporic and conidial states were not

observed on the same thallus, the perithecial and pycnidial morphotypes had

been treated as different species until their taxonomic status could be clarified.

Finally, based on the then-available specimens, Liicking (2008) proposed three

taxa with perithecia and three with pycnidia.

We have been able to study further fresh collections of foliicolous lichens

from Bolivia and Brazil and discovered more and more thalli of the otherwise

rare Lyromma species. These new findings form the basis of our re-evaluation

of the species concept based on conidial-ascosporic relationships.

Material & methods

Morphology and anatomy were examined by using NIKON SMZ800 and a NIKON

EcuipsE 80i (DIC), as well as OLympus SZX9 and Otympus BX50 (DIC) microscopes.

Preparations were studied in water and KOH solution (K), and for ascus structure in

Lugol's iodine solution without (I) or with pre-treatment with KOH (K/J). All anatomical

measurements were made in water.

Handling the sporocarps required great care to establish exact sizes and numbers of

setae. Anatomy, measurements, and number of setae should be examined on sectioned

or squash mounts in water under a compound research microscope.

Results

Lyromma coronatum Flakus & Farkas, sp. nov. PLATE 14, B

MycoBank MB 802289

Differs from other Lyromma species by its reduced perithecial appendages composed of

numerous individual clavate hyphae that form a regular crown around the ostiole.

Lyromma revised taxonomy and key ... 129

Type — Bolivia, Dept. Beni, Prov. Ballivian, near Rio Pircayo, 14°18'56"S 67°26'34"W,

190 m, lowland Amazon forest, 4 Dec. 2004, A. Flakus 4315 (Holotype, KRAM; isotype,

LPB).

EryMoLocy — The species is named after its reduced appendages forming a characteristic

crown around ostiole.

THALLUS foliicolous, epiphyllous, ecorticate, formed by rounded patches,

smooth, 0.4-3 mm wide, olive-green; PHOTOBIONT Phycopeltis, cells rectangular,

12-20 x 4-6 um, arranged in radiate plates; PERITHECIA sessile, globose, 50-100

um in diam., and 50-80 um high, pale brown, around ostiole furnished with

crown of almost reduced appendages; OSTIOLE apical, 20-35 um wide; SETAE

dark brown, almost reduced to 5-7(-12) x (4—)5-7 um, forming a very regular

crown around ostiole, composed of numerous individual clavate, apically

thickened (apical walls c. 1 um thick) hyphae (sometimes branched apically);

PERIDIUM pale brown, 3-5 um thick, composed of rectangular cells (3-5 um

wide), thick walled (c. 1-1.5 um thick), arranged irregularly to almost parallel;

PARAPHYSES Sparse in mature perithecia, simple, c. 1 um thick; ascr bitunicate,

8-spored, 30-50 x 10-15 um; ascosporess fusiform, hyaline, 3-septate, with

slight constrictions at septa, upper second cell usually slightly enlarged, 17-20

x 4-5 um; PYCNIDIA not seen.

DISTRIBUTION & ECOLOGY — The new species is known from two localities

in Bolivia, one of which is located in the protected area Reserva Nacional de

Vida Silvestre Amazonica Manuripi. It grows on leaves of vascular plants in

lowland Amazon forest.

ADDITIONAL SPECIMENS EXAMINED — BOLIVIA. DEpT. PANDO. PRov. MANURIPI,

Reserva Nacional de Vida Silvestre Amaz6onica Manuripi, Bajada colony near Chive

village, near Rio Madre de Dios, 12°24'03"S 68°26'45"W, 170 m, 2006, A. Flakus 6018

(herb. Flakus).

COMMENTS — Lyromma coronatum is characterized mainly by pale brown

perithecia and the presence of reduced perithecial appendages that form a

characteristic crown around the ostiole composed of numerous very short

clavate hyphae. The species can be confused with the ascosporic state of

L. palmae, which produces appendages composed of similar clavate hyphae.

Therefore, we initially regarded the new species as the juvenile form of

L. palmae with not fully developed appendages. However, a detailed study

showed that L. coronatum is mature species with fully developed asci and the

largest ascospores yet known in the Lyromma (Licking 2008). Thus, L. palmae

can be distinguished from the new species by its darker perithecia (especially

in the upper part), and the perithecial appendages that are larger [10-30(-40)

uum long], erect to horizontal, composed of about 20-60 individual hyphae, and

usually grouped in 3-6 brush-shaped appendages (then faintly agglutinated

in basal part). The two species also differ in the size of the photobiont cells

(12-20 x 4-6 um in L. coronatum, and 10-13 x 5-6 um in L. palmae), ascospores

130 ... Flakus & Farkas

(17-20 x 4-5 um in L. coronatum, and 14-18 x 3-5 um in L. palmae), and the

ostiole width (25-35 um in L. coronatum, and 15-20 um in L. palmae).

Lyromma dolicobelum Cavalc., in Cavalcante et al., Publ. Inst. Micol. Univ. Fed.

Pernambuco 647: 39. 1972. PLATE 1C

Type — Brazil. Roraima: Mucajai-Caracarai, II 1962, de Lima 19682 (URM 37178 -

holotype; INPA- isotype).

“Lyromma nectandrae var. dolicobelum” Bat. & Cavalc., in Bezerra et al., Atas Inst.

Micol. Univ. Fed. Pernambuco 5: 386, 389, 397, 409, 421. 1967, nom. nud.

[See Lticking (2008) for a detailed description of pycnidia. ]

THALLUS foliicolous, epiphyllous, ecorticate, formed by rounded patches,

smooth, 2-8 mm wide, olive-green; PHOTOBIONT Phycopeltis, cells rectangular,

10-17 x 4-7 um, arranged in radiate plates; PERITHECIA sessile, subglobose

to globose, 50-80 um in diam., and 60-80 um high, dark brown, around

ostiole furnished with 2-3(-4) setae; sETAE dark brown to black, tubular,

almost horizontal to recurved, 60-200(-300) um long and 20-30 um broad

in basal part, composed of agglutinated hyphae; PERrpIuM dark brown, lower

part slightly paler, 2-4 um thick, 3-5 um thick, composed of rectangular cells

(2-5 um wide), thick walled (c. 1-1.5 um thick), arrangement irregular to

almost parallel; PARAPHYSES sparse in mature perithecia, simple, c. 1 um thick;

ASCI bitunicate, 8-spored, 30-35 x 10-15 um; ascospores fusiform, hyaline,

3-septate, with slight constrictions at septa, 16-18 x 4-5 um.

DISTRIBUTION & ECOLOGY — ‘This species, known previously only from

lowland Amazon forest in Brazil and lowland rainforest in Mexico (Cavalcante

et al. 1972, Licking et al. 1998, Herrera-Campos et al. 2004, Liicking 2008), is

now reported for Bolivia.

SELECTED SPECIMENS EXAMINED — BOLIVIA. DEPT. PANDO. PRov. MANURIPI, Reserva

Nacional de Vida Silvestre Amazénica Manuripi, Puerto Rico village, 11°06'12''S

67°31'14"'W, 191 m, 2006, A. Flakus 6570 (LPB); Prov. FEDERICO ROMAN, near Santa

Cruzito colony, 10°43'03''S 65°55'05''W, 149 m, 2006, A. Flakus 7107, 7160, 7111, 7257,

7127 (KRAM, LPB, herb. Flakus).

ComMENTs — Lyromma dolicobelum is distinguished from all other Lyromma

species by the few but very long appendages, up to 200-300(-400) um long and

composed of agglutinated hyphae.

Lyromma multisetulatum Flakus & Farkas, sp. nov. PLATE 1D; 2A, J

MycoBank MB 802290

Differs from Lyromma pilosum by its more numerous, larger, and strongly recurved

perithecial appendages.

Type — Bolivia, Dept. Santa Cruz, Prov. Guarayos, Reserva Vida Silvestre Rios Blanco y

Negro, Plan de Manejo AISU, 15°01'58"S 62°46'36"W, 242 m, lowland Amazon forest,

24 July 2009, A. Flakus 13961 & P. Rodriguez (Holotype, KRAM; isotype, LPB).

Lyromma revised taxonomy and key... 131

PiaTE 1. Morphology of perithecial appendages of Lyromma. A-B: L. coronatum, reduced, forming

a crown around ostiole (holotype). C: L. dolicobelum, tubular, horizontal to recurved, composed

of agglutinated hyphae. D: L. multisetulatum, setose, recurved, composed of individual acicular

hyphae (holotype). E: L. nectandrae, tubular, horizontal to recurved, composed of agglutinated

hyphae. F—H: L. ornatum, triangular to irregularly lobate (H, deeply divided), horizontal, composed

of agglutinated hyphae. I-J: L. palmae, setose to brush-shaped, erect, composed of individual clavate

hyphae or agglutinated at the base (J, brush-shaped). K: L. pilosum, setose, recurved, composed of

individual hyphae. Scale bars = 25 um.

132 ... Flakus & Farkas

EryMoLocy — The species is named after its strongly recurved appendages produced in

large numbers near the top of perithecia.

THALLUS foliicolous, epiphyllous, ecorticate, formed by rounded patches,

smooth, 1-2 mm wide, olive-green; PHOTOBIONT Phycopeltis, cells rectangular,

18-22 x 6-8 um, arranged in radiate plates; PERITHECIA sessile, subglobose to

globose, 70-80 um in diam., and 70-90 um high, dark brown, around ostiole

furnished with (10-)15-30(-40) setae; seTAE dark brown, septate, strongly

recurved, composed of individual hyphae, 30-50(-60) um long and 3-4 um

broad (2-3 um broad in apical part); PERIDIUM dark brown, lower part slightly

paler, 3-4 um thick, composed of rectangular cells (3-4 um wide), thick walled

(c. 1 um thick), arrangement irregular to almost parallel; PARAPHYSES sparse

in mature perithecia, simple, c. 1 um thick; asci bitunicate, 8-spored, 30-35 x

10-13 um; AscosporEs fusiform, hyaline, 3-septate, with slight constrictions

at septa, 14-18 x 4-5 um; pycnip1a elongate, barrel-shaped with tapering top,

100-130 um high and 80-90 um wide, dark brown, upper part around ostiole

furnished with 15-30 setae; seTAE dark brown, septate, strongly recurved,

composed of individual hyphae, (20-)30-50(-60) um long and 3-4 um broad

(1-2 um broad in apical part); conrp1a (microconidia) bacilliform, non-

septate, 2-5 x 0.8-1.2 um.

DISTRIBUTION & ECOLOGY — The new species is known from the Reserva

Vida Silvestre Rios Blanco y Negro, Bolivia and the Private Nature Conservation

Reserve founded by Karoly Kégl near Sao Paulo, Brazil. It grows on vascular

plant leaves in lowland Amazon forest and Atlantic submontane rainforests.

ADDITIONAL SPECIMENS EXAMINED — BRAZIL. EsTapo SAo Pauto. SAO ROQUE, sobre

la Lagunita, al Norte de la Laguna Sapucaia, ca 850 m, 1995, A. Borhidi s.n. (VBI).

CoMMENTS — Lyromma multisetulatum is characterized by its numerous

[(10-)15-30(-40)], long [30-50(-60) um], and strongly recurved perithecial

appendages, each composed of an individual hypha. It is the first Lyromma

species known to produce microconidia. Perithecia of the most similar species,

L. pilosum, differ in forming less numerous appendages [(5-)10-15] that are

less curved and almost twice as short (10-15 um) (Liicking 2008).

Lyromma nectandrae Bat. & H. Maia, Atas Inst. Micol. Fed. Univ. Pernambuco 2:

360. 1965. PLATE IE

Type — Brazil. Pernambuco: Recife, Dois Irmdos, III 1960, Soares da Silva 13832 (URM

18764 — holotype); Brazil: Amazonas: Manaus, Itacoatiara, VII 1967, Omar 40677 (URM

69375 - epitype, designated by Liicking et al.: 145. 1998).

= Lyromma confusum Licking & Sérus., in Licking, Fl. Neotrop. Monogr. 103: 187. 2008.

Type — Papua New Guinea, Madang prov. Balek Wildlife Reserve, c. 15 km S of Madang

along road to Lae, 5°18'S 145°43’E, c. 20 m, in tropical lowland rainforest, on tree trunk/

on shaded limestone cliff (former coral reef), 29 July 1992, E. Sérusiaux 13555-17 (LG

- holotype!).

Lyromma revised taxonomy and key ... 133

PLaTE 2. Morphology of pycnidial appendages and anatomy of conidia of ae A: L. multi-

setulatum, setose, recurved, composed of individual acicular hyphae (holotype). B-C: L. ornatum,

tubular (simple to furcate), horizontal to recurved, composed of agglutinated hyphae (B, young

pycnidia). D-G: L. palmae, setose, erect, composed of individual clavate hyphae (D, young,

glabrous pycnidium). H-I: L. pilosum, setose, recurved, composed of individual acicular hyphae

(H, young, glabrous pycnidium). J: L. multisetulatum, short, bacilliform microconidia (holotype).

K: L. pilosum, large, filiform macroconidia. Scale bars: A-I, K = 25 um; J = 10 um.

“Lyromma confusum” Liicking, in Herrera-Campos et al.,

Lichenologist 36: 315. 2004, nom. nud.

[For detailed descriptions see Batista & Maia (1965) and Liicking (2008)].

DISTRIBUTION & ECOLOGY — This pantropical species is rather widespread in

the Neotropics, where it is known from Argentina, Brazil, Costa Rica, Ecuador,

French Guyana, Mexico, and Panama (Sipman 1997, Ferraro & Licking 2000,

Herrera-Campos et al. 2004, Liicking 2008), and is now also reported from

Bolivia, where it was found in lowland rainforests.

134 ... Flakus & Farkas

SELECTED SPECIMENS EXAMINED — BOLIVIA. Dept. BENI. PRov. BALLIVIAN, near

Pircayo river, 14°18'56"S 67°26'34"W, 190 m, 2004, A. Flakus 4174.1 (LPB). DEpT.

PANDO. PRov. FEDERICO ROMAN, near Santa Cruzito colony, 10°43'03"S 65°55'05" W,

149 m, 2006, A. Flakus 7193.1, 7298 (KRAM, LPB). DEPT. SANTA Cruz, PRov.

Guarayos, Reserva Vida Silvestre Rios Blanco y Negro, Virgen de Pilar near Chonta

village, 15°38'54"S 62°57'37"W, 229 m, 2009, A. Flakus 13481, 13381.1 & P. Rodriguez

(KRAM, LPB); Plan de Manejo AISU, 15°01'58"S 62°46'36"W, 242 m, 2009, A. Flakus

13911 & P. Rodriguez (LPB). BRAZIL. Estapo ToCANTINS. TONCINOPOLIS, 3 km al

NE de la ciudad, Fazenda de José Borges cerca del Riacho Francisco, 1995, A. Borhidi

s.n. (VBI). ESTADO RIO DE JANEIRO. SERRA DO MANTIQUEIRA, Parque Nacional de

Itatiaia, en el alrededor del Cachueiro Veu da Noiva, ca 1300-1500 m, 1995, A. Borhidi

s.n. (VBI). PAPUA NEW GUINEA. MADANG PROVINCE. PONT DE LA GOGOL RIVER,

route de Lae a env. 15 km de Madang, 5°20’S 145°43’E, 30 m, 1987, J. Lambinon 87/370

(LG); RAMU VALLEY. Brahman, along road to Bundi, 5°46'S 145°21'E, 200 m, 1992, E.

Sérusiaux 14200-2 (LG); FOTHILLS OF FINISTERRE RANGE, along road Madang-Lae, km

39, c. 230 m, 1992, E. Sérusiaux 14303-48 (LG). CENTRAL PROVINCE. N of Brown River,

3 km NE of Hiritano highway, 30-50 m, 1987, J.R. Sloover 87 L 23 (LG). INDONESIA.

BALI. GIANYAR DistTrRIcT, Gununk Kawi, 8°28'S 115°18’E, 1991, 500 m, L. Hoffmann

91-115 (LG).

Comments — Although Liicking (2008) considered the perithecial appendages

of Lyromma confusum morphologically very similar to those on the pycnidia

of L. nectandrae, he treated the two as separate taxa because there was no proof

of a conidial-ascosporic connection. We found thalli bearing pycnidia typical

of L. nectandrae mixed together with perithecia of L. confusum and so regard

L. confusum as a synonym of L. nectandrae.

Mature perithecia of L. nectandrae have 3-5, horn-shaped, recurved

(rarely horizontal), 30-70 um long and 10-15(-20) um broad (at the base)

appendages and are sometimes 2-3-furcate at the ends (Lticking 2008). Young

undeveloped L. nectandrae specimens can be confused with L. ornatum, which

has short perithecial appendages but which can be distinguished from mature

L. nectandrae material by the irregularly lobate to triangular, horizontal (rarely

slightly recurved), comparatively shorter [15-30(-40) um long], perithecial

appendages (Liicking & Kalb 2000, Lticking 2008).

Likewise, the pycnidia of L. nectandrae are morphologically similar to those

of L. ornatum (shown on PLATE 2C) with (2-)3-5 cylindrical to irregular

appendages that are 40-60(-100) um long and 10-15 um broad (at the base)

and may be simple to 2-3-furcate at the ends (Batista & Maia 1965, Liicking

2008).

Lyromma ornatum Licking, Kalb & Sérus., in Liicking & Kalb, Bot. Jahrb. Syst. 122:

39. 2000. PLATE 1F-H; 2B, C

Type — Brasilien. Sao Paulo, Serra do Mar, Serra do Garraozinho, zwischen Mogi das

Cruzes und Bertioga, in einem primaren Regenwald, 23°45'S 46°10'W, 300-600 m,

6.1X.1980, K. Kalb 32601 & G. Plobst (herb. K. Kalb - holotype!).

[For detailed descriptions of the thallus and perithecia, see Licking & Kalb

(2000) and Licking (2008)].

Lyromma revised taxonomy and key ... 135

PycnipiA elongate barrel-shaped with a tapering top, 100-120 um high

and 35-60 um in wide, dark brown, upper part around the ostiole furnished

with 2-4 setae; SETAE dark brown to black, recurved, composed of agglutinated

hyphae, cylindrical to irregular, simple to 2-4-furcate on the ends, 30-80 um

long and 10-20 um broad (at the base); conip1a (macroconidia) filiform,

multiseptate, 50-80 x 0.8-1.2 um.

DISTRIBUTION & ECOLOGY —This species, rather widespread in the

Neotropics, has been reported from Argentina, Brazil, Ecuador, Peru, Mexico,

and Surinam (Ferraro & Liicking 2000, Licking & Kalb 2000, Herrera-Campos

et al. 2004, Liicking 2008). These are the first records from Bolivia, where it is

known only from lowland Amazon forest.

SELECTED SPECIMENS EXAMINED — BOLIVIA. Dept. BENI. PRov. BALLIVIAN near

Pircayo river, 14°18'56"S 67°26'34"W, 190 m, 2004, A. Flakus 4173 (LPB). Dept.

PANDO. PRov. MANuRIPI, Reserva Nacional de Vida Silvestre Amazonica Manuripi,

Bajada colony near Chive village, 12°24'03"S 68°26'45"W, 170 m, 2006, A. Flakus 6019.1

(LPB); Chive village, 12°23'19"S 68°34'39"W, 157 m, 2006, A. Flakus 6407 (KRAM,

LPB); Puerto Rico village, 11°06'12"S 67°31'14"W, 191 m, 2006, A. Flakus 6554, 6565

(KRAM, LPB); Prov. NICOLAS SUAREZ, near Cobija village, 11°02'16"S 68°45'31”"W,

191 m, 2006, A. Flakus 6455, 6457 (KRAM, LPB, herb. Flakus); Prov. MADRE DE Dios,

Reserva Nacional de Vida Silvestre Amazonica Manuripi, Puerto Madre de Dios village,

11°30'56"S 67°16'07"W, 153 m, 2006, A. Flakus 6802, 6865 (KRAM, LPB); Prov.

FEDERICO ROMAN, near Santa Cruzito colony, 10°43'03"S 65°55'05"W, 149 m, 2006,

A. Flakus 7275 (LPB). DEPT. SANTA Cruz. PRov. GuARAYOS, Reserva Vida Silvestre

Rios Blanco y Negro, Virgen de Pilar near Chonta village, 15°38'54"S 62°57'37"W, 229

m, 2009, A. Flakus 13381, 13382 & P. Rodriguez (KRAM, LPB); Plan de Manejo AISU,

130 km from Ascencién de Guarayos village, 15°03'35"S 62°45'20" W, 258 m, 2009, A.

Flakus 13841, 13842 & P. Rodriguez (KRAM, LPB, herb. Flakus). BRAZIL. Estapo

TOCANTINS. TONCINOPOLIS, 3 km al NE de la ciudad, Fazenda de José Borges cerca del

Riacho Francisco, 1995, A. Borhidi s.n. (VBI).

COMMENTS — Studies on a wide range of material show that Lyromma

ornatum exhibits considerable variation in the shape and size of its perithecial

appendages. ‘The species forms 3-6 horizontal to (rarely) slightly recurved

appendages composed of distinctly agglutinated hyphae, which are triangular to

irregularly lobate but sometimes also deeply divided at the apex, 15-30(-40) um

long and 7-15(-20) um broad (at the base). Morphotypes with strongly incised

appendages are externally similar to the perithecia of L. palmae. However,

L. palmae has erect to horizontal appendages, composed of 20-60 clavate hyphae

(apically <4-7 um broad), usually grouped into 3-6 brush-shaped appendages

(then faintly agglutinated at the base). Lyromma nectandrae, another similar

species (see above), has much larger recurved perithecial appendages (30-70

um long) (Lticking 2008).

Only a few poorly developed (probably juvenile) pycnidia were observed

on thalli of L. ornatum together with perithecia typical for the species. They

most closely resemble those of L. nectandrae in having recurved simple to

136 ... Flakus & Farkas

2-4-furcate appendages composed of agglutinated hyphae, but they are less

numerous [2-3(-4)] and shorter (30-80 um long). The relationship between

these two species can be clarified only by studying more complete material or

by molecular analyses.

Lyromma palmae (Cavalc. & A.A. Silva) Licking & Sérus., in Licking et al.,

Lichenologist 30: 136. 1998. PLATE LI, J; 2D-G

= Anconomyces palmae Cavalc. & A.A. Silva, in Cavalcante et al.,

Publ. Inst. Micol. Univ. Fed. Pernambuco 647: 26. 1972.

Type — Brazil. Amazonas: Manaus, Manaus, Rodovia AM-1, km 55, Aug 1961, Carvalho

17065 (URM 29109 - holotype; INPA - isotype); Brazil. Maranhao: Zé Doca, IX 1965,

Peres 25270 (URM 52420 - epitype, designated by Liicking et al.: 136. 1998).

THALLUS foliicolous, epiphyllous, ecorticate, forming rounded patches,

smooth, 0.3-3 mm diam, olive-green; PHOTOBIONT Phycopeltis, cells

rectangular, 8-21 x 4-7 um, arranged in radiate plates; PERITHECIA sessile,

subglobose to globose, 40-80 um in diam., and 40-60 um high, dark brown,

around ostiole furnished with 20-60 individual setae or 3-6 brush-shaped

appendages; sETAE dark brown, erect to horizontal, composed of about 20-60

individual, clavate hyphae, 10-30(-40) um long and (3-)4-6(-7) um broad

(at the apex), setose but usually grouped in 3-6 brush-shaped appendages,

then faintly agglutinated at the base; PERIDIUM dark brown, lower part slightly

paler, 3-4 um thick, composed of rectangular cells (2-3 um wide), thick walled

(c. 1 um thick), arrangement irregular to almost parallel; PARAPHYSES sparse

in mature perithecia, simple, c. 1 um thick; asc bitunicate, 8-spored, 30-35 x

10-14 um; ascospores fusiform, hyaline, 3-septate, with slight constrictions

at septa, second cell from above sometimes slightly enlarged, 14-18 x 3-5 um;

PYCNIDIA elongate barrel-shaped with a tapering top, 100-130 um tall and 40-

55 um wide, dark brown, upper part around the ostiole furnished with 20-25

setae; SETAE dark brown, erect, usually clavate, septate, composed of individual

hyphae, (10-)20-30(-40) um long, 3-4 um broad in basal part and (3-)4-6

(-7) um broad in apical part; conipIA (macroconidia) filiform, multiseptate,

40-100 x 0.8-1.2 um.

DISTRIBUTION & ECOLOGY — This species is known from Argentina, Bolivia,

Brazil, Costa Rica, and Mexico (Cavalcante et al. 1972, Liicking et al. 1998,

Ferraro & Licking 2000, Herrera-Campos et al. 2004, Flakus & Licking 2008,

Liicking 2008). It is found mostly in lowland rainforests, and also in northern

Argentinean forests.

SELECTED SPECIMENS EXAMINED — BOLIVIA. DEPT. BENI. PRov. BALLIVIAN, Nuevos

Reyes colony, 14°13'17"S 67°28'25"W, 190 m, 2004, A. Flakus 3811 (LPB); near Pircayo

river, 14°18'56"S 67°26'34"W, 190 m, 2004, A. Flakus 4174.1, 4195.1 (KRAM, LPB).

Dept. Panpo. Prov. MANnurRIpPI, Reserva Nacional de Vida Silvestre Amazonica

Manuripi, Bajada colony near Chive village, 12°24'03"S 68°26'45"W, 170 m, 2006, A.

Lyromma revised taxonomy and key ... 137

Flakus 5989.3 (KRAM); Puerto Madre de Dios village, 11°27'38"S 67°15'56"W, 166

m, 2006, A. Flakus 6662, 7046 (KRAM, LPB). DEPT. SANTA CRUZ. PRov. GUARAYOS,

Reserva Vida Silvestre Rios Blanco y Negro, Virgen de Pilar near Chonta village,

15°38'54"S 62°57'37"W, 229 m, 2009, A. Flakus 13332, 13491 & P. Rodriguez (KRAM,

LPB); Plan de Manejo AISU, 130 km from Ascencién de Guarayos village, 15°03'35"S

62°45'20"W, 258 m, 2009, A. Flakus 13842.1, 13842.2 & P. Rodriguez (KRAM, LPB); 160

km from Ascencién de Guarayos village near Rio Negro, 14°58'50"S 62°36'19"W, 242 m,

2009, A. Flakus 14021 & P. Rodriguez (herb. Flakus).

ComMENTS — ‘The perithecia of L. palmae are characterized by the erect to

horizontal setae composed of 20-60 clavate hyphae grouped usually into 3-6

brush-shaped appendages, 10-30(-40) um long and (3-)4-7 um broad at the

apex. This distinguishes it from the most similar species, i.e. L. coronatum,

L. ornatum (see also below), and L. pilosum.

The structure of the pycnidia and pycnidial appendages of L. palmae were

variously interpreted before 2008. According to Liicking et al. (1998), the species

was characterized by pycnidia with reduced apical setae, compared to those of,

for example, L. nectandrae or L. dolicobelum. In his subsequent monograph,

Liicking (2008) described L. palmae as having glabrous pycnidia without

appendages. However, the original description and illustration (Cavalcante

et al. 1972: 26, fig. 5), as well as a detailed study of the epitype by Lticking

et al. (1998: 136) established that the species develops small appendages near

the pycnidial apex. The rich collections now available show that L. palmae is

characterized by pycnidia with 20-25 erect usually clavate setae composed of

individual hyphae that are (10-—)20-30(-40) um long, 3-4 um broad at the base,

and (3—)4—6(-7) um broad at the apex (PLATE 2 FE, G). Pycnidia of L. palmae are

somewhat reminiscent of those of L. pilosum but differ in having acicular, horn-

shaped, and strongly recurved appendages (1-3 um broad at the apex).

Lyromma pilosum Liicking, Fl. Neotrop. Monogr. 103: 187. 2008. PLATE 1K; 2H, I, K

Type — Costa Rica. Puntarenas: Monteverde Biological Reserve, 1600-1700 m, Feb

2000, Liicking 00-418 (CR — holotype).

[For a detailed description of the thallus and perithecia, see Licking (2008). ]

PycnipiA elongate barrel-shaped with tapering top, 100-130 um tall and

40-60 um in wide, dark brown, upper part around ostiole furnished with 5-10

setae; SETAE dark brown, acicular, horn-shaped, strongly recurved, septate,

composed of individual hyphae, (10-—)20-30(-40) um long, and 4-5 um broad

(2-3 um broad in apical part); conrp1a (macroconidia) filiform, multiseptate,

40-70 x 0.8-1.2 um.

DISTRIBUTION & ECOLOGY — This species was described from Argentina

and Costa Rica (Liicking 2008). Here we report it from Bolivia and Brazil for

the first time. It grows in northern Argentinean forests, montane forest, lowland

Amazon forest, and Atlantic submontane rainforest.

138 ... Flakus & Farkas

SELECTED SPECIMENS EXAMINED — BOLIVIA. DEPT. PANDO. PRov. MANURIPI, Reserva

Nacional de Vida Silvestre Amazonica Manuripi, Chive village, 12°23'47"S 68°35'28" W,

179 m, 2006, A. Flakus 5900, 5903.2 (KRAM, LPB); Bajada colony near Chive village by

Rio Madre de Dios, 12°24'03"S 68°26'45"W, 170 m, 2006, A. Flakus 5975, 6019.2 (LPB,

herb. Flakus); Prov. NicoLas SUAREZ, near Cobija village, 11°02'16"S 68°45'31"W,

191 m, 2006, A. Flakus 6440 (LPB); Prov. MADRE DE Dios, Reserva Nacional de Vida

Silvestre Amazonica Manuripi, near Puerto Madre de Dios village, near Rio Madre de

Manupare, 11°31'37"S 67°17'29"W, 155 m, 2006, A. Flakus 6719 (KRAM). BRAZIL.

EstapDo SAo PauLo. SAO ROQUE, cascada sobre la Laguna Sapucaia, ca 800 m 1995,

A. Borhidi s.n. (VBI); sobre la Laguna Sapucaia, arriba del camino, ca 850 m, 1995, A.

Borhidi s.n. (VBI).

ComMENTs — Lyromma pilosum was the first species of the genus described as

having short perithecial appendages composed of an individual hypha (Licking

2008). It is most similar to L. multisetulatum and L. palmae, which differ in the

size, shape, and orientation of the perithecial appendages. For more details, see

remarks below those species.

The pycnidia of L. pilosum and L. palmae have similar appendages, each

composed of individual hypha that is 3-5 um broad across the base. Juvenile

pycnidia of both species are smooth (without appendages) and can be

distinguished only in the mature stage. Pycnidia of L. pilosum have 5-10 setae,

which are acicular, strongly recurved, (10—)20-30(-40) um long, and 2-3 um

broad at the apex. In contrast, those of L. palmae produce 20-25 setae, which

are erect, usually clavate, (10—)20-30(-—40) um long, and (3-)4-6(-7) um broad

at the apex.

Key to species of Lyromma based on ascosporic and conidial states

la. Sporocarps perithecia, globose to very short barrel-shaped, usually <100 um high . . .2

1b. Sporocarps pycnidia, elongate barrel-shaped, usually >100 um high............. 8

2a (la). Perithecial appendages (5-)10-30(-60), setose (strongly recurved) to brush-

shaped (erect to horizontal), each composed of individual hypha (sometimes

faintly agglutinated at the base), (5—)10-30(-40) um long and 3-5 um broad

at the base) or almost reduced, forming a crown ................ esse ee eee 5

2b. Perithecial appendages 2-6, horn-shaped or triangular to irregularly lobate,

horizontal to recurved, composed of agglutinated hyphae (sometimes deeply

divided at the apex), 15—200(-300) tum long and 7-30 um broad (at the base) . . . .6

3a (2a). Perithecial appendages reduced, forming a crown, composed of individual

clavate hypha, 5-7(-12) um long and (4—)5-7 um broad

Ci dita ho 4 ce eae en a ee TO enn Te Co Oa toe COR en eo L. coronatum

3b. Perithecial appendages larger, strongly recurved, setose 10-50(-60) um long or

erect to horizontal, composed of 20-60 individual hyphae usually grouped

into 3-6 brush-shaped appendages, 10-30(-40) um long.................... 4

Lyromma revised taxonomy and key ... 139

4a (3b). Perithecial appendages erect to horizontal, composed of 20-60 clavate

hyphae usually grouped into 3-6 brush-shaped appendages (then faintly

agglutinated at the bases), 10-30(-40) um long and (3-)4—7 um broad

bal CHOP OK) tanh snags Mostra N nan eN cal haan hN cwky Carl cot 0) cu cea Wes an L. palmae

4b. Perithecial appendages (5-)10-30(-40) strongly recurved setae composed

of acicular hyphae, 10-50(-60) um long and 2-3 um broad (at the apex)....... 5

5a (4b). Perithecial appendages (5—) 10-15 setae, 10-30(-40) um long ..... L. pilosum

5b. Perithecial appendages (10-)15-30(-40) setae, 30-50(-60) um long

Dice ys alone Sle New salle NeW tells bens olloa bi alls basllbs ns ala basa L. multisetulatum

6a (2b). Perithecial appendages 3-6, triangular to irregularly lobate (sometimes

deeply multi-furcated on the apex), horizontal to rarely slightly recurved,

15-30(-40) um long and 7-15(-20) um broad (at the base) ......... L. ornatum

6b. Perithecial appendages 2-5, cylindrical to irregular, horn-shaped (sometimes

2-3-furcate on the ends), recurved or rarely almost horizontal, 30-200 um

long-and:10225; une broad: Gat the: Dase yy a. ih thie od oie hk g og Ow ae aha ele 7

7a (6b). Perithecial appendages 3-5; 30-70 um long, simple to 2-3-furcate

OMIGEI CMA POX ay cin yk a eee eg didte peg AiO eg: d aber G4 ager PQ abrt-Fo Ober Ss artes L. nectandrae

7b. Perithecial appendages 2-3(—4); 60-200(-300) um long ........... L. dolicobelum

8a (1b). Pycnidia glabrous (when young) or with 5-30 appendages composed of

mdividual- hyphaey3—o- umn broad (atthe base): sss B, stowa at ona Move Bae ava kav 9

8b. Pycnidia with 2-5 appendages composed of agglutinated hyphae,

LO 2 Onin DOA Abate ASO pr wath cm crete ore see ann cos ee owe el entwa sce ciewe ate 11

9a (8a). Appendages 20-25, clavate, erect to almost vertical, (10—)20-30(-40)

uum long and (3—)4-6(-7) um broad (at the apex)................0.. L. palmae

9b. Appendages acicular, horn-shaped, strongly recurved, 1-3 um broad

CAE TTS ARO aes esg Psat od eal s of tose gg amas ersten eM eae RRS ee ae oa, teh EG ate 10

10a (9b). Appendages 5-10, (10—)20-30(-40) um long; macroconidia

filiform, multiseptate, 40-60 x 0.8-1.2 um.... eee eee eee L. pilosum

10b. Appendages 15-30, (20-)30-50(-60) um long; microconidia bacilliform,

THONESe Prater? =a J liis). brea es tesla tase Neusat Ne cae L. multisetulatum

lla (8b). Appendages (2-)3-5, simple to 2—-4-furcate on the ends,

cylindrical to irregular, 30-60(-100) um long and 10-20 um

broad (at the base) ... 0.0.0... cc cece ce eee ee L. nectandrae or L. ornatum

11b. Appendages 2(-3), simple, cylindrical, 200-400 um long and 10-20 um

broad: (atthe bases aby. s dics.eg diou.e z dinsee a diners o dineee a drosee s deed L. dolicobelum

Discussion

During the current study, we discovered undescribed ascosporic states for

Lyromma dolicobelum and L. palmae and conidial states for L. ornatum and

L. pilosum. For the first time in these species both perithecia and pycnidia were

observed associated on the same thallus under natural conditions. A conidial-

140 ... Flakus & Farkas

ascosporic connection for Lyromma nectandrae suggests that L. confusum

should be treated as a synonym of that species. Additionally, two new species

of the genus, Lyromma coronatum and L. multisetulatum, were discovered in

material from Bolivia and Brazil.

Since all Lyromma taxa have very similar thalli, their relationships have

been based on the morphology and anatomy of their sporocarps, with the

most useful characters provided by the variable appendages produced near

the ostiole both in the ascosporic and the conidial states. From our taxonomic

revision of additional, recently collected material, we propose a new concept

of the conidial-ascosporic relationships in this foliicolous genus. Despite the

many differences between perithecia and pycnidia noted by Liicking (2008),

our results show similarities between appendage types that connect ascosporic

and conidial states for each species.

Our hypothesis on the generic circumscription, which is probably temporary

due to the still limited knowledge on this overlooked genus, can be expected to

evolve as new collections and/or molecular sequence data becomes available.

The size of these organisms (with a mean thallus diameter normally <5 mm,

and sporulating structures around 100 um high) also limits further studies. We

hope, however, that our revised key to Lyromma species using both perithecial

and pycnidial characters will encourage further research into this fascinating

foliicolous lichenized genus.

Acknowledgments

We are very grateful to Prof. David Hawksworth (London), Dr. Robert Liicking

(Chicago), and Dr. Shaun Pennycook (Auckland) for reviewing the manuscript, and

for valuable and constructive comments on previous versions of this paper. Mr. Ken

Hudson (Egham) and Dr. David Minter (Egham) are thanked for clarifying bibliographic

problems concerning to cited publications. We are also greatly indebted Prof. Klaus Kalb

(Neumarkt) and Prof. Emmanuél Sérusiaux (Liege) for loan of the type material for

study, Dr. Martin Kukwa (Gdansk) for valuable comments, as well as to Rosa I. Meneses

Q., the Director of Herbario Nacional de Bolivia, Universidad Mayor de San Andrés, La

Paz for generous cooperation. This research received support from the NCN in Poland

for the years 2008-2011 (no. N N303 345335), the NCBiR in Poland under the LIDER

Programme for the years 2010-2013 (no. 92/L-1/09), the W. Szafer Institute of Botany

of the Polish Academy of Sciences through the program supporting research of young

scientists, and the Hungarian Scientific Research Fund (OTKA K81232).

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einer Checkliste und Bemerkungen zu Coenogonium und Dimerella (Gyalectaceae). Bot. Jahrb.

Syst. 122: 1-61.

Licking R, Sérusiaux E, Maia LC, Pereira CG. 1998. A revision of the names of foliicolous lichenized

fungi published by Batista and co-workers between 1960 and 1975. Lichenologist 30: 121-191.

http://dx.doi.org/10.1017/S0024282992000124

Matzer M. 1996. Lichenicolous ascomycetes with fissitunicate asci on foliicolous lichens. Mycol.

Pap. 171: i-x, 1-202.

Sipman HJM. 1997. Observations on the foliicolous lichen and bryophyte flora in the canopy of a

semi-deciduous tropical forest. Abstracta Botanica 21: 153-161.

Upadhyay HBP. 1964. Three new hyperparasites for Mazosia phyllosema (Nyl.) A. Zahlbr. from

Amazonas Valley. Publ. Inst. Micol. Univ. Fed. Pernambuco 402: 1-11.

MYCOTAXON

http://dx.doi.org/10.5248/124.143

Volume 124, pp. 143-148 April-June 2013

Scolecobeltrania,

an interesting new microfungus from Venezuela

TERESA ITURRIAGA', ROBERTO FERNANDEZ’, RAFAEL F. CASTANEDA-RUIZ?,

Davip W. MINTER* & ALISSON CARDOSO RODRIGUES DA CRUZ>*

"Departamento de Biologia de Organismos, Universidad Simon Bolivar,

Apartado 89000 Sartenejas, Baruta, Edo Miranda, Venezuela

?Instituto de Investigaciones Fundamentales en Agricultura Tropical ‘Alejandro de Humboldt’

(INIFAT), Académico Titular de la Academia de Ciencias de Cuba,

Calle 1 Esq. 2, Santiago de Las Vegas, C. Habana, Cuba, C.P. 17200

‘CABI,Bakeham Lane, Egham, Surrey, TW20 9TY, United Kingdom

°Departamento de Ciéncias Biolégicas, Laboratorio de Micologia, Universidade Estadual

de Feira de Santana, BR116 KMO3, 44031-460, Feira de Santana, Brazil

*CORRESPONDENCE TO: iturriagamariateresa@gmail.com

ABSTRACT — A new microfungus collected on dead plant material from the “Cerro El Avila”

mountain range and “Parque Nacional Canaima’ tropical forest in Venezuela is described and

illustrated. Scolecobeltrania vermispora gen. & sp. nov. is characterized by macronematous

branched conidiophores, polyblastic conidiogenous cells, and unicellular vermiform to

sigmoid pale brown conidia with up to 11 hyaline transverse bands.

KEY worpDs — anamorphic fungi, systematics, leaf litter

Introduction

During two mycological surveys of anamorphic fungi in the cloud and

tropical forests in Venezuela, we collected an interesting conspicuous fungus.

It has some characters that are present in Beltrania and some allied genera,

but the conidia differ morphologically from those in all previously described

anamorphic genera of this group. We propose a new genus to accommodate

this new fungus described here.

Plant samples were placed in paper and plastic bags, taken to the laboratory,

and treated according to Castafieda (2005). Mounts of fungal material were

prepared in polyvinyl alcohol-glycerol (8 g in 100 ml of water, plus 5 ml of

glycerol) and diagnostic characters were measured at x1000. Micrographs were

obtained with a Zeiss Axioskop 40.

144 ... Iturriaga & al.

Taxonomy

Scolecobeltrania Iturr., R.F. Castafieda & Rob. Fernandez, gen. nov.

MycoBank MB801469

Differs from Beltraniella by polyfasciate scolecoconidia and from Pseudobeltrania by

setae intermixed with conidiophores and polyfasciate scolecoconidia.

TyPE SPECIES: Scolecobeltrania vermispora Iturr. et al.

Erymotoey: from Scoleco-, referring to conidia with a length/width radio >15:1 +

beltrania, name of a similar anamorphic fungal genus.

CoLoniegs on the natural substratum amphigenous, effuse, coalescent, felted

to compact floccose, forming conspicuous patches, brown to olivaceous or

black. Mycelium superficial and immersed. SETAE erect, straight or sinuate,

septate with or without radially lobed basal cell, smooth or verruculose,

brown. CONIDIOPHORES macronematous, mononematous, erect, branched,

septate, brown to olivaceous, smooth or verruculose. CONIDIOGENOUS CELLS

monoblastic and polyblastic, discrete, determinate or indeterminate, with

holoblastic sympodial proliferations. Conidial secession schizolytic. CONIDIA

scolecosporous, solitary, unicellular, sigmoid, vermiform to long cylindrical,

curved or sinuate, brown to olivaceous, always with several hyaline or subhyaline

transverse bands, smooth or verruculose. Teleomorph unknown.

Scolecobeltrania vermispora Iturr., R.F. Castafieda & Rob. Fernandez, sp. nov.

Figs. 1 -2

MycoBank MB801470

Differs from Beltraniella spp. in its polyfasciate scolecoconidia and from Pseudobeltrania

spp. in its multiseptate setae intermixed with conidiophores and multifasciate

scolecoconidia.

Type: Venezuela, Bolivar State, tropical forest, Luepa, Sector Gran Sabana, Parque

Nacional Canaima, 5°50'N 61°27'W, 880-1250 m alt., on decaying leaves of unidentified

plant, 25.IV.2005, coll. Teresa Iturriaga (Holotype: VEN108367).

ErymMo_oey: Latin, vermispora, referring to worm-shaped conidia.

CoLoniEs on the natural substrate, effuse, felted forming extensive parches, often

coalescence, olivaceous-brown. Mycelium mostly superficial and immersed.

Hyphae septate, 2-4 um diam, profusely branched, densely anastomoses,

sometimes forming a rudimentary stroma with somewhat similar “textura

intricata’, smooth, brown to pale brown. SETAE erect, straight or curved to

sinuate, cylindrical, acerose or lanceolate at the apex, 7-12-septate, 150-250

uum tall, 10-15 um diam. at the radially lobed base, smooth, dark brown below,

brown towards the apex. CONIDIOPHORES macronematous, mononematous,

erect, dichotomous, trichotomous or irregularly branched, forming more or

less a cluster at the high level, arising from superficial hyphae, 20-35 x 10-12

um, smooth, pale brown to pale olivaceous-brown. CONIDIOGENOUS CELLS

Scolecobeltrania vermispora gen. & sp. nov. (Venezuela) ... 145

Fic. 1. Scolecobeltrania vermispora (ex holotype VEN108367).

A-B. Conidia. C. Seta. D. Conidiophores and conidiogenous cells.

Scale bars: A~B, D = 10 um, C=25um.

146 ... Iturriaga & al.

Fic. 2. Scolecobeltrania vermispora (ex holotype VEN108367).

A. Conidia. B. Conidiophore and conidiogenous cells. C. Seta.

Scale bars: A~B = 10 um, C= 25 um.

polyblastic, discrete, cylindrical, but slightly attenuate near the apex, denticulate

with conspicuous denticles, somewhat opaque at the conidiogenous loci,

indeterminate, with several sympodial proliferations, 8-12 x 3-3.5 um, pale

brown. Conip1a solitary, vermiform, sigmoid, attenuate and truncate at the

base, obtuse to rounded at the apex unicellular, 40-65 x5.5-8.5 um, smooth,

dry, fasciate, pale brown to pale olivaceous brown, with (2-)5-11 transverse

hyaline bands across the length of conidial body. Teleomorph unknown.

Scolecobeltrania vermispora gen. & sp. nov. (Venezuela) ... 147

ADDITIONAL SPECIMEN EXAMINED: VENEZUELA, Caracas, “Cerro El Avila’, Parque

Nacional El Avila, 10°33'N 66°51'W, on decaying leaves of Clusia hilariana Schltdl,

20.11.2008, coll. R Fernandez INIFAT 2461.

NoTE: Seifert et al (2011) have provided a key, descriptions, and illustrations of

Beltrania and similar anamorphic genera. Among these fungi the conidiophores

and conidiogenous cells of Beltraniella Subram. and Pseudobeltrania Henn.

are morphologically similar to Scolecobeltrania. Several Beltraniella species

have irregular branched conidiophores with branches arising near the bases

of straight thick-walled dark brown setae; similar conidiophores also occur in

Pseudobeltrania, but it lacks setae. The conidia in Beltraniella are unicellular,

turbinate, and oblageniform (or nearly so) with a hyaline transverse band

frequently arising from separating cells or from conidiogenous loci (Pirozynski

1963, Castafieda Ruiz et al. 1996, Kendrick 2003, Seifert et al. 2011), and

Beltraniella species with supra-equatorial subhyaline or hyaline (pore-like)

spots have recently been segregated into the genus Porobeltraniella (Gusmao

2004). Pseudobeltrania has unicellular biconical olivaceous brown conidia

with a transverse hyaline band near the widest part arising directly from

conidiogenous loci (Heredia et al. 2002). Scolecoconidia with several transverse

hyaline bands originating directly from conidiogenous loci are conspicuous

unique characters that clearly separate Scolecobeltrania from the other genera.

Acknowledgments

The authors express their sincere gratitude to Dr. Xiu-Guo Zhang and Dr. De-Wei

Li for their critical review of the manuscript. The authors are very grateful to Fondo

Nacional de Ciencia, Tecnologia e Innovacion (Fonacit, Venezuela) for funding Project

.N° $1-2001000663 “Sistematica Molecular y Morfoldgica de Hongos Ascomycetes y

Basidiomycetes Presentes en las Sucesiones Fungicas Descomponedoras de Madera en

Bosques Tropicales”; to the Cuban Ministry of Agriculture for facilities. Collecting in

Venezuela has been possible through permits obtained from INPARQUES-0721 and

MARNR- 1-096, institutions to whom we are indebted. The authors thank Drs Uwe Braun,

Pedro Crous, De-Wei Li, and Gregorio Delgado for their generous and valued assistance

with literature not otherwise available. We thank Beatriz Ramos and Aliana Sosa for

technical assistance. We also acknowledge the facility provided by Dr P.M. Kirk and

Drs V. Robert and G. Stegehuis through the Index Fungorum and Mycobank websites.

Dr Lorelei L. Norvell’s editorial review and Dr Shaun Pennycook’s nomenclature review

are greatly appreciated.

Literature cited

Castafieda Ruiz RF. 2005. Metodologia en el estudio de los hongos anamorfos. Anais do V

Congresso Latino Americano de Micologia. Brasilia: 182-183.

Castafieda Ruiz RE, Guarro J, Cano J. 1996. Notes on conidial fungi VII. Two new species of

Beltraniella from Cuba. Mycotaxon 58: 243-251.

Gusmao LFP. 2004. Porobeltraniella gen. nov. to accommodate two species of Beltraniella. Mycologia

96(1): 150-153. http://dx.doi.org/10.2307/3761996

148 ... Iturriaga & al.

Heredia G, Arias RM, Reyes M, Castafieda-Ruiz RE. 2002. New anamorph fungi with rhombic

conidia from Mexican tropical forest litter. Fungal Diversity 11: 99-107.

Kendrick B. 2003. Analysis of morphogenesis in hyphomycetes: new characters derived from

considering some conidiophores and conidia as condensed hyphal systems. Can. J. Bot. 81(2):

75-100. http://dx.doi.org/10.1139/b03-008

Pirozynski, KA. 1963. Beltrania and related genera. Mycological Papers 90: 1-37.

Seifert K, Morgan-Jones G, Gams W, Kendrick B. 2011. The genera of hyphomycetes. CBS

Biodiversity Series 9: 1-997. http://dx.doi.org/10.3767/003158511X617435

MY COTAXON

http://dx.doi.org/10.5248/124.149

Volume 124, pp. 149-154 April-June 2013

A new species of Pisolithus from Spain

Maria P. MarRTIN*?, FATIMA DURAN’,

CHERDCHAI PHOSRI? & ROY WATLING?

‘Dept. Micologia, Real Jardin Botanico. CSIC, Plaza de Murillo 2, 28014 Madrid, Spain

’ Biology programme, Pibulsongkram Rajabhat University, Phitsanulok, 65000, Thailand

>Caledonian Mycological Enterprises, Edinburgh EH 4 3 HU, Scotland UK

CORRESPONDENCE TO *: maripaz@rjb.csic.es

AsstRACT — Pisolithus calongei is described as a new species based on our morphological

observations and comparison of ITS nrDNA sequences.

Key worps — Basidiomycota, Cistus, Boletales, taxonomy, Pisolithus tinctorius

Introduction

During their overview of the donkey dung fungus (Pisolithus Alb. & Schwein.)

from many regions of the world using molecular and classical methods, Phosri

et al. (2012) analysed collections from Europe. All generated molecular trees

grouped Spanish specimens into three clades, of which two represent the classic

Pisolithus tinctorius (Pers.) Coker & Couch and P. arhizus (Scop.) Rauschert.

However, four sequences obtained from Pisolithus specimens associated with

Cistus ladanifer L. (Cistaceae) formed a separate group more closely related to

P. marmoratus (Berk.) E. Fisch. (occurring in Eucalyptus spp. plantations from

Australia, China, and Kenya) than to other Spanish specimens.

Cistaceae is well documented as an ectomycorrhizal phanerogam

family (Harley & Harley 1987), although many species also form VA and

ectendomycorrhizas. Association-forming species are widespread amongst

the larger fungi, e.g. Leccinum arenicola Watling & Redhead, found in North

America with Hudsonia L. (Redhead & Watling 1979), and Terfezia alsheikhii

Kovacs et al., found in Spain with Helianthemum Mill. (Kovacs et al. 2011).

In Europe a number of fungi associate with Helianthemum, including Boletus

spp. (Agueda et al. 2006, 2008; Ponce et al. 2011), Laccaria laccata (Scop.)

Cooke (Torres et al. 1995), and Cortinarius spp. and Hebeloma spp. (Watling

1988; Moreau 2002; Henrici 2003, 2005). Even Astraeus hygrometricus (Pers.)

150 ... Martin & al.

Morgan, a close relative of Pisolithus, has been associated with Cistus in Spain

(Martin 1988, specimens in BCN; M. Jeppson personal collection with duplicate

specimens in E).

Recently, three Pisolithus specimens were collected near Cistus ladanifer

in Spain. Phylogenetic analyses performed according to Phosri et al. (2012)

separated the specimens into two distinctive taxa (Fic. 1): one collection

(MA-Fungi 47734 (duplicates at BH & E): J.C. Campos & E. Ramirez MPM

3320 collectors near El Robledillo, 435-465 m alt. on 4-Dec-2010; GenBank:

HE578142) grouped in the ‘tinctorius’ clade and the other two collections (MA-

Fungi 47732, MA-Fungi 47733) grouped in the “Pisolithus species 3” subclade

from Spain together with sequences AF228641-AF228644 (Diez et al. 2001)

from specimens from Alcala de Henares Univ. Herbarium (AH) and FR748140/

FR748142 (Phosri et al. 2012) from the MJ6192 collection (M. Jeppson personal

herbarium) revised here.

MA-Fungi 47734

P. albus complex

99 64

88 ——] Pisolithus sp. 8

7 MA-Fungi 47732

83 ; MA-Fungi 47733

100

P. abditu.

<—] Pisolithus sp. 1

100 5 P. aurantioscabrosus

indicus

Outgroup

Fic. 1. Pisolithus calongei and other Pisolithus spp. One of the 100 most parsimony trees obtained

under heuristic search (PAUP* 4.0b10) using the sequences included in Phosri et al. (2012) and

three newly generated sequences from our Pisolithus collections from Spain. The parsimony

bootstrap values (%) are indicated on the branches. Bar = 20 changes.

Pisolithus calongei sp. nov. (Spain) ... 151

Identification of microscopic features distinctive to MA-Fungi 47732 and

MA-Fungi 47733 associated with C. ladanifer further support our belief that

the specimens represent a new species, which we describe here.

Fic. 2. Pisolithus calongei. a~-b (MA-Fungi 47732): Turbinate gastrocarp. c-d (Holotype, MA-Fungi

47733): Basidiospores (SEM) coarsely ornamented by broad connate structures that form irregular

pyramid-like spines. Scale bars: a = 1 cm; b = 0.5 cm; c-d = 1 um.

Taxonomy

Pisolithus calongei M.P. Martin, Phosri & Watling, sp. nov. Fics 2-3

MycoBANnk MB 563437

Differs from Pisolithus tinctorius and P. arhizus by its turbinate habit and from all

Pisolithus spp. in its ITS nrDNA sequence.

Type: Spain. Avila: Candeleda, Las Atalayas, 30TU-00477, 355 m alt., close to Cistus

ladanifer, 11-Dec-2010, leg. E. Ramirez & J. C. Campos MPM 3319 (Holotype, MA-

Fungi 47733; isotypes, BH, E; GenBank, HE578141).

EryMoLoGcy: Honouring Francisco D. Calonge, who has worked so diligently on the

Iberian gasteromycete flora.

GASTROCARP turbinate, pulvinate, 32-40 mm broad, 28-45 mm high, with

shortened stipe 6-14 x 10-13 mm, with abrupt base, + attached to soil mass,

main primary peridial layer 15-24 mm deep. PERIDIUM irregular, at most 0.5

152... Martin &al.

Fig. 3. Pisolithus calongei (M. Jeppson MJ6192). Turbinate gastrocarp.

mm thick, but soon thinning to show outline of peridioles below, even stretching

to accommodate complete peridioles, finally disintegrating and leaving small

islands of original ferruginous peridium above yellow-ochre peridiole surfaces

or bruising to form lacquer-like blackened smudges. PERIDIOLES 1-1.5 mm

diam. (rarely <2 mm), ochraceous yellow at first, later ferruginous brown,

compressed in uppermost part, often forming in stipe apex or even developing

within the entire stipe to produce irregular rugulose outer surface on the stipe,

concolorous with and non-differentiated from the peridial surface; upper

peridium finally eroding away to expose the disintegrating peridioles as a dry

ferruginous powdery mass. BasipD1osPoREs globose 8.6-11 um diam., coarsely

ornamented with broadly connate structures that form irregular pyramid-like

spines, 1.5 um high, rarely collapsing to give broader structures.

ADDITIONAL SPECIMENS EXAMINED: SPAIN. Avia: Candeleda, Las Atalayas, 30TU-

00477, 350 m alt., close to Cistus ladanifer, 4-Dec-2010, leg. E. Ramirez & J. C. Campos

MPM3318 (MA-Fungi 47732; duplicate, BH; GenBank, HE578140). Huetva: Las

Palmas, 1-Jan-2003, leg. M. Jeppson MJ6192 (M. Jeppson personal herbarium).

Pisolithus calongei is distinctive in the field in its turbinate habit, close fruiting

to the soil-surface and therefore being concealed by the Cistus with which

it is thought to associate. The collectors observed no Pinus species in the

vicinity. The new species may have been overlooked in the past or identified

as P. arhizus or P. tinctorius. Nonetheless, the ITS nrDNA sequence analysis

(Fic. 1) clearly separates P calongei from the other three European species

Pisolithus calongei sp. nov. (Spain) ... 153

(P. arhizus, P. capsulifer (Sowerby) Watling et al., and P. tinctorius), as well as

from the other species previously analyzed: P. albus complex, P abditus Kanch. et

al., P aurantioscabrosus Watling, P indicus Natarajan & Senthil., P marmoratus

(Berk.) E. Fisch., PR microcarpus (Cooke & Massee) G. Cunn., and P orientalis

Watling et al. (Watling et al. 1995, Martin et al. 2002, Kanchanaprayudh et al.

2003a,b, Moyersoen et al. 2003, Reddy et al. 2005, Phosri et al. 2012).

Acknowledgments

We thank M. Jeppson (Sweden) and A.J.S. Whalley (John Moores University, Liverpool)

for presubmission review. Thanks to E. Ramirez, J.C. Campos, and J.C. Zamora for

providing the specimens included in this study, to J. Jeppson for permission to reproduce

Fic. 3 (specimen M. Jeppson MJ6192), and to Y. Ruiz for her technical assistance with

the SEM pictures of the holotype. This work was supported in part by project REN2001-

1842/GLO.

Literature cited

Agueda B, Parladé J, De Miguel AM, Martinez-Pefia F. 2006. Characterization and identification

of field ectomycorrhizae of Boletus edulis and Cistus ladanifer. Mycologia 98(1): 23-30.

http://dx.doi.org/10.3852/mycologia.98.1.23

Agueda B, Parladé J, Fernandez-Toiran LM, Cisneros O, De Miguel AM, Modrego MP, Martinez-

Pefia F, Pera J. 2008. Mycorrhizal synthesis between Boletus edulis species complex and rockroses

(Cistus sp.). Mycorrhiza 18(8): 443-449. http://dx.doi.org/10.1007/s00572-00

Diez J, Anta B, Manjon JL, Honrubia M. 2001. Genetic variability in Pisolithus isolates associated

with native hosts and exotic eucalyptus in the western Mediterranean region. New Phytol.

149(3): 577-587.

Harley JL, Harley EL. 1987. A check-list of mycorrhiza in the British flora. New Phytologist 105(2):

1-102. http://dx.doi.org/10.1111/j.1469-8137.1987.tb00674.

Henrici A. 2003. Notes and records. Field Mycology 4: 30-31.

Henrici A. 2005. Notes and records. Field Mycology 11: 141-143.

Kanchanaprayudh J, Zhou Z, Yomyart S,. Sihanonth P, Hogetsu T, Watling R. 2003a. A new species,

Pisolithus abditus, an ectomycorrhizal fungus associated with dipterocarps in Thailand.

Mycotaxon 88: 463-467.

Kanchanaprayudh J, Zhou Z, Yomyart S, Sihanonth P, Hogetsu T. 2003b. Molecular phylogeny of

ectomycorrhizal Pisolithus fungi associated with pine, Dipterocarp, and Eucalyptus trees in

Thailand. Mycoscience 44: 287-294. http://dx.doi.org/10.1007/s10267-003-0110-7

Kovacs GM, Balazs TK, Calonge FD, Martin MP. 2011. The diversity of Terfezia desert truffles: new

species and a highly variable species complex with intrasporocarpic nrDNA ITS heterogeneity.

Mycologia 103(4): 841-853. http://dx.doi.org/10.3852/10-312.

Martin MP. 1988. Aportacién al conocimiento de las Higroforaceas y los gasteromicetes de

Catalufia. Edicions Especial Societat Catalana de Micologia. Volum 2. 508 p.

Martin F, Diez J, Dell B, Delaruelle C. 2002. Phylogeography of the ectomycorrhizal Pisolithus

species as inferred from nuclear ribosomal DNA ITS sequences. New Phytologist. 153:

345-357. http://dx.doi.org/10.1046/j.0028-646X.2001.00313.x

Moreau P-A. 2002. A la découverte des champignons de zone alpine (numero special). Bulletin

mycologique et botanique Dauphiné-Savoie 167: 5-42.

154 ... Martin & al.

Moyersoen B, Beever RE, Martin F 2003. Genetic diversity of Pisolithus in New Zealand

indicates multiple long-distance dispersal from Australia. New Phytologist. 160(3): 569-579.

http://dx.doi.org/10.1046/j.1469-8137.2003.00908.x

Phosri C, Martin MP, Suwannasai N, Sihanonth P, Watling R. 2012. A new species from SE Asia and

a new combination in Pisolithus. Mycotaxon 120: 195-208. http://dx.doi.org/10.5248/120.195.

Ponce RA, Agueda B, Agreda T, Modrego MP, Aldea J, Fernandez-Toiran LM, Martinez-Pefa F 2011.

Rockroses and Boletus edulis ectomycorrhizal association: realized niche and climatic suitability

in Spain. Fungal Ecology 4(3): 224-232. http://dx.doi.org/10.1016/j.funeco.2010.10.002

Rambaut A. 2002. Se-al: sequences alignment editor v2.0al1. Edinburgh: Inst. Evolutionary

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Reddy MS, Singla S, Natarajan K, Senthilarasu G. 2005. Pisolithus indicus, a new species of

ectomycorrhizal fungus associated with Dipterocarps in India. Mycologia 97(4): 838-843.

http://dx.doi.org/10.3852/mycologia.97.4.838

Redhead S, Watling R. 1979. A new psammophilic Leccinum. Canadian Journal of Botany 57:

117-119.

Torres P, Roldan A, Lansac AR, Martin A. 1995. Ectomycorrhizal formation between Cistus

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MY COTAXON

http://dx.doi.org/10.5248/124.155

Volume 124, pp. 155-163 April-June 2013

A reassessment of excavated Tuber species from China

based on morphology and ITS rDNA sequence data

Li FAN’, JIN-ZHONG CAO? & Yu LY?’

" College of Life Science, Capital Normal University,

Xisanhuanbeilu 105, Haidian, Beijing 100048, China

? Institute of Mycology, Jilin Agricultural University, Changchun 130118, China

CORRESPONDENCE TO *: fanli@mail.cnu.edu.cn

ABSTRACT — A composite study was made of the morphology and phylogeny of Chinese

Tuber species that produce ascomata with a cavity at the base (excavated ascomata). One

new species, T: neoexcavatum, was found and is here described. It is characterized by brown

ascomata with distinctly verrucose surface and prosenchymatous peridium. The presence of

T: excavatum in China is still in doubt. A key is provided for all excavated species of Tuber.

Key worps — Ascomycota, molecular analysis, taxonomy, truffle

Introduction

Species of Tuber that produce distinct cavities at the base of ascomata are

often referred to as ‘excavated species. The ascomal surface within the cavity of

such species differs considerably from the surface covering the outer ascomata

(Cao 2010). For example, T. pseudohimalayense (= T: pseudoexcavatum)

produces larger pyramidal warts inside the cavities but smaller ones outside

the cavities, even forming a distinct area with the same large pyramidal

warts in some abnormal ascomata that lack cavities. The only exception is

T: mesentericum Vittad., in which pyramidal warts completely cover the ascomal

surface (Riousset et al. 2001).

Occasionally other Tuber species (e.g., T: nitidum Vittad. and T. taiyuanense

B. Liu in the Rufum group; T. liyuanum L. Fan & J.Z. Cao in the Puberulum

group) produce an umbilicus or a distinct pit at the base of an ascoma, giving

them the appearance of an excavated species. However, these Tuber species

could never be mistaken for excavated species, because there is no clear

difference between the cavity interior and exterior inside and a cavity is not

consistently present within all ascomata in the collection.

156 ... Fan, Cao & Li

Tuber species with true cavities were first described by Vittadini in 1831

(T. excavatum and T. mesentericum) and Quélet in 1880 (T. fulgens). Two Chinese

species — T. pseudoexcavatum (Wang et al. 1998) and T. sinoexcavatum (Fan et

al. 2011) — also produce ascomata with cavities. During intensive investigation

of the Chinese Tuber species with excavated ascomata, the authors found a new

excavated species. Here we describe the new species, discuss all three Chinese

excavated species, and reassess their taxonomic status.

Materials & methods

Morphological studies

Fresh specimens of the new species described here were collected from Baoshan City,

Yunnan Province, P.R. China and deposited in BJTC (Herbarium Biology Department,

Capital Normal University in Beijing). The dried specimens were deposited in BJTC and

HKAS (Herbarium of Cryptogams, Kunming Institute of Botany, Chinese Academy of

Sciences). Macro- and microscopic characters were described from fresh specimens and

rehydrated specimens. Sections were made witha razor blade and mounted in 3% KOH

for observation. Specimens were dried for archival purposes, and permanent slides were

prepared by staining in Melzer’s reagent, rinsing in water and mounting in polyvinyl

lactic glycerol. For scanning electron microscopy (SEM), ascospores were scraped from

fresh or dried gleba onto doubled-sided tape mounted directly on an SEM stub, coated

with gold—palladium, and examined and photographed with a Hitachi S—4800 SEM.

Molecular methods

Samples from herbarium material were crushed by shaking for 3 min at 30 Hz

(Mixer Mill MM 301, Retsch, Haan, Germany) in a 1.5 ml tube together with one 3

mm diameter tungsten carbide ball. Total genomic DNA was then extracted using

the PeqLabE.Z.N.A._Fungal DNA kit following the manufacturer’s protocol. The ITS

regions were amplified with PCR using the primers ITS1/ITS4 (White et al. 1990). PCR

was performed in 50 ul reactions containing DNA template 2 ul, primer (10 uM) 2 ul

each, and 2 x Master Mix (Tiangen Biotech (Beijing) Co. Ltd.) 25 ul. PCR reactions

were run as follows: an initial denaturation at 95 °C for 3 min, followed by 30 cycles at

95 °C for 2 min, 55 °C for 25 s, 72 °C for 2 min and a final extension at 72 °C for 10 min.

The PCR products were sent to Invitrogen Biotechnology Co. Ltd. (Beijing, China) for

purifying, sequencing, and editing. The other sequence data of ITS rDNA included in

this study were downloaded from GenBank. GenBank numbers are shown in TABLE 1.

Phylogenetic analyses

DNA sequences were aligned with Clustal X (Thompson et al. 1997). The alignment

was manually adjusted with Se-Al v.2.03a (Rambaut 2000). The aligned dataset was

analyzed with maximum parsimony (MP) using PAUP*4.0b10 (Swofford 2002).

Maximum parsimony analysis was conducted using heuristic searches with 1000

replicates of random-addition sequence, tree bisection reconnection (TBR) branch

swapping algorithm. All characters were equally weighted and unordered. Gaps were

treated as missing data to minimize homology assumptions. A bootstrap (BS) analysis

was performed with 1000 replicates, each with 10 random taxon addition sequences.

TABLE 1. Sources of sequences for molecular analysis

Tuber neoexcavatum sp. nov. (China) ... 157

GEOGRAPHICAL ITS

SPECIES VOUCHER SPECIMEN GRiGIn

Terfezia boudieri Chatin mot08 Algeria AF276673

DDtb1 Tunisia GU474809

Tuber aestivum Vittad. El Italy AF516788

E17 Italy AY226042

T. borchii Vittad. GB62 Italy HM485342

GB45 Italy HM485344

T. excavatum Vittad. Texc-eu02 France DQ329362

BM100 Spain FJ748899

°T. excavatum HKAS 52006 China GQ217540

T. fulgens Quél. M2435 Italy HM485358

SFI: TUBFUL/041008B Slovenia FN433150

T. magnatum Picco Tm4 Italy AJ586308

_— Italy EU807975

T. melanosporum Vittad. A59 France AF106878

Tm13 France AF132501

T. mesentericum TmsW095-W029 (UPS) Sweden AJ888047

TmsW088-W047 (UPS) Sweden AJ888044

T. neoexcavatum BJTC FAN 184 (Holotype) China JX458715

T. pseudoexcavatum Tpse-yn05 China DQ329374

T. pseudohimalayense BJTC FAN120 China JX458716

T. rufum Picco Morphotype 5 Italy AY940646

1506 Italy AY112894

T. sinoexcavatum BJTC FAN 130 (Holotype) China JX458717

BJTC FAN166 Chinan JX458718

TBR branch swapping was employed. The Bayesian analysis was performed with

MrBayes 3.1.2 (Huelsenbeck et al. 2001; Ronquist & Huelsenbeck 2003) with two sets

of four chains (one cold and three heated) and the stoprule option in effect, halting

the analyses at an average standard deviation of split frequencies of 0.01. The sample

frequency was set to 100, and the first 25% trees were removed as burn-in. Bayesian

posterior probabilities (PP) were obtained from the 50% majority rule consensus of the

remaining trees. Two sequences derived from Terfezia boudieri were used as outgroup.

Results

Molecular phylogenetics

480 of 688 characters were found to be parsimony-informative. The

maximum parsimony analysis of sequences resulted in one most parsimonious

tree (Fic. 1) with a length (TL) = 1422 steps, consistency index (CI) = 0.6582,

retention index (RI) = 0.8002, homoplasy index (HI) = 0.3418, and rescaled

consistency index (RC) = 0.5267.

The phylogenetic analysis (Fic. 1) reveals that the Tuber species with

excavated ascomata are scattered in different clades with strong support.

The new species T. neoexcavatum clusters in a clade with other three other

158 ... Fan, Cao & Li

Tuber fulgens HM485358

Tuber fulgens FN433150

Tuber sinoexcavatum FAN 166

Tuber sinoexcavatum FAN 130

Tuber excavatum GQ217540

Tuber neoexcavatum FAN184

Tuber excavatum FJ748899

Tuber excavatum DQ329362

Tuber aestivum AY 226042

100

1.00

Tuber aestivum AF516788

100

1.00)

Tuber mesentericum AJ888047

100

1.00

Tuber mesentericum AJ888044

Tuber magnatum EU807975

100

1.00

Tuber magnatum AJ586308

Tuber borchii HM485344

100

ie Tuber borchii HM485342

Tuber pseudohimalayense FAN120

100

1.00

Tuber pseudoexcavatum DQ329374

80)

1.00

Tuber melanosporum AF 132501

100

70 1.00

T.00 Tuber melanosporum AF 106878

Tuber rufum AY940646

100)

1.00

Tuber rufum AY 112894

Terfezia boudieri GU474809

Terfezia boudieri AF276673

_10

Fic. 1. Phylogeny derived from maximum parsimony analysis of the ITS rDNA sequences of some

Tuber species with real excavated ascomata, using Terfezia boudieri as outgroup. Bootstrap values of

more than 70% from 1000 replications are shown above the respective branches. Bayesian posterior

probabilities are estimated and clades with PP>0.95 (95%) are marked under the branches.

Tuber neoexcavatum sp. nov. (China) ... 159

excavated species: T: excavatum, T. fulgens, and T! sinoexcavatum. Furthermore,

four strongly supported subclades (each represented by two sequences)

correlate exactly with individual taxa as defined using morphological methods,

indicating that the four excavated species are closely related but distinct. The

other two excavated species are in two separate clades: T. pseudohimalayense

grouping with T. melanosporum and T. mesentericum with T: aestivum.

Taxonomy

Tuber neoexcavatum L. Fan &Yu Li, sp. nov. Pre. 2

MycoBank MB 801128

Differs from other Tuber species by its brown ascoma with a distinctly verrucose surface

and prosenchymatous peridium.

Type: China. Yunnan Province, Baoshan City, in soil under forest of P yunnanensis

Franch., 28 Dec. 2011, Jin-Zhong Cao 511 (Holotype, BJTC FAN184).

ETYMOLOGY: neoexcavatum (Lat.), referring to the similarity to Tuber excavatum.

AscoMata hypogenous, subglobose, 3-4 cm in diam., with typical basal cavities

(excavated ascomata), covered with distinctly verrucose warts on the surface,

yellow brown to brown with shadow of olive green; interior cavity surface

covered with many fine, irregular scales and large warts, pale white to yellow-

white when fresh. Odor and flavour not recorded. PERIDIUM 250-350 um thick

including the 100-150 um high verrucose warts, prosenchymatous, composed

of interwoven hyphae, light brown and slightly thick-walled towards the outside,

3-6 um in diam., hyaline and thin-walled towards the inside, 2.5-5 um in diam.

GLEBA yellow brown to brown, with the shadow of olive green at maturity, veins

white to cream white, more or less radiate from the cavity. Asc1 subglobose

or long oval, hyaline, thin walled, 75-100 x 62.5-80 um, with a short stalk,

2-4-spored. Ascosporgs elliptic or long elliptic, occasionally broadly elliptic,

hyaline at first, light brown or brown at maturity, reticulate ornamentation on

the surface, 30-47.5 x 25-30 um excluding the ornamentation, meshes 5-6 um

deep, 2-4 across the spore width.

ADDITIONAL SPECIMEN EXAMINED — CHINA. SICHUAN PROVINCE, HUIDONG

COUNTY, in soil under forest of P armandii and P. yunnanensis, 3 Nov. 2006, Juan Chen

411 (HKAS 52006, as T: excavatum)

COMMENTS — Ascomata of Tuber neoexcavatum resemble those of T. pseudo-

himalayense (= T. pseudoexcavatum), including the color and small distinct

surface warts, but T: neoexcavatum is easily distinguished by its spino-reticulate

ornamented ascospores. Tuber excavatum, T: fulgens, and T. sinoexcavatum also

produce similar ascomata, but T’ excavatum is distinguished by its pale and

smooth or finely papillar ascomata and T! fulgens and T: sinoexcavatum produce

globose or subglobose ascospores. The European species, T: mesentericum, is

also excavated but differs from T: neoexcavatum in its blackish ascomata that

160 ... Fan, Cao & Li

Fic. 2. Tuber neoexcavatum (BJTC FAN184, holotype). a Ascomata; b Asci and ascospores

observed under light microscope; c Ascospore observed under SEM.

are completely covered with large pyramid warts. In addition, the peridium

structure in T: neoexcavatum is typically prosenchymatous, which is completely

(including the verrucose warts) composed of interwoven hyphae. The ITS

rDNA phylogeny (Fic. 1) places T. neoexcavatum sequences within an evidently

independent clade (BP = 97, PP = 1.00) and clearly supports the new species

as independent.

Chen et al. (2008) reported T: excavatum in China based on specimen HKAS

52006 collected in 2006 from Sichuan Province (also see Cao 2010). However,

our ITS rDNA sequence analysis of HKAS 52006 and other excavated Tuber

species (Fic. 1) places sequence GQ217540 (from HKAS 52006) in a clade with

our T. neoexcavatum but not with the T: excavatum sequences from France

and Spain. In examining HKAS 52006, we could find no ascomal surface

and peridial differences between the specimen of Chen et al. (2008) and the

type specimen of T: neoexcavatum. We therefore redetermine HKAS 52006 as

T. neoexcavatum.

As all other reports of T! excavatum in China are not linked to specimens,

there is no firm evidence for T: excavatum being present in China. Whether

T. excavatum occurs in China or not needs further investigation.

Tuber neoexcavatum sp. nov. (China) ... 161

Tuber sinoexcavatum L. Fan & Yu Li, Mycotaxon 116: 352 (2011)

SPECIMENS EXAMINED — CHINA. SICHUAN PROVINCE, PANZHIHUA CITY, under the

soil of Pinus sp., 20 Dec. 2007, De-fu Liu (Holotype, BJTC Fan130); YUNNAN PROVINCE,

Hu1zHE County, in soil under forest, 12 Nov. 2011, Shao-ping Li 005 (BJTC Fan166).

DISTRIBUTION: China: Sichuan Province; Yunnan Province.

CoMMENTS— Tuber sinoexcavatum was originally described from one collection

(BJTC Fan130), but in 2011 an additional specimen (BJTC Fan166) was found

in Yunnan Province, China. The sequence analyses (Fic. 1) support the two

specimens as conspecific. It should be pointed out that the ascospores are near

regularly globose in the type specimen, but in the new specimen about half the

ascospores are subglobose or broadly elliptic. This phenomenon also occurs in

T. latisporum Juan Chen & P.G. Liu and T. subglobosum L. Fan & C.L. Hou (Fan

et al. 2013). The T! latisporum ascospores are broadly elliptic to subglobose,

but also sometimes globose in some ascomata in the same collection, while in

some ascomata T: subglobosum may occasionally produce a large proportion of

broadly elliptic ascospores rather than typical subglobose ones.

Tuber sinoexcavatum is morphologically similar to the European species

T. excavatum, which, however, produces elliptic ascospores. Furthermore, the

peridium in T: sinoexcavatum is normally covered with fine papillary warts

while T. excavatum produces smooth or only finely scurfy warts (Pegler 1993,

Riousset et al. 2001). Another European species, T: fulgens, also produces

globose ascospores, but its ascoma is brightly colored and a small hole opens

to the outside (Lange 1956, Riousset et al. 2001), unlike the large opening in

T. sinoexcavatum. Here also the ITS rDNA sequence analyses strongly support

three separate species (Fic. 1).

Tuber pseudohimalayense G. Moreno, Manjon, J. Diez & Garcia-Mont., Mycotaxon

63: 218 (1997)

= T. pseudoexcavatum Y. Wang, G. Moreno, Riousset, Manjon &

G. Riousset, Cryptog. Mycol. 19(1-2): 115 (1998)

SPECIMENS EXAMINED — CHINA. SICHUAN PROVINCE, HUIZHE COUNTY, in soil under

trees, 19 Oct. 2003, Juan Chen 127 (HKAS 44346); 3 Nov. 2006, Juan Chen 412 (HKAS

52010); 5 Nov. 2006, Juan Chen 416 (HKAS 52017); 20 Oct. 2003, Juan Chen 134 (HKAS

44325); 19 Oct. 2003, Juan Chen (HKAS 44346); in soil under P. armandii, 15 Dec. 2008,

Li Fan 020 (BJTC FAN120); YUNNAN PROVINCE, KUNMING CITY, in soil under forest,

25 Nov. 2002, Zhu-liang Yang 3603 (HKAS 41313); 8 Nov. 2004, Juan Chen 234 (HKAS

47619); 8 Nov. 2002, Juan Chen 287 (HKAS 49744); 9 Mar. 2003, Xing-hua Wang 1477

(HKAS 42399); JINNING CouNTY, in soil under trees, 20 Sep. 2006, Juan Chen 427

(HKAS 52032); YONGSHENG COUNTY, in soil under trees, 29 Oct. 2006, Juan Chen 407

(HKAS 52027); Juan Chen 408 (HKAS 52036).

DISTRIBUTION: China: Sichuan Province; Yunnan Province.

CoMMENTS—Moreno et al. (1997) described T: pseudohimalayense for a

black truffle with spino-reticulate ornamented ascospores. Although the type

162 ... Fan, Cao & Li

specimen was found in a Spanish truffle market, the authors suspected it was

probably imported from China (Moreno et al. 1997). The next year, Wang et

al. (1998) published T. pseudoexcavatum to accommodate a very common

black truffle species in China. It and T. pseudohimalayense produce identical

ascospores, but T: pseudohimalayense is characterized by a distinct cavity at the

ascomal base. Manjon et al. (2009) and Chen & Liu (2011) concluded that the

two ‘species’ were definitely conspecific based on their analysis of molecular

data. As the earlier published name, T. pseudohimalayense has priority over

T. pseudoexcavatum. We have found it is widely distributed in Yunnan and

Sichuan provinces of southwest China during autumn and early winter, and

Song (2005) has cited it from Hunan Province. Its spino-reticulate ascospores

and 1-8-spored asci separate P pseudohimalayense from all other excavated

Tuber species.

Discussion

Six total species of Tuber with excavated ascomata have been described thus

far, including three species from China. As noted above, the European species

T. excavatum has not yet been confirmed in China.

The ITS rDNA-based phylogeny supports four closely related species

— T. excavatum, T. fulgens, T. sinoexcavatum, T. neoexcavatum — within

a well-supported clade and shows that T! pseudohimalayense is close to

T. melanosporum and T. mesentericum is close to T: aestivum. Although Tuber

species with excavated ascomata appear morphologically similar, however,

their rDNA sequence analyses imply that they are definitely polyphyletic.

Key to Tuber species with excavated ascomata

la. Ascomata blackish; surface with pyramidal warts ................ T. mesentericum

1b. Ascomata not blackish; surface smooth, verrucose, or with small warts .......... g

2a. Ascomata distinctly verrucose or with small warts ............ 0.0... e eee eee 3

2b: Ascomata’smooth or minutely verrucose: 1... baa ew ene ee ge eek os 4

3a. Ascospores with spino-reticulate ornamentation ........... T. pseudohimalayense

3b. Ascospores with reticulate ornamentation ..................04. T. neoexcavatum

Ad. ASCOSPOTES THOSE CIM PLIC-< ree tonaeg! 2 wstiete oat eta oe one ies T. excavatum

4b. Ascospores mostly globose or subglobose. ......... 0... e cece eee eee 5

5a. Ascomata golden brown, opening to outside by a small hole............ T. fulgens

5b. Ascomata yellow brown to light brown, typically excavated....... T. sinoexcavatum

Acknowledgments

We are grateful to Prof. Guozhong Li and Dr. Ian Hall for reviewing the manuscript.

The study was supported by the National Natural Science Foundation of China (No.

31270058, 30770005), the Beijing Natural Science Foundation (No. 5072006). We wish

Tuber neoexcavatum sp. nov. (China) ... 163

to extend a special thanks to the Herbarium of Cryptogams, Kunming Institute of

Botany, Chinese Academy of Sciences, for providing specimens.

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MY COTAXON

http://dx.doi.org/10.5248/124.165

Volume 124, pp. 165-171 April-June 2013

A new species and a new record of the genus Entoloma

from China

JiaN-Ru1 WANG & TOLGOR Bau*

Institute of Mycology/Engineering Research Center of

Chinese Ministry of Education for Edible and Medicinal Fungi,

Jilin Agricultural University, Changchun 130118, P.R. China

* CORRESPONDENCE TO: Junwusuo@126.com

ABSTRACT —Two omphalinoid Entoloma species were collected from Shandong Province in

China. Entoloma shandongense is described as new to science, and E. rusticoides is recorded for

the first time in China. Detailed descriptions and illustrations of the two species are provided.

In addition, molecular phylogenetic analysis based on ITS sequences is also presented.

Key worps —Entolomataceae, new taxon, morphology, taxonomy

Introduction

More than one thousand species of Entoloma have been described worldwide

(Kirk et al. 2008). Major works on this genus have been published in the past

twenty years (Largent 1994, Noordeloos 2004, Noordeloos & Hausknecht 2007,

Gates & Noordeloos 2007, Horak 2008, Noordeloos & Gates 2009, 2012). In

China, 137 species of Entoloma have been recorded, including several new taxa

and new records in recent systematic studies (Li et al. 2008, 2009; He et al. 2010,

2011, 2012, 2013).

Recently, two omphalinoid Entoloma species were collected in Shandong

Province. One with a blue pileus is described as new to science, and the other

represents a first record for China. Morphological characters and the results of

ITS sequence analyses are provided for both species.

Materials & methods

Materials were collected from Shandong province, north China and were deposited

in the Herbarium of Mycology of Ludong University (HMLD). Morphological characters

of the two species were described and illustrated using traditional taxonomic methods.

For morphological studies, color notations follow Kornerup & Wanscher (1978).

Microscopic characters were examined using water to observe the color of the tissues,

166 ... Wang & Bau

5% KOH solution as a mounting solvent, 1% Congo Red solution as a staining agent,

and Melzer’s reagent to detect amyloidy. At least 20 basidiospores, basidia, and cystidia

per specimen were measured.

The molecular confirmation was derived from ITS sequences. DNA was extracted

according to Sun et al. (2009). ITS rDNA was amplified using primers ITS1 and ITS4.

Amplification was performed in 25 uL volumes containing 0.5 uL template DNA,

2.5 uL PCR-buffer (10x), 2 wL Mg2+ (25 mmol/L), 0.2 uL Taq enzyme (5 U/uL), 0.5

uL dNTP mix (10 mmol/L), 0.5 wL of each primer and 17.8 wL ddH,O. The reaction

was carried out with initial denaturation (94°C, 3 min), 35 cycles by the following

conditions: denaturation (94°C, 50s), annealing (50°C, 30s), extension (72°C, 50s), final

extension (72°C, 10min). The PCR amplification products were sequenced by Sangon

Co., Shanghai, China. The target gene sequences were checked using Bioedit (Version

7.0.9; Hall 1999). The final BLASTN was done in Genbank.

Entoloma shandongense T. Bau & J.R. Wang, sp. nov. Fic. 1

MycoBank MB 802463

Differs from Entoloma ater by its blue pileus and its smaller basidiospores.

Type: China, Shandong Province, Dezhou City, Botanical Garden of Dezhou,

2 September 2011, Jian-Rui Wang (Holotype, HMLD 1796; GenBank, KC257440).

EryMo oey: shandongense refers to the type locality, Shandong Province.

BASIDIOMATA small, omphalinoid. Piteus 10-18 mm broad, hemispherical,

depressed in the center to infundibuliform, not hygrophanous, slightly

translucent-striate almost to center, smooth, mostly blue with somewhat purple

tinge (22B5-22B4) over the whole surface, slightly deeper (21C5-21C6) or deep

dull blue-violet (20C5-20C6) in the center, tomentose. LAMELLAE decurrent or

adnate with short decurrent tooth, 1.5-2 mm broad, distant, pale pinkish beige

(7A1-7A2) to pale pinkish red (11A1-11A2); margin entire. LAMELLULAE

present, 2-3 tiers. CONTEXT thin, whitish to pale brown. STIPE central to

somewhat eccentric, 10-15 mm in length, 1-2 mm in width, cylindrical to

subclavate, gradually narrowing towards base, concolorous with lamellae, pale

beige pink, light beige to white at base. ODOR AND TASTE not distinctive.

BASIDIOSPORES 5.6-7.2 X (7.5—)8.3-10(-10.5) um, Q = 1.3-1.5, with obvious

apiculus, 5-8-angled in side view, thick-walled. Basip1a 29-36 x 8-10 um,

clavate, 4-spored, hyaline, thick-walled when mature, homogeneous or with

granular contents. PLEUROCYSTIDIA & CHEILOCYSTIDIA absent. LAMELLAR

TRAMA regular, made up of cylindrical, narrow elements 30-70 x 8-12 um.

PILEIPELLIS a trichoderm of cylindrical hyphae, often narrower than lamellar

hyphae, with terminal elements 30-70 x 5-8 um, pigment intracellular,

plasmatic, dark blue with somewhat purple tinge, sometimes forming clots,

which dissolve in 5% KOH. SuBPELLIS composed of subhyaline or very pale

blue plasmatic pigment. PILEITRAMA made up of cylindrical elements, hyaline,

thin-walled. StrPITIPELLIs composed of hyphae 5-8 um in diameter, hyaline,

Entoloma shandongense sp. nov. (China) ... 167

Zum 8888 21) ;

Fic. 1. Entoloma shandongense (holotype): 1. Basidiomata (in situ).

2. Line drawings: a. Basidiospores; b. Basidia; c. Young basidia; d. Pileipellis; e. Pileitrama.

3. SEM: Basidiospores. Scale bars: 1 = 10 mm; 2 = 10 um.

thin-walled, without intracellular pigment. STIPE TRAMA regular, made up of

longitudinally cylindrical hyphae up to 5-10 um in diameter. CAULOCYSTIDIA

absent. Oleiferous hyphae scattered in lamellar trama and pileal trama. Brilliant

granules abundant in all tissues. CLAMP CONNECTIONS absent.

EcoLocy — on soil in Poa pratensis grassland.

COMMENTARY — ‘The main characteristics of Entoloma shandongense are the

omphalinoid blue pileus, the many-angled to nodulose spores, and the absence

of cystidia and clamp connections.

There are many species with a blue pileus in Entoloma, but they are rarely

omphalinoid. Only E. ater (Hongo) Hongo & Izawa (Hongo 1958) resembles

E. shandongense in its small omphalinoid basidiomata and grassland

association, but the pileus of E. ater is often purplish and its basidiospores

are larger. Some Entoloma species with blue caps lacking the violet hues

have recently been described from China (Li et al. 2009, He et al. 2012), e.g.,

E. azureosquamulosum T.H. Li & Xiao Lan He, E. subaltissimum T.H. Li & Chuan

H. Li, and E. dinghuense T.H. Li & Chuan H. Li. Entoloma azureosquamulosum

168 ... Wang & Bau

differs from E. shandongense in its convex or conical cap and possession of

hymenial cheilocystidia and caulocystidia on the stipe. The spores of Entoloma

subaltissimum are isodiametric and those of E. dinghuense are quadrate, neither

heterodiametric as in E. shandongense.

Other eastern Asian species with blue-violet pilei (e.g., E. cyanonigrum

(Hongo) Hongo, E. violaceum Murrill, E. glutiniceps (Hongo) Noordel. &

Co-David, E. kujuense (Hongo) Hongo) all differ in larger size (pilei often >2 cm

broad) and a dark purplish (not blue) coloration. In addition, E. cyanonigrum

spores are larger (10.4-11.7 x 6.5-9.lum; Katumoto 2010) and E. glutiniceps

spores are isodiametric (7-8 um diam.; Co-David 2009), distinguished from

the smaller ellipsoid spores of E. shandongense. Finally, pleurocystidia and

cheilocystidia, absent in E. shandongense, are present in both E. kujuense

(Hongo 1985) and E. violaceum (Murrill 1917).

Entoloma rusticoides (Gillet) Noordel., Persoonia 11(2): 150, 1981. Fic. 2

BASIDIOMATA small, mycenoid or omphalinoid. PrLeus 8-14 mm broad,

convex to hemispherical, sometimes with a small papilla, becoming depressed

to umbilicate in the center, hygrophanous, translucent-striate almost to

center, smooth, dark gray to umber brown, light beige brown when dry,

finely tomentose, center always whitish fibrillose-scaly, margin incurved and

undulating. LAMELLAE pale beige to pale brown-pink, decurrent; margin entire.

CONTEXT thin, cream-colored to whitish. Stipe central, 10-15 mm in length,

1-1.5 mm in width, cylindrical to subclavate, surface smooth, gray to brown,

whitely tomentose at base. Opor faintly herbaceous, Taste mild.

BASIDIOSPORES (8.2—)8.5-10.5(-11.0) x (7.6—)8.0-10.3(-11.7) um, ellipsoid

to sub-isodiametric, 5-6-angled in side view, thick-walled, straw coloured.

BASIDIA 28-41 x 9.5-10.5(-12) um, cylindrical to clavate, 2 or 4-spored, hyaline,

thick-walled when mature, clampless. PLEUROCYSTIDIA & CHEILOCYSTIDIA

absent. LAMELLAR TRAMA regular, made up of cylindrical elements, 50-100 x

5-15 um. PILEIPELLIS made up of cylindrical elements 8-20 um in diameter,

septa without clamps, occasional hyphal ends slightly inflated, up to 30 um

in diameter, thin-walled, without clamp connections, externally encrusting

pigments brown. STIPITIPELLIS composed of clampless hyphae 8-15 um,

thin-walled, encrusting pigments brown. STIPE TRAMA regular, made up of

longitudinal cylindrical hyphae, up to 5-15 um in diameter, without clamp

connections. CAULOCYSTIDIA absent.

EcoLocy — in soil alongside paths or associated with Poa pratensis or

Cynodon dactylon grasslands.

SPECIMENS EXAMINED — CHINA, SHANDONG PROVINCE, HEZE: Zhaowanghe Garden,

7 August 2011, Jian-rui Wang (HMLD 1442; GenBank, KC257438); LiAoCcHENG:

Dongchanghe, 1 September 2011, Jian-rui Wang (HMLD 1490; GenBank, KC257441).

Entoloma shandongense sp. nov. (China) ... 169

Fic. 2. Entoloma rusticoides (HMLD1442): 1. Basidiomata (in situ).

2. Line drawings: a. Basidiospores; b. Basidia; c. Pileipellis.

Scale bars: 1 = 10 mm; 2 = 10 um.

COMMENTARY — Entoloma rusticoides is similar to E. rhodocylix (Lasch)

M.M. Moser, E. phaeocyathus Noordel., and E. flocculosum (Bres.) Pacioni in

its omphalinoid shape and sub-isodiametric spores. However, E. rhodocylix

(Moser 1978) and E. phaeocyathus (Noordeloos 1985) both have cheilocystidia

in the lamellar edge, and E. flocculosum (Pacioni 1987) differs in its non-striate,

densely floccose-scaly pileus.

Discussion

The morphology-based taxonomic differences of Entoloma shandongense,

E. rusticoides, and related species have been confirmed by ITS sequence

analyses.

The ITS sequence of the E. shandongense holotype is similar to those of

E. platyphylloides (Romagn.) Largent (GenBank JF908003) and E. undatum

(Gillet) M.M. Moser (GenBank JF908007), with higher query coverage values

(<99%) and max identity (>90%) than other GenBank sequences. However,

the three species obviously differ in fruitbody and spore color, shape, and size.

Entoloma platyphylloides, which occurs in Europe, has a brownish pileus >2 cm

in diameter (Kithner & Romagnesi 1954), while E. undatum has a gray-white

pileus and sometimes possesses cheilocystidia (Moser 1978).

Two ITS sequences from the two E. rusticoides specimens are close to

those of E. infundibuliforme Hesler (GenBank HQ179671), E. sericeoides

(J.E. Lange) Noordel. (GenBank JF908006), and E. defibulatum Arnolds &

Noordel. (GenBank JX454928), with higher query coverage values (<98%) and

max identity (<93%) than other GenBank sequences. However, the spores of

E. defibulatum (rounded-angular; Arnolds & Noordeloos 1979) and

170 ... Wang & Bau

E. sericeoides (Noordeloos 1980) are smaller than in E. rusticoides. Entoloma

infundibuliforme has a squamulose pileus and brown cheilocystidia (Hesler

1967) not found in E. rusticoides.

Acknowledgments

We thank Dr. T.H. Li (Guangdong Institute of Microbiology, China) and Dr.

Genevieve Gates (School of Agricultural Science, University of Tasmania, Australia)

for their reviews of the manuscript. We also thank Dr. X.L. He (Sichuan Academy of

Agricultural Sciences, China) for providing valuable suggestions and references in

the study. Financial support for this work was provided by Changjiang Scholars and

Innovative Research Team in University (No. IRT1134).

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MY COTAXON

http://dx.doi.org/10.5248/124.173

Volume 124, pp. 173-188 April-June 2013

Russula atroaeruginea and R. sichuanensis spp. nov.

from southwest China

Guo-Jrz Li’”, Qi ZHAO?, DONG ZHAO’, SHUANG-FEN YUE",

SAI-FEI L?, HUA-AN WEN™ & XING-ZHONG Liu”

'State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences,

No. 1 Beichen West Road, Chaoyang District, Beijing 100101, China

*University of Chinese Academy of Sciences, Beijing 100049, China

°Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany,

Chinese Academy of Sciences, Kunming 650204, Yunnan, China

‘College of Life Science, Capital Normal University,

Xisihuanbeilu 105, Haidian District, Beijing 100048, China

* CORRESPONDENCE TO: * wenha@im.ac.cn ® liuxz@im.ac.cn

ABSTRACT — Two new species of Russula are described from southwestern China based

on morphology and ITS1-5.8S-ITS2 rDNA sequence analysis. Russula atroaeruginea (sect.

Griseinae) is characterized by a glabrous dark-green and radially yellowish tinged pileus,

slightly yellowish context, spores ornamented by low warts linked by fine lines, and numerous

pileocystidia with crystalline contents blackening in sulfovanillin. Russula sichuanensis,

a semi-sequestrate taxon closely related to sect. Laricinae, forms russuloid to secotioid

basidiocarps with yellowish to orange sublamellate gleba and large basidiospores with warts

linked as ridges. The rDNA ITS-based phylogenetic trees fully support these new species.

KEY worps — taxonomy, Macowanites, Russulales, Russulaceae, Basidiomycota

Introduction

Russula Pers. is a globally distributed genus of macrofungi with colorful

fruit bodies (Bills et al. 1986, Singer 1986, Miller & Buyck 2002, Kirk et al.

2008). As ectomycorrhizal symbionts, they play an important beneficial role

in forest ecosystems (Richardson 1970, Bills et al. 1986, Villeneuve et al. 1991,

Claridge & May 1994, Buyck et al. 1996, Gardes & Bruns 1996). China has a

long history of using Russula mushrooms as traditional food and medicine.

Twenty-two medicinal species and 82 edible species have been reported in the

country (Ying et al. 1982, 1987; Wang & Liu 2002; Yang 2002; Wang et al. 2004;

Dai et al. 2009; Li et al. 2010a), and their ectomycorrhizal capacity, population

genetics, and mycochemistry have been studied in recent years (Gao et al. 2001;

174 ... Li & al.

Tan et al. 2001, 2004; Liang et al. 2004; Liu 2004, 2005; Li et al. 2010b; Zhao et

al. 2010). So far, only 14 new species and three new varieties have been reported

from China (Singer 1935; Chiu 1945; Ying 1983, 1989; Bi & Li 1986; Zang &

Yuan 1999; Wen & Ying 2001; Wang et al. 2009; Li et al. 2011, 2012). Although

southwestern China represents one of the world’s biodiversity “hotspots” and

has a high diversity of macrofungi (Yang & Zang 2003; Dai et al. 2004, 2007; Dai

& Yuan 2008), only a few russulacean species have been reported in this region

and the adjacent Himalayas (Wang & Liu 2009, 2010; Das et al. 2010; Buyck &

Atri 2011; Das & Verbeken 2011; Wang et al. 2012). During a systematic survey

of Russula species in southwestern China from 2004 to 2009, two interesting

species were found, which we propose here as new species.

Materials & methods

Specimens were photographed, and the macroscopic characteristics of fresh mature

fruit bodies were recorded under daylight in the field. Specimens were tested chemically

in sulfovanillin (SV) solution. They were then kept 50-60 °C until completely dried and

were subsequently deposited in the Herbarium of Cryptogams, Kunming Institute of

Botany, Chinese Academy of Sciences (HKAS). Color names and codes were assigned

based on Ridgway (1912). Tissues of dried specimens were immersed in 5% KOH for

10-20 seconds to prepare for microscopic observation in Congo Red using a Nikon

E80i microscope with a 100x oil immersion objective lens. Basidiospores were observed

and measured in Melzer’s reagent. Spore measurements do not include apiculus and

ornamentation, and sterigma lengths were excluded from basidium measurements.

Sections from the pileipellis were anticlinally cut through the pileus centre to the

margin. Cystidia with incrustations were observed in distilled water. Scanning electron

microscopic images were captured with an FEI Quanta 200 microscope. Microscopic

observation and statistical citation of measure technicalities follow Yang (2000) and

Wang et al. (2009).

Protocols for DNA extraction, PCR, and sequencing followed those in Li et al.

(2012). The internal transcribed spacer (ITS) regions were amplified with the primer

pairs ITS1/ITS4 (Gardes & Bruns 1993). PCR products were purified with the Bioteke

DNA Purification Kit (Bioteke, Beijing, China). The ITS regions were sequenced with

the ABI 3730 DNA analyzer and ABI Bigdye 3.1 terminator cycle sequencing Kit

(BGI, Beijing, China), and new sequences were deposited in GenBank (see Fic. 5 for

accession numbers). Sequences of representative and closely related Russula taxa, with

an emphasis on samples of the russuloid to secotioid forms (Eberhardt 2002, Miller &

Buyck 2002, Lebel & Tonkin 2007), were selected from GenBank and included in this

analysis. Assembly and editing of sequences of each region were performed with Clustal

X and BioEdit (Thompson et al. 1997, Hall 1999) and were manually adjusted when

necessary. Some poorly aligned terminal sites were excluded from further analysis.

Based on previous results (Lebel & Tonkin 2007), Albatrellus ovinus (Schaeff.) Kotl.

& Pouzar was chosen as outgroup (Ryman et al. 2003). Sequences of datasets including

the ITS1-5.8S-ITS2 region were analyzed with maximum parsimony (MP) and Bayesian

analysis (BA) methods.

Russula spp. nov. (China) ... 175

MP analysis of the phylogenetic relationships were performed in PAUP* v.4.01

(Swofford 2004). Gaps in alignment were treated as missing data, and all sites were treated

as unordered and unweighted. The tree bisection-reconstruction (TBR) algorithm was

performed using with the heuristic search option, and bootstrap analysis was conducted

with 1000 replicates (Felsenstein 1985). Consistency index (CI), retention index (RI),

and tree length (TL) were also calculated. Trees were displayed with Treeview 1.6.6

(Page 1996).

Bayesian analysis was carried out in MrBayes 3.1.2 (Ronquist & Huelsenbeck 2003).

The best-fit model of Bayesian posterior probabilities (PP) were calculated with the

Markov Chain Monte Carlo (MCMC) algorithm (Larget & Simon 1999). One cold and

three heated Markov chains were run for 2,000,000 generations, the trees were sampled

every 100" generation, and the run was terminated once the average standard deviation

of split frequencies fell below 0.01. A discard of 25% trees was set in the Burn-in option

(Hall 2004).

Taxonomy

Russula atroaeruginea GJ. Li, Q. Zhao & H.A. Wen, sp. nov. Figs 1-2

MycoBank MB804644

Differs from Russula aeruginea by its non-fading green pileus lacking scattered rusty

spots and whitish to pale cream spore print.

Type: China. Sichuan Province, Daofu County, 25.VII.2007, Z.W. Ge 1540 (Holotype,

HKAS53626, GenBank JX391970)

ETYMOLOGY: atroaeruginea (Latin) = dark green. The epithet refers to the dark-green

basidioma pileus.

BASIDIOMATA small to medium sized. PiLeus 3-7 cm in diam., first

hemispherical, then convex to applanate, sometimes slightly depressed above

the stipe, blackish-green tinged, intermixed with radial greenish yellow fringe,

Dull Blackish Green (XLI29""'m) at centre, Sepia (XXIX17'"m) when dry, never

viscous; margin incurved first, straight when mature, sometimes undulate, not

striate, Meadow Green (VI35k) to Antique Green (VI33m) with yellowish tinge

of Oil Yellow (V25i) towards the margin, Verona Brown (XXIX13"k) when dry.

LAMELLAE 10-15/cm at the edge, <7 mm wide, adnate to almost free, forked

around the stipe, occasionally near the pileus margin, not interveined, cream

to yellowish, Cream Color (XVI19’f), brittle; edge even, slightly narrowing

towards the pileus margin; lamellulae occasional. StrpE 4-6 x 1-2 cm, central

to slightly eccentric, smooth, not pruinose, cylindrical, ventricose toward the

base, without annulus, whitish, often with pale-greenish tinge, part turning

pale yellow when aged, sometimes with rusty spots, first unchanging, stuffed,

hollow when mature. CONTEXT 3-5 mm thick from the lamellae attachment

to the stipe, compact under pileus, white to whitish, no color change when

bruised, turning slightly pale yellowish in age; taste mild; no distinct odor.

SPORE PRINT whitish to pale cream (Romagnesi Ib-IIa).

176 ... Li & al.

edge sterile. SUBHYMENIUM 15-20 um thick, composed of slender cylindrical

cells 4-7 um wide under basidia and inflated cells (15-)20-35(-45) um in

diam. next to trama. PILEIPELLIS a trichoderm 200-300 um thick, composed

of 4-6 um thick, slender hyaline hyphae without intracellular pigmentation;

pileocystidia numerous, 40-70 um long, sometimes inflating up to 7-10 um

wide, fusiform, with crystal contents but no septa, sharply blackened in SV.

STIPITIPELLIS not well developed, a cutis composed of thin-walled, septate,

cylindrical hyphae 3-6 um wide; caulocystidia absent. CLamp CONNECTIONS

absent in all tissues.

ECOLOGY & DISTRIBUTION: Single or gregarious in conifer forest (Picea spp.)

in July and August in China (Sichuan, Xizang, and Yunnan).

ADDITIONAL SPECIMENS EXAMINED: CHINA, SICHUAN PROVINCE, DAOFU COUNTY,

25.VII.2007, Z.W. Ge 1532 (HKAS53618); YUNNAN PROVINCE, YULONG, Laojunshan,

9. VIII.2008, Q. Zhao 8238 (HKAS55220); XIZANG AUTONOMOUS REGION, CHANGDU

County, Zhuge Village, 31°04'N, 96°58’E, alt. 4200 m, 7.VIII.2003, Z.L. Yang 4305

(HKAS45684).

ComMENTs: Russula atroaeruginea is characterized by its dark-green intermixed

with radially yellowish-tinged (striate) pileus, whitish to pale-cream spore

print, and basidiospores with thin low fine ornamentations (<1.0 um tall).

The dark-green pileus contrasting with the whitish (often green-tinged) stipe

is a good field character, although it is sometimes difficult to distinguish this

species against from the green forest surroundings. This species should be

placed in the Russula sect. Griseinae (Jul. Schaff.) Romagn. based on its mild-

flavored context, pileipellis lacking primordial hyphae and red pigments but

with typical multiseptate (often subulate) hyphal extremities, and unicellular

pileocystidia that blacken in SV.

Romagnesi (1967) recognized 5 greenish tinged species in sect. Griseinae:

R. pseudoaeruginea (Romagn.) Kuyper & Vuure, R. aeruginea Fr., R. medullata

Romagn., R. stenotricha Romagn., and R. anatina Romagn. The morphologically

similar R. aeruginea, R. anatina, R. cyanoxantha f. peltereaui Singer, R. medullata,

and R. pseudoaeruginea have been reported in eastern Asia (Tai 1979, Ying et

al. 1982, Imazeki & Hongo 1989, Ying 1989, Shimono et al. 2004). Russula

pseudoaeruginea, however, has a gray-tinged green olive or somewhat cream and

pale-brown intermixed pileus and an ochreous-cream spore print (Romagnesi

1967, Sarnari 1998); it often grows in temperate to boreal deciduous forests

dominated by Tilia and Quercus spp. (Romagnesi 1967, Hansen & Knudsen

1992), while R. atroaeruginea grows in conifer forests dominated by Picea spp.

in alpine subtropical regions.

Another taxon superficially resembling R. atroaerugineais R. aeruginea,avery

common species originally described from Europe which grows in subalpine

coniferous to mixed forests containing Betula, Pinus, and Picea. However,

R. aeruginea has a green-tinged pileus with a darker pileus centre, whitish

Russula spp. nov. (China) ... 177

1 Re > | . . = ff 4

Fic. 1: Russula atroaeruginea: a. HKAS 45684; b. HKAS 53626 (Holotype)

stipe with rusty spots, a slightly acrid taste, cream-colored spore deposits, and

spores with many fine lines formed by connected low warts (Schaeffer 1952,

Romagnesi 1967, Roger 1981, Woo 1989, Hansen & Knudsen 1992, Sarnari

178 ... Li & al.

1/19/2011 WD

10 um

av ( BN Li i

(UE A\

S(T / GN NNT

Wks, D \S

LICE

Fic. 2: Russula atroaeruginea (HKAS 53626): a. Basidiospores (SEM); b. Basidiospores;

c. Pleurocystidia; d. Basidia; e. Hyphal extremities in pileipellis; f. Pileocystidia; g. Pileipellis

Basip1osPores [80/4/3] (6.3-) 6.8-8.1 (-9.0) x (5.9-) 6.1-7.4 (-7.8) um,

Q = (1.01-)1.04-1.23(-1.32) (Q =1.15 + 0.07), subglobose to broadly ellipsoid

or (sometimes) ellipsoid, verrucose; ornamentation amyloid, composed of

warts mostly fused into fine lines, cristulate to subreticulate, with some isolated

verrucae, <1.0 um high; plage not or indistinctly amyloid. Bastp1a 40-48 x 9-11

um, clavate, inflated towards upper half, 4-(rarely 2-) spored, projecting 7-13

uum beyond hymenium, hyaline in KOH; sterigmata 3-5 um, pointed, straight to

slightly tortuous. PLEUROCYSTIDIA numerous, 72-115 x 9-13 um, originating

in subhymenium, projecting 35-50 um beyond hymenium, thin-walled, clavate

to subfusiform, sometimes with refractive contents; apices subacute to acute, at

times with a moniliform appendage. CHEILOCYSTIDIA not observed; lamellar

Russula spp. nov. (China) ... 179

1998). Russula aeruginea can be distinguished from R. atroaeruginea by its more

or less discolored pileus, which is often spotted with scattered, rusty spots.

Russula atroaeruginea is also easily distinguished from three other species

originally described from France: R. medullata grows under deciduous trees

of Corylus, Populus, Fraxinus, Betula, Fagus, Castanea, and Quercus, has an

ochre (III a, b-c) spore print, spores with isolated warts, and rare pileocystidia

(Romagnesi 1967, Hansen & Knudsen 1992); R. stenotricha has a grayish-green

pileus, slightly spicy lamellae, short obtuse pileipellis terminal cells, and short

wide pileocystidia 25-50 x 5-9 (12) um (Romagnesi 1967, Hansen & Knudsen

1992); and R. anatina has long cystidia 60-130 x 6.7-12 um (Romagnesi

1967).

Also similar to R. atroaeruginea is the green form of R. cyanoxantha

(Schaeff.) Fr. (= R. cyanoxantha f. peltereaui), which differs in its elastic lamellae,

white (Ia) spore mass, basidiospores with scattered low warts that are never

connected with fine lines, and slender cystidia 65-100 x 5.7-7 um (Romagnesi

1967, Roger 1981, Hansen & Knudsen 1992).

Among the Russula species originally described from China, the

morphologically related R. viridirubrolimbata J.Z. Ying differs from R. atro-

aeruginea by its finely rimose pileus, unchanging white context, isolated spore

ornamentations, hyaline pleurocystidia, and a pileipellis without pileocystidia

(Ying 1983).

Russula sichuanensis G.J. Li & H.A. Wen, sp. nov. FIGS 3-4

MycoBAank MB804645

Differs from Macowanites yunnanensis by its larger gastrocarp, dirty white to pale

tinged pileus, cream to yellowish lamellae, and larger basidiospores with larger

ornamentations.

Type: China. Sichuan Province, from Rangtang County to Seda County, banks of Duke

River, 5.VHI.2007, Z.W. Ge1707 (Holotype, HKAS53792, GenBank JX391969)

ErymMo_ocy: Referring to the type locality region.

BASIDIOMATA semi-hypogeous to epigeous, russuloid to secotioid, small.

Piteus 3.1-4.4 cm in diam., irregularly globose to subglobose when young,

hemispheric, barely expanded, sometimes pulvinate when mature, centre slightly

depressed with age, whitish, dirty-white to pale-pinkish tinge of Pale Salmon

Color (XIV9’f) to Pale Flesh Color (XIV7’f), rarely with greenish tinge of Dark

Olive Buff (XL21'”’), intermixed with brown to olivaceous brown tinge of Wood

Brown (XL17"”), Brussels Brown (III15m) when dry; margin whitish to cream,

White (LIII) to Cream color (XVI119’f), often glabrous, dry, slightly viscid when

moist, strongly incurved and indented around the stipe, sometimes slightly

radially rugose, with slightly pinkish tinge of Pale Flesh Color (XIV7’'f), not

striate. CONTEXT <3 mm at the disc, whitish, fragile; odor not distinctive; taste

mild. LAMELLAE/GLEBA 3-6 mm high, sublamellate to lamellate, composed of

180 ... Li & al.

US cn. a = q : vars

Fic. 3: Russula sichuanensis: a. HKAS 45645; b. HKAS 53792 (Holotype)

sinuate, contorted, very crowded, convoluted lamellae, often exposed near the

stipe, cream to yellowish tinge of Pale Orange-Yellow (III15f) when juvenile,

Russula spp. nov. (China) ... 181

ss a = —_

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Fic. 4: Russula sichuanensis (Holotype HKAS 53792): a. Basidiospores (SEM); b. Basidiospores;

c. Basidia; d. Hyphal extremities in pileipellis; e. Pileocystidia; f. Caulocystidia; g. Pleurocystidia.

with pale orange tinge of Orange Buff (III15d) to Light Orange- Yellow (II117d)

when mature. STIPE 4.0-6.0 x 1.0-1.5 cm, subcylindrical, surface dry, rugulose

longitudinally, dull, without annulus, slightly attenuate upwards, whitish,

<3 mm at pileus diameter, stuffed first, becoming hollow when old. SpoRE

PRINT Ocher to yellow (Romagnesi IIc-IVa).

BASIDIOSPORES [100/3/2] (8.8—)9.4-14.1(-15.5) x (7.6-)7.9-12.8(-13.1)

um, Q = (1.01—-)1.02-1.26(-1.32), (Q = 1.11 + 0.08), globose to subglobose,

slightly yellowish; ornamentation cristulate to subreticulate, composed of

amyloid warts linked as small crests and ridges, forming a partial to nearly

complete network, rarely intermixed with isolated verrucae, warts often >1 um

high (but no more than 1.5 um); suprahilar plage not well defined. Basip1A

182 ... Li & al.

24-35 x 11-15 um, mostly with two sterigmata 4-7 um long, rarely 4-spored,

claviform to clavate, short and rather voluminous, rarely cylindrical, hyaline,

or sometimes yellow in KOH. PLEuRocystipiA rare, 45-72 x 10-15 um,

distinctly projecting 10-30 um beyond the basidia, ventricose, sometimes

clavate to sphaeropedunculate, thin-walled, rarely thick-walled, often empty,

at times with refringent contents, no reaction in SV. SUBHYMENIUM a 20-35

uum thick cellular layer composed of inflated cells 7-13 um in diam., hyaline,

sometimes pale yellowish in KOH. PILEIPELLIS an ixotrichoderm 125-150

uum thick, slightly gelatinized, composed of thin-walled hyphae 3-6 um wide,

cylindrical, hyaline; pileocystidia septate, clavate to cylindrical, 6-10 um wide,

apex sometimes inflated, with refractive contents. STIPITIPELLIS filamentous

hyphae 3-6 um in diam., intermixed with inflated cells, hyaline, some hyphae

yellowish to pale ocher in KOH; caulocystidia infrequent, 35-44 x 8-11 um,

cylindrical, septate, with round apex. CLAMP CONNECTIONS AND LATICIFEROUS

HYPHAE absent from all tissues.

ECOLOGY & DISTRIBUTION: Single or gregarious in conifer forests (Picea

spp.) at altitude of 3300-3900m during July and August, China (Xizang and

Sichuan).

OTHER SPECIMENS EXAMINED: CHINA, XIZANG AUTONOMOUS REGION, JIANGDA

County, Tongpu Township, 31°35'N, 98°23’E, alt. 3300 m, 2.VIII.2004, Z.L. Yang 4266

(HKAS45645); RrwocHE County, Sangduo Township, 31°05’N, 96°29’E, alt. 3900

m, 11.VIII.2004, Z.W. Ge335 (HKAS46115); ZuoGoNG County, Wangda Township,

alt. 3700 m, 18.VII.2009, Z.L. Yang 5285 (HKAS 57828); SICHUAN PROVINCE, ABA

County, Angiang Township, Ping’ an Village, 11. VIII.2007, Z.W. Ge 1800 (HKAS53885,

GenBank JX391971).

ComMENTSs: Until Russula species were emended by Lebel & Tonkin (2007),

Russula sichuanensis was treated in Macowanites Kalchbr., a widespread

sequestrate genus containing diverse species phylogenetically close to Russula

(Miller et al. 2001, Lebel & Castellano 2002, Eberhardt & Verbeken 2004).

That very little knowledge is available concerning Macowanites in China may

result partly from its semi-hypogeous habit; only M. yunnanensis M. Zang has

been described, based on materials collected from Zixi Mountain, Chuxiong,

Yunnan Province (Zang & Yuan 1999). Its morphology conspicuously differs

from that of R. sichuanensis: M. yunnanensis has a smaller gastrocarp (0.7-1.2

cm broad), a pinkish- to reddish-tinged pileus surface, whitish lamellae, and

smaller basidiospores (10-12.5 x 6-7 um) with 0.3-0.5 um ornamentations

(Zang & Yuan 1999).

Russula_ sichuanensis resembles a medium-sized, distorted or aborted

agaricoid Russula, because it has a curly pileus, a white percurrent stipe often

covered by the pileus, and tightly contorted sinuate cream to yellow lamellae.

Other characters include a cream-tinged pileus (often olivaceous brown at the

centre), a yellowish to orange sublamellate gleba, and stipitate basidiomata.

Russula spp. nov. (China) ... 183

The following microscopical characters are distinctive: the partially reticulate

globose to subglobose basidiospores, short voluminous basidia, very rare

pleurocystidia, and septate pileocystidia with crystal contents. In addition, the

association of R. sichuanensis with Picea spp. in coniferous forests of the eastern

Qinghai-Tibet plateau is a distinctive ecological and biogeographical feature.

A number of species have a similar sublamellate gleba and percurrent stipe:

Macowanites citrinus Singer & A.H. Sm., M. chlorinosmus A.H. Sm. & Trappe,

M. luteolus A.H. Sm. & Trappe, M. subolivaceus A.H. Sm., Russula galbana T.

Lebel, R. kermesina T. Lebel, R. luteirosea (Bougher) T. Lebel, and M. arenicola

S.L. Mill. & D. Mitch. However, M. citrinus, originally described from Idaho

(in the northwestern U.S.A.), differs from R. sichuanensis in having smaller

subglobose to short ellipsoid basidiospores ornamented by conical spines and

short flattened ridges and with a small indistinct suprahilar plage (Pegler &

Young 1979), and M. chlorinosmus (from Oregon, U.S.A.) has a rimose pileus

cutis, a strong chlorine odor, a very unpleasant taste, and basidiospores with

low warts (Smith 1963). Macowanites luteolus (also from Oregon) differs by

its short thin stipe, smaller basidiospores with scattered to close 0.3-0.7 um

ornamentations, subhymenium cells that are hyaline in KOH, and numerous

macrocystidia (Smith 1963). Macowanites subolivaceus (from Idaho) has a

lobed to very irregular pileus margin, a very fragile thin context, basidiospores

7-9 um in diam., and a peridial epicutis consisting of a turf of gelatinous

branched hyphae with 60-200 um long pseudocystidia (Smith 1963). Russula

galbana (from Queensland, northeastern Australia) has smaller basidiomata,

a short slender stipe, and common, dimorphic hymenophoral cystidia with

obtuse or mucronate apices (Lebel & Tonkin 2007); R. kermesina, (from

Nelson, New Zealand) is characterized by its carmine red to dark-red-tinged

pileus, chalk-white gleba, large cystidia not or only slightly projecting beyond

the basidia, and pileipellis without pileocystidia (Label & Castellano 2002).

Russula luteirosa (from western Australia) has a pileus with intermixed cream

to pale yellowish and rosaceous colors, large basidia, and an association

with Allocasuarina, Acacia, and Eucalyptus (Bougher 1997). Macowanites

arenicola (from northwestern Florida, U.S.A.) has a yogurt- or soy sauce-type

odor, smaller broadly elliptical to elliptical spores with low ornamentation

(8.8-10.4 x 6.8-7.2 um, Q = 1.37, verrucae 0.2-0.4 um high), long slender

cystidia (80-120 x 6-7.5 um), and grows in white quartzite sands of costal

scrub forests (Miller 2004).

Russula sichuanensis also shows some superficial affinities with Russula sect.

Laricinae Romagn. for its small-sized basidiocarps, ocher to yellow spore print,

short basidia, rare pleurocystidia, and pluriseptate dermatocystidia (Romagnesi

1985), but its russuloid to secotioid basidiocarps and large basidiospores

obviously distinguish it from the agaricoid sect. Laricinae.

184 ... Li & al.

Albatreilus ovinus AY198202

R adusta AY061652

R ingwa EU019919 Sect. Mgricantinae

R nigricans AY061695

& joetens AY061677 | Sect. Foetentinae

R galbana EU019936

& neerimea EU019915

(—— & albobrunnea EU019918 |

R caerulea AY¥061661 Sect. integroidinae

R xerampelina AY061734 | Sect. Miridantinae

95IL) R. cessans AY061685

& laricina AY061730

R. sichuanensis JX391971 HKAS5S3885 Sect. Larcinae

RX curtipes AY061668

R nauseosa JF908642

R decolorans AY061670 | Sect. Decolorantinae

R alivacea AY061699 |. Sect. Ofvaceinae

R violecea AY061725_— | Sect. Violacinae

R kalimna EU019927

R rubrolutea EU019940

“— RB tapawera EU019935

R purpureofiava EU019917

R wollumbina EU019921

R emetica AY061673 7

R persanguinea EU019916 | Sect. Bmeticinae

R nobilis AF418620

R. exalbicans AF418622 I Sect. Bxalbicantinae

R fellea AY061676 | Sect. Felleinae

-10.99

RX queleti AYO61711 | Sect. Sardoxinae

oe! R aeruginea AF418202

- R grisea AY061679 Sect. Griseinae

R. atroaeruginea TX391970 HEAS53626 Holotype

il eyanoxantha AY061669 | Sect. Indalentinae

Rilicis AY061682 Gare Heternho th

MY R werner? DQ422021 a re Ae aati

R crustosa EW598194

R virescens AY061727

& iterika EU019929

R mustelina AY061693

& vesea AF418610

R rostraticystidia EU019938

& variispora EU019934

R marangania EU019930

100/1 & delica AY061671 | Sect. Plarantinae

R pumicoidea EU019931

| Sect. Virescentinae

| Sect. Heferophyiinae

—— 10 changes

Fic. 5. Phylogenetic relationship tree of Russula species inferred from the dataset of ITS1-5.8S-

ITS2 sequences. Consistency index (CI) = 0.461, retention index (RI) = 0.665, and tree length (TL)

= 1030. MP bootstrap support values (>50%) and PP (>0.94) from Bayesian Analysis are indicated

above or below the branches as MP BS/BA PP. Sectional names are from Romagnesi (1987).

Phylogenetic results

The ITS1-5.8S-ITS2 dataset included sequences representing 44 taxa

representing nine Russula subgenera. ‘The aligned sequence comprised 471

nucleotide sites. In the MP analysis, 195 characters were constant, 69 variable

characters were parsimony-informative, and 207 characters were parsimony-

uninformative. In the ITS phylogenetic tree (Fic. 5), R. atroaeruginea clustered

with R. aeruginea and R. grisea with strong statistical support (98% boot strap,

1.0 posterior probability). A Blast comparison of R. atroaeruginea (JX391970)

and R. aeruginea (AF418612) through the NCBI databank shows a 93% max

identification and 96% coverage. Another ITS blast with R. atroaeruginea

(JX391970) and R. grisea (AY061679) shows a 90% max identification of 99%

coverage, confirming that R. atroaeruginea is related to R. grisea / aeruginea

group in Russula sect. Griseinae. The separation between R. atroaeruginea and

Russula spp. nov. (China) ... 185

R. cyanoxantha is also well supported. These phylogenetic results suggest that

R. atroaeruginea and R. aeruginea are closely related but different species.

The clade in which R. sichuanensis is located is highly supported (Fie. 5).

Members of Russula subgen. Tenellula sect. Larcinae cluster together, suggesting

that R. sichuanensis is closely related to the R. laricina / cessans group. However,

given the lack of stability in the topologies due to GenBank’s limited number of

ITS sequences from closely related secotioid Russula taxa, we hesitate making a

definitive statement about the phylogenetic position of R. sichuanensis. Broader

collections from East Asia, particularly of russuloid to secotioid Russula taxa,

are required in future work.

Acknowledgments

This study was supported by the National Natural Science Foundation of China

(No. 30770013), International Cooperation Program of Ministry of Science and

Technology of the People’s Republic of China (No. 2009DFA31160) and the Hundred

Talents Program of the Chinese Academy of Science. The authors are very grateful to

Dr. Y.C. Dai (Institute of Applied Ecology, Chinese Academy of Sciences) and Dr. J.P. Xu

(Department of Biology, McMaster University) for reviewing the manuscript; Dr. Z.W.

Ge for assistance in specimen collecting; Dr. L.S. Wang, Dr. E.D. Liu, and Dr. H. Peng

(Kunming Institute of Botany, Chinese Academy of Sciences) for the loan of herbarium

specimens; Mr. S. Shi (Institute of Botany, Chinese Academy of Sciences) for providing

references; Ms. X.F. Zhu for inking in line drawings; to Dr. X.L. Wang (Institute of

Microbiology, Chinese Academy of Sciences) for providing advice and suggestions

regarding phylogenetic analysis; Dr. B.A. Jaffee (University of California Davis), Dr.

Z.L. Yang (Kunming Institute of Botany, Chinese Academy of Sciences), Dr. J.Y. Zhuang

(Institute of Microbiology, Chinese Academy of Sciences), Dr. S.R. Pennycook (Landcare

Research) and Dr. L.L. Norvell (Pacific Northwest Mycology Service) for improving

the manuscript; and Dr. C.L. Li and Ms. J.N. Liang (Institute of Microbiology, Chinese

Academy of Sciences) for providing assistance in SEM photography.

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JUAN-JULIAN BORDALLO’, ANTONIO RODRIGUEZ’, JUSTO M. MuNOz-

MOHEDANO’!, LAURA M. Suz 7, MARIO HONRUBIA! & ASUNCION MorRTE"

"Departamento de Biologia Vegetal (Botanica), Facultad de Biologia, Universidad de Murcia,

Murcia, 30100, Spain

? Mycology section, Jodrell Laboratory, Royal Botanic Gardens,

Kew, Richmond, Surrey, TW93DS, UK

* CORRESPONDENCE TO: amorte@um.es

ABSTRACT — A phylogenetic analysis of Iberian Terfezia collections indicates eight clades, of

which three correspond with previously described species (T: alsheikhii, T. fanfani, T. olbiensis)

and five are described as new species — T. albida, T. eliocrocae, T. extremadurensis, T: pini,

and T: pseudoleptoderma. These results are supported by the morphology of the examined

Terfezia ascomata that were identified by their ecological patterns and chorology, as well as

by phylogenetic analyses. Morphological features and ITS-rDNA sequence analyses from

identified Terfezia specimens are described. A key to these Terfezia species is also provided.

Key worps — desert truffle, hypogeous, mycorrhizal fungi, Pezizaceae

Introduction

The genus Terfezia (Tul. & C.Tul.) Tul. & C.Tul. includes twelve species (Kirk

et al. 2008) of hypogeous ascomycetes formerly placed in the family Terfeziaceae

within the order Pezizales (Trappe 1979). However, based on molecular and

morphological characters, Terfezia is now accepted in the Pezizaceae (Norman

& Egger 1999; Percudaniet al. 1999; Laessoe & Hansen 2007). These hypogeous

fungi are edible and are known as “desert truffles” due to their habitat in

typically arid and semi-arid ecosystems, mostly in the Mediterranean region

(Morte et al. 2009).

Most Terfezia species establish mycorrhizal symbiosis with plants from

family Cistaceae, mainly with perennial and annual Helianthemum species

(Dexheimer et al. 1985; Fortas & Chevalier 1992; Gutiérrez et al. 2003) or with

trees from different phyla (Diez et al. 2002; Taylor et al. 1995). These plants

and their associated fungi play a major role in maintaining Mediterranean

shrub lands and xerophytic grasslands and thus in preventing erosion and

190 ... Bordallo & al.

desertification (Honrubia et al. 1992). This mycorrhizal association is well

adapted to semiarid climates through the physiological mechanism of drought

avoidance (Morte et al. 2000, 2010).

Six Terfezia species have been reported so far from the Iberian Peninsula:

T. claveryi Chatin, T: boudieri Chatin, T: arenaria (Moris) Trappe, T: leptoderma

Tul. & C. Tul., T. olbiensis, and T. alsheikhii. Terfezia claveryi, T. boudieri, and

T. olbiensis have been recorded from basic soils and T: arenaria from acid soils,

but all four in mycorrhizal association with different Helianthemum species.

However, T: leptoderma has been found in association with Tuberaria guttata

L. Fourr. in acid soils, with Cistus in slate-derived soils, and with Quercus ilex

L. and Pinus halepensis Mill. in basic soils (Diez et al. 2002). Isolations from

Cistus, Pinus, and Quercus could belong to distinct species with different host

or/and edaphic specialization. These authors emphasized that the sampling in

these ecosystems is scanty and further study is necessary. Moreover, the small

spores of T: leptoderma specimens collected under pine and evergreen holm

oak fit those of T. olbiensis, which was described as morphologically similar to

T. leptoderma, except for slightly smaller spores and shorter spines (Diez et al.

2002). There is a certain consensus that T: olbiensis is an immature form and a

synonym of T leptoderma (Diez et al. 2002; Moreno et al. 1986).

The main objective of this study was to describe five new Terfezia species. For

this purpose, we conducted classical morphological studies complemented by

phylogenetic analyses based on ITS-rDNA sequences from Terfezia specimens

collected throughout the Iberian Peninsula.

Materials & methods

Fungal specimens

Ascomata of Terfezia spp. were collected in different years and from different

locations in the Iberian Peninsula (Spain and Portugal). The ascomata were collected

from cracks in the soil close to host plants. Characterized and sequenced specimens are

listed in TaBLE 1. Generally, three specimens per collection were used when the material

was fresh. Furthermore, conserved dried material was processed. Soil characters were

obtained from the Spanish Geological Map, according to FAO-CEE, 1998 from the

“Atlas Nacional de Espafia” (IGN). The different host plants were identified using Flora

Ibérica keys (Mufioz-Garmendia & Navarro 1993).

External ascocarp characteristics (shape, colour, appearance) were recorded in detail.

Ascomata were then cut and the morphology of the peridium and gleba was described.

Asci and ascospores were examined using an Olympus BX51 microscope equipped with

a digital camera (Canon PSprol). A minimum of 100 asci, ascospores, and peridial warts

was measured per species. The ascospores of Terfezia eliocrocae and T. pseudoleptoderma

were stained with acid fuchsine solution (0.01% acid fuchsine in acetic acid, ethylene

glycol, and lactic acid, 1:1:1, v/v/v) to improve visualization.

Samples of the collected specimens are stored in the Mycological Herbarium of the

Botany Area of the University of Murcia (MUB).

TABLE 1. Terfezia collections analyzed

CLADE

DNDAD UU uu wo Be BP BP BP BP BP BP BPW WW WW WN NO RR RR Re Re Re Re Be eS Re

NNDNNDDAAHATA AHN HD AW

COLLECTION” #

tO9*

tO10*

tO11*

tO17*

tO18

tO19

tO20*

tO21

tL9

tL10*

tL1l

tL12

tO4*

tO7*

tTP1

tTP2*

tL13

ORIGIN 4“ & Host

Belvis (Cc). Tuberaria guttata

Belvis (Cc). T: guttata

Valdecafias (Cc). T. guttata

Castuera (Ba). T: guttata

Valdecafias (Cc). T. guttata

C. Arafiuelo (Cc). T. guttata

Lorca (Mu). Helianthemum

Montesihno. Portugal. Cistaceae

Quintana (Ba). Cistaceae

Sieteiglesias (Va). Quercus

Ronquines (Va). Pinus

Cornudilla (Bu). Pinus

Madrid. Pinus

Villafranca (Bu). Cistaceae

Valdecafias (Cc). Pinus

Madrid

Belvis (Cc). T: guttata

Castuera (Ba). Cistaceae

Garrovilla (Cc). T. guttata

Valdecafias (Cc). T. guttata

Cortas Blas (Va). Cistaceae

Quintana (Ba). T. guttata

Cafiaveral (Hu). Cistaceae

Terfezia spp. nov. (Iberia) ... 191

YEAR, COLLECTOR

2009. J. Mohedano

2009. M. Romero

2007. J. Mohedano

2002. J. Diez

2001. A. Morte

2007. A. Morte

2006. A. Rodriguez

2008. M. Romero

2011. G. Kovacs

2003. A. Garcia

1998. A. Garcia

2008. FE Sainz

2008. E Sainz

2008. Barriuso et al.

2008. E Sainz

2008. FE Sainz

2010. J. Mohedano

2008. A. Rincon

2009. J. Mohedano

2009. M. Romero

2004. A. Mateos

2001. J. Mohedano

1998. A. Garcia

2009. M. Romero

2002. J. Diez

GENBANK N°

HM056199

HM056200

HMO056201

HM056202

HM056203

HM056204

AF276678

HM056205

HM056206

HM056207

HM056208

HQ698098

HQ698100

HM056209

HM056210

DQ386140

HM056211

HM056212

HM056213

FJ013064

HM056214

HM056215

HM056216

HM056217

HM056218

HM056219

AF396862

AF276676

192... Bordallo & al.

TABLE 1, concluded

CLADE COLLECTION” # ORIGIN “ & Host YEAR, COLLECTOR GENBANK N°

6 #* Madrid. 2008. A. Rincén FJ013087

— #* Valencia. Pinus 2002. J. Diez AF396864

7 tO15* Ballestero (Ab). Helianthemum 2009. A. Rodriguez HM056220

7 tO16* Albacete (Ab). Helianthemum 2009. A. Rodriguez HM056221

7 #* Murcia. Helianthemum 2001. A. Gutiérrez AF387655

8 tO12* Masegoso (Ab). Quercus 2009. A. Rodriguez HM056222

8 tO13 Masegoso (Ab). Quercus 2009. A. Rodriguez

8 tO14* Masegoso (Ab). Quercus 2009. A. Rodriguez HM056223

8 tO34* Santa Espina (Va). Pinus 1998. A. Garcia HM056224

8 tO39* Onteniente (V). Pinus 2009. EF Garcia HM056225

8 #* Murcia. Pinus 2001. Gutiérrez et al AF387656

8 #* — 2002. J. Diez AF276677

8 #* France. Quercus 2002. J. Diez AF396863

— #* Jarada. Morocco 1994. M. Achouri AF301421

= #* C. Arafiuelo (Cc). 2002. J. Diez AF276674

— #* Zeelim. Israel 1996. Bedouins AF092098

* = included in the cladistic analysis; # = external sequence.

S Spanish provinces: Ab: Albacete; Ba: Badajoz; Bu: Burgos; Cc: Cacerese; Hu: Huelva;

Mu: Murcia; V: Valencia; Va: Valladolid.

Macroscopic and microscopic characterization

For identification, ascomata were compared with descriptions from Mattirolo (1900,

1906, 1907, 1922), Lazaro Ibiza (1908), Malencon (1938, 1973), Ceruti (1960), Cerutti et

al. (2003) and keyed according to Montecchi & Lazzari (1993) and Montecchi & Sarasini

(2000). Trappe (1971, 1979), Calonge et al. (1977) and Honrubia et al. (1992) were

also taken into account. The descriptions of Tuber lutescens Lazaro Ibiza (= Terfezia

lutescens (Lazaro Ibiza) Malencon) and T: pallidum Lazaro Ibiza (= T! pallida (Lazaro

Ibiza) Malencon) were also consulted (Lazaro Ibiza 1908, Malencon 1938). In addition,

descriptions of T’ leptoderma, T: fanfani, T. cadevalli Font Quer, T: hafizi Chatin,

T. berberiodora Lesp. ex Tul. & C.Tul., and T. goffartii Chatin were also checked.

DNA analyses

Genomic DNA was isolated from 150-200 mg of the inner gleba of the ascocarps using

the E.Z.N.A. Fungal DNA kit (Omega Bio-Tek, Doraville, GA, USA) and following the

manufacturer's instructions. The Internal Transcribed Spacer (ITS) region of the rDNA,

including the 5.8S ribosomal gene, was amplified using the universal ITS5 and ITS4

primers (White et al. 1990). All PCR amplifications were carried out in a final volume

of 25 uL containing 0.2 mM of each dNTP, 0.6 uM of each primer, 3.75 mM MgCL, 1X

PCR buffer and 1.25 U of Taq DNA polymerase (Invitrogen, California, USA).

PCR reactions were performed in a Mastercycler Gradient thermocycler (Eppendorf,

Hamburg, Germany) with the following cycling parameters: an initial denaturalization

step for 2 min at 94°C, 30 cycles consisting of 30 s at 94°C, 1 min at 65°C, and 1 min at

72°C, and a final extension at 72°C for 4 min.

PCR products were purified using the E.Z.N.A. Cycle-Pure kit (Omega Bio-Tek)

following the manufacturer's instructions. Clean PCR products were sequenced in both

directions at the Molecular Biology Service (University of Murcia). The BioEdit (Hall

Terfezia spp. nov. (Iberia) ... 193

1999) program was used to obtain the consensus sequence of each sample. Sequences

were deposited on GenBank (NCBI) under accession numbers indicated in TaBLE 1.

Phylogenetic analyses

Nucleotide Basic Local Alignment Search Tool (BLAST) searches (megablast) were

used to compare the sequences obtained in this study with other DNA sequences in

GenBank (NCBI, Altschul et al. 1997) to provisionally identify the specimens prior to

phylogenetic analyses. The sequence alignment was carried out with ClustalW (MEGA

software, version 4; Tamura et al. 2007), following the default options. A minimal

manual adjustment was made with the MEGA v.4 editor. Alignments are available in

TreeBase (http://purl.org/phylo/treebase/phylows/study/TB2:S 10906?x-access-code=ea

dd311aaa59cd96424d699af26cb699&format=html)

Evolutionary history was inferred based on the Maximum Parsimony (MP; Eck

& Dayhoff 1966) and the Minimum Evolution (ME; Rzhetsky & Nei 1992) methods

using MEGA v.4. The closest species not belonging to the sister group were selected as

outgroups. The bootstrap consensus tree inferred from 1,000 replicates was taken to

represent the evolutionary history of the taxa analyzed. The branches corresponding

to the partitions reproduced in less than 50% bootstrap replicates were excluded. The

percentages of replicate trees in which the associated taxa clustered together in the

bootstrap test (1000 replicates) are shown next to the branches (Felsenstein 1985).

The MP tree (PLATE 1) was obtained using the Close-Neighbour-Interchange

algorithm at search level 3 with which the initial trees were obtained with the random

addition of sequences (10 replicates). The tree was drawn to scale, and branch lengths

were calculated using the average pathway method (Nei & Kumar 2000). The number

of changes over the whole sequence is presented in units. All positions with gaps and

missing data were eliminated from the dataset (the Complete Deletion option). The final

dataset contained a total of 429 positions, of which 69 were parsimony informative.

The ME tree was drawn to scale, with branch lengths in the same units as those of the

evolutionary distances used to infer the phylogenetic tree. The evolutionary distances

were computed using the Maximum Composite Likelihood method (Tamura et al.

2004) and are presented in the units of the number of base substitutions per site. The

ME tree was searched using the Close-Neighbour-Interchange (CNI) algorithm (Nei &

Kumar 2000) at search level 1. The Neighbour-joining algorithm (Saitou & Nei 1987)

was used to generate the initial tree. All the positions with gaps and missing data were

eliminated from the dataset (the Complete Deletion option). The final dataset contained

a total of 429 positions.

The sequences from T. claveryi, T. boudieri and T: arenaria were chosen as

outgroup.

Phylogenetic results

Sequence analyses of the ITS-rDNA from the examined samples produced

two trees based on the Maximal Parsimony (MP) and the Minimum Evolution

(ME) methods, both with a virtual sampling or bootstrap of 1000 replicas.

In the ME phylogeny, the 38 sequences are clearly distributed in eight well-

separated lines (PLATE 1) that match the eight phenetically described species.

194 ... Bordallo & al.

tO17 Terfezia extremadurensis

tO11Terfezia extremadurensis

tooTerfezia extremadurensis

CLADE 1

AF276678 Tuberaria guttata, acid soils,

spined ascospores

99/83 tL10Terfezia extremadurensis

tO20Terfezia extremadurensis

94/46

‘— tO10Terfezia extremadurensis

CLADE 2

tO7Terfezia eliocrocae Helianthemum spp, basic soils,

reticulated ascospores

99/99 tO4Terfezia eliocrocae

100/100 facet tae alsheikhii CLADE 3

a Cistaceae, acid soils,

99/96 tTP2 Terfezia alsheikhii teliculdted ascospores

91/81 DQ386140

oe CLADE 4

93/90 tO32Terfezia pini Pinus spp, Quercus: app; sandy soils,

spined ascospores

— tO40Terfezia pini

9/81

;——— tL17 Terfezia pseudoleptoderma

tO42Terfezia pseudoleptoderma ices

P Py Cistaceae, sandy soils,

89/81 é

U spined ascospores

> FJO13064

99/97 — tL16Terfezia pseudoleptoderma

AF396862

tL7Terfezia fanfani

CLADE 6

tLiTerfezia fanfani Tuberaria guttata, acid soils,

spined ascospores

97/93 99/97 tO23Terfezia fanfani

tL8Terfezia fanfani

tL14Terfezia fanfani

FJO13087

cea AF276676

85/90 tO22

AF396864

tO1STerfezia albida

99/98

48/03 a CLADE 7

tO16Terfezia albida Ctstaceae, marl soils,

[| spined ascospores

AF387655

tO14Terfezia olbiensis

72/55

tO34Terfezia olbiensis

AF276677

97/74

; | CLADE 8

AF396863 Pinus spp, Quercus spp, marl soils,

spined ascospores

'— tO39Terfezia olbiensis

U AF387656

tO12Terfezia olbiensis

AF092098Terfezia boudieri

AF301421Terfezia claveryi

AF276674Terfezia arenaria

0.005

Terfezia spp. nov. (Iberia) ... 195

The MP phylogenetic topology is consistent with that of the ME analysis in

which the same nodes and clusters are reproduced (PLATE 1). The cladogram

distinguishes eight clades (TABLE 2).

TABLE 2. Diagnostic characters of clades and Terfezia spp.

CLADE SPECIES BOOTSTRAP % SOIL Host

1 T. extremadurensis 94 Acid Tuberaria guttata

2 T. eliocrocae 94 Alkaline Helianthemum spp.

3 T. alsheikhii 89 Acid Cistaceae

4 T. pini 91 Acid Pinus spp., Quercus spp.

5 T. pseudoleptoderma 91 Acid Cistaceae

6 T. fanfani 70 Acid Tuberaria guttata

7 T. albida 70 Alkaline Helianthemum spp.

8 T. olbiensis 70 Alkaline Pinus spp., Quercus spp.

Taxonomy

The morphology and distributions of the examined specimens support eight

phenetic species, described below (presented in clade order; PLATE 1).

Terfezia extremadurensis Mufioz-Mohedano, Ant. Rodr. & Bordallo, sp. nov.

MycoBank MBs561591 PLATE 2A,B

Differs from all other spiny-spored Terfezia spp. by its larger spores with wider spines

and its Tuber-like glebal morphology.

Type: Spain, Extremadura, Caceres, Belvis de Monroy, 17 March 2009, leg Mufioz-

Mohedano (Holotype, MUB Fung-0026).

EryMo_oey: referring to Extremadura, the western Spanish region where the species

was first identified

AscomMatTa hypogeous to partially emergent at maturity, 2-5 cm, subglobose,

sometimes furrowed and nodulose, often cracked, often with a small basal

depression, rarely with pseudostipe, cream colour at first, becoming brown,

black spots on the sun-exposed parts or where handled, smooth. PERIDIUM

300-600 um thick, well-defined, concolorous with surface in cross section,

pseudoparenchymatous, composed of subglobose cells, hyalines and thin-

walled in the innermost layers, yellowish and with thicker walls in the outermost

layers. GLEBA solid, fleshy, succulent, whitish at first, soon becoming salmon

pink, darkening with age, greenish grey at maturity, marbled with thin, white,

PiaTE 1. Minimum Evolution (ME) and Maximum Parsimony (MP) consensus tree showing

evolutionary relationships of 38 sequences derived from the ITS-rDNA region. On each branch, %

of 1000 bootstrapping replicates supported by ME is shown first (in bold) and by MP second (in

plain font). Host plant, soil, and ascospore information is presented for each clade. Non-significant

bootstrap values under 70 are not included.

196 ... Bordallo & al.

meandering veins, sometimes arising from the base and inconspicuous in very

mature specimens; frequently with small holes indicating mycophagous activity.

Odour faint, not distinctive. Asci inamyloid, subglobose to ovate, sessile or

short-stipitate, 60-80 x 50-65 um, walls 1-2 um thick, with 6-8 irregularly

disposed spores, randomly arranged in the gleba. Ascospores globose, (21-)

22-26(-27) um diam (median = 24 um) including ornament [(16-)17-19(-20)

um (median = 18 um) without ornament], hyaline, smooth and uniguttulate

at first, by maturity yellow and ornamented with conical, blunt, thick spines,

sometimes truncated, sometimes finger-like, often joined at the base, 3-4(-5)

uum long, 1-3 um wide at the base.

ECOLOGY & DISTRIBUTION — Widely distributed in the western half of

the Iberian Peninsula, common in grassland of Extremadura in sandy, acid

soils, associated exclusively with Tuberaria guttata, from late winter to early

spring. This is the earliest Terfezia species to appear (in March) associated with

T. guttata.

ADDITIONAL COLLECTIONS EXAMINED: SPAIN: EXTREMADURA, CACERES: Valdecajfias,

2007, Mufioz-Mohedano (MUB Fung-0012); 2009, Mufioz-Mohedano (MUB Fung-

0035); Bapayoz: Castuera, 2009, M. Romero (MUB Fung-0036).

CoMMENTS — Terfezia extremadurensis differs from other spiny-spored

Terfezia species in its Tuber-like gleba, with meandering veins of the gleba not

completely surrounding the fertile tissue and not forming pockets as is typical

for all other Terfezia species. Terfezia fanfani, described below, shares the same

habitat but has reddish tones, a different gleba, and smaller spores (19-22 um)

with thin spines 1 um wide at the base.

Clade 1 comprises T: extremadurensis and clearly differs from the other

Terfezia species in the shape of the gleba (reminiscent of Tuber) and the

ascospores with truncated crested spines. As far as we are aware, these two

characteristics have not been described to date for Terfezia. The phylogenetic

analyses clearly support with a high bootstrap value this taxon in an independent

clade (PLATE 1). Despite the similarity of T. lutescens (Malencon 1938) to

T. extremadurensis, the absence of any description of spines with crossed crests

(an undoubtedly visible character) by Lazaro Ibiza (1908) leads us to think that

it is actually a different species. However, we were unable to find the original

samples of Lazaro Ibiza to compare their ITS-rDNA sequences.

Terfezia eliocrocae Bordallo, Morte & Honrubia, sp. nov. PLATE 2C,D

MycoBank MB561592

Differs from other reticulated spored Terfezia spp by its friable and crumbly gleba that

remains milky white when exposed.

Tye: Spain, Murcia, Lorca, Zarzadilla de Totana, 21 April 2007, leg A. Morte (Holotype,

MUB Fung-0015).

Terfezia spp. nov. (Iberia) ... 197

Erymo oey: referring to Eliocroca, old Roman name for the city of Lorca (Murcia,

Spain), and its surrounding area, where the type specimen was collected.

ASCOMATA more or less crushed shapes, roughened, embossed, >5 cm.

Cream colour, blackish aspect when ripe with a rough, cracked texture.

Sticky mycelial remains and pseudo-stipe. PERIpIUM thin, well delimited,

inseparable from the gleba, cream to blackish-brown colour, partially rough

when ripe; pseudoparenchymatous, <200 um thick, cells round, 30 x 15 um;

external layer with hyphal extensions. GLEBA intensely white, remaining white

and not darkening when mature or in contact with air; both fertile and sterile

tissues milky white with small islets of fertile tissue surrounded by very thin

ramified sterile veins. Ascr 8-spored, subglobose, ovoid or pyriform elongated.

Ascosporss spherical with a well-developed reticulum, regular, 17-19 um

diam. including ornamentation; at first hyaline and smooth, in age becoming

yellow with a very marked (<1 um tall) reticulum.

HasBitatT — Growing in grasslands on calcareous soils with Helianthemum

almeriense and H. violaceum (Cistaceae).

ADDITIONAL COLLECTIONS EXAMINED: SPAIN: Murcia: Totana, 2001, A. Morte (MUB

Fung-0005); 2007, A. Morte (MUB Fung-0016).

CoMMENTS — Terfezia eliocrocae (Clade 2), distinguished by inner milky white

colour that persists in contact with air, differs from T: berberiodora (characterized

by hazelnut-shaped ascocarps, an atypical gleba in islets, and a characteristic

flavour of Berberis vulgaris) in its smaller spores and larger ascocarps (Castro

& Freire 1982). Terfezia hafizi also produces slightly reticulate but larger (18-20

um) ascospores (Chatin 1892). The markedly reticulate ascospores distinguish

T. eliocrocae from T. hafizi and T: alsheikhii (see below).

Terfezia alsheikhii Kovacs, M.P. Martin & Calonge, Mycologia 103: 848. 2011.

PLATE 2E,F

AscoMata regular, globose, 0.5-1.5 cm, smooth, firm; no strong odour

detected. PERrpDIuM smooth, thin, inseparable from the gleba; brown-

ochre; pseudoparenchymatous throughout, with thick-walled cells <50 um

in diameter. GLEBA formed by big pinkish islets of fertile tissue, varying

in size and surrounded by a scarce sterile white tissue, stained by brown-

reddish zones when in contact with air. Asci primarily 8-spored, subglobose,

ovoid, pyriform, 60-100 x 40-70 um, sessile or with a short thick peduncle.

Ascosporss spherical, reticulate, 15-18 um diam. (including ornamentation);

at first hyaline, yellow when mature; decorated by an irregular, well-developed

reticulum with thickish (nearly 1 um) net; very mature spores with roundish to

flat warts <2 um x 2 um.

Hasitat — Terfezia alsheikhii grows in sandy acidic soils associated with

cistaceous plants.

198 ... Bordallo & al.

COLLECTIONS EXAMINED: SPAIN: EXTREMADURA, BaADaAjoz: Castuera, 2009, M.

Romero (MUB Fung-0034). PORTUGAL: BRraGANza: Portelo, Montesinho Natural

Park, 2006, A. Rodriguez (MUB Fung-0009).

COMMENTS — Gregarious specimens have been found by raking under Cistus

monspeliensis L. on the border of a forest track.

Terfezia pini Bordallo, Ant. Rodr. & Mufioz-Mohedano, sp. nov. PLATE 2G,H

MycoBank MB561594

Differs from T: fanfani and T: pseudoleptoderma by its spores with long spines joined at

their bases to form a pseudo-reticulum.

Type: Spain, Burgos, Cornudilla, 8 March 2008, leg F. Sainz (Holotype, MUB Fung-

0027).

ETyMo_ocy: referring to pinewoods, its preferred habitat.

AScCOoMATA globose, round, regular, <2 cm diam., smooth, base often with

residual mycelium. PERIDIUM smooth, slightly tomentose, thin, not clearly

delimited and difficult to separate from the gleba, 200-400 um thick; at first

cream colour, becoming ochre, grey in cross-section grey, partially covered

by a whitish film; pseudoparenchymatous, cells thick-walled, <40 um diam.,

pigmented in the outer part. GLEBA initially whitish; when mature, the fertile

tissue forming round islets of various sizes, initially pale pinkish becoming

greenish brown and greyish when very mature and always surrounded by a

sterile tissue of white veins. Asc1 6-8-spored, ovoid, ellipsoid, subglobose,

sessile, 60-90 x 45-60 um, walls lum thick. Ascosporgs spherical with spines,

20-23(-25) um (including ornamentation); initially hyaline and smooth with

a big central drop, when mature, yellowish-ochre and decorated by cylindrical

spines (3-4(-5) x 1 um) with a round tip, joined basally to form crests, often in

a pseudo-reticulum.

EcoLocy— The species grows mainly in sandy pine forests from November

to May. It is rarely observed on the surface and is often found at a depth of 3-5

cm, usually under mosses.

ADDITIONAL COLLECTIONS EXAMINED: SPAIN: CASTILE & LEON, VALLADOLID:

Sieteiglesias, 2003, Aurelio Garcia (MUB Fung-0007); Ronquines, 1998, Aurelio Garcia

(MUB Fung-0010).

COMMENTS — Terfezia pini (represented by Clade 4) is diagnosed by its

occurrence on burnt areas under pine and oak and lack of association with

cistaceous plants and its spore ornamentation comprising long spines joined at

the bases to forma pseudo-reticulum. A high bootstrap value in the phylogenetic

analyses strongly supports its separation from other species (PLATE 1).

The spine crests of T. pini resemble the lamelliform folds of the non-reticulate

episporium of T: cadevalli (Sola 1925), which is distinguished in that it forms

neither a reticulum nor clearly identifiable spines). The larger ascospores of

Terfezia spp. nov. (Iberia) ... 199

PLaTE 2. Terfezia extremadurensis (holotype, MUB Fung-0026): A, ascocarps; B, ascospores.

T. eliocrocae (MUB Fung-0016): C, ascocarps; D, ascospores. T. alsheikhii (MUB Fung-0009):

E, ascocarps; F, ascospores. T. pini (holotype, MUB Fung-0027): G, ascocarps; H, ascospores. Bars

= 10 um.

200 ... Bordallo & al.

T. goffartii (25 um without ornamentation) differentiates that species from

T: pini. Despite the ascocarps of T: pini and T. leptoderma resemble each other,

their spore ornamentations differ: the Tulasne brothers described the spicules

of T. leptoderma as isolated (Tulasnes & Tulasne 1951), not crested nor forming

a sub-reticulum on the spore surface. Moreover, the exclusive preference for

burned oak or pine habitats has not been described for T: leptoderma.

Terfezia pseudoleptoderma Bordallo, Ant. Rodr. & Mufioz-Mohedano, sp. nov.

MycoBank MB561595 PLATE 3A,B

Differs from T. leptoderma by its spore spicules with symmetric bases.

TyPE: Spain, Burgos, Villafranca Montes de Oca, Puerto de la Pedraja, 12 May 2008, leg

E Sainz (Holotype, MUB Fung-0028).

EryMo_oey: referring to the morphological similarity with Terfezia leptoderma.

Ascomarta globose, round, quite regular, <2 cm diam., smooth when immature,

partially rough when mature; initially cream colour, maturing to reddish-

brown. PERtpium smooth or slightly rough, inseparable from the gleba; cream

colour, darkening where exposed to air. GLEBA initially whitish, with the fertile

tissue forming translucent greyish-blue islets surrounded by white veins or

sterile tissue. Asc1 8-spored; globose to ovoid. Ascospores spherical, 19-23

um diam. (including ornamentation), spines separate, blunt, 2-5 um long with

bases asymmetric, <1 um diam.; initially hyaline, yellowing in age.

HABITAT — associated with cistaceous plants near pine and oak forests.

ADDITIONAL COLLECTION EXAMINED: SPAIN: EXTREMADURA, CACERES: Valdecajfias,

2010, Mufioz-Mohedano (MUB Fung-0039).

ComMENTS — The ascocarps of T: pseudoleptoderma (Clade 5) are similar in size

to T: leptoderma, which differs in the symmetric bases of the spore spicules.

In general, we noticed considerable confusion among authors who identify

specimens as T: leptoderma that are much bigger than those described by

Tulasne brothers.

Terfezia fanfani Mattir., Malpighia 14: 71. 1900. PLATE 3C,D

Ascomara globose, round, regular, occasionally lobed, usually with mycelial

remnants, 2-5 cm diam., smooth or slightly rough; firm; usually odourless.

PERIDIUM smooth, slightly rough, inseparable from the gleba, thin, 200-700

um thick; initially white, soon becoming reddish brown, darkening in maturity

with black maculae present in sun-exposed zones exposed; white in cross-

section; pseudo-parenchymatous structure formed by rounded prismatic cells.

GLEBA initially white, then fertile tissue in islets becoming pale pink, then olive

green, finally blackish grey when very mature. The gleba is always surrounded

by white sterile tissue. Asci primarily 8-spored, sessile; subglobose, elongated

Terfezia spp. nov. (Iberia) ... 201

and ovoid, 70-80 x 55-70 um diam. with 1 um thick walls. AscospoREs

spherical with spines, 19-23(-25) um diam. (including ornaments), initially

hyaline, smooth, with a large central drop; in age ochre yellow and decorated

with sharp thin elongated conic spines (2-—)3-4(-5) um long with 1 um diam.

bases (the spines not joined through the bases).

HasitatT — acidic grasslands associated with Tuberaria guttata from the

end of March to the end of April.

COLLECTIONS EXAMINED: SPAIN: EXTREMADURA, CACERES: Belvis, 2009, Mufioz-

Mohedano (MUB Fung-0032); Garrovilla, 2004, A. Mateos (MUB Fung-0008);

Valdecafias, 2001, Mufioz-Mohedano (MUB Fung-0004); Bapayoz: Quintana de Serena,

2009, M. Romero (MUB Fung-0033); CASTILE & LEON, VALLADOLID: Cortas de Blas,

1998, Aurelio Garcia (MUB Fung-0011).

ComMENTS — Terfezia fanfani is recognized by its reddish colour, its growth

with Tuberaria guttata, and its spores decorated with long and isolated spines.

Initially hypogeous, it later it rises to the surface. Sharing both habitat and

fruiting season with T. arenaria, T. fanfani fruits before T. arenaria, usually

preferring saltier and less deep soils.

In size, T. fanfani seems better to match the Clade 6 specimens that we

examined as well as those cited by other authors (Calonge et al. 1977; Ceruti

1960; Janex-Favre et al. 1988; Moreno et al. 1986). Thus for both phylogenetic

and morphological reasons, we believe this species is independent.

Terfezia albida Ant. Rodr., Mufioz-Mohedano & Bordallo, sp. nov. PLATE 3E,F

MycoBank MB561596

Differs from all other spiny-spored Terfezia spp. in its larger ascomata, its white

peridium, and its spermatic odour.

TyPeE: Spain, Albacete, El Ballestero, 2 May 2009, leg A. Rodriguez (Holotype, MUB

Fung-0029).

ETyMo_ocy: referring to the external whitish colour.

Ascomarta hypogeous to partially emergent at maturity, 2-4 cm across, 3-4 cm

high, subglobose to turbinate, pulvinate, often with tapered, sterile base, white

at first, becoming light cream, often black spots on the sun-exposed parts or

where handled, greenish with age on injured areas, smooth. PERIDIUM 200-500

uum thick, poorly delimited, white in cross section, pseudoparenchymatous,

composed of subglobose cells of variable size, hyaline and thin-walled in the

innermost layers, yellowish and with thicker walls in the outermost layers.

GLEBA Solid, fleshy, succulent, white at first, maturing to grayish green pockets

of fertile tissue separated by whitish, sometimes with pink spots, sterile veins.

Spermatic odour, stronger in young specimens. Asc inamyloid, subglobose to

ovate, sessile or short-stipitate, 70-85(-90) x 55-70 um, walls 1 um thick, with

6-8 irregularly disposed spores, randomly arranged in the gleba. AscosPoREs

202 ... Bordallo & al.

globose, (18-)19-22(-23) um diam (median = 20 um) including ornament,

14-17(-18) um (median = 16 um) without ornament, hyaline, smooth and

uniguttulate at first, by maturity yellow ochre and ornamented with conical,

blunt, straight spines, sometimes cylindrical and curved, sometimes truncated,

separate, 2-3 um long, 1-2 um wide at the base, sometimes connected to form

a pseudo-reticulum.

ECOLOGY & DISTRIBUTION— southeastern Iberian Peninsula, limited to arid

and semiarid areas in calcareous alkaline soils, associated with Helianthemum

spp., from late April to mid May.

ADDITIONAL COLLECTIONS EXAMINED: SPAIN: CasTILE-LA MANCHA, ALBACETE:

Albacete, 2009, A. Rodriguez (MUB Fung-0037).

CoMMENTS— Terfezia albida (Clade 7) differs from other spiny-spored Terfezia

species in its larger average size, white peridial colour, and spermatic odour. It

is the only spiny-spored Terfezia species associated with Helianthemum spp.

in alkaline soils. Terfezia eliocrocae (described above) and T! claveryi share the

same habitat but differ in their reticulate spores.

Clade 7 includes taxa with different morphologies, distributions, and host

plants from its sister clade, Clade 8 (where T: olbiensis is found). The two clades

are separated by a 72% bootstrap value (PLATE 1). Only after wide sampling we

were able to place T: albida in Clade 7 (associated with cistaceous plants) and

T. olbiensis in Clade 8 (associated with pine and oak hosts).

Terfezia olbiensis Tul. & C. Tul., G. Bot. Ital.1(2(7-8)): 60. 1845. PLATE 3G,H

AscomatTa globose, round, regular, rarely presents pseudostipe, 2-5 cm

across, smooth. PERrpIuM smooth, inseparable from the gleba, thin, 300-500

um thick, not clearly delimited; initially cream, becoming brown, frequently

with black maculae where exposed to the sun or bruised, white in cross-

section; pseudoparenchymatous structure formed by + rounded thin-walled

hyaline cells of different sizes that yellow and become prismatic towards the

periphery. GLEBA initially white, then fertile tissue forming small grey (later

greenish grey) islets surrounded by salmon-tinged white sterile tissue. Asc1

sessile to occasional on a short thick peduncle, 8-spored, ellipsoidal to ovoid,

citrus-shaped, 60-90 x 50-60 um with 1-2 um thick walls; dextrinoid when

immature. Ascosporgs spherical and spiny, 15-19 um (including ornament)

diam., initially hyaline, smooth, and with a great central drop, when mature

ochre yellow and covered by pointy thin conical 1-2 (-2.5) um long (base = 1

um) spines (some truncated) that are sparse and not joined through the base.

Hasitat — Terfezia olbiensis grows in limestone and clayey pine and oak

woodlands without Helianthemum spp. from mid-March to mid-April. It is the

first Terfezia species to produce ascocarps in limestone soils.

Terfezia spp. nov. (Iberia) ... 203

PLaTE 3. Terfezia pseudoleptoderma (holotype, MUB Fung-0039): A, ascocarps; B, ascospores.

T. fanfani (MUB Fung-0032): C, ascocarps; D, ascospores. T. albida (holotype, MUB Fung-0029):

E, ascocarps; EF, ascospores. T. olbiensis (MUB Fung-0031): G, ascocarps H, ascospores. Bars =

10 um.

204 ... Bordallo & al.

COLLECTIONS EXAMINED: SPAIN: CASTILE-LA MANCHA, ALBACETE: Masegoso, 2009,

A. Rodriguez (MUB Fung-0031); CASTILE & LEON, VALLADOLID: Santa Espina, 1998,

Aurelio Garcia (MUB Fung-0013); VALENCIA: Onteniente, 2009, E Garcia (MUB Fung-

0030).

COMMENTS — Initially hypogeous, T: olbiensis later rises to the surface, where

it remains firm. The fruitbodies usually are parasitized underground by larvae

or eaten by rabbits, probably because they appear early in the year, when there

is greater humidity and less sunlight. The odor is distinctive (somewhat rotten);

the flavour is less than other edible Terfezia species. The most distinguished

characteristic is the reduced length of its spines, always shorter than 2.5 um.

Although similar to T: leptoderma, T. olbiensis has a bigger ascocarp

(hazelnut- to walnut-sized) and smaller ascospores. Clade 8 taxa have different

morphological and distribution characteristics from the other clades, which is

also supported by the phylogenetic analyses with a 72% bootstrap value (PLATE

1). Therefore, we conclude that Clade 8 is formed by T: olbiensis.

Discussion

All the species described herein produce similar sized spiny to reticulate

ascospores, which has led to the belief that they represent only one species. Our

exhaustive comparison of the ascocarps and ascospores and comprehensive

ITS-rDNA sequence analyses support recognition of several species.

Our results agree with those of Diez et al. (2002), who recognized three clades

separated by a high statistical support (89%, 98%) based on morphological

(gleba structure, size of ascospore spicules, ascospore size) and biological

(acidic vs. alkaline soils, cistaceous vs. pine-oak hosts) characters. We propose

Clade 4 for Terfezia pini based on data from Barriuso et al. (2008; see GenBank

DQ386140). Moreover, a new sequence deposited by Rincén (unpublished data:

GenBank FJ013064) belongs to Clade 5. In their later phylogenetic analyses of

Terfezia spp., Ferdman et al. (2005) clearly separate DNA sequences AF276679,

AF276678, AF387657, and AF387648 into two strongly supported different

clades (100/99 bootstrap value) as T: leptoderma and T: olbiensis (Clade 8). This

scenario is contrary to Malencon’s opinion (see Diez et al. 2002, Moreno et al.

1986) that T: olbiensis represents an immature form of T: leptoderma.

The group covering species diagnosed by a spiny episporium (Clades 1,

4, 5, 6, 7 and 8) is paraphyletic, suggesting that episporium ornamentation

is not a homologous character. Therefore, we do not consider episporium

ornamentation a good taxonomic character, in contrast to other authors such

as Mattirolo (1900).

Moreover, although AF396864 was classified as T: leptoderma (Diez et al.

2002), our analyses place it in a different clade by a relatively high bootstrap

value (73/77). More sequences are needed to confirm its identity.

Terfezia spp. nov. (Iberia) ... 205

Our phylogenetic analyses corroborate the conclusion by Diez et al. (2002)

that fungus-host specialization and soil tolerance are the keys that allow us

to separate different species (clades). The monophyly of the proposed clades

(species) (PLATE 1) is congruent with the morphological (PLATEs 1 and 2) and

biological (TABLE 1) data reported for the studied taxa.

Distributed throughout temperate and subtropical regions of the northern

hemisphere, Cistaceae show the highest genus and species diversity in the

Mediterranean floristic region (Guzman & Vargas 2009). The Iberian Peninsula

hosts a high diversity of Cistaceae and high number of mycorrhizal fungal

species. Recent studies suggest that Helianthemum almeriense strongly depends

on the presence of a fungal symbiont in its roots for survival (Morte et al. 2010).

The Iberian Peninsula seems to be a desert truffle reservoir and further study

and sampling are necessary in these ecosystems.

As recently highlighted by Bidartondo et al. (2008), we also emphasize

the importance of an accurate identification of specimens whose DNA

sequences have been subsequently registered in public databases, such as

GenBank. Mistakes made in species identification can lead to erroneous and

false conclusions. Checking the databases with contrasted data is necessary to

prevent the perpetuation of errors when identifying new specimens.

Key to examined Terfezia species

Ascospore measurements include ornamentation.

la. Fertile tissue does not create islets or reticulate ascospores ...........-----2 000 2

1b. Fertile tissue creates islets and spiny ascospores ............0. 0s eee e eee eee ees 3

Za TASCOSPORes: WILLS PINGS 55.5.0 S ca yensqee teens tweens T. extremadurensis (Clade 1)

2D IASCOSPOTESPELGUIAIE it Date tReet D tien Rit Ee ete eee ene ee Ure Fe Rae tit tetsA ae +

3a. Ascospores <20 um and spines <2.5um ..............0006- T. olbiensis (Clade 8)

SUPASCOSP ORES 220 (i yas. wt tor ne nea ek cee Re rane A a MD, RE Rena on, Sac we 5

Aare Vite le Dat Rat Aueet ARG ARO ARE Ne Lae T. eliocrocae (Clade 2)

ABS PHU Olea, | we. ttan h oeatin chain Gaeta ttaa a estan ares atten andeabtin anseablagh are T. alsheikhii (Clade 3)

Dai oo PUES Fs PTI. era lel Neceath Meerastey eamaces teens ste iene te Mera ee ttens cea T. albida (Clade 7)

SU, Spinesero Wille, ft, dh ie ek ole wk old hee ee a Eee Suet) Cte Eos 6

GarCrestedispiiies™ wo. eSuis oe oe ee ee 28 ees 2 ied oh eis Sie T. pini (Clade 4)

OE: SGI ated is pie Set ee hate SR chat late UE hai OR Reto OM et UR Ate le OM en UL, | eel a 7

7a. Spines blunt with asymmetric base; ascomata <2 cm diam

Su Ree sles te dlad x lag Ho Mig sete $e 324 T. pseudoleptoderma (Clade 5)

7b. Spines pointed with symmetric base; ascomata 2-5 cm diam .. T. fanfani (Clade 6)

Acknowledgments

This work has been supported by project CGL2011-29816 (MINECO-FEDER, Spain).

The authors are grateful to Prof. Gabriel Moreno (Univ. Alcala de Henares, Madrid) and

206 ... Bordallo & al.

Prof. Giuseppe Venturella (Universita di Palermo, Italy) for their presubmission reviews

and Dr. Shaun Pennycook and Dr. Lorelei L Norvell for their huge effort and help in the

text edition. They also thank Mr. A. Bordallo for his help with the English revision of

the manuscript.

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MYCOTAXON

http://dx.doi.org/10.5248/124.209

Volume 124, pp. 209-217 April-June 2013

Hypochnicium pini,

a new corticioid basidiomycete in East Asia

YEONGSEON JANG’, SUNG WOOK LEE’, YOUNG WOON LIM’,

JIN SUNG LEE’, NILS HALLENBERG*‘ & JAE-JIN KIM’*

‘Division of Environmental Science & Ecological Engineering,

College of Life Sciences & Biotechnology, Korea University

5-1 Anam-dong, 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

‘Department of Biological and Environmental Sciences,

Box 461, S-405 30 Goteborg, Sweden

*CORRESPONDENCE TO: jae-jinkim@korea.ac.kr

ABSTRACT — A new species, Hypochnicium pini, is described based on morphological

and molecular characters. This species was collected in Seoul, Korea, and Hubei, China. It

belongs to the H. punctulatum group and has a morphology quite similar to its sister taxon,

H. cremicolor. However, the basidiome thickness, cystidial size, and ITS region sequences

differentiate H. pini from H. cremicolor. Detailed description and illustration of H. pini are

presented and a key to Korean species of Hypochnicium is provided.

Key worps — phylogeny, Polyporales, taxonomy

Introduction

Hypochnicium J. Erikss. is a basidiomycete genus of wood-rotting fungi. It is

characterized by resupinate effused adnate basidiomes, a margin that is usually

not especially differentiated, monomitic hyphal system, hyaline thin- to thick-

walled hyphae, cystidia present or absent, more or less suburniform 4-spored

basidia normally with a basal clamp, and ellipsoid to subglobose smooth or

ornamented basidiospores with thickened cyanophilous walls (Eriksson &

Ryvarden 1976). More than 30 species have been described worldwide, and

a core group around the type, H. bombycinum (Sommerf.) J. Erikss., has been

shown to be monophyletic when Hyphoderma species are used as outgroup

(Paulus et al. 2007; Telleria et al. 2010).

210 ... Jang & al.

Traditionally, northern European Hypochnicium species were subdivided

into six morphological groups (Eriksson & Ryvarden 1976), and within

each group, species delimitation was troublesome because of rather similar

morphological features. Recently, molecular phylogenetic analysis has been

used to delimit species in Hypochnicium. Nilsson & Hallenberg (2003) first

used ITS region sequences to differentiate species within the H. punctulatum

group [H. albostramineum (Bres.) Hallenb., H. cremicolor (Bres.) H. Nilsson &

Hallenb., H. punctulatum (Cooke) J. Erikss., H. wakefieldiae (Bres.) J. Erikss.]

and clearly distinguished the species which complemented morphology and

crossing test results. Several additional species belonging to this complex

have been described: H. cystidiatum Boidin & Gilles from Africa (Boidin &

Lanquetin 1971), H. aotearoae B.C. Paulus et al. from New Zealand (Paulus

et al. 2007), H. guineense Telleria et al. from Equatorial Guinea (Telleria et

al. 2010), and Hypochnicium patagonicum Gorjon & Hallenb. from Chilean

Patagonia (Gorjon & Hallenberg 2012).

Six Hypochnicium species have been reported from Korea: H. bombycinum,

H. detriticum (Bourdot) J. Erikss. & Ryvarden, H. eichleri (Bres. ex Sacc. & P.

Syd.) J. Erikss. & Ryvarden, H. lundellii (Bourdot) J. Erikss., H. punctulatum,

and H. vellereum (Ellis & Cragin) Parmasto (Jung 1991, 1994, 1996; Lim et

al. 2010). Hypochnicium eichleri and H. punctulatum were reported by Jung

(1991, 1996), who identified the species referring to CORTICIACEAE OF NORTH

Europe (Eriksson & Ryvarden 1976). However, Nilsson & Hallenberg (2003)

proved that “H. punctulatum” as described in Eriksson & Ryvarden (1976) was

actually H. cremicolor (‘The type of H. punctulatum has bigger basidiospores

than H. cremicolor). Moreover, H. eichleri has been synonymised with authentic

H. punctulatum. Therefore the Korean report of “H. punctulatum” should be

corrected to H. cremicolor; and the Korean record of H. eichleri should be

updated to H. punctulatum.

Recently, the national biological inventory project (governed by the

National Institute of Biological Resources) is underway in Korea. During the

investigation of indigenous fungi, we found a Hypochnicium specimen sharing

features with H. cremicolor but with smaller cystidia. Our further investigation

revealed two specimens from China with the same deviating characteristics.

Based on morphological and molecular data, we describe these three specimens

as a new species.

Materials & methods

Collection and morphological examination

The corticioid basidiome was collected from Gungsan (Mt.), Seoul, Korea and

air-dried. The dried specimen was used for macro- and microscopic examination.

The specimen was mounted in 1% cotton blue, 5% KOH, 1% phloxine, and Melzer’s

Hypochnicium pini, sp. nov. (Asia) ... 211

reagent (IKI) (Largent et al. 1977) and observed at up to 1000x using Olympus BX51

light microscope. Munsell color (2009) was followed as the color standard. More than

30 basidiospores were examined. The specimen was deposited at National Institute of

Biological Resources, Korea (KB). The Chinese specimens were borrowed from National

Museum of Natural Science, Taiwan (TNM) and examined as above.

Phylogenetic analysis

For the Korean specimen, genomic DNA extraction, ITS region PCR, and

sequencing were carried out according to Jang et al. (2012). For the Chinese specimens,

the revised method of Cenis (1992) was used for genomic DNA extraction. PCR and

sequencing were performed according to Jang et al. (2012). The sequences obtained

in this study were deposited under the GenBank accession no. JX217823, KC282470-

282472. The sequences were proofread and compared with the reference sequences

in GenBank. Closely related Hypochnicium sequences were retrieved from GenBank.

Only one sequence was selected when two or more sequences had the same nucleotides;

H. geogenium (Bres.) J. Erikss. (GenBank AF429426) was included as outgroup based on

the phylogenetic tree in Telleria et al. (2010). The sequences were aligned using MAFFT

6.885 (Katoh & Toh 2008) with L-INS-i method. The aligned dataset was viewed and

manually edited using MacClade 4.08 (Maddison & Maddison 2005). Phylogenetic

tree was constructed using MrBayes 3.2 (Ronquist et al. 2012). Two independent runs

with 8 chains were operated with 1,000,000 generations and every 100th tree was saved.

In this process, the best-fit model was searched by MrModeltest 2.3 (Nylander 2004)

under AIC criterion and GTR + I model was applied. The graphical presentation of log

likelihood value was checked and the first 25% of the trees were discarded. The 50%

majority rule consensus tree was produced with the rest 75% of the trees. The potential

scale reduction factors (PSRF) were reasonably close to 1.0 for all parameters. Posterior

probabilities were calculated to support nodes. The tree was viewed using FigTree 1.3.1

(http://tree.bio.ed.ac.uk/software/figtree/).

Taxonomy

Hypochnicium pini Y. Jang & J,J. Kim, sp. nov. FIGs 1-3

MycoBank MB 800807

Differs from Hypochnicium cremicolor by its thinner basidiome and its smaller cystidia.

Type: Korea. Seoul, Gangseo-gu, Gungsan (Mt.), 37°34'25"N 127°50'25"E, on bark

of Pinus densiflora Siebold & Zucc., 23 Sep 2008, Yeongseon Jang KUC20081023-04

(Holotype, KB NIBRFG0000107453; GenBank JX217823).

ETryMOoLoGcy: Growing on pines (Pinus).

Basidiome resupinate, effused, membranaceous, pale yellow (2.5Y8/2,2.5Y8.5/2)

when dry, up to 0.1 mm thick; hymenophore smooth to porulose (hypochnoid);

margin not differentiated. Hyphal system monomitic; hyphae with clamps,

ramified, hyaline, 3.8-5.8 um wide; subhymenial hyphae thin-walled while

the basal ones are thick-walled, walls up to 1.5 um thick, loosely interwoven.

Cystidia subcylindrical, slightly tapering but with an obtuse apex, enclosed

212 ... Jang & al.

Fic. 1. Hypochnicium pini (NIBRFG0000107453, holotype).

Basidiocarp. Scale bar = 1 cm.

within the hymenium or projecting up to 40 um above the basidial layer, thin-

walled, variable in size, 40-80 x 5.5-8.5 um. Basidia clavate, sinuous, 28-39

x 5.5-7.5 um, with four sterigmata and a basal clamp. Basidiospores broadly

ellipsoid to subglobose, 5.5-6.9 x 4.6-5.9 um, mean 6.1 x 5.2 um, verrucose in

1% cotton blue and 1% phloxine, smooth in 5% KOH, thick-walled, some with

a few guttula, spore wall cyanophilous, IKI-.

ECOLOGY & DISTRIBUTION — China and Korea, on Pinus spp.

Additional specimens examined:

Hypochnicium pini: CHINA, HuBeE1, Wuhan, Mt. Lion, alt. 30 m, 30°29'N 114°21'E,

on trunk of Pinus sp., 12 November 2008, S.H. Wu 0811-75 (TNM F0023763; GenBank

KC282471); Wu 0811-77 (TNM F0023765; GenBank KC282472).

Hypochnicium cremicolor. CHINA, YUNNAN, Tali, Tsangshan, alt. 2800 m, on

trunk of Pinus armandii Franch., 19 October 2000, S.H. Wu & S.C. Wu 0010-180 (TNM

F0012210; GenBank KC282470).

REMARKS — Hypochnicium pini is very similar to H. cremicolor. However,

H. cremicolor has a thicker basidiome (0.1-0.3 um) and larger cystidia (70-150

x 7-10 um; Eriksson & Ryvarden 1976, as “H. punctulatum”). Phylogenetic

analysis also revealed the close relationship between the two species, but there

was sufficient genetic differentiation to separate them (Fie. 3).

Hypochnicium pini, sp. nov. (Asia) ... 213

Fic. 2. Hypochnicium pini (NIBRFG0000107453, holotype).

A, basidia; B, basidiospores; C, cystidia; D, thick-walled basal hyphae.

Phylogeny

The amplified ITS region sequences of Hypochnicium pini NIBRFG0000107453

from Korea, and F0023763 and F0023765 from China were 578 bp. There were

two nucleotide differences between the Korean specimen and the Chinese

specimens of H. pini and the sequences of Chinese specimens were identical.

214 ... Jang & al.

Hypochnicium pini F0023765 (KC282472)

4 Hypochnicium pini F0023763 (KC282471)

Hypochnicium pini NIBRFG0000107453 (JX217823)

4sHypochnicium cremicolor FCUG 160 (AF429425)

Hypochnicium cremicolor FCUG 2151 (AF 429424)

Hypochnicium cremicolor F0012210 (KC282470) II-E

Rael al aotearoae FCUG 3120 (GQ906536)

Hypochnicium aotearoae FCUG 2972 (DQ309071)

Hypochnicium guineensis MA-Fungi 79156 (FN552536)

Hypochnicium cystidiatum FCUG 3086 (DQ658163)

Hypochnicium cystidiatum FCUG 3087 (DQ658164)

Hypochnicium punctulatum FCUG 1065 (AF429413)

4 |Hypochnicium punctulatum FCUG 1921 (AF 429410)

Hypochnicium punctulatum FCUG 1794 (AF 429414)

Hypochnicium punctulatum FCUG 1362 (AF429411)

Hypochnicium wakefieldiae MA-Fungi 79157 (FN552532)

9.991 Hypochnicium wakefieldiae MA-Fungi 23913 (FN552533)

Hypochnicium wakefieldiae MA-Fungi 7675 (FN552531)

$38 Hypochnicium wakefieldiae FCUG 2437 (AF429420)

Hypochnicium wakefieldiae FCUG 2755 (AF429418) II-F

Hypochnicium wakefieldiae FCUG 1709 (AF429419)

espe albostramineum FCUG 269 (AF429422)

0.98

lI-D

Hypochnicium albostramineum FCUG 1865 (AF429421)

Hypochnicium albostramineum FCUG 1772 (AF429423)

Hypochnicium geogenium FCUG 2052 (AF429426)

0.1

Fic. 3. 50% majority rule consensus tree of the Hypochnicium punctulatum group inferred by

ITS region sequences using bayesian analysis. The tree was rooted by H. geogenium FCUG 2052.

Numbers above branches indicate posterior probabilities. H. pini is indicated by bold type.

The clades presented in the tree follow those resolved by Telleria et al. (2010).

By the nucleotide BLAST, the closest match was H. cremicolor FCUG160 from

Denmark AF429425 (93%) and H. cremicolor FCUG2151 from Spain AF429424

(93%) (555 out of 596 positions are identical for Korean specimens and 557

out of 596 positions for Chinese specimens). In order to fully understand the

relationship within the H. punctulatum group, H. pini sequences were aligned

with 22 closely related Hypochnicium sequences obtained from GenBank

including H. cremicolor Wu 0010-180 from China, and with H. geogenium

FCUG2052 as outgroup. The aligned ITS dataset of 25 taxa comprised 586

characters including gaps. The 50% majority rule consensus tree using bayesian

analysis is shown in Fie. 3.

Each species was well resolved in the tree as indicated by high posterior

probability values. Three main groups were recovered which correspond to the

subclades II-D, II-E, and II-F in Telleria et al. (2010). They all have ornamented

basidiospores, but there are just minute morphological characteristics which

distinguish each subclade. Hypochnicium pini was included in subclade

I]-E with four formerly described species - H. aotearoae, H. cremicolor,

H. guineense, and H. cystidiatum. Hypochnicium pini NIBRFG0000107453 from

Hypochnicium pini, sp. nov. (Asia) ... 215

Korea was monophyletic with H. pini F0023763 and F0023765 from China

with high posterior probability (1.0 p.p.); and H. cremicolor Wu 0010-180 from

China was monophyletic with formerly sequenced H. cremicolor FCUG160 and

FCUG 2151, also with high support (1.0 p.p.), and it was 98% similar to them in

BLAST search (588 out of 596 positions are identical).

Discussion

Morphologically, species within II-E clade have similar characteristics.

Hypochnicium pini is characterized by its pale yellow basidiocarp, hypochnoid

hymenophore, thick-walled basal hyphae, one type of cystidium and broadly

ellipsoid to subglobose spores, which are very similar to H. cremicolor (Fics 1,

2). They both have the basal thick-walled hyphae, one type of cystidium, and

ornamented basidiospores which are similar in size [H. pini: 5.5-6.9 x 4.6-5.9

um; and H. cremicolor: (5-)5.5-6.5 (-7.5) x 4.5-5 um (Eriksson & Ryvarden

1976) or 6-6.5 x 5-5.5 um (Nilsson & Hallenberg 2003)]. The hosts of H. pini

are Pinus spp. while H. cremicolor has been reported from both broadleaved and

coniferous trees including Pinus spp. (Eriksson & Ryvarden 1976, Bernicchia

& Gorjén 2010). The obvious morphological differences are the thickness of

basidiome and the size of cystidia (TaBLE 1). Hypochnicium aotearoae and

H. cystidiatum differ from H. pini by having two types of cystidia, and H.

guineense differs by larger basidiospores (Telleria et al. 2010).

TABLE 1. Morphological characteristics of Korean species of Hypochnicium s.1.

SPECIES CYsTIDIA (um) BASIDIA (um) BASIDIOSPORES (Lm)

H. bombycinum (Jung 1994) None 40-50 x 6-8 Smooth, 8-12 x 6-8

H. cremicolor (Jung 1996, as 60-130 x 9-12 25-30 x 6.5-7.5 Ornamented, (5—)6-6.5 x 4.5-5.5

“H. punctulatum”)

H. detriticum (Jung 1994) 60-100 x 6-10 15-20 x5 Smooth, 4.5-6 x 3.5-4

H. lundellii Jung 1994) None 25-30 x 5-6 Smooth, 5.5-6 x 4.5-5

H. pini (this paper) 40-80 x 5.5-8.5 28-39 x 5.5-7.5 | Ornamented, 5.6-6.9 x 4.8-5.9

H. punctulatum (Jung 1991, 90-110 x 6-10 25-35 x 7.5-9 Ornamented, 8-9(-10) x 6-7.5

as “H. eichleri”)

H. vellereum (Lim et al. 2010) None 42-65 x 4-7.42 Ornamented, 6.8-7.4 x 5.7-6.2

In conclusion, we propose that the unknown Hypochnicium species collected

from Gungsan (Mt.) in Korea and from Hubei in China represents a new

species, Hypochnicium pini, minutely distinguished by morphological data, but

with distinct genetic differences towards other species in this complex. We also

update the list of Korean species of Hypochnicium sensu lato (TABLE 1).

216 ... Jang & al.

Key to the Korean species of Hypochnicium s.I.

1 PSV OUE OY SUCIA 4325 aioe cl fata whee ele Saee ole Boe ule See ile Out edie baie s Gedy ahs 2

Ie WILE VS LILA ed ie Ee cis cy a Ere Me Bh a ON aN Soi aha 4

PAS OLES SUNOOL He Aats Pere Ra ata couig Alain Baia hhh hit AM he 2 OM het Pade te Ree. ek 3

2. Spores globose, with irregularly uneven or finely rough surface........ H. vellereum

3. Spores broadly ellipsoid to ovoid, 8-12 x 6-8 um.............000. H. bombycinum

3.. Spores ellipsoid; 5.5=6- 4:5=5: With. see ye ssee yes teye eniege # end Bonet Eee ge H. lundellii

He SPORES: GHIOOUN 2, See 255 le os solave Ws colaps On Salers les 4 Sere el Marty Sle AS H. detriticum

A SPORES OLMAIMeTiLed bat) ied iS ed hed i AU AU AU ALE ALS oe ier oe 5

Ds ppores longer: thyary, 8, iia. Spay 6 eaps Lh aeape Pape Piaget Page H. punctulatum

SES POLES SHOr be Tel MAM Ba Lis 25! Mecwats Mecwantel Mecwn <r etewe (Me secu ee nate ee eae (Ste nt Saath aes 2 6

6. Basidiome 0.1-0.3 mm thick, cystidia 60-130 x 9-12 um............ H. cremicolor

6. Basidiome <0.1 mm thick, cystidia 40-80 x 5.5-8.5 um................0004 H. pini

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 grateful to Dr. Sheng-Hua Wu (National Museum of Natural Science,

Taiwan) for providing Chinese specimens. We are grateful to Dr. Sergio Pérez Gorjon

and Dr. Leif Ryvarden for their valuable suggestions on the manuscript.

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http://dx.doi.org/10.3852/09-242

MY COTAXON

http://dx.doi.org/10.5248/124.219

Volume 124, pp. 219-229 April-June 2013

Taxonomic studies on Mucor inaequisporus,

isolated for the first time in South America

ANDRE Luiz C.M. DE A. SANTIAGO’, ANDRE RODRIGUES?,

Enzo M. CANEDO? & EDSON R. FILHO?

‘Universidade Federal Rural de Pernambuco - Unidade Académica de Serra Talhada,

Fazenda Saco, s/n, 56900-000, Serra Talhada, Pernambuco, Brazil

?Universidade Estadual Paulista - UNESP, Departamento de Bioquimica e Microbiologia,

Av. 24A, 1515, Bela Vista, 13506-900, Rio Claro, Sao Paulo, Brazil

>Universidade Federal de Sao Carlos, Departamento de Quimica,

Rodovia Washington Luiz, km 235, 13565-905, Sado Carlos, Sdo Paulo, Brazil

* CORRESPONDENCE TO: andrelcabral@msn.com

ABsTRACT— Mucor inaequisporus (Mucorales, Mucoromycotina) was isolated from Syzygium

cumini, a common introduced tree species in Brazil. It is distinguished from similar species

by producing straight or undulate sporangiophores with randomly distributed irregular

swellings. The columellae are mostly pyriform at 25 °C, but oblong, conic, ellipsoid, and

obovoid columellae were also found. The sporangiospores vary in size and shape, with a

minor proportion exhibiting an irregular shape. ITS-based phylogenetic analyses reveal that

M. inaequisporus is closely related to M. amphibiorum and M. nederlandicus. This species can

grow between 20 and 30 °C with optimal development at 25 °C, but variations in the shape

of sporangiospores and columellae caused by incubation under different temperatures were

observed. No growth was detected at 10 and 40 °C.

Key worps— DNA sequencing, growth rate, zygomycete

Introduction

Mucor Fresen., which belongs to the subphylum Mucoromycotina (Hibbett et

al. 2007), is characterized by the formation of non-apophysate sporangia (with

wet or dry walls when mature) on top of simple or branched sporangiophores

that emerge from the substrate (Benny 2006). Taxa of this genus have been

isolated from soil, dung, decaying fruits, and plants (White et al. 2006, Santiago

& Souza-Motta 2008, Santiago et al. 2011), while others are considered the

causal agent of cutaneous zygomycosis in humans (Alvarez et al. 2011). Several

species are able to produce enzymes with biotechnological applications (Alves

et al. 2002a), and some are used to prepare fermented food (Hesseltine 1983,

Abe et al. 2004).

220 ... Santiago & al.

Previous studies have treated Mucor as polyphyletic and highlighted the

difficulty of establishing subgeneric groups (O’Donnell et al. 2001, Voigt &

Wostemeyer 2001, Jacobs & Botha 2008). Although over 300 species are cited

in the literature (Jacobs & Botha 2008), the actual number of species may range

only from 50 to 75 (Gherbawy et al. 2010). After revising several Mucor species,

Schipper (1978) reduced the proposed taxa to 39 species, four varieties, and

11 forms. Subsequently, another 17 species have been described as new taxa

(Mehrotra & Mehrotra 1979; Mirza et al. 1979, Subrahamanyam 1983, Chen

& Zheng 1986, Schipper 1989, Schipper & Samson 1994, Watanabe 1994, Pei

2000, Alves et al. 2002b, Jacobs & Botha 2008, Alvarez et al. 2011, Madden et

al. 2012).

Mucor inaequisporus was first described by Dade (1937) from Spondias

mombin L. fruits in Aburi, Ghana. Since then, a few records of this species

from several fruits have been reported. Morphologically, M. inaequisporus is

mainly characterized by the highly variable form and size of columellae and

sporangiospores (Schipper 1978).

The present study describes Mucor inaequisporus isolated from a fruit of

Syzygium cumini (L.) Skeels (= Eugenia jambolana Lam.; Myrtaceae), commonly

found in the State of Sao Paulo and known locally as ‘Jambolao’ or ‘Jamelao: We

discuss the influence of temperature, light, and culture media on growth and on

morphology of M. inaequisporus.

Materials and methods

Isolation and morphological identification of M. inaequisporus

Mature fruits of S. cumini were collected on the campus of the Federal University of

Sao Carlos (21°59'02"S 47°52'55"W), Sao Paulo, Brazil. In February 2009 (URM 6532)

and February 2010 (URM 6533), mycelium fragments were removed directly from

growing fruits under a stereomicroscope (Leica EZ4) and transferred to Petri dishes with

potato dextrose agar (PDA) (Benny 2008) supplemented with chloramphenicol (Neo

Fenicol - Neo Quimica) (80 mg L’"). Plates were left on a bench at room temperature

(28 + 2 °C) for 7 days in alternating light and dark periods. The strains were subcultured

in order to obtain pure cultures. Identification was based on macroscopic (color, aspect

and diameter of the colonies) and microscopic characters according to Dade (1937) and

Schipper (1978).

Influence of temperature, culture media, and light on the growth and

morphology of M. inaequisporus

Pure cultures were grown in malt extract agar (MEA) and PDA (Benny 2008) at 15,

20, 25, 30, 35, and 40 °C for 7 days under light and dark periods. Mycelial fragments

were removed from cultures to prepare wet mounts using KOH (3%) and studied

under bright-field microscopy (BFM) (Carl Zeiss Axioscope 40). Color designation

followed Watling (1969). The morphological characteristics were also studied using

Environmental Scanning Electron Microscopy (ESEM). Agar blocks retrieved from

Mucor inaequisporus in South America ... 221

TABLE 1. Cokeromyces and Mucor species included in the molecular analysis

SPECIES ISOLATE GENBANK No.

Cokeromyces recurvatus Poitras CBS 158.507 DQ118986

M. amphibiorum Schipper CBS 763.74" FJ455861

M. bainieri B.S. Mehrotra & Baijal CBS 293.63 JF299222

M. circinelloides Tiegh. CBS 195.68%7 DQ118991

(as M. circinelloides f. circinelloides)

M. ellipsoideus Ed. Alvarez et al. UTHSC 02-2090" FN650647

M. flavus Bainier CBS 230.35 EU484282

M. fragilis Bainier CBS 236.35 FN650655

M. fuscus Bainier CBS 132.227 FN650653

M. genevensis Lendn. CBS 114.087 EU484275

M. guilliermondii Nadson & Filippov CBS 174.277 JF299231

M. hiemalis f. corticola (Hagem) Schipper CBS 106.09 AY243950

M. hiemalis Wehmer f. hiemalis CBS 201.65 DQ118992

M. inaequisporus CBS 255.367 JN206177

CBS 351.50 JN206178

URM 6532 JQ014007

M. indicus Lendn. CBS 226.29" DQ118994

M. irregularis Stchigel et al. CBS 103.937 DQ119006

M. lusitanicus Bruderl. CBS 108.177 FN650644

(as M. circinelloides f. lusitanicus)

M. mucedo Fresen. CBS 109.16 EU484199

M. nederlandicus Vanova CBS 735.70 JF299219

M. nidicola Madden et al. CBS 638.67 JF299220

(as M. hiemalis)

M. piriformis A. Fisch. CBS 169.25 EU484276

M. plumbeus Bonord. CBS 111.07 AF412290

M. racemosus Fresen. CBS 260.68" DQ118996

M. ramosissimus Samouts. CBS 135.65‘7 FN650643

M. variosporus Schipper CBS 837.70' HM623322

M. velutinosus Ed. Alvarez et al. UTHSC 06-1667 FN650652

M. zonatus Milko CBS 148.697 JF299232

CBS = Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; UTHSC = Fungus Testing

Laboratory, University of Texas Health Science Center, San Antonio, Texas, USA; URM = University

Recife Mycology, Recife, Brazil

NT = neotype strain; ’ = type strain

cultures were mounted in microscope slides for direct observation with a scanning

electron microscope (FEI Quanta™ 250).

Phylogenetic analyses

DNA was extracted following Gerardo et al. (2004). Cultures of M. inaequisporus

URM 6532 were cultured in malt broth (2%) at 25 °C for seven days and the mycelium

was freeze-dried and disrupted using liquid nitrogen. Genomic DNA was recovered

using CTAB and purified with 70% ethanol. PCR primer pair ITS4 and ITS5 was used to

amplify the ITS1-5.8S rDNA-ITS2 region (White et al. 1990). Both forward and reverse

sequences were generated in ABI3500 (Applied Biosystems). Sequences were assembled

in Bioedit v. 7.0.5.3 (Hall 1999) and the consensus was aligned with sequences of several

Mucor species retrieved from GenBank using Muscle v. 3.6 (Edgar 2004) (TABLE 1).

A neighbor-joining tree was inferred in MEGA5 (Tamura et al. 2011) under the

222 ... Santiago & al.

a Mucor variosporus

Mucor indicus

100 Re

Mucor ellipsoideus

Mucor nederlandicus

Mucor amphibiorum

Mucor inaequisporus CBS 255.36

100] Mucor inaequisporus URM 6532

Mucor inaequisporus CBS 351.50

Mucor fuscus

Mucor genevensis

98 ae Mucor plumbeus

Mucor racemosus

Mucor circinelloides

Mucor velutinosus

Mucor fragilis

Mucor bainien

Mucor ramosissimus

60 |_ Mucor lusitanicus

Mucor flavus

63 Mucor zonatus

Mucor hiemalis

= Mucor irregulans

Mucor hiemalis f. corticola

Mucor hiemalis f. hiemalis

m Mucor mucedo

Mucor pinformis

Mucor guilliermondii

Cokeromyces recurvatus

Mucor inaequisporus in South America ... 223

maximum-composite likelihood algorithm to calculate genetic distances. Robustness

was calculated using 1000 bootstrap pseudo-replicates, and the tree was rooted with

Cokeromyces recurvatus (TABLE 1).

Results

Phylogenetic Analysis

The size of the ITS region of M. inaequisporus URM 6532 is 536 bp. BLASTn

comparisons with sequences deposited at the GenBank revealed that the ITS

sequence of this strain is 92% identical with a sequence labeled as Mucoraceae

LM042 (EF060714), 91% identical with another undescribed Mucor species

(AB638465), 90% identical to the sequence of M. amphibiorum (FJ455861),

95% identical with M. inaequisporus (CBS 255.36), and 100% identical with

M. inaequisporus (CBS 351.50). The phylogenetic relationship among Mucor

species based on the ITS region is illustrated in Fic. 1. Mucor guilliermondii

appears as the basal species, phylogenetically distant from other species in the

genus. Mucor inaequisporus was genetically distant from M. mucedo, the type

species of the genus, and clustered in a well-supported clade (100% bootstrap

value) with M. nederlandicus, M. amphibiorum, M. ellipsoideus, M. variosporus,

and M. indicus. Another well-supported clade (98% bootstrap value) included

M. ramosissimus, M. lusitanicus, M. bainieri, M. fragilis, M. circinelloides,

M. velutinosus, M. plumbeus, M. racemosus, and M. genevensis. Phylogenetic

analysis indicates only distant relatedness within two pairs of morphologically

similar species, M. genevensis/M. hiemalis and M. nederlandicus/M. guilliermondii.

Taxonomy

Mucor inaequisporus Dade, Trans. Br. Mycol. Soc. 21(1): 25. 1937. Pid

Colonies grow rapidly on MEA, filling the entire Petri dish (9 cm diam.

and 10 mm height) after 72 hours at 25 °C in the dark. The colony ranges

from an obverse intense yellow (8G to luteus 51) with yellow to olive-gray

spots, corresponding to sporangia, and reverse yellow (8G), particularly

at the point of inoculum. Rhizoids were short to long (<600 um in length),

poorly branched, with or without septa. A sweet aroma was produced.

SPORANGIOPHORES 9-30 um in diam., with or without yellow droplets, simple

or with long and short sympodial branches (3-10 times, mostly after 6 days of

incubation), erect, undulant, curved, sometimes with one or several randomly

spaced septa and sometimes constricted next to the sporangia; phototropic,

Fic. 1. Neighbor-joining tree of Mucor spp. inferred from the ITS-rDNA region. The maximum-

composite likelihood algorithm was used to calculate genetic distances. Numbers on branches

are bootstrap support values obtained from 1000 pseudo-replicates (values under 50% are not

shown). A sequence of Cokeromyces recurvatus was used to root the tree. Mucor inaequisporus

examined in this work is denoted in bold. Scale bar indicates the number of base substitutions

per site.

224 ... Santiago & al.

with yellow to chestnut contents and with incrusted walls. Some sporophores

exhibit randomly distributed irregular swellings (<47 um in diam.), frequently

gradually tapering towards the base. Sterile branches are common. SPORANGIA

globose and subglobose to lightly depressed, yellow and turn brown with

age, with finely echinulated, transparent and deliquescent walls that are more

persistent in the smaller sporangia, (47.5-)70-130(-180) x (45-)60-120

(-165) um; COLUMELLAE highly variable in shape, frequently pyriform (mostly

in the larger ones), conic or oblong, but also ellipsoid, obovoid, subglobose,

colorless or slightly yellow to chestnut; smooth walled with prominent collars,

(45-)60-100(-140) x (35-)50-80(-120) um; SpoRANGIOsPORES hyaline

with granular contents, highly variable in shape and size: majority elliptical

(sometimes flattened at one side), 5—14(-22.5) x 3.5-10(-15) um, also globose,

subglobose (<15 um diam.), some irregular (<24 x 15 um). CHLAMYDOSPORES

and ZYGOSPORANGIA not observed.

Influence of temperature, culture media and light on the growth and

morphology of M. inaequisporus

Macroscopic and microscopic features on PDA were similar to those on

MEA. The optimum growth temperature was 25 °C (colonies filled the entire

Petri plate after 72 hours) with good growth and sporulation between 15 and

30 °C. Limited growth was recorded at 35 °C and no growth was observed

at 10 and 40 °C. Colonies incubated at 30 °C grew well (9 cm diam. after 72

hours), but were shorter in height (6 mm) and the sporangiophores were more

branched than at 25 °C. The columellae were mainly oblong and conical, rarely

pyriform, and the sporangiospores were irregular in shape, predominantly

globose and subglobose. Colonies incubated at 20 °C reached 9 cm in diam.

after 72 hours. Such colonies were irregularly zonate, at raised in the center

and then touching the entire lid of the Petri dish after 7 days. Abundant twisted

sterile and coiled hyphae were observed in the aerial mycelium. Colonies were

also irregularly zonate at 15 °C, reaching 9 cm diam. and 2 mm in height after

96 hours. Abundant twisted sterile hyphae were also observed in the aerial

mycelium. Influence of light was not detected.

SPECIMENS EXAMINED — BRAZIL, SAo PauLo: Campus of the Federal University of

Sao Carlos, (21°59'02"S 47°52'55"W), on mature fruits of Syzygium cumini, 23.01.2009,

leg. EM. Canedo (URM 6532); 12.02.2010, leg. EM. Canedo (URM 6533).

Hasitat — Growing on fruits of Artocarpus glaucus, Bouea macrophylla,

Diospyros kaki, Flacourtia inermis, Musa paradisiaca, M. sapientum, Spondias

mombin, Syzygium cumini, and Theobroma cacao (Dade 1937, Boedijn 1959,

CBS Database, IMI Database) and in the rhizosphere of root-knot nematode

host plants (Zangeneh et al. 2007).

DISTRIBUTION — Ghana, Indonesia, Iran, Malaysia, and Brazil. This is the

first report for South America.

Mucor inaequisporus in South America ... 225

2, oh 4

peo 2° Fe gre 2

‘ : ee oe » ie

Bo Sate — nce Seg

+1,

PL. 1. Mucor inaequisporus grown in MEA at 25 °C for 7 days. A. Colony growing in a Petri dish.

B-D. Branched sporangiophores, some with randomly distributed irregular swellings with sporangia

and columellae. E. Single undulate sporangiophore with columella. F Young sporangiophore.

G. Columellae. H-I. Sporangiospores. Scale bars. A = 5 cm; B, D-F, H = 50 um; C, G =100 um;

I=5 um. (A-E, G, H = pictures under BFM; F, I = pictures under ESEM.)

226 ... Santiago & al.

Discussion

Several studies have reported the variability of nucleotide sequences of the

ITS-rDNA region as a reliable indicator to differentiate taxa of Mucorales at

the species level, including taxa of Mucor (Iwen et al. 2002, Schwarz et al. 2006,

Hoffmann et al. 2009). Strain URM 6532 shared an identical ITS sequence with

strain CBS 351.50. According to our phylogenetic analysis, M. inaequisporus

is genetically distinct from other described Mucor species. This species

exhibited low percentages of similarity with M. nederlandicus (91%), with

M. amphibiorum and M. variosporus (90%), and with M. ellipsoideus (89%).

According to Walther et al. (2013), the ITS is a barcode marker for mucoralean

species identification, despite the wide intraspecific variations found for some

taxa. Mucor inaequisporus appears to have some ITS variability because strain

URM 6532 showed a 95% similarity with strain CBS 255.36. Thus, our phylogeny

is in agreement with the phylogeny presented in Walther et al. (2013).

Morphologically, M. inaequisporus is distinguished from other Mucor

species by the production of sporophores that are straight, curved, or undulant,

and with randomly distributed irregular swellings. The columellae are variable

in form, being predominantly pyriform at 25 °C. Sporangiospores are extremely

variable in size and shape at 25 °C. However, some features of our isolate, such

as curved or undulant sporophores with irregular swellings, were not described

by Dade (1937) and Schipper (1978). Columellae sizes reported in the literature

(<100 x 90 um, Dade 1937; 83-75 um, Schipper 1978) were smaller than our

maximum measurements, whereas sporangiospores (<25 x 20 um, Dade 1937;

<30 x 23 um, Schipper 1978) were larger than our maximum measurements.

According to Schipper (1978), different strains of M. inaequisporus may exhibit

variable morphologic and physiologic features.

Our results show that M. inaequisporus exhibited growth from 15 to 30

°C. However, colonies incubated at 30 °C showed an increased number of

branches. The columellae were mostly oblong and conical, rarely pyriform, and

the sporangiospores were mostly globose, subglobose, and irregular in shape,

different from the observed at 25 °C. Schipper (1975, 1976) has noted the

influence of temperature on the shape of columellae and/or sporangiospores in

other Mucor species such as M. plasmaticus Tiegh., M. psychrophilus Milko, and

M. racemosus f. chibinensis (Neophyt.).

We report for the first time the occurrence of M. inaequisporus in Brazil,

thereby expanding the knowledge of Mucorales distribution. Strains URM 6532

and URM 6533 were isolated from a common introduced tree species in Brazil,

S. cumini, which belongs to the Myrtaceae. As this is the first report of a Mucor

species in fruits in this country, this record adds to the twenty-four species

of Mucor that have been isolated from soil and/or dung in Brazil (Santiago

2012).

Mucor inaequisporus in South America ... 227

Acknowledgments

This work was financially supported by Fundacao de Amparo a Ciéncia e Tecnologia

do Estado de Pernambuco (FACEPE). The authors would like to thank Fundacao de

Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) for a scholarship to Enzo M.

Canedo. We are also grateful to Rafael José Vilela, Diogo Xavier Lima and Dr. Luciana

Bueno dos Reis Fernandes for technical assistance. We are in debt to Dra. Maria Inés

Salgueiro Lima for the identification of plant specimens and to Dr. José Ivanildo de

Souza and Dr. Matias Cafaro for manuscript review.

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MY COTAXON

http://dx.doi.org/10.5248/124.231

Volume 124, pp. 231-238 April-June 2013

Molecular phylogeny reveals Megacollybia virosa

is a Cantharocybe

T.K. ARUN KUMAR’ & P MANIMOHAN?

‘Department of Botany, The Zamorin’s Guruvayurappan College, Kerala, 673014, India

?Department of Botany, University of Calicut, Kerala 673635, India

* CORRESPONDENCE TO: thakumar@gmail.com

ABSTRACT — Nuclear LSU rDNA sequence analysis unequivocally places an agaric currently

designated as Megacollybia virosa in Cantharocybe. Based on this molecular evidence,

Cantharocybe virosa comb. nov. is proposed. This transfer results in the recognition of a third

distinct species of Cantharocybe. The collections from India form the first record of the genus

outside the North American continent. Phenetically, C. virosa shares with the type species

of the genus, C. gruberi, the clitocyboid habit, distinct cheilocystidia, convex pileus with an

inrolled margin, subdecurrent to decurrent lamellae, thickset stipe, ellipsoid, thin-walled,

hyaline, smooth and inamyloid basidiospores, clamped hyphae, subregular to regular lamellar

trama, a cutis-type pileipellis disrupted to form trichodermal patches, pileipellis hyphae with

plasmatic pigment, and cystidioid terminal cells. A key to the known species is provided.

Key worps — Agaricales, Basidiomycota, systematics, taxonomy

Introduction

Cantharocybe is a little studied agaric genus (Agaricales, Basidiomycota) so

far known only from North and Central America. The genus was described

by Bigelow & Smith (1973) to accommodate a clitocyboid agaric with robust,

yellowish basidiomata, lecythiform or mucronate cheilocystidia, oblong or

subcylindric, smooth, inamyloid basidiospores, and clamped hyphae. The

genus was originally typified by Clitocybe gruberi A.H. Sm. (Smith 1944)

from Idaho based on a single sporocarp but subsequently collected from New

Mexico, California, and Washington as well. According to Bigelow & Smith

(1973), the combination of yellow pigmentation, the unusual cheilocystidia,

and subcylindric basidiospores of this species warranted its transfer from

Clitocybe to a new genus. Cantharocybe remained a monotypic genus for nearly

three decades, but very recently, another species, C. brunneovelutina Lodge

et al., was described from Belize (Ovrebo et al. 2011). Ovrebo et al. (2011)

232 ... Kumar & Manimohan

emended the genus concept by widening the range of states of characters such

as the texture and color of pileus surface and shape of both basidiospores

and cheilocystidia. Although the distinctiveness of Cantharocybe as a genus

is supported by molecular analyses within the Agaricales, the family within

which Cantharocybe should be placed is uncertain (Binder et al. 2010, Ovrebo

et al. 2011). Cantharocybe is currently placed among the basal genera of the

hygrophoroid clade (i.e., Hygrophoraceae) based on molecular analyses with

low support (Binder et al. 2010; Ovrebo et al. 2011).

Repeated collections of an agaric from various parts of Kerala State, India

have been made in the last two decades. As these particular collections defied

identification even to the genus level, it remained unpublished for quite a

long time. Eventually it was described as Megacollybia virosa based solely on

morphology (Manimohan et al. 2010). Lingering doubts about its identity

prompted us to undertake a molecular analysis, which revealed that it is closely

allied to the two species of Cantharocybe mentioned above. The results of our

molecular analysis are presented and discussed here.

Materials & methods

DNA was extracted from authentic material (TENN063483) of Megacollybia virosa

deposited at the Herbarium of the University of Tennessee. Detailed voucher information

is available in Manimohan et al. (2010). DNA was extracted from dried tissue using the

procedure described in Mata et al. (2007). The nuclear large subunit (LSU) ribosomal

RNA region was amplified and sequenced with primers LROR and LR5. Sequences were

edited in Sequencher 4.10.1 (Gene Codes Corp., Ann Arbor, Michigan), the segments

with ambiguous bases trimmed from the ends, and similar sequences were searched

in GenBank with the BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) algorithm.

Initial BLAST queries showed close affinities with Cantharocybe brunneovelutina

(92% sequence identity) and C. gruberi (91% sequence identity). An LSU sequence

dataset was assembled with sequences of taxa that showed >90% similarities with the

newly generated sequence, additional sequences of representative members of genera

(including outgroup Phyllotopsis nidulans (Pers.) Singer) selected based on Ovrebo et

al. (2011), and sequences of representative species of Megacollybia (Hughes et al. 2007)

were downloaded from GenBank and assembled into a dataset. The newly generated

sequence was deposited in GenBank with accession number JX101471.

The LSU sequence dataset was manually aligned in CLUSTALX 2.1 and then

examined in Se-Al 2.0all (Andrew Rambaut, University of Edinburgh, U.K.

http://tree.bio.ed.ac.uk/software/seal). The region analyzed contained a total of 1452 bp.

The alignment was deposited in TreeBase with submission number 12767 (http://purl.

org/phylo/treebase/phylows/study/TB2:S12767). Maximum Parsimony (MP) analysis

was conducted in PAUP* 4.0b10 (Swofford 2002) using a heuristic search option with

TBR branch swapping, 1000 replicates using random step-wise addition, and holding

one tree at each step. All minimal length trees were saved, and maxtrees was set as

unrestricted. Bootstrap analysis (Felsenstein 1985) was carried out to evaluate support for

Cantharocybe virosa comb. nov. (India) ... 233

91 Cantharocybe virosa Jx101471

90 | 2% Cantharocybe brunneovelutina HM588721

Cantharocybe gruberi DQ234540

Hygrocybe pratensis AF261457

Ampulloclitocybe clavipes —U522769

99 Hygrocybe coccinea AM946460

93 Hygrocybe conica 19071739

Camarophyllus griseorufescens GU233423

aa 100 Camarophyllus basidiosus DQ457651

98 Camarophyllus canescens DQ457652

Xeromphalina campanella ay207312

Lichenomphalia umbellifera AF261445

Gliophorus laetus AF261446

88 Cortinarius distans AF261595

92 Cortinarius metallicus ay219582

Chrysomphalina chrysophylla DQ457656

Hygrophorus chrysodon 0Q071733

Hygrophorus sordidus AF042562

98 Megacollybia subfurfuracea EU623750

96 Megacollybia rodmanii £u623792

91 Megacollybia texensis —U623738

88 Megacollybia fusca —U623743

96 Megacollybia platyphylla EU623712

74 94 Megacollybia marginata —u623689

99 | Megacollybia fallax —u623723

Megacollybia clitocyboidea —U623679

Phyllotopsis nidulans DQ071736

FiGurRE 1. One of four equally parsimonious trees generated during the phylogenetic analyses of

LSU rDNA sequences. Numbers above branches indicate MP bootstrap values and numbers below

branches indicate BI clade credibility (posterior probability) values. Only values greater than 70%

are shown. GenBank accession numbers are provided.

the branching topologies with a full heuristic search option on 1000 replicates. Bayesian

inference (BI) analysis was done using MrBayes 3.1.2 (Ronquist & Huelsenbeck 2003).

The general time reversible model with gamma distributed rate variation option was

selected. Multiple independent analyses were done for 1 million generations with trees

saved every 100 generations. Options for incremental heating scheme, chain number,

and priors were set to MrBayes default settings. The first 1000 samples were burned

before calculating posterior probabilities.

Manimohan et al. (2010) is followed for the morphological description. Color codes

refer to Kornerup & Wanscher (1978).

234 ... Kumar & Manimohan

Results

Megacollybia virosa is resolved in a clade with C. brunneovelutina and

C. gruberi with strong branch support in both Bayesian and parsimony

analyses (Fic. 1). The analysis also places Hygrocybe pratensis (Fr.) Murrill

(= Camarophyllus pratensis (Fr.) P. Kumm.) as the sister group to Cantharocybe

but with no support. Three species of Camarophyllus (= Cuphophyllus) were

included in the analysis group with species of Xeromphalina, Ampulloclitocybe,

and Hygrocybe. Although a relationship of these taxa with Cantharocybe is

indicated, the placement is not well supported. Eight representative species

of the genus Megacollybia form a distinct clade with strong support in all the

maximum parsimony and maximum likelihood analyses. Cantharocybe virosa

shows no close phylogenetic affinities with members of Megacollybia.

Discussion

DNA sequencing and analysis of the nLSU gene region supports a close

phylogenetic relationship between Megacollybia virosa and the two known

species of Cantharocybe. Although initially described asa species of Megacollybia,

the distinctiveness of M. virosa had been recognized based on its abundant

cheilocystidia of a unique morphology and the distinctive terminal elements of

the pileipellis (Manimohan et al. 2010). The cheilocystidia in M. virosa feature a

clavate, cylindrico-clavate, or ventricose basal part and an upper part extending

into an elongated neck with or without a rounded capitulum (Fic. 2b). While

this unique cystidial morphology is not characteristic of Megacollybia, it is

seen in the type species of Cantharocybe, C. gruberi. Phenetically, apart from

the morphology of cheilocystidia, M. virosa shares the following characters

with Cantharocybe: robust clitocyboid basidiomata (Fic. 2a), convex pileus

with an inrolled margin, subdecurrent to decurrent lamellae, thickset stipe,

ellipsoid, thin-walled, hyaline, smooth and inamyloid basidiospores (Fic. 2c),

clamped hyphae, subregular to regular lamellar trama, a cutis-type pileipellis

disrupted to form trichodermal patches, and pileipellis hyphae with plasmatic

pigment and cystidioid terminal cells. At the same time, it differs from both

C. gruberi and C. brunneovelutina in having a grayish brown pileus surface.

Additionally, C. gruberi differs in having much larger, narrowly elliptic to

oblong basidiospores, whereas C. brunneovelutina has a unique cystidial

morphology with a resemblance to basidia. According to Matheny et al. (2006),

C. gruberi belongs to pluteoid clade and in Moncalvo et al’s (2002) analysis,

the relationships of C. gruberi remained unresolved although it formed a

distinct clade with Camarophyllus pratensis. The placement of Camarophyllus

(= Cuphophyllus) and Cantharocybe in the hygrophoroid clade has been

indicated by Binder et al. (2010) and Ovrebo et al. (2011). The collections of

M. virosa from India form the first report of a Cantharocybe species outside the

North American continent.

Cantharocybe virosa comb. nov. (India) ... 235

Based on the results of the present molecular analysis, the following new

taxonomic combination is proposed:

Cantharocybe virosa (Manim. & K.B. Vrinda) T.K.A. Kumar, comb. nov. Fic. 2

MycoBank MB800480

= Megacollybia virosa Manim. & K.B. Vrinda, Mycotaxon 111: 364. 2010.

BASIDIOMATA medium-sized, fleshy, clitocyboid. Piteus 45-100 mm broad,

convex, becoming broadly convex; surface light brown (6D5), brown (6E4),

grayish brown (7E3), or dark gray (7F8), slightly darker at the center, pruinose

to somewhat granular to the naked eye, with fine appressed scales under a

lens, dry; margin inrolled when very young, becoming incurved and finally

becoming straight, initially entire, becoming fissile with age. LAMELLAE adnate

to decurrent, moderately crowded, with lamellulae in four to eight tiers, up to

9 mm deep, whitish to yellowish white (1A2); edges smooth to finely fimbriate

under a lens. StrpE 20-75 x 5-23 mm, central or at times slightly excentric,

terete to slightly compressed, almost equal with a dilated apex, solid, with

white basal mycelium; surface dull white with fine, light brown (6D5), grayish

FIGURE 2. Cantharocybe virosa (TENN063485, holotype).

a. Basidiomata; b. Cheilocystidium; c. Basidiospore. Scale bars: a = 1 cm; b, c = 10 um.

236 ... Kumar & Manimohan

brown (7E3), or dark gray granular squamules concentrated towards the base

and disappearing easily when handled. ConTEXT <20 mm thick, white. ODOUR

strong and unpleasant. SPORE PRINT white.

BASIDIOSPORES 6.5-11(-12) x 5-7 um, subglobose to ellipsoid, thin-

walled, smooth, with refractive guttules, inamyloid. Basip1a 23-56 x 7.5-11

um, cylindrico-clavate to clavate, thin-walled, hyaline, with granular contents,

4-spored; sterigmata <5 um long. CHEILocystTip1A abundant, 20-63 x 5.5-9

um, clavate, cylindrico-clavate, or ventricose, majority with a small capitellum

on a slender neck up to 35 um long, often septate, thin-walled, hyaline to pale

yellowish, apex sometimes covered with glutinous exudates; edges of lamellae

sterile. PLEUROCYSTIDIA absent. LAMELLAR TRAMA regular to subregular,

hyphae 3-15 um wide, hyaline to pale yellowish, thin-walled, inamyloid.

PILEAL TRAMA interwoven; hyphae 2-20 um wide, slightly inflated, hyaline to

pale yellowish, thin-walled. PILEIPELLIS mostly a cutis of highly interwoven

agglutinated hyphae, occasionally disrupted with trichodermal patches,

hyphae 3-10 um wide, thin- to slightly thick-walled, often with a grayish

brown plasmatic pigment; terminal elements cystidioid, 23-85 x 3-10 um,

similar to cheilocystidia in all aspects, often with a greyish brown plasmatic

pigment. STIPITIPELLIs a highly disrupted and irregular cutis with agglutinated

trichodermal patches of ascending to erect hyphal elements, hyphae 2-21 um

wide, thin- to slightly thick-walled, with gray to dark grayish plasmatic and

membrane pigment; terminal elements cystidioid, similar to cheilocystidia in

all aspects. CLamps frequent on all hyphae.

Hasirat: On soil or on mud walls, often associated with roots of coconut

trees in a tropical climate, solitary or in caespitose clusters, May to August.

SPECIMENS EXAMINED — INDIA, KERALA STATE, CaLicuT District, Puthiyangadi,

31 July 2005, T.K. Arun Kumar AK373 (TENN063483, GenBank, JX101471); 10

August 2005, T.K. Arun Kumar AK378 (TENN063476); 12 June. 2006, T.K. Arun

Kumar AK395 (TENN063485, holotype); 21 June 2006, T.K. Arun Kumar AK397

(TENN063486); THIRUVANANTHAPURAM District, Plamood, 30 May 1998, C.K.

Pradeep TBGT4310 (TENN063480); Vizhinjam, 11 December 2000, C.K. Pradeep

TBGT5249 (TENN063479); 23 May 2002, C.K. Pradeep TBGT5526 (TENN063482);

Muttada, 9 July 2006, K.B. Vrinda TBGT9804 (TENN063481); 28 August 2006, K.B.

Vrinda TBGT9938 (TENN063484).

Key to the species of Cantharocybe

la. Pileus dark brown; cheilocystidia resembling basidia with 1-4 sterigma-like

apical appendages, extending at oblique angles and frequently swollen or

capitate at the apex; basidiospores 9-9.5 x 5.5-6 tum, Belize, Central America

REPO PCa) YabeesTe crag Eh: wal Coke wat Be Oh cata AS cna sow oh Lets cw Rt crt C. brunneovelutina

1b. Pileus yellow or pale grayish brown; cheilocystidia lecythiform or

SOUIS TIES HNALIY, AATIUCE OTALE APEX * Weert, wavy las cln Mre nl ined adhe heyy whem 2

Cantharocybe virosa comb. nov. (India) ... 237

2a. Pileus yellow; basidiospores (13.5)14-15(16) x 3 5.5-6 um, narrowly elliptic to

oblong, western North America (temperate) ...................006. C. gruberi

2b. Pileus pale grayish brown; basidiospores 6.5-11(-12) x 5-7 um, subglobose to

broddh-eéllipsoid: Indias (itopical):. tt esl h ew vee dn we onete « oloveld eatin > A C. virosa

Cantharocybe virosa is known to be toxic, capable of inducing severe gastro-

intestinal upset when eaten (Manimohan et al. 2010). Nothing is known about

the potential toxicity of the two American species.

Acknowledgments

We are greatly indebted to Prof. Karen W. Hughes and Prof. Ronald H. Petersen

for their kind help in sequencing the DNA of Cantharocybe virosa. We thank Dr.

M. Catherine Aime for valuable comments on the identity of the species and for a critical

review of the manuscript. We are also grateful to Dr. P. Brandon Matheny for a critical

review of the manuscript.

Literature cited

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http://dx.doi/org/10.2307/3758121

Binder M, Larsson K-H, Matheny PB, Hibbett DS. 2010. Amylocorticiales ord. nov. and Jaapiales

ord. nov.: early diverging clades of Agaricomycetidae dominated by corticioid forms. Mycologia

102: 865-880. http://dx.doi/org/10.3852/09-288

Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution

39: 783-791.

Hughes KW, Petersen RH, Mata JL, Psurtseva, NV, Kovalenko AE, Morozova OV, Lickey EB,

Cifuentes Blanco J, Lewis DP, Nagasawa E, Halling RE, Takehashi $, Aime MC, Bau T, Henkel

T. 2007. Megacollybia (Agaricales). Reports of the Tottori Mycological Institute 45: 1-57.

Kornerup A, Wanscher JH. 1978. Methuen handbook of colour, 3rd edn. London, Methuen.

Manimohan P, Kumar TKA, Vrinda KB, Pradeep CK. 2010. Megacollybia virosa, a new species with

toxic basidiomata from India. Mycotaxon 111: 363-368. http://dx.doi/org/10.5248/111.363

Mata, JL-, Hughes, KW, Petersen, RH. 2007. An investigation of Omphalotaceae (Fungi: Euagarics)

with emphasis on the genus Gymnopus. Sydowia 58: 191-289.

Matheny PB, Curtis JM, Hofstetter V, Aime MC, Moncalvo JM, Ge ZW, Yang ZL, Slot JC, Ammirati

JE, Baroni TJ, Bougher NL, Hughes KW, Lodge DJ, Kerrigan RW, Seidl MT, Aanen DK, DeNitis

M, Daniele GM, Desjardin DE, Kropp BR, Norvell LL, Parker A, Vellinga EC, Vilgalys R,

Hibbett DS. 2006. Major clades of Agaricales: a multilocus phylogenetic overview. Mycologia

98: 982-995. http://dx.doi/org/10.3852/mycologia.98.6.982

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

seventeen clades of euagarics. Mol Phylogenet Evol 23: 357-400. http://dx.doi/org/10.1016/

$1055-7903(02)00027-1

Ovrebo CL, Lodge DJ, Aime MC. 2011. A new Cantharocybe from Belize with

notes on the type of Cantharocybe_ gruberi. Mycologia 103: 1102-1109.

http://dx.doi/org/10.3852/10-360

Ronquist F, Huelsenbeck JP. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed

models. Bioinformatics 19: 1572-1574.

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Smith AH. 1944. New North American agarics. Mycologia 36: 242-262.

http://dx.doi/org/10.2307/3754821

Swofford DL. 2002. PAUP*: phylogenetic analysis using parsimony (*and other methods). 4.

Sunderland, Massachusetts: Sinauer Associates.

MY COTAXON

http://dx.doi.org/10.5248/124.239

Volume 124, pp. 239-245 April-June 2013

New record of Scedosporium dehoogii from India

ROHIT SHARMA’, GIRISH KULKARNI,

MAHESH S. SONAWANE!’ & YOGESH S. SHOUCHE’

"Microbial Culture Collection, National Centre for Cell Science &

?Molecular Biology Unit, National Centre for Cell Science

NCCS Complex, Ganeshkhind, Pune-411 007 (Maharashtra), India

* CORRESPONDENCE TO: rohit@nccs.res.in

ABSTRACT — Three strains of Scedosporium were isolated from Pimpri (Pune), Maharashtra,

India, during a survey of soil fungi in industrial areas. Morphological and sequence (ITS1-

5.8S-ITS2 rDNA) analyses confirm them to be Scedosporium dehoogii (reported here as a new

record for India) and S. apiospermum.

Key worps — disease, Microascaceae, phylogeny, scedosporiosis, taxonomy

Introduction

Scedosporium Sacc. ex Castell. & Chalm. (teleomorph Pseudallescheria

Negroni & I. Fisch.; Microascaceae) are cosmopolitan fungi found in soil,

sewage, and water, some of which can cause infection in humans. Five medically

important Scedosporium species — S. apiospermum Sacc. ex Castell. & Chalm.,

S. aurantiacum Gilgado et al., S. boydii (Shear) Gilgado et al., S. dehoogii, and

S. prolificans (Hennebert & B.G. Desai) E. Guého & de Hoog (Gilgado et al.

2005, 2008) — cause various diseases (broadly termed as scedosporiosis;

Guarro et al. 2006, Cortez et al. 2008) that mostly infect immunocompromised

patients, causing mycetoma, septic arthritis, osteomyelitis, meningitis, brain

abscesses, etc. (Cooley et al. 2007). In India, there have been several reports

of Scedosporium infection (Lingappa & Lingappa 1962, Acharya et al. 2006,

Gopinath et al. 2010, Nath et al. 2010, Mathew et al. 2012). During the present

study we isolated one Scedosporium apiospermum strain and two S. dehoogii

strains. Our survey of the literature and fungal checklists (Bilgrami et al. 1991,

Jamaluddin et al. 2004) indicates that S. dehoogii is a new record for India.

Materials & methods

The fungi were isolated from soil collected from industrial region of Pimpri (Pune),

Maharashtra, India, on 23 December 2011. Isolated strains were cultured on five media

240 ... Sharma & al.

obtained from Hi-Media (India) — 2% malt extract agar (MEA), potato dextrose

agar (PDA), Czapek Dox agar (CDA), potato carrot agar (PCA), oatmeal agar (OA)

— at 10—40+0.2°C in the dark. Morphological characteristics were observed on PDA

at 25+0.2°C (7 d). Cultures have been deposited at the Microbial Culture Collection

(MCC), National Centre for Cell Science, Pune, India as S. apiospermum MCC 1042 and

S. dehoogii MCC 1043 and MCC 1044, and a specimen of MCC 1044 has been deposited

in the Ajrekar Mycological Herbarium (AMH), Agharkar Research Institute, Pune,

India. Microscopic characters were studied in lactophenol stained with cotton blue (Hi-

Media, India) using Nikon YS100 microscope (Nikon, Japan). Microphotographs were

taken on Olympus BX53 (Olympus Corporation, Japan) fitted with ProgRes C5 camera

(Jenoptik, USA). Samples were coated with platinum on Jeol minor coater (Jeol-JFC

1600) for examination on a Jeol-JSM 6360A scanning electron microscope at 10 kV.

Fungal DNA was isolated from pure 7 d old cultures with beads and lysis buffer in 2

ml Eppendorf tubes according to Sharma & Graser (2011). Total DNA was checked on

gel and NanoDrop spectrophotometer (NanoDrop, USA). PCR was conducted with a

Gene Amplifier PCR System 9700 (Perkin Elmer, USA) using ITS1 and ITS4 primers

for ITS1-5.8S-ITS2 region (White et al. 1990). A 25 ul mixture containing 10 U Taq

polymerase buffer (New England Biolabs), 2 mM dNTPs, 10 pM primers, 1 unit Taq

polymerase (New England Biolabs), and 10 ng DNA was amplified following a protocol

of initial denaturation at 94°C for 2 min, 35 cycles of denaturation at 94°C for 1 min,

annealing at 55°C for 1 min and extension at 72°C for 1 min, final extension at 72°C

for 10 min. The PCR product was checked on 1.0% agarose gel and purified by PEG-

NaCl (Polyethylene glycol-NaCl). Sequencing was done on ABI 3730xl Automated

Sequencer using ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit’

(Perkin Elmer Applied Biosystems Division, Foster City, CA). An NCBI Blastn search

for ITS region was conducted for sequence similarity (Altschul et al. 1990). Sequences

of MCC 1042, MCC 1043 and MCC 1044 were generated in lab and other sequences

retrieved from GenBank and deposited in European Molecular Biology Laboratory

(EMBL) database. The sequences were aligned and edited in Clustal W of Molecular

Evolutionary Genetics Analysis (MEGA 5.0) computer program (Higgins et al. 1994,

Tamura et al. 2011). Phylogenetic analyses were done by Kimura 2-parameter model

using Neighbour Joining (NJ) statistical method. Gaps and missing data was removed

by complete deletion option of the software. The bootstrap consensus tree was inferred

from 1000 replicates and bootstrap values <50% were removed from the tree (Saitou &

Nei 1987; Tamura et al. 2011).

Taxonomy

Scedosporium dehoogii Gilgado, Cano, Gené & Guarro, J. Clin. Microbiol. 46(2):

768. 2008. PLATE 1

The PDA colonies, which reached 32 mm diam. after 7 d at 25+0.2°C, were

radially zonate, dense, cottony to lanose, grayish-white to white and with a

whitish lobate or irregular and fimbriate margin; the reverse was yellowish

fading towards margin and with radial zones also visible. Other strain was

effused, white to pale gray, fast growing, 75 mm after 7 d at 25+0.2°C on PDA

Scedosporium dehoogii new to India... 241

Fic. 4. SEM of mycelial mat with conidia. Fic. 5. SEM of sessile conidia. Scale bars: 1-3 = 50 um;

4=10um;5=2 um.

with colorless reverse. Solitary conidiophores were subhyaline smooth-walled,

10—54 x 1-1.4 um, and produced pale brown, obovoid or ellipsoidal, 5.9-6.2 x

3.6—4.4 um conidia. Sessile conidia subhyaline, thick-walled, mostly obovate,

(6.9-)7.3(-7.8) x (3.5-)3.8(-4.0) um. Hyphae hyaline to subhyaline, 1.2-2.5

uum. A teleomorph was not observed even after 60 d of incubation.

DISTRIBUTION & HABITAT: Pimpri (Pune), Maharashtra, India (soil from industrial

area). (First described from garden soil in Barcelona, Spain.)

MATERIAL EXAMINED: INDIA, Manarasutra State, Pimpri (Pune), 18°37(07.04(N

73°48(13.43(E, soil, 23.X1I.2011, Rohit Sharma, culture S9-H (AMH-9561; MCC 1044;

GenBank HF570054); culture S9-D (MCC 1043; GenBank HF570053).

Results & discussion

Both Scedosporium dehoogii strains had similar optimum and maximum

growth temperatures, but the MCC 1043 PDA colonies grew around two times

faster than those of MCC 1044 (PLATE 2). Scedosporium apiospermum MCC

242 ... Sharma & al.

S. dehoogii MCC 1043

= 60 ——CDA

8 50

a —™—MEA

>, 40

5 —*- OMA

& 30

Y 20 =—PCA

10 ——PDA

0 ’

10 15.20 25 30 35.40

Temperature (°C)

ma S. dehoogii MCC 1044

2 40 —-—CDA

A 30 —a—MEA

Pont

E —— OMA

2 20

é) —— PCA

10 S—PDA

10 15 20 25 30 35 40

Temperature (°C)

PLATE. 2. Temperature-growth relationship of Scedosporium dehoogii MCC 1043 and MCC 1044

on Malt Extract Agar (MEA), Czapek Dox Agar (CDA), Oatmeal Agar (OA), Potato Dextrose Agar

(PDA), Potato Carrot Agar (PCA) (mm diam. of 7-d old colonies).

1042 had an optimum temperature of 35+0.2°C on OMA (15+0.2°C minimum

and 40+0.2°C maximum). The sessile conidia of S. dehoogii MCC 1044 are

narrower than in the ex-type culture CBS 117406 (5—6 um; Gilgado et al. 2008).

ITS sequences of 62 strains retrieved from GenBank were aligned with the

sequences from our S. apiospermum and S. dehoogii strains. Of the 545 bp used

to construct the phylogeny, 434 were conserved, 107 variable, 76 parsimony-

informative, and 31 singletons. The NJ-phylogenetic analysis clustered MCC

1043 and MCC 1044 in the S. dehoogii species group, which forms a separate

branch clearly distinguishing it from other Scedosporium species (PLATE 3).

Scedosporium apiospermum and S. prolificans have been reported several

times from India and are associated with various types of infections in both

immunocompromised and immunocompetent patients (Gopinath et al. 2010,

Nguyen & Raychaudhuri 2011, Patil et al. 2011, Mathew et al. 2012, Kaur et al.

2013, Matlani et al. 2013); however, S. dehoogii is reported for the first time from

Scedosporium dehoogii new to India ... 243

edosporitun apiospenmum THEM 15642 (AJ888403)

edosporium apiospermum RKI 2956/93 (AJ888415)

edosporium apiospennum THEM 15149 (AJ888402)

edospor ium apiospermum THEM 14754 (AJ888399)

edospor itm apiospermum IHEM 14462 (AJ888395)

edospor ium apiospermum FMR. 8535 (AJ888409)

edospor ium apiaspermum FMR. 8534 (AJ888408)

edosporitum apiospermum FMR. 8521 (AJ888430)

Scedosporium apiospermum FMR. 6922 (AJ888390)

edosporium apiospermum FMR 6918 (AJ888433)

edosporium apiospermum FMR 8619 (AJ8884 16)

cedosporium apiospermum FMR. 8625 (AJ888420)

edosporium apiospermium IHEM 14268 (AJ888393)

edosporium apiospermum MUCL 6106 (AJ888404)

‘Scedosporium apiospermum MUCL 8302 (AJ888442)

Scedosporium apiospermum MCC 1042 (HF570052)

\Scedosporiwn apiospermum FMR 6919 (AJ888388)

\Scedosporitun apiospermum FMR. 6920 (AJ888434)

\Scedosporitum apiospermum FMR. 8522 (AJ888431)

\Scedosporiwm apiospermum FMR 8530 (AJ888406)

\Scedosporiwn apiospermum FMR. 8621 (AJ888418)

\Scedosporiwun apiospermwn IHEM 14464 (AJ888396)

\Pseudallesche ria baydii IHEM 14758 (AJ888400)

Scedosporiwm apiospermum IHEM 15144 (AJ888401)

Pseudallescheria fusoidea CBS 106.53 (AJ888428)

Pseudailescheria fusoidea MUCL 14092 (AJ888429)

Pseudallescheria baydii MUCL 18784 (AJ888405)

Pseudallescheria boydii THEM 14467 (AJ888397)

Pseudallescheria boydit THEM 14354 (AJ888437)

Pseudallesche ria ellipsoidea FMR. 8623 (AJ888427)

Pseudallescheria boydii FMR. 8539 (AJ888411)

3q|Pseudallesche ria boydit FMR 7885 (AJ888392)

Pseudallesche ria boydii FMR 4167 (AJ888385)

‘Pseudallescheria boydii FMR. 7884 (AJ888391)}

Pseudallescheria baydii IHEM 14451 (AJ888394)

Pseudallesche ria boydit IHEM 14263 (AJ888436)

Pseudallescheria baydii FMR 8620 (AJ888417)

6\ Pseudallescheria baydii FMR. 8538 (41888443)

Pseudallescheria boydii FMR. 6697 (AJ888387)

Pseudatlescheria boydit FMR. 6694 (AJ888386)

Pseudallescheria baydii FMR. 8537 (AJ888410)

Pseudallesche ria boydit FMR 8540 (AJ888412)

Pseudallescheria boydii RV 43605 (AJ888383)

Pseudallesche ria boydit IHEM 14358 (AJ888438)

Pseudallesche ria boydit IHEM 14638 (AJ888398)

95) Pseudallescheria baydii CBS 101.22 (AJ888435)

Pseudallescheria boydii MUCL 14009 (AJ888422)

Pseudailescheria elipsoidea CBS 418.73 (AJ888426)

da ahaa mirutispora FMR. 4072 (AJ888384)

Pseudallesche ria mirutispora MUCL 29258 (AJ888424)

oo [ipoenalesonets angusta FMR 8541 (AJ888413)

89 Pceudallescheria angusta CBS 254.72 (AJ889414)

Scedosporium dehoogii MCC 1044 (HF570054)

\Scedosporitum dehoogii FMR. 6921 (AJ888389)

\Scedosporitum dehoogii FMR 8532 (AJ888407)

“pascedosporium dehoogii FMR. 8622 (AJ888419)

Scedosporium dehoogii MUCL 8522 (AJ888421)

[— Scedosporium dehoogii MCC 1043 (HF570053)

Scedosporium dehoogii MUCL 20263 (AJ888423)

cedosporium aurantiacwn FMR. 8630 (AJ888440)

cedosporitum aurantiacum IHEM 15458 (AJ88844 1)

9lscedosporium aurantiacum IHEM 15579 (AJ888439)

Scedosporium aurantiacwn RKI 2782/95 (AJ888432)

‘Pseudailescheria africana CBS 311.72 (AJ888425)

‘Scedosporiwn prolificans FMR. 7294 (AJ888444)

100

-—H

0.01

PiaTE. 3. Neighbor-joining tree derived from the ITS region rDNA sequences of Scedosporium spp.

Bootstrap values were calculated from 1000 replications. Bootstrap below 50% were deleted.

244 ... Sharma & al.

India and outside Europe. Isolation of these strains from a single soil sample

is an important factor as there are growing numbers of immunocompromised

patients in Pune, India due to immunopulmonary infections (Salvi & Barnes

2009), AIDS, H1N1, and other viral infections caused by compromised

hygiene in many hospitals. Although Gilgado et al. (2009) has shown that S.

dehoogii is pathogenic to immunocompetent mice, detailed surveys of different

natural and human dominated habitats are required to confirm distribution

of Scedosporium in India as noted by Kaltseis et al. (2009) for Austria and the

Netherlands. Research on secondary fungal infections of immunocompromised

patients may also determine whether the victims are actually succumbing to

secondary infection caused by Scedosporium or similar fungi.

Acknowledgments

The authors thank DBT, India for funding and Director, NCCS, India for laboratory

facilities. Dr. S.V. Shinde, University of Pune, India is acknowledged for SEM microscopy.

Authors also thank Dr. J. Guarro, Universitat Rovira i Virgili, Spain and Dr. G.S. de

Hoog, CBS, Netherlands for reviewing the manuscript and suggestions.

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Cooley L, Spelman D, Thursky K, Slavin M. 2007. Infection with Scedosporium apiospermum

and S. prolificans, Australia. Emerging Infectious Diseases 13(8): 1170-1177.

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Cortez KJ, Roilides E, Quiroz-Telles FE, Meletiadis J, Antachopoulos C, Knudsen T, Buchanan W,

Milanovich J, Sutton DA, Fothergill A, Rinaldi MG, Shea YR, Zaoutis T, Kottilil S, Walsh TJ.

2008. Infections caused by Scedosporium spp. Clinical Microbiology Reviews 21(1): 157-197.

http://dx.doi.org/10.1128/CMR.00039-07

Gilgado F, Cano J, Gené J, Guarro J. 2005. Molecular phylogeny of the Pseudallescheria boydii

species complex: proposal of two new species. Journal of Clinical Microbiology 43(10):

4930-4942. http://dx.doi.org/10.1128/JCM.43.10.4930-4942.2005

Gilgado F, Cano J, Gené J, Sutton DA, Guarro J. 2008. Molecular and phenotypic data supporting

distinct species statuses for Scedosporium apiospermum and Pseudallescheria boydii and the

proposed new species Scedosporium dehoogii. Journal of Clinical Microbiology 46(2): 766-771.

http://dx.doi.org/10.1128/JCM.01122-07

Gilgado FE, Cano J, Gené J, Serena C, Guarro J. 2009. Different virulence of the species of the

Pseudallescheria boydii complex. Medical Mycology 47: 371-374.

http://dx.doi.org/10.1080/13693780802256539

Gopinath M, Cherian A, Baheti NN, Das A, Antony M, Sarada C. 2010. An elusive diagnosis:

Scedosporium apiospermum infection after near-drowning. Annals of Indian Academy of

Neurology 13: 213-215. http://dx.doi.org/10.4103/0972-2327.70878

Scedosporium dehoogii new to India... 245

Guarro J, Kantarcioglu AS, Horre R, Rodriguez-Tudela JL, Estrella MC, Berenguer J, de Hoog

GS. 2006. Scedosporium apiospermum: changing clinical spectrum of a therapy-refractory

opportunist. Medical Mycology 44: 295-327. http://dx.doi.org/10.1080/13693780600752507

Higgins D, Thompson J, Gibson T, Thompson JD, Higgins DG, Gibson TJ. 1994. CLUSTAL W:

improving the sensitivity of progressive multiple sequence alignment through sequence

weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research

22(22): 4673-4680. http://dx.doi.org/10.1093/nar/22.22.4673

Jamaluddin, Goswami MG, Ojha BM. 2004. Fungi of India: 1989-2001. Scientific Publishers,

Jodhpur, India. 326 p.

Kaltseis J, Rainer J, de Hoog GS. 2009. Ecology of Pseudallescheria and Scedosporium species in

human-dominated and natural environments and their distribution in clinical samples 47(4):

398-405. http://dx.doi.org/10.1080/13693780802585317

Kaur R, Kashyap B, Bhalla P. 2013. A five-year survey of onychomycosis in New Delhi, India:

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59. http://dx.doi.org/10.1186/1471-2334-12-S1-P59

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immunocompetent child, misdiagnosed as tubercular osteomyelitis. Indian Journal of

Dermatology 58(1): 80-81. http://dx.doi.org/10.4103/0019-5154.105319

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733-743. http://dx.doi.org/10.1016/S0140-6736(09)61303-9

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methods and applications. Academic Press, Inc., New York.

MY COTAXON

http://dx.doi.org/10.5248/124.247

Volume 124, pp. 247-254 April-June 2013

Inocybe nitidiuscula and its ectomycorrhizae

associated with Alnus nitida from Galyat, Pakistan

S. Ityas*, A. RAZAQ & A.N. KHALID

Department of Botany, University of the Punjab, Lahore. 54590, Pakistan

*CORRESPONDENCE TO: fungalcommunity@gmail.com

ABSTRACT — Inocybe nitidiuscula and its ectomycorrhizae are characterized from Pakistan

morphologically and using rDNA internal transcribed spacer sequences. The sequences of

above- and below-ground structures exhibit >97% similarity. This represents a new species

record for Pakistan and the first description of ectomycorrhiza formed by I. nitidiuscula.

Keyworps — alders, euagarics, Himalayan moist temperate forest, morphotype

Introduction

Inocybe (Fr.) Fr. (Agaricales, Basidiomycota) is a large, widely distributed

genus of gilled fungi with more than 500 species worldwide (Kirk et al. 2008).

This genus is characterized by a fibrillose pileus, equal or bulbous stipe,

metuloids or thin walled cystidia, and yellowish brown smooth to angular-

nodulose ellipsoid to amygdaliform spores. Many Inocybe species have thick

walled pleuro-cheilocystidia (metuloids) that others lack (Heim 1931, Kiihner

& Romagnesi 1953, Kiihner 1980, Kuyper 1986, Singer 1986, Stangl 1989,

Kobayashi 2002).

Inocybe species play a very important role in the ecology of coniferous

and deciduous trees by forming ectomycorrhizal associations (Larsson et

al. 2009). The salient features of Inocybe ectomycorrhizae (EcM) are shiny

ramifications, absence of rhizomorphs and cystidia, emanating hyphae

with clamp connections, and a short exploration type (Agerer 2006). EcM

have been reported for Inocybe appendiculata Kihner, I. avellana P. Kumm.,

I. fuscomarginata Kiihner, I. heimii Bon, I. lacera (Fr.) P. Kumm., I. lanuginella

(J. Schrot.) Konrad & Maubl., I. obscurobadia (J. Favre) Grund & D.E. Stuntz,

I. petiginosa (Fr.) Gillet, and I. terrigena (Fr.) Kuyper. So far, ECM have not been

described for I. nitidiuscula with any host plant.

Galyat, a narrow 250,000 acre strip of Himalayan moist temperate forest

(HMTF) northeast of IsLamabad extending on both sides of KPK-Punjab border,

248 ... Ilyas, Razaq & Khalid

is considered the best representative of HMTF with a high species diversity

(JJamal & Khadija 2009, Irshad & Khan 2012). The region is characterized by

mixed vegetation of deciduous and coniferous trees. Niazi (2008) and Niaziet al.

(2006, 2007, 2009, 2010) have reported on previous studies on ectomycorrhizal

fungi from this area.

During fieldwork in Koza Gali and Jhika Gali in Galyat, Inocybe nitidiuscula

was collected along with belowground ectomycorrhizae associated with Alnus

nitida. Anatomical and molecular characterization of both above- and below-

ground parts determined its distribution with coniferous and deciduous

vegetation.

Materials & methods

Basidiomata were collected, photographed and dried in the field. Collected material

was characterized anatomically and using DNA sequence data. For microscopic

observation, free hand sections were stained with Congo Red and Melzer’s reagent.

Drawings were made using a camera lucida attached to a compound microscope.

Soil blocks of 10 x 12 cm under selected trees were dug out and washed to isolate,

characterize, and identify ECM. Morphotypes were characterized morphologically

following Agerer (1987-2006) and have been deposited in the Herbarium of the Botany

Department, University of the Punjab, Lahore (LAH).

DNA was extracted using the Extract-N-Amp™ Plant kit (Sigma, St Louis, MO, USA).

The rDNA internal transcribed spacer (ITS) region was amplified using the primer pair

ITS1F (Gardes et al. 1991) and ITS 4 (White et al. 1990) with denaturation at 94°C for 4

min, followed by 35 cycles of 45 sec at 94°C, 45 sec at 54°C, and 1 min 30 sec at 72°C, anda

final extension at 72°C for 2 min. PCR products from basidiomata and EcM morphotypes

were sent for bidirectional sequencing (Macrogen, Korea). Sequences were queried to

GenBank using the NCBI BLAST interface for sequence comparison and identification

of both basidiomata and fungi on EcM root tips. Sequence alignment and phylogenetic

analysis were performed using Molecular Evolutionary Genetics Analysis (MEGA)

software (Tamura et al. 2011). Maximum Likelihood (ML) optimization was conducted

with a Jukes-Cantor substitution model and Nearest-Neighbor-Interchange (NNI) as

the heuristic search method. Branch support was assessed using 1000 nonparametric

bootstrap replicates. Selected sequences were aligned using Clustal W and corrected

manually. The specimens are deposited in the Herbarium of the Department of Botany,

University of the Punjab, Lahore (LAH) and the Herbarium of Hokkaido University

Museum, Sapporo, Japan (SAPA). Nucleotide sequences are deposited in GenBank as

accession numbers HE862959 (basidiome), and HF565509 (ectomycorrhizal root).

Results

Inocybe nitidiuscula and its ectomycorrhizae were identified morpho-

anatomically and molecularly. The ITS sequences from basidiomata and

EcM morphotypes obtained in the present study exhibited 97-99.8%

sequence identity, with identification confirmed by phylogenetic analysis. In

both maximum likelihood and neighbor-joining methods, all sequences of

Inocybe nitidiuscula mycorrhizae (Pakistan) ... 249

95 ,AM882765.2 Inocybe bulbosissima

FJ904159.1 Inocybe bulbosissima

JF908118.1 Inocybe rimosa

AM882766.2 Inocybe nmosa

FJ904165.1 Inocybe umbrinella

FJ904163.1 Inocybe umbnnella

100 | F3904166.1 Inocybe umbnnella

AM882781.2 Inocybe mimica

FJ904124.1 Inocybe mimica Clade |

FJ904134.1 Inocybe arenicola

AM882772.2 Inocybe perlata

AM882783.2 Inocybe squamata

AM882785.2 Inocybe squamata

AM882779.1 Inocybe flavella

JQ724027.1 Inocybe flavella

°° | AM882782.2 Inocybe flavella

100 , GU234082.1 Mallocybe dulcamara

GU234129.1 Mallocybe dulcamara

HQ650752. 1 Mallocybe terngena

HQ604783.1 Mallocybe terigena

HQ604779.1 Mallocybe terigena Clade Il

HQ604780.1 Mallocybe terrigena

GU980653. 1 Inocybe arthrocystis

93 | GU980654.1 Inocybe arthrocystis

AB211269.1 Inocybe lacera

AB327181.1 Inocybe lacera

AJ893275.1 ectomycormiza Inocybe

39, JF908176.1 Inocybe nitidiuscula

pe AB244791.1 Inocybe nitidiuscula

JF908249.1 Inocybe nitidiuscula

JF908088.1 Inocybe nitidiuscula Clade Il

100 |; Ml HF565508.1 Inocybe nitiduscula SD-2 Pakistan

M HF565509.1 Inocybe nitidiuscula SD-3 Pakistan

89 || HI HE862959.1 Inocybe nitidiuscula KP-17 Pakistan

91 | BM HE863671.1 Inocybe nitidiuscula SD-5 Pakistan

@ HE863670.1 Inocybe nitidiuscula SD4 Pakistan

100

Fic. 1. Phylogenetic relationship of I. nitidiuscula (MM) with other members of Inocybe based on

Maximum Likelihood analysis of nrITS sequences. Bootstrap values based on 1000 replicates are

shown above the branches (values <50 are not shown). The analysis included 35 sequences. All

positions containing gaps and missing data were eliminated, yielding a total of 616 nucleotide

positions in the final dataset. Clade I contains members of Inocybe sect. Rimosae, Clade II

members of Inocybe subg. Mallocybe, and Clade III members of Inocybe having metuloid pleuro-

cheilocystidia.

I. nitidiuscula from Pakistan clustered with sequences of I. nitidiuscula (Fie. 1,

clade III) with significant bootstrap support.

Taxonomy

Inocybe nitidiuscula (Britzelm.) Lapl., Dict. Iconogr. Champ. Sup.: 523, 1894. Fie. 2

Prteus 2.2-3.5 cm broad, light brown to dark brown from margins to

centre, conical when young and plano-convex at maturity, sharply umbonate,

shiny, smooth or minutely woolly at very center, umbo dark brown to blackish,

outwards radially fibrous, uniformly aligned brown fibrils move toward margins

which lighten in colour, margins not smooth, somewhat dentate. LAMELLAE

cream to light brown, sinuate in attachment to stipe, margins dentate, lamellulae

two-tiered, alternating with lamellae. Stipe 3.4-5.4 x 0.35-0.42 cm, central,

250 ... Ilyas, Razaq & Khalid

Cc D

Fic. 2: Inocybe nitidiuscula. A. Basidiomata; B. Basidia; C. Basidiospores; D. Metuloids (cheilocystidia

and pleurocystidia). Scale bars: A = 1 cm, B-C = 3.45 um, D = 6.25 um.

light brown, smooth, cylindrical, slightly swollen at base. ODoR not distinctive.

TASTE not recorded.

Basidiospores 10-11 x 6.5-8um, smooth, amygdaliform in side view,

elliptical to ellipsoid in front view, yellowish brown, thick walled, with a

prominent apiculus. Bastp1A 29-31 x 9.0-10.5 um, tetra sterigmate, clavate,

with olive green droplets in 5% KOH. PLeurocystip1a metuloids, 47.0-71.5 x

12.0-17.5 um, abundant, cylindrical to fusiform, contents hyaline to pale yellow

in 5% KOH with thick brown walls, apices heavily encrusted with colourless

crystals. CHEILOCysTIDIA similar to pleurocystidia, abundant, 38-41 x

11.8-13 um, hyaline, clustered, thick walled, with encrusted apices. PILEIPELLIS

a cutis of loosely arranged hyphae, light yellowish-brown, 7-9 um wide. CLAMP

CONNECTIONS abundant in all tissues.

MATERIAL EXAMINED: PAKISTAN: KHYBER PAKHTUNKHWA, Galyat, Ayubia, Koza

Gali, 2250 m a.s.l., gregarious, on moist ground under Alnus nitida, 23 August 2010,

Abdul Razaq, KP-17 (LAH 230817, SAPA 100000; GenBank, HE862959).

Morpho-anatomical description of ectomycorrhizae Fic. 3

ECTOMYCORRHIZAL SYSTEM dichotomous, axis 3-6 x 1.0-1.5 mm., in close

contact with surrounding soil due to gelatinous mantle. UNRAMIFIED ENDS

straight to beaded, 1-2 mm long and 0.5-1 mm in diam.; younger tips creamy

Inocybe nitidiuscula mycorrhizae (Pakistan) ... 251

Fic. 3: Inocybe nitidiuscula A,B. Ectomycorrhizae. C. Outer mantle layer D. Inner mantle layer.

E. Emanating hyphae. Scale bars: A = 5.4 mm, B = 3 mm, C-E= 9 um

white, older tips light brown. Texture of system smooth with shiny luster; host

tissue visible under the sheath. RH1zomMoRpPHs absent. EMANATING HYPHAE

frequent, straight, frequently clamped; clamps large, half or more than a half

252 ... Ilyas, Razaq & Khalid

semicircle; anastomoses H-shaped. Cystip1a absent, COLOR REACTION absent

with lactic acid & KOH.

MANTLE plectenchymatous in all layers. OuTER MANTLE loosely

plectenchymatous with netlike hyphal arrangement (Agerer 1987-2006, type

A), hyphae 2-3 um diameter, lacking clamps and cystidia, transparent, no cell

contents visible, anastomoses present. INNER MANTLE densely plectenchymatous

(Agerer 1987-2006, type A), hyphae 2-3 um diameter, colorless to light

yellowish, cell contents clear, no clamps or cystidia observed (FIG. 3).

MATERIAL EXAMINED: PAKISTAN: KHYBER PAKHTUNKHWA, Galyat (Koza Gali &

Jhika Gali), roots of Alnus nitida, 23 August 2010, Sobia Ilyas, SD-3 (LAH; GenBank,

HF565509).

Discussion

Inocybe is an integral part of almost all ectomycorrhizal communities in

association with coniferous and deciduous trees from different areas of the

world (Cullings et al. 2001, Yamada & Katsuya 2001, Obase 2006, Tedersoo

et al. 2009, Walbert et al. 2010, Kennedy et al. 2011, Ma et al. 2012). Pakistani

forests are well represented by different ectomycorrhizal fungi associated with

wide host range (Niazi 2008, Niazi et al. 2006, 2007, 2009, 2010, Jabeen et al.

2012), but no published data on Inocybe is available even though it seems to

be the most abundant group in this area among all euagarics. Basidiomata of

I. nitidiuscula are also reported from different forests associated with Picea,

Pinus, Larix, Quercus, Corylus, Carpinus, Fagus, Salix, and Dryas but no

description of its mycorrhizae has yet been published (Kuyper 1986).

Inocybe nitidiuscula and I. lacera are closely related as both clustered together

in phylogenetic analyses (Fic. 1, Clade III). Both species produce metuloid

cystidia on lamellar face and edges and form ectomycorrhizal symbioses.

The sequence (HE862959.1) from the I. nitidiuscula Pakistani basidiome

clustered with I. nitidiuscula isolates from other regions of the world. The

Pakistani sequence shows a 94-95% match with I. nitidiuscula (FN550920.1,

HQ604085.1), although its morphological details are same as given by Obase et

al. (2006). The genetic variation may be due to geographic distance.

The I. nitidiuscula ectomycorrhiza is characterized by smooth ramification,

white to light brown color, lack of rhizomorphs and cystidia, prominent

clamped emanating hyphae, and a plectenchymatous mantle. These resemble

the ectomycorrhizae of I. appendiculata associated with Picea and Abies and

I. fuscomarginata associated with Salix spp., except that the ramification type

more closely resembles I. terrigena, reported with Pinus (Agerer 1987-2006).

Phylogenetic analyses are a useful tool for identifying unknown Inocybe

sequences to the species level (Ryberg et al. 2008). Both above- and below-

ground parts of the Pakistani collections cluster in the I. nitidiuscula clade

(Fic.1, Clade III). Although Inocybe has been reported to associate with

Inocybe nitidiuscula mycorrhizae (Pakistan) ... 253

alders (Tedersoo et al. 2009), to our knowledge this is the first report of ECM

of I. nitidiuscula with that host. In one study, Tedersoo et al. (2006) reported

ectomycorrhiza of Inocybe sp. 5 (AJ893275.1) that showed maximum sequence

identity with I. nitidiuscula (AJ534934), although with only 77% sequence

similarity. In our phylogenetic analysis, this sequence clustered in the same clade

as I. nitidiuscula but significantly distant from sequences from Pakistan, Japan,

and Italy. Previous studies have shown that alder ectomycorrhizal communities

tend to be host specific (Rochet et al. 2011). Tedersoo et al. (2009) indicated

that ascomycete EcM symbionts of Alnus might not be host-limited, although

their results suggested high Alnus specificity in basidiomycete symbionts. A

detailed study on Alnus ECM communities from Pakistan will determine the

level of host specificity.

Acknowledgements

We are thankful to Dr. Abdul Rehman Khan Niazi and Dr. Todd Osmundson for

acting as expert reviewers to improve the manuscript and Dr. Takahito Kobayashi,

Curator of SAPA Herbarium, Japan, for the identification of specimens. Higher

Education Commission, Pakistan, provided financial support for this research work.

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relationships within species of section Rimosae (Inocybe) based on ITS, LSU and mtSSU

sequence data. Persoonia 23:86-98. http://dx.doi.org/10.3767/003158509X475913

Ma D, Zang S, Wan L, Zhang D. 2012. Ectomycorrhizal community structure in chronosequences

of Pinus densiflora in eastern China. African Journal of Microbiology Research 6(32):

6204-6209. http://dx.doi.org/10.5897/AJMR12.902

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of Pakistan. Ph.D. Thesis, Department of Botany, University of Punjab, Lahore, Pakistan.

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brevipes Peck and its ectomycorrhiza, a new record from Himalayan moist temperate forests of

Pakistan. Pakistan J. Bot. 38(4): 1271-1277.

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Himalayan moist temperate forests. Mycotaxon 101: 375-383.

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spruce (Picea smithiana) from Pakistan. Mycotaxon 107:73-80.

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MY COTAXON

http://dx.doi.org/10.5248/124.255

Volume 124, pp. 255-262 April-June 2013

Xerocomus porophyllus sp. nov., morphologically intermediate

between Phylloporus and Xerocomus

WEN-JUAN YAN, Tal-Hu1 Li’, MING ZHANG & TING LI

State Key Laboratory of Applied Microbiology, South China

(The Ministry—Province Joint Development), Guangdong Institute of Microbiology,

Guangzhou 510070, Guangdong, China

“CORRESPONDENCE TO: mycolab2011@163.com

ABSTRACT— Xerocomus porophyllus, discovered from Dinghushan Biosphere Reserve in

Guangdong province (southern China), is described as a new species. It is a morphologically

intermediate taxon between Phylloporus and Xerocomus since its hymenophore is lamellate

around the stipe but strongly anastomosing to distinctly faveolate or poroid towards pileus

margin. Molecular analyses of nuclear rDNA ITS and LSU sequences, however, indicate that

it has a closer genetic relationship with Xerocomus than with Phylloporus.

Key worps— Basidiomycota, Boletaceae, Boletales, phylogeny, taxonomy

Introduction

Phylloporus Quél. and Xerocomus Quél. belong to the family Boletaceae

and have a close relationship, whether Phylloporus has been considered as

a primitive group in the family (Corner 1972) or as a derived genus next to

Xerocomus (Binder 1999; Binder & Hibbett 2006). Molecular data have shown

the Xerocomus subtomentosus complex to be closely related to Phylloporus

(Binder 1999). The lamellate hymenophore in Phylloporus and the wide poroid

or tubular one in Xerocomus are still used as the main morphological characters

for differentiating the two genera (Neves & Halling 2010; Neves et al. 2012).

Worldwide, Phylloporus encompasses about 70 named species (Neves et al.

2012), 21 of which have been reported from China (Teng 1963; Zang & Zeng

1978; Li et al. 1992; Bi et al. 1993, 1997; Dai & Li 1994; Zang et al. 1996; Zeng

et al. 2012). However, two Chinese records should be excluded because the

type specimen of Phylloporus scabrosus M. Zang is a Lentinus sp., and Chinese

exsiccatae labelled as “Phylloporus rhodoxanthus” were misidentified (Zeng et

al. 2012).

Although some mycologists have incorporated Xerocomus into Boletus

(Corner 1972, Kirk et al. 2008), other authors still treat it as an independent

256 ... Yan & al.

genus (Watling & Hills 2005, Hills 2008, Neves et al. 2012, Zeng et al. 2012).

Until now, more than 210 species and varieties of Xerocomus have been

registered (www.indexfungorum.org), of which at least 46 species have been

reported from China (Zhang et al. 2012).

Recently, an interesting bolete was recorded in Dinghushan Biosphere

Reserve in Guangdong province (southern China) and found to differ from all

known taxa. Although its hymenophore is consistently lamellate (Phylloporus-

like) around the stipe, it is also obviously faveolate or tubular (Xerocomus-like)

towards the pileal margin. Molecular analyses of nuclear rDNA ITS and LSU

sequences, however, indicated it to have a closer genetic relationship with

Xerocomus than with Phylloporus. Further investigation revealed that it is a new

species to science, and therefore it is formally described here. The holotype is

deposited in the Fungal Herbarium of Guangdong Institute of Microbiology

(GDGM).

Materials & methods

Specimens were annotated and photographed in the field, and dried in an electric

drier. Color descriptions were made according to Kornerup & Wanscher (1978). Tissue

sections were mounted in 5% KOH or 1% Congo Red for microscopic examination.

Dimensions of basidiospores are given using the notation (a—)b-c(-d), where “b-c’

represents 90% or more of the measured values of at least 30 basidiospores, and the

parenthetical ‘a and ‘d’ are the extreme values. Lm refers to the average length of all

basidiospores standard deviation; Wm refers to the average width of all basidiospores

standard deviation; Qm refers to the average Q of all basidiospores standard deviation;

and n refers to the number of spores measured per specimen. Basidiospores were also

observed under a scanning electron microscope (SEM). Light micrographs were taken

using an Olympus BX51 trinocular phase contrast microscope and the scanning electron

micrographs were taken on a Philips FEI-XL30 scanning electron microscope.

Genomic DNA was isolated from dried specimens and the ITS1-5.8S-ITS4 segment

from the ribosomal DNA (rDNA) was amplified with primer sets and ITS1 (5'-crr

GCG TTG ATT ACG-3') and ITS4 (5'-Tcc Tcc GCT TAT TGA TAT GC-3’) by polymerase

chain reaction (PCR) techniques (White et al. 1990). A portion of the nuclear ribosomal

large subunit (nrLSU) was amplified with the primers LR6 (5’-cGc CAG TTC TGC TTA

cc-3') and LROR (5'-acc CGC TGA ACT TAA GC-3’) (Vilgalys & Hester1990). Amplified

products were examined with agarose gel electrophoresis using a 2kb DNA marker.

The amplified fragments were cloned into pCR2.1 TOPO (Invitrogen) to generate, the

vector pCR2.1 TOPO containing the targeting fragment was transformed into its host

Escherichia coli strain JM105 (Lopez-Garcia et al. 2003). The E. coli with the cloned

fragments was sequenced by BGI Company and the data were deposited in GenBank. For

the analysis of phylogenetic placement of the new species, the rDNA sequences of other

related Phylloporus and Xerocomus taxa were downloaded from Genbank. Phylogenetic

tree inferred from LSU rDNA sequences was constructed using maximum likelihood

(ML) in Mega 5.05. Bootstrap analysis was implemented with 1000 replicates.

Xerocomus porophyllus sp. nov. (China) ... 257

Fic. 1. Xerocomus porophyllus (GDGM 30303, holotype): a-c. basidiomes; d. pileipellis; e. basidia;

f. basidiospores; g. hymenium with basidia, pleurocystidia and basidiospores; h. pleurocystidium;

i. basidiospores under SEM. Scale bars: a~c = 1 cm; d-h = 10 um.

Taxonomy

Xerocomus porophyllus T.H. Li, WJ. Yan & Ming Zhang, sp. nov. FIGs. 1-2

MycoBank MB 563347

Differs from Phylloporus cingulatus by its larger basidiospores and non-cyanescent

context on exposure; and from all species of Xerocomus s. str. by the largely lamellate

hymenophore near the stipe.

Type: China, Guangdong Province, Zhaoqing City, Dinghushan Biosphere Reserve,

23°10.11'N 112°31.50’E, alt. 300 m a.s.l., 17 May 2012, Tai-Hui Li, Wen-Juan Yan & Hao

Huang (Holotype, GDGM 30303; GenBank KC168089, KC561775).

EryMoLocy: porophyllus refers to the partially poroid and partially lamellate

hymenophore of the new species.

PiLEus 50-80 mm broad, fleshy, convex, becoming plane to revolute with age,

dry, even, subtomentose, matted subtomentose to slightly scurfy, often faintly

and finely rimulose; grayish-ruby (12D5-6, 12E5). FLesH 5-20 mm thick

at stipe, white to buff, sometimes with a very faint pinkish tint, unchanging

258 ... Yan & al.

when injured, with mild odor and flavor. Hymenophore brownish-orange

(5C4-6, 6C5-6), 2-5 mm deep, decurrent, lamellate near the stipe and strongly

anastomosing, poroid or faveolate towards pileus margin, with 48-76 lamellae;

pores 1.5-3 mm wide near pileus margin. STIPE cylindrical, 33-45 mm long,

5-15 mm wide at apex, attenuated at the base, dry, pale yellow to light yellow

(4A3-4), solid when young, becoming hollow with age, with white basal

mycelium; STIPE CONTEXT white (1A1), unchanging or with pale pinkish flush

when exposed.

BASIDIOSPORES (5.5—)7.5-10.5(-12.8) x (4.0-)5.0-5.8(-7.0) um [n = 30,

Lm = 9.57 + 1.17, Wm = 5.15 + 0.86, Qm = 1.79 + 1.41], ellipsoid, smooth

under light microscope but nearly smooth to finely rugulose or weakly bumpy

under SEM, yellow to olivaceous-yellow in KOH, inamyloid in Melzer’s reagent.

Basip1A clavate, 28-34 x 8.0-11 um, 2-4-spored, hyaline; sTERIGMATA (2-)

3-5(-8) um long. PLEUROCysTIDIA 42-68 x 9.0-12.5 um, fusiform, thin-

walled, hyaline. CAULOCysTIDIA and CHEILOCYSTIDIA absent. HYMENOPHORAL

TRAMA subparallel to nearly bilateral with hyphae of the lateral strata loosely

arranged, hyaline, 4.5-19 um broad. PILEIPELLIS composed of tufts of erect

hyphal elements 4-21 um broad, terminal cells 11-55 x 4.5-20 um, clavate,

brownish-yellow to yellowish. CLAMP CONNECTIONS absent in all tissues.

ECOLOGY AND DISTRIBUTION — Solitary or clustered on soil under Schima

superba Gardner & Champ. (Theaceae) in a mixed forest with Pinus massoniana

Lamb. (Pinaceae) and other frondose trees. May to June. Known only from the

type locality in southern China (Guangdong).

COMMENTS — ‘The new species is considered morphologically intermediate

between Phylloporus and Xerocomus because of its dominantly lamellate

hymenophore near the stipe, with the strongly anastomosing gills forming

a faveolate or poroid pattern towards the pileus margin. The nearly smooth

to finely rugulose spores observed under SEM are quite common in both

Phylloporus and Xerocomus subtomentosus complex (Sutara 2008, Neves &

Halling 2010, Zeng et al. 2012), which also implies that the two genera are

closely related.

Many species of Phylloporus have a partially poroid hymenophore. The

species most similar to X. porophyllus is P. cingulatus Corner with strongly

anastomosing lamellae or poroid hymenophore, but differing in the slowly

cyanescent context on exposure and the smaller spores (7.5-9 x 5-5.5 um;

Corner 1970). Among other species of Phylloporus with strongly anastomosing

lamellae or poroid hymenophore are: P albocarnosus Heinem., P. ater (Beeli)

Heinem., P. boletinoides A.H. Sm. & Thiers, P carmineus Heinem., P. fibulatus

Singer et al., P ochraceobrunneus Corner, P. pumilus M.A. Neves & Halling,

P. purpureus var. ambiguus Heinem., and P. testaceus Heinem. & Gooss.-Font.,

which can all be easily distinguished from X. porophyllus by their pileus colors

Xerocomus porophyllus sp. nov. (China) ... 259

MiRY JOS

Wate

Fig. 2. Xerocomus porophyllus (GDGM 30303, holotype): A. basidiomes; B. basidiospores;

C. pileipellis. Scale bars: A-B = 10 um; C = 20 um.

and sizes of basidiospores or basidiomes. Phylloporus albocarnosus has a white

to straw-colored pileus and smaller spores (7-8 x 3-4 um); P. ater is a blackish

species; P. boletinoides shows a color variability ranging from cinnamon-brown

or dark vinaceous-brown to cocoa-brown; P carmineus is a carmine-red

species; P. fibulatus has a much smaller (11-35 mm) and yellow to dull yellow

pileus; P. ochraceobrunneus has fuscous-brown to brownish-yellow pileus and

260 ... Yan & al.

TABLE 1. Sequence identity comparisons of Xerocomus porophyllus

with species of Xerocomus and Phylloporus.

SPECIES SEQUENCE BLAST MAX SCORE

ITS

Xerocomus sp. ? FJ197009.1 : 695

i (88% query coverage)

(85% max identity)

Xerocomus sp. i EU569235.1 P7605

(88% query coverage)

(85% max identity)

Boletus dryophilus : :

ati ation dryophilus) ot

BODES IODINE ng | AY185183.1 686

(= Xerocomus dryophilus)

Phylloporus sp. : JQ003620.1 : 368

Phylloporus sp. : JQ003650.1 : 359

Phylloporus rhodoxanthus DQ533980.1 346

sen Ehylloporus foliiporus : JQO03641 ip Weta Marra

LSU

Xerocomus fennicus : AF514820.1 : 1127

: (100% query coverage)

: (94% max identity)

Xerocomus ripariellus i AF514818.1 aL

(100% query coverage)

: (94% max identity)

Phylloporus centroamericanus : JQ003663.1 1075

Phylloporus purpurellus JQ003672.1 1016

larger spores (11-15 x 4-4.7 um); P. pumilus has a very small (5-9 mm) cocoa

brown or dark-brown pileus; P. purpureus var. ambiguus has a carmine-purple

pileus and larger spores (9.3-12 x 4.2-5.3 um); and P testaceus has a rose-

ochre to deep carmine-brown pileus and much larger spores (11.5-14.0 x

5.0-6.0 um) (Heinemann 1951, 1955, Corner 1970, Singer et al. 1990, Neves

& Halling 2010, Neves et al. 2012). Although some species of Xerocomus, such

as X. armeniacus (Quél.) Quél. (= Xerocomellus armeniacus (Quél.) Sutara)

and_X. chrysenteron (Bull.) Quél. (= Xerocomellus chrysenteron (Bull.) Sutara),

may have a subtomentose and finely rimulose pileus with similar color as

X. porophyllus, their entirely poroid hymenophore is easily differentiated from

the largely lamellate pattern of the latter.

The low max identities of the sequences demonstrate that the species

compared are obviously different, and the higher max scores with the Xerocomus

species also indicate that X. porophyllus has a closer genetic relationship with

Xerocomus than with Phylloporus (TABLE 1). In a phylogenetic tree constructed

with the LSU sequences of the new species, eight Phylloporus species and eight

other Xerocomus species, with Auricularia auricula-judae (Bull.) Queél. as

outgroup, X. porophyllus is grouped with the other Xerocomus species (FIG. 3).

Xerocomus porophyllus sp. nov. (China) ... 261

65 Xerocomus porosporus (AF050645)

33 Xerocomus chrysenieron (AF 514809)

30 Aerocomus pruinatus (AF 050644)

Xerocomus cisalpinus (AF 514814)

63 Xerocomus dryophiius (AF5i4823)

Xerocomus

Xerocomus fennicus (AF514820)

85 Xerocomus ripariellus (AF 514818)

70 Xerocomus rubellus (AF050649)

Xerocomus porophyllus (KC561775)

” Pinytloporus rhodoxanthus (1Q003675)

Piylloporus pelletieri (AF 456818)

60 Piylioporus centroconericanus (1Q003664)

Phylloporus bogoriens is (10003680) Phylloporss

49] 30 Piylloporus belius (1Q003661)

Pinlioporus purpurelius (1Q003672)

Piylioporus orientalis (JQ003701)

Piylioporus rubiginosus (IQ003694)

Auricularia auricula-judae (AF291289) Outgroup

0.02

Fic.3. Phylogenetic placement of Xerocomus porophyllus inferred from LSU rDNA data. The

bootstrap values (1000 replicates) are shown next to the branches. Taxa are labeled by their names

and GenBank accession numbers.

Therefore, the novel entity is described as a new species of Xerocomus

according to morphological and molecular analyses.

Acknowledgments

The authors are very grateful to Dr. Beatriz Ortiz-Santana (US-Forest Service,

Northern Research Station, Center for Forest Mycology Research, US) and Dr. Matteo

Gelardi for reviewing the manuscript. The study is supported by the National Natural

Science Foundation of China (No. 30970023, 31070024, 30870019) and the Natural

Science Foundation of Guangdong Academy of Sciences for Young Scholars, China (No.

qnjj201202), Science and Technology Planning Project of Guangdong Province, China

(No. 2012B091100258, 2012B091100072).

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697-702. http://dx.doi.org/10.1073/pnas.0235779 100

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MY COTAXON

http://dx.doi.org/10.5248/124.253

Volume 124, pp. 263-268 April-June 2013

Glomus mume and Kuklospora spinosa: two new species of

Glomeromycota from China

BANG-PING CAI’, LIANG-DONG GUO’, JUN-YU CHEN?! & QI-XIANG ZHANG?

‘Xiamen Botanical Garden, Xiamen 361003 China

Systematic Mycology & Lichenology Laboratory, Institute of Microbiology,

Chinese Academy of Sciences, Beijing 100101, China

College of Landscape Architecture of Beijing Forestry University, Beijing 100083 China

*CORRESPONDENCE TO: cbangping@163.com

ABSTRACT — Two new arbuscular mycorrhizal fungal species, Glomus mume (Glomerales)

and Kuklospora spinosa (Diversisporales), were isolated from rhizosphere soil of Prunus mume

in China. Glomus mume resembles G. taiwanense, but differs by its spore walls swelling to a

globose to subglobose structure in or near the middle of the spore apex. Kuklospora spinosa

resembles K. colombiana but differs by the fine spines covering the middle-layer of the outer

spore wall.

KEY worps —morphotaxonomy, Guizhou, Szechwan, HMAS

Introduction

During a survey of arbuscular mycorrhizal fungi associated with Prunus

mume (Siebold) Siebold & Zucc. (Rosaceae) in China, spores of an undescribed

species of Glomus Tul. & C. Tul. were found in the soil samples collected from

the Maolan National Nature Reserve (25°14'-25°18'N 107°56'-108°10'E) of

subtropical woodland in southeast Guizhou province. A second undescribed

species, representing Kuklospora Oehl & Sieverd., was found in the soil samples

collected from the brushy mountain of Muli county (27°58'N 101°15’E) in

Szechwan province. The two arbuscular mycorrhizal fungi are described herein

as Glomus mume and Kuklospora spinosa, based on spore morphology.

Materials & methods

Soils were collected from the rhizosphere of Prunus mume, from 5-20 cm soil depth.

The soil samples of ca. 1000 g were placed in sterilized cotton-cloth bags, labeled, and

+ Prof. Jun-yu Chen, a senior member of Chinese Academy of Engineering, died on 8 June 2012. a

264 ... Cai & al.

air-dried in shade. Then they were cleaned from plant debris, sieved with a 2 mm sieve,

stored at 4°C, until processed.

Spores were isolated according to Gerdemann & Nicolson (1963) with the wet

sieving and decanting method as modified by Daniels & Skipper (1982). Arbuscular

mycorrhizal fungal spores were identified following the descriptions of Schenck &

Pérez (1988), Morton & Redecker (2001), and more recent species descriptions of

Glomeromycota. We follow the classification proposed and summarized by Oehl et al

(2011). Colors were referred to http://invam.caf-wvu.edu/otherinfo/articles/colorchart.

jpg, the INVAM color chart. Specimens were deposited at the Herbarium Mycologicum

Academiae Sinicae (HMAS) in Beijing.

Taxonomy

Glomus mume B.P. Cai, Jun Y. Chen, Q.X. Zhang & L.D. Guo, sp. nov. PLATE 1:A-C

FUNGAL NAME 570036

Differs from Glomus taiwanense by its spore walls swelling to a globose to subglobose

structure, in or near the middle of the spore apex.

Type: China, Guizhou, Libo, Maolan National Nature Reserve, from the rhizosphere of

Prunus mume, 17 Sep. 2005 (Holotype, HMAS143348).

ErymMo.ocy: Referring to the plant species with which this fungus was originally

associated.

SPOROCARPS yellow-brown (20-20-20-0) to dark brown (0-30-40-0), globose

to subglobose, 350-600 x 380-650 um, with spores formed radially in a single,

tightly packed layer around a central plexus of hyphae. Peridium absent. Spores

clavate, cylindro-clavate, triangular or irregular, 90-165 x 30-50 um, tapering

to a cylindric subtending hypha 6-12 um. Chlamydospore wall laminate,

yellow-brown (20-20-20-0) to dark brown (0-30-40-0), 1.5-4 um thick on

the side walls, swelling to a globose to subglobose structure, 13-20 x 15-25 um,

in or near the middle of the spore apex, and thickened to 3-7 um at spore base.

Spore base at hyphal attachment with a small pore, open or occluded by wall

thickening. Reaction to Melzer’s reagent not distinctive.

EcoLocy — Spores of Glomus mume were isolated from soil sampled from

the rhizosphere of Prunus mume growing at 700-800 m a.s.l in the karst forest

area of southwest China (25°19'N 107°57'E). The chemical properties of the soil

sample were: pH 6.62, organic matter 146.27 g.kg’, total N 6.5 g.kg"', organic N

0.51 g.kg’, total P 0.48 g.kg', organic P 0.025 g.kg’, and exchangeable K 0.13

g.kel.

Glomeralean species occurring with G. mume were Claroideoglomus

etunicatum (W.N. Becker & Gerd.) C. Walker & A. Schiissler, Funneliformis

geosporus (T.H. Nicolson & Gerd.) C. Walker & A. Schiissler, Glomus australe

(Berk.) S.M. Berch, G. clavisporum (Trappe) R.T. Almeida & N.C. Schenck, G.

multicaule Gerd. & B.K. Bakshi, G. rubiforme (Gerd. & Trappe) R.T. Almeida &

N.C. Schenck, and Septoglomus deserticola (Trappe et al.) G.A. Silva et al.

Glomus & Kuklospora spp. nov. (China) ... 265

PLATE 1. Glomus mume and morphologically similar species. Glomus mume: A. Broken sporocarp

showing the central hyphal plexus (pl) in Melzer’s reagent; B. Spore in Melzer’s reagent; C. Structure

of the spore wall (sw) in Melzer’s reagent. Glomus clavisporum: D. Portion of the sporocarp. Glomus

taiwanense E. Portion of the sporocarp. Glomus liquidambaris: F. Spores with “paraphysis-like”

structures. Scale bars: A, B, D, E, F = 20 um; C = 10 um.

Kuklospora spinosa B.P. Cai, Jun Y. Chen, Q.X. Zhang & L.D. Guo sp. nov. PLATE 2

FUNGAL NAME 570037

Differs from Kuklospora colombiana by fine spines covering the middle-layer of the

outer spore wall.

Type: China, Szechwan, Muli country, from the rhizosphere of Prunus mume, 5 Sep.

2005 (Holotype, HMAS142950).

266 ... Cai & al.

PLATE 2. Kuklospora spinosa. A. Spore with a distal hypha (hy) in water; B. Spore connections

to the sporiferous saccule (sac) and the cicatrix proximal to saccule (PC) in Melzer’s reagent;

C. Cicatrix proximal to saccule (PC) in Melzer’s reagent; D. Broken spore with three-layered

outer wall (sw1-3), two layers of the middle wall (mw1 & mw2) and the inner wall (iwl & iw2);

iw2 stained by Melzer’s reagent. Scale bars: A, B = 20 um; C, D = 10 um.

Etymo ocy: Latin, spinosa, referring to the ornamentation on the surface of the outer

laminate spore wall layer (sw2).

SPOROCARPS unknown. Spores formed by inflating the hyphal stalk in some

distance to a terminally or intercalary formed sporiferous saccule. Spores

hyaline, subhyaline to yellowish-white (0-10-20-0), globose to subglobose,

100-250 um diam. Spores have three walls: an outer spore wall (sw), a middle

wall (mw) and an inner wall (iw). The outer wall (sw) tri-layered (sw1, sw2 and

sw3): swl hyaline, 1-1.5 um thick, evanescent, usually completely degraded or

sloughed in mature spores; sw2 laminate, yellowish-white (0-10-20-0), 3-5

um thick, covered with fine spines, 2-3 high x 0.5 um in diam, which grow into

swl; sw3 hyaline, 0.5-1 um thick, tightly adherent to sw2, usually separated

from sw2 in Melzer’s reagent, reaction to Melzer's reagent not distinctive. The

middle wall (mw) hyaline, bilayered (mw1 and mw2), thin, less than 1 um thick,

no reaction in Melzer’s reagent. The inner wall (iw) bilayered (iwl and iw2): iwl

membranous, granular, beaded, 0.5-1 um thick, granules sometimes scattering

in crushed spores; iw2 plastic, 0.5-1 um thick, staining pink (0-30-20-0) to

Glomus & Kuklospora spp. nov. (China) ... 267

reddish purple (0—40-60-10) in Melzer’s reagent. Sporiferous saccule hyaline,

globose, 90-150 um diam, collapsing at maturity and leaving a cicatrix proximal

to saccule and a distal hypha or cicatrix. Cicatrix proximal to saccule, 8-15

um diam, distal hypha, 1.5-3 um wide at the spore base. Wall of soporiferous

saccule bilayered, 1.5-3 um thick.

EcoLocy — Spores of Kuklospora spinosa were isolated from soil sampled

under Prunus mume growing at 2450-3100 m a.s.l. in the brushy mountain

of southwest China (27°58’N 101°15'E), The chemical properties of the soil

sample from which G. mume was isolated were: pH 6.48, organic matter 56.32

g.kg', total N 2.5 g. kg’, organic N 0.15 g.kg", total P 0.48 g.kg’, organic P 0.037

g.kg', and exchangeable K 0.2 g.kg".

Glomeralean species occurring in the same samples were Acaulospora

tuberculata Janos & Trappe, Claroideoglomus claroideum (N.C. Schenck &

G.S. Sm.) C. Walker & A. Schiissler, C. luteum (L.J. Kenn. et al.) C. Walker

& A. Schiissler, Diversispora trimurales (Koske & Halvorson) C. Walker &

A. Schissler, Funneliformis geosporus, F. mosseae (T.H. Nicolson & Gerd.)

C. Walker & A. Schiissler, Glomus clavisporum, and Septoglomus deserticola.

Discussion

Glomus mume is morphologically similar to G. clavisporum, G. taiwanense

(C.G. Wu & Z.C. Chen) Y.J. Yao, G. liquidambaris (C.G. Wu & Z.C. Chen) YJ.

Yao, and G. coremioides (Berk. & Broome) D. Redecker & J.B. Morton. The

sporocarpic spores of G. mume can be easily separated from these four species

by the swelling in the middle of the spore apex (PLATE 1A, B). The spore wall

of G. clavisporum is 1-1.5 um thick at the sides, thickened to 17-25 um at the

apex and to 5-8 um at the base (Wu & Chen, 1987; Almeida & Schenck, 1990;

PLaTE 1D). Spore wall of G. taiwanense is 1-1.5 um thick at the sides, thickens

to 4-25 um at the apex and to 1-5.5 um at the base (Wu & Chen, 1987; Almeida

& Schenck, 1990; PLATE 1E). Spore wall of G. liquidambaris is 2-5 um thick at

the sides, thickens to 7.5-25 um at the apex and to 6-10 um at the base, and the

prominent characteristic of this latter species is a “paraphysis-like” structure

protruding from the central plexus of hyphae to the outer part of the sporocarp

(Wu & Chen, 1987; Almeida & Schenck, 1990; PLaTE 1F). Spore wall of

G. coremioides is 2-2.5 um thick, up to 4 um thick near the spore base (Almeida

& Schenck, 1990).

Kuklospora was erected by Sieverding & Oehl (2006), to accommodate

two species previously described in Entrophospora: K. colombiana (Spain &

N.C. Schenck) Oehl & Sieverd. and K. kentinensis (C.G. Wu & Y.S. Liu) Oehl

& Sieverd. Kuklospora spinosa has a morphology typical for the genus based

on the formation of spores within the stalk of the sporiferous saccule and the

presence of a beaded inner wall layer (iw1). Spores of K. spinosa differ clearly

268 ... Cai & al.

from those of K. colombiana and K. kentinensis in size, colour, and outer spore

wall ornamentation.

Acknowledgments

The authors acknowledge the help of Dr. Tiezheng Wei and Yi-Jian Yao, Systematic

Mycology & Lichenology Laboratory, Institute of Microbiology, Chinese Academy of

Sciences, Beijing, China. We especially thank Dr. Ewald Sieverding (Institute of Plant

Production and Agroecology in the Tropics and Subtropics, University of Hohenheim,

Stuttgart, Germany), Prof. Dr. Janusz Blaszkowski (Department of Plant Pathology,

Academy of Agriculture, Szczecin, Poland) and Prof. Dr. Runjin Liu (Institute of

Mycorrhizal Biotechnology, Qingdao Agricultural University, Shandong, China), for

their valuable comments and revisions on the manuscript. We appreciate the corrections

by Shaun Pennycook, Nomenclatural Editor, and suggestions by Lorelei L. Norvell,

Editor-in-Chief. This study was supported in part by the National Natural Science

Foundation of China Grants 30470006, the science and technology item of Xiamen

(3502220072010 and 3502Z20102004).

Literature cited

Almeida RT, Schenck NC. 1990. A revision of the genus Sclerocystis (Glomaceae, Glomales).

Mycologia 82: 703-714. http://dx.doi.org/10.2307/3760157

Daniels HBA, Skipper HD. 1982. Methods for the recovery and quantitative estimation of

propagules from soil. 29-37, in: NC Schenck (ed.). Method and Principles of Mycorrhiza

Research. American Society for Phytopathology. St Paul, Minn.

Gerdemann JW, Nicolson TJ. 1963. Spores of mycorrhizal Endogone species extracted from soil by

wet sieving and decantation. Transactions of British Mycological Society 46: 235-244.

Morton JB, Redecker D. 2001. Two new families of Glomales, Archaeosporaceae and Paraglomeraceae,

with two new genera Archaeospora and Paraglomus, based on concordant molecular and

morphological characters. Mycologia 93(1): 181-195. http://dx.doi.org/10.2307/3761615

Oehl F, Sieverding E, Palenzuela J, Ineichen K, da Silva GA. 2011. Advances in Glomeromycota

taxonomy and classification. IMA Fungus 2: 191-199.

Schenck NC, Pérez Y (eds). 1988. Manual for identification of vesicular-arbuscular mycorrhizal

fungi, INVAM. University of Florida.

Sieverding E, Oehl F. 2006. Revision of Entrophospora and description of Kuklospora and Intraspora,

two new genera in the arbuscular mycorrhizal Glomeromycetes. Journal of Applied Botany and

Food Quality 80: 69-81.

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Taiwan. Transactions of the Mycological Society of the Republic of China 2(2):73-83.

MY COTAXON

http://dx.doi.org/10.5248/124.269

Volume 124, pp. 269-278 April-June 2013

Russula changbaiensis sp. nov. from northeast China

Guo-Jrz Li’”, DONG ZHAO’, SAI-FEI Li’, HUAI-JUN YANG',

Hua-AN WEN™ & XING-ZHONG Liu!™

'State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences,

No 3 'st Beichen West Road, Chaoyang District, Beijing 100101, China

?University of Chinese Academy of Sciences, Beijing 100049, China

° Shanxi Institute of Medicine and Life Science,

No 61 Pingyang Road, Xiaodian District, Taiyuan 030006, China

CORRESPONDENCE TO *: “wenha@im.ac.cn, *liuxz@im.ac.cn

Asstract —Russula changbaiensis (subg. Tenellula sect. Rhodellinae) from the Changbai

Mountains, northeast China, is described as a new species. It is characterized by the red tinged

pileus, slightly yellowing context, small basidia, short pleurocystidia, septate dermatocystidia

with crystal contents, and a coniferous habitat. The phylogenetic trees based on ITS1-5.8S-

ITS2 rDNA sequences fully support the establishment of the new species.

Key worps —Russulales, Russulaceae, taxonomy, morphology, Basidiomycota

Introduction

Theworldwide genus of Russula Pers. (Russulaceae, Russulales) is characterized

by colorful fragile pileus, amyloid warty spores, abundant sphaerocysts in a

heteromerous trama, and absence of latex (Romagnesi 1967, 1985; Singer 1986;

Sarnari 1998, 2005). As a group of ectomycorrhizal fungi, it includes a large

number of edible and medicinal species (Li et al. 2010). The genus has been

extensively investigated with a long, rich and intensive taxonomic history in

Europe (Miller & Buyck 2002). Although Russula species have been consumed

in China as edible and medicinal use for a long time, their taxonomy has been

overlooked (Li & Wen 2009, Li 2013). Only 14 new species and three new

varieties have been reported from China (Singer 1935; Chiu 1945; Ying 1983,

1989; Bi & Li 1986; Zang & Yuan 1999; Wen & Ying 2001; Wang et al. 2009; Li et

al. 2011, 2012), and a systematic investigation of the Russulaceae in China has

also been limited until recently (Wang & Liu 2009, 2010; Das et al. 2010; Buyck

& Atri 2011; Das & Verbeken 2011; Wang et al. 2012).

The Changbai Mountain range on the borders of China, Korea, and Russia,

is considered a region highly diverse in macro-fungi (Xie et al. 1986). During

270 ... Li & al.

a foray in the Changbai Mountains, several interesting Russula specimens were

collected; after thorough examination, they are described here as a new species.

Materials & methods

Fresh specimens were photographed using a Sony Cyber Shot DSC-S75 digital camera,

and macroscopic characteristics of only the intact fresh fruitbodies were recorded under

sunlight in the field. After being dried at 50-60 °C in a SIGG Dorrex food dehydrator,

the specimens were deposited in the Mycological Herbarium, Institute of Microbiology,

Chinese Academy of Sciences (HMAS). Small samples of fresh fruit bodies were

dried in silica gel for DNA extraction. Color names and codes follow Ridgway (1912).

Microscopic characters were examined under a Nikon Eclipse 80i microscope according

to Yang (2000), Wang et al. (2009) and Li et al. (2012). Sulfovanillin (SV) solution was

used to test chemical reactions of the rehydrated specimens.

Protocols for DNA extraction, PCR, and sequencing followed those in Li et al. (2012)

and references therein. Sequences of the internal transcribed spacer (ITS) region were

amplified with the primer pairs ITS1/ITS4 (Gardes & Bruns 1993). PCR products were

purified with the Bioteke DNA Purification Kit (Bioteke, Beijing, China). ITS regions

were sequenced with the ABI 3730 DNA analyzer and ABI Bigdye 3.1 terminator cycle

sequencing Kit (BGI, Beijing, China). Sequences obtained were submitted to GenBank

(accession numbers are shown in Fig 3). Other sequences of representative and closely

related Russula taxa (Eberhardt 2002; Miller & Buyck 2002; Li et al. 2011, 2012; Li 2013)

were retrieved from GenBank. Assembly and editing of sequences of each region were

performed with Clustal X and BioEdit (Thompson et al. 1997, Hall 1999) and were

manually modulated when necessary. Some ambiguously aligned terminal sites were

excluded in sequence alignment.

Based on previous phylogenetic works (Eberhardt 2002, Miller & Buyck 2002,

Ryman et al. 2003, Lebel & Tonkin 2007), Albatrellus flettii Morse ex Pouzar and

Gloeocystidiellum aculeatum Sheng H. Wu were chosen as outgroup taxa.

Maximum Parsimony (MP) analysis of the phylogenetic relationships among the

taxa were performed in PAUP* v.4.01 (Swofford 2004). Gaps in alignment were treated

as missing data, and all sites were treated unordered and unweighted. ‘The tree bisection-

reconstruction (TBR) algorithm was performed with the heuristic search option, and

bootstrap analysis was conducted with 1000 replicates (Felsenstein 1985). Consistency

index (CI), retention index (RI), and tree length (TL) were also calculated. Trees were

displayed with Treeview 1.6.6 (Page 1996).

Taxonomy

Russula changbaiensis G.J. Li & H.A. Wen, sp. nov. Fics 1-2

INDEX FUNGORUM IF 550187

Differs from Russula puellula by wider pileocystidia with crystal contents.

Type: China, Jilin Province, Antu County, Erdaobaihe, Heping Forest Farm, 43°07'N

128°54’E, alt. 1014 m, in coniferous forest dominated by Abies nephrolepis, 22.V1I.2010,

L.D. Guo, X. Sun, G.J. Li & L.J. Xie CBS20100344 (Holotype, HMAS262369, GenBank

KC412162); CBS20100431 (Isotype, HMAS262394; GenBank KC412165).

Russula changbaiensis sp. nov. (China) ... 271

Fic. 1: Russula changbaiensis A. Holotype HMAS 262369; B. HMAS 262355.

272.00 al.

EryMoLoey: named after the mountains of the type locality.

BASIDIOMATA small to medium sized, slender and fragile. PILEUS 2-6 cm

in diam., first hemispherical, then convex to applanate, sometimes slightly

depressed above the stipe, or even shallow infundibuliform, Dark Vinaceous

(xxvuil’’) to Corinthian Red (xxv113"’) at centre rarely with small patches of

Pale Yellow-Orange (11115f), glossy, Prussian Red (xxvu5''k) to Indian Red

(xxv113"'k) when dry, viscous when humid; margin incurved first, straight to

slightly up-curled when mature, sometimes undulate and dehiscent, slightly

striate, Pale Vinaceous (xxvul''f) to Hydrangea Pink (xxvitl’'i), Deep

Corinthian Red (xxv113’'i) to Ocher Red (xxvu15''b) when dry; cutis 1/2-3/4

separable from pileus edge, sometimes with small part exfoliating. LAMELLAE

up to 5 mm height in middle, adnate to slightly subfree, rarely forked near

the stipe, slightly interveined, Cream Color (xv119’f), brittle, Naples Yellow

(x19'd) when dry; edge even, narrowing towards the pileus margin, 11-13

pieces per cm in the edge; lamellulae absent. StrpE 3-5 x 1-1.5 cm, central,

smooth, subclavate to cylindrical, sometimes a little ventricose toward the base,

without annulus, White (L111), partly turning Baryta Yellow (1v21f) when aged

and dry, rarely with small Cinnamon (xx1x15’’) spots at the base, not pruinose,

fragile, hollow when mature. CONTEXT 2-4 mm thick at the pileus center,

White (x11), turning slightly Sulphur Yellow (v25f) when old and injured; taste

mild to slightly acrid, moderately acrid in gills; no distinct odor. SPORE PRINT

Pale Cream to Cream (Romagnesi lamb).

BASIDIOSPORES [100/5/5] (6.6-)6.9-7.9(-8.1) x (5.8-)6.0-6.6(-7.3)

um, Q = (1.03-)1.11-1.24(-1.28) (Q =1.19 + 0.05), subglobose to broadly

ellipsoid, verrucose; ornamentation amyloid, composed of warts mostly

fused into fine lines, cristulate to subreticulate, with some isolated verrucae,

less than 1.0 um high; plage distinctly amyloid. Basrp1a 26-34 x 8-13 um,

clavate, inflated towards upper half, 4-spored, projecting 5-10 um beyond

hymenium, hyaline, smooth; sterigmata 2-4 um, pointed, straight to slightly

curved. PLEUROCYSTIDIA few, 43-56 x 6-10 um, originating in subhymenium,

projecting 25-30 um beyond hymenium, thin-walled, clavate to subfusiform,

with refractive contents grayish in SV in middle and lower parts; apices obtuse,

often with a moniliform to papillate appendage. CHEILOCysTIDIA not observed;

lamellar edge sterile. PILEIPELLIS two layered. EprcuTis a trichoderm 150-250

uum thick, composed of 3-4 um thick, slender hyaline hyphae, occasionally

with intracellular pigmentation particles. PILEOCYSTIDIA numerous, 70-130 x

6-8 um, originating in subcutis, clavate, slender, septate, with crystal contents

ash black in SV; apices obtuse. SUBCUTIS a cutis composed of recumbent,

hyaline, 2.5-4 um diam hyphae. TRAMA composed of connective hyphae 2-5

um diam and hyaline, globose to subglobose, 25-40 um diam sphaerocyststs.

STIPITIPELLIS a cutis not well developed, composed of thin-walled, septate,

Russula changbaiensis sp. nov. (China) ... 273

/ Z Zp oNas ss

S 7A SSN

Fic. 2 Russula changbaiensis HMAS 262369 (holotype).

A. Basidiospores (SEM); B. Basidia; C. Pleurocystidia; D. Pileipellis.

cylindrical, hyaline hyphae 3-6 um wide; caulocystidia absent. CLAMP

CONNECTIONS absent in all tissues.

ECOLOGY & DISTRIBUTION: Solitary in conifer and conifer-broadleaf forest

(often has a preference for Abies spp.). DISTRIBUTION: China (Jilin). SEASON:

July.

274 ... Li & al.

OTHER SPECIMENS EXAMINED: CHINA, JILIN PROVINCE, ANTU CouNTY, Hepingyingzi,

43°07'N 128°54’E, alt. 882 m, in mixed coniferous-broadleaf forest, 21.VII.2010, L.D.

Guo, X. Sun, GJ. Li & LJ. Xie CBS20100044 (HMAS262381, GenBank KC412164);

CBS20100514 (HMAS262376, GenBank KC412163); Changbai Mountains Forest

Ecological System Research Center, 43°23''N 128°05’E, alt. 811 m, in mixed coniferous-

broadleaf forest, 25.VII.2010, X. Sun & G.J. Li CBS20100299(HMAS262355, GenBank

KC412161).

Comments: The combination of slender basidiomata, red tinged pileus

with slightly striate margin, separable pileipellis, small basidia, few short

pleurocystidia, and pluriseptate dermatocystidia placed this new species

in sect. Rhodellinae Romagn. in Russula subg. Tenellula Romagn. Russula

changbaiensis is characterized by its slightly yellowing context, cream spore

print, dermatocystidia with crystal contents, and coniferous habitat. The nine

other species with a reddish tinged pileus in sect. Rhodellinae are R. arpalices

Sarnari, R. conviviales Sarnari, R. font-queri Singer, R. impolita (Romagn.) Bon,

R. melzeri Zvara, R. pseudoimpolita Sarnari, R. puellula Ebbesen et al., R. rhodella

E.-J. Gilbert, and R. zonatula Ebbesen & Jul. Schaff. (Romagnesi 1967, 1985;

Sarnari 2005). All are differentiated from R. changbaiensis as follows: Russula

arpalices has a strongly yellowing context and distinctive Pelargonium-like odor;

R. conviviales has an ocher spore print, partly subulate epicutis terminal cells,

and wider pileocystidia (8-12 um, Sarnari 2005) with golden yellow contents;

R. font-queri has obviously yellowish-orange tinged pileus and lamellae, pale red

tinged stipe, yellow spore print, context with a taste of old fruit, longer basidia

(28-50 x 8.5-12.5 um, Romagnesi 1967), and pleurocystidia (40-85 x 7-12 um,

Romagnesi 1967); R. impolita has an opaque pileus cutis, fruity odor, spores

with isolated warts, and finely encrusted dermatocystidia; R. melzeri has an

epicutis composed of large and short hyphal cells growing from a subcutis made

up of sphaerocyst-like cells; R. pseudoimpolita has a velutinous pileus, barely

changing context, ampullaceous pileipellis hyphal terminals, and association

with Quercus; R. puellula has larger hyaline basidia, slender dermatocystidia

(3-6 um, Sarnari 2005), and an association with beech and oak deciduous

forest; R. rhodella has an ocher to pale yellow spore print, and isolated spore

ornamentation; R. zonatula is recognized for its obviously discolored pileus,

yellowish spore print, isolated spore ornamentation, pleurocystidia over 90

um long, and an association with beech forest (Romagnesi 1967, 1985; Sarnari

2005).

Russula taxa with reddish tinged pilei originally described from China are

R. chichuensis W.F. Chiu, R. griseocarnosa X.H. Wang et al., R. handelii Singer,

R. jilinensis G.J. Li & H.A. Wen, R. minutula var. minor Z.S. Bi, R. taliensis

W.E. Chiu, R. pseudovesca J.Z. Ying, R. punicea W.F. Chiu, and R. zhejiangensis

GJ. Li & H.A. Wen. It is easy to discriminate between these species and

R. changbaiensis: R. chichuensis has a white spore print, large pleurocystidia <130

Russula changbaiensis sp. nov. (China) ... 275

um long, and no pileocystidia; R. griseocarnosa differs in the larger pileus (5-12

cm diam.), slightly pinkish stipe, and water-soaked grayish context; R. handelii

has hyaline pleurocystidia and a stipitipellis with numerous caulocystidia; R.

jilinensis has a yellowish green tinged pileus center, mild tasting context, and

ocher to yellow spore print; R. minutula var. minor differs in the small (0.8-2.0

cm) pileus and pileipellis with primordial hyphae but without pileocystidia;

R. taliensis is recognized by its pruinose opaque pileus, yellowish spore print,

and spores with rare and isolated ornamentation; R. pseudovesca can be clearly

perceived by its whitish spore print, numerous pleurocystidia >80 um in

length, and pileocystidia with 0.9-1.8 um thick cell walls; R. punicea has a

strongly acrid context that does not change color when old and injured; and

R changba@ensis KC 412161 HMAS262355

R changbaensis KC 412162 HMAS262369 Holotype

R changbdensis KC 412163 HMAS262376

R changba@ensis KC 412164 HMAS262331

R changbaiensis JX425401 HMAS262394

R odorata AY061698 mubg, feels

R versicolor JX425394 HMAS250966

9 R versicolor JX425372 HMAS131941

R versicolor JX425401 HMAS262394

R versicolor AY061722

R versicolor JX425372 HMAS131941

R nauseosa AY061733

cy) R abiefina EU598179

R solaris AF418627

R pueliuia AY061710

R puellaris AY061709

R messapica AY061692

69 R azurea AY061660

R lilacea AY061731

100 R cessans AY061730

R laricina AY061685

109 R jilinensis HQ693524 HMAS262364

R pilinensis GU966632 HMAS 194253

R curfipes AY061668

100_ R aduiterina AY061651

R cuprea AY061667

81 R meizeri AY061691

R zhejiangensis GQ344559 HMAS 187071

R paludosa AY061703

72 R risigalina AY061713

R furet AY061720

7Q R amoenipes AY061656

R pascua AY061705

R xerampelina AY061734

R nifida AY061696

R sphagnophiia AY061719

R maculata AY061688

100 R auranfiaca AY061658

R rosea AY061686

R lepidicolor AY061687

74) R amethystina AY061653

R rubra AY061717

94 R claroflava AY061665

R vinosa A¥061724

72 R infegriformis AY061684

100 R velenovskyi AY161721

R pseudointegra AY061708

A flefit AY061733

G aculeafum AY061739

— 5changes

Fic. 3: Phylogenetic relationship tree of Russula changbaiensis and other related Russula species

inferred from the dataset of ITS1-5.8S-ITS2 sequences. Consistency index (CI) = 0.486, retention

index (RI) = 0.647, and tree length (TL) = 842. MP bootstrap support values >50% are indicated

above the branches.

276 ... Li & al.

R. zhejiangensis has a bright red pileus with dark center, dark cream to ocher

spore print, longer (<74 um) pleurocystidia, and a habitat of subtropical

evergreen forest (Singer 1935, Chiu 1945, Bi & Li 1986, Ying 1989, Wang et al.

2009, Li et al. 2011, 2012).

Phylogenetic results

The dataset comprised 38 Russula taxa with particular attention paid to

closely related Russula subg. Tenellula members and other red-capped Russula

species. The sequence length was 534 nucleotides. In the MP analysis of ITS1-

5.8S-ITS2 sequences, 264 characters were constant, 185 variable characters were

parsimony-informative, and 85 characters were parsimony-uninformative.

In the phylogenetic tree (Fic. 3), R. changbaiensis sequences formed a

conspicuous clade, showing enough phylogenetic distance to support them

as an independent taxon. Russula changbaiensis and most members of subg.

Tenellula form a clade with a moderate bootstrap support of 52%, the same

support as cited in Miller & Buyck (2002). The molecular analyses do support

R. changbaiensis within subg. Tenellula. However, the phylogenetic relationships

between R. changbaiensis and other subg. Tenellula taxa are still unclear; no

stable topology can be established because there are so few corresponding

sequences from dependably morphologically identified specimens.

Acknowledgments

The authors are grateful to Dr. T. Lebel (National Herbarium of Victoria, Royal

Botanic Gardens Melbourne) and Dr. K. Das (Botanical Survey of India, Sikkim

Himalayan Regional Centre) for carefully reviewing the manuscript; to Ms. H.M. Li,

Ms. A.R. Yan and Dr. T.Z. Wei (HMAS) for providing the herbarium specimens; to

Ms. X.F. Zhu for inking in line drawings, B.A. Jaffee (University of California Davis),

Dr. S.R. Pennycook (Landcare Research), Dr. L.L. Norvell (Pacific Northwest Mycology

Service), and Dr. J.Y. Zhuang for improving the manuscript; to Dr. L.D. Guo, Dr. X.

Sun, Dr. X.L. Wang, Mr. Z.M. Li, Ms. S.F. Yue, Mr. D. Zhao, Ms. J.X. Yang (Institute

of Microbiology, CAS), Ms. L.J. Xie, Dr. C.-Y. Sun and Ms. J. Zhou (National Institute

of Occupational Health and Poison Control, China CDC) for assistance in collecting

specimens and lab work; to Dr. C.L. Li and Ms. J.N. Liang (Institute of Microbiology,

CAS) in SEM photographing. This project was supported by the National Natural

Science Foundation of China (No. 30770013) and International Cooperation Program

of Ministry of Science and Technology of the People’s Republic of China (No.

2009DFA31160).

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MY COTAXON

http://dx.doi.org/10.5248/124.279

Volume 124, pp. 279-300 April-June 2013

Studies on three rare coprophilous plectomycetes from Italy

FRANCESCO DOVERI”*, SABRINA SARROCCO, & GIOVANNI VANNACCI

Department of Agriculture, Food and Environment, University of Pisa,

80 via del Borghetto, 56124 Pisa, Italy

* CORRESPONDENCE TO: f.doveri@sysnet.it

ABSTRACT — The concept of plectomycetes is discussed and their heterogeneity emphasised.

Three ascohymenial cleistothecial ascomycetes, collected or isolated from herbivore or

omnivore dung in damp chamber cultures, are described. Emericella quadrilineata and

Lasiobolidium orbiculoides are discussed and compared morphologically with similar taxa.

A key to Lasiobolidium and the related Orbicula is provided. The importance of the second

worldwide isolation of Cleistothelebolus nipigonensis and the difficulties of distinguishing it

from Pseudeurotium species are stressed. The Italian collection of C. nipigonensis from canid

dung is compared with the original strain from wolf, and its epidermoid peridial tissue is

regarded as one of the main morphological differentiating features from Pseudeurotium

ovale. The morphological characteristics of the monospecific genus Cleistothelebolus are

discussed and compared with those of Pseudeurotiaceae and Thelebolaceae, particularly with

Pseudeurotium and Thelebolus. [TS and LSU rDNA sequences of the Cleistothelebolus isolate

support its placement in Thelebolaceae.

Key worps — coprophily, phylogeny, Pyronemataceae, Thelebolales, Trichocomaceae

Introduction

Our twenty-year study on fungi growing on faecal material (Doveri 2004),

both in the natural state and in damp chamber cultures, has allowed us to

record from Italy several species of plectomycetes, which must be regarded as

an assemblage of heterogeneous Ascomycota characterised by small, globose

or subglobose, prototunicate asci, irregularly disposed in a centrum of

cleistothecial or gymnothecial ascomata (Ulloa & Hanlin 2000, Geiser et al.

2006, Stchigel & Guarro 2007, Kirk et al. 2008). Most are pleomorphic, often

with a hyphomycetous asexual state, whose features have a primary taxonomic

value in separating genera and families and in establishing their phylogenetic

relationships. Many are saprotrophic, growing on decaying wood or vegetables,

soil, and dung, while some are parasites of human and mammal tissues

(Udagawa 1987).

280 ... Doveri, Sarrocco & Vannacci

Plectomycetes are found in different orders and families of discomycetes,

pyrenomycetes, and loculoascomycetes (Cain 1956, Benny & Kimbrough

1980). Phylogenetic studies resulting from gene sequencing combined with

cladistics (Sugiyama et al. 1999, Suh & Blackwell 1999) have confirmed their

great heterogeneity.

The aim of this work is to provide a detailed morphological description of

Emericella quadrilineata and Lasiobolidium orbiculoides, two plectomycetous

fungi recently isolated by us from dung, and to analyse their relationships

with similar taxa. We also include an extensive study of the second collection

worldwide of the very rare Cleistothelebolus nipigonensis, attempting to

determine, by morphological, physiological and preliminary molecular data

(ITS and LSU rDNA sequences), its connection with Thelebolaceae Eckblad

and other families with an uncertain position in Leotiomycetes O.E. Erikss. &

Winka.

Material & methods

Collection of Emericella quadrilineata and Lasiobolidium orbiculoides

Specimens of E. quadrilineata (on dried sheep dung) and L. orbiculoides (on dried

goat dung) were collected in the Tuscan archipelago (North Tirreno sea) and placed

in a non-sterilised damp chamber according to Richardson & Watling (1997) and

Richardson (2001) as slightly modified by Doveri (2004). Cultured material, incubated

at room temperature (20-25°C) under natural light but not exposed to direct sunlight,

was examined every day under a stereomicroscope. Ascomata of both species grew

fast, maturing after 7 days incubation, those of E. quadrilineata directly on dung, while

those of L. orbiculoides on blotting paper at the dung base, partly obscured by the thick

meshes of an aerial mycelium. Only three specimens of E. quadrilineata were produced,

which were picked up from dung by a sterile needle, utilised for the microscopic

study, preserved later in a luted slide. Specimens of L. orbiculoides were produced

in a large quantity; however, all the attempts to isolate this fungus in axenic culture

were unsuccessful. Part of the material was utilised for the microscopic study and for

preservation as dried material.

Isolation of Cleistothelebolus nipigonensis

Cleistothelebolus nipigonensis was obtained from dried canid dung collected in

northern Italy, placed in a non-sterilised damp chamber, and cultured following the

same methods utilised for the other two species. Mature ascomata were hardly noticed

after 20 days incubation but, owing to their smallness and staying in hiding, they may

have appeared a few days earlier. Ascomata were picked up from dung by a sterile

needle and crushed on PDA (Potato Dextrose Agar, Difco Lab.) amended by 2 mg L”

streptomycin, for primary isolation. Plates were incubated for 14-15 days at room

temperature (20-25°C) under natural light. After incubation, plates were observed at

the stereomicroscope (up to 100x) and germinating groups of ascospores were selected,

Emericella, Lasiobolidium, & Cleistothelebolus spp. (Italy) ... 281

picked out of the agar and transferred to PDA plates using a glass needle. Pure cultures

on PDA were used to obtain single-spore isolates.

100 ul of a water suspension of ascospores were placed on WA (Water Agar, 15 g

dissolved in 1 L of distilled water) plates by a sterile micropipette and incubated at 25 +

2°C 12h/12h light/darkness. Monoascosporic isolations were transferred to PDA.

Morphological studies

Emericella quadrilineata, L. orbiculoides and C. nipigonensis were examined

microscopically in mounts of water, Congo red, and methyl blue or cotton blue in lactic

acid. Fifty ascospores discharged from mature asci derived from at least two ascomata

were measured in water.

Cultural studies

To assess the effect of different substrates on the growth of C. nipigonensis, a 6 mm

diameter agar disk, collected from the actively growing margin of PDA colonies, was

placed in the centre of 90 mm Petri dishes containing CMA (Corn Meal Agar, Difco

Lab.), MA (Malt Agar, Difco Lab.), NA (Nutrient Agar, Difco Lab.), PDA, V8 agar (Gams

et al. 1998), and Emerson YpSs agar (Yeast protein Soluble starch Agar) (Gams et al.

1998). Five plates of each medium were incubated at 24 + 2°C 12h/12h light/darkness.

Radial growth of the colonies was recorded at different times from the second day of

incubation. Regression and variance analysis of growth curves were performed by using

SigmaPlot package (SPSS Inc., Chicago, IL) and Graphpad Prism 5.02 Software. P<0.05

was assumed as significant level.

Molecular studies

DNA EXTRACTION, AMPLIFICATION AND SEQUENCING — Mycelium for DNA

extraction, produced on PDA plates overlaid with a cellophane membrane at 25°C,

was scraped from the plates with a sterile scalpel, placed in a 1.5 ml tube (0.15 g) and

extracted using the DNeasy® Plant Mini Kit (QIAGEN) according to the manufacturer's

instructions. Polymerase Chain Reaction (PCR) was used to amplify the LSU and the

ITS regions of the nuclear ribosomal DNA, employing the following primers: LROR

and LR3 for the first 650 kb of the LSU gene and ITS1 and ITS4 for the ITS region

(Gardes & Bruns 1993, White et al. 1990). Amplification reaction mixtures contained

25-50 ng of template DNA, GoTaq*Green Master Mix (Promega) 1X and 0.5 mM of

each primer in a volume of 50 uL (Doveri et al. 2010). Amplification was performed in

a GeneAmp® PCR System 2400 (Perkin Elmer) using the following parameters: for LSU

initial denaturation step at 94°C for 5 min, 35 cycles consisting of denaturation at 94°C

for 1 min, annealing at 52°C for 1 min and extension at 72°C for 2 min, final extension

of 72°C for 7 min; for ITS initial denaturation step at 94°C for 1 min, 30 cycles consisting

of denaturation at 94°C for 30 s, annealing at 54°C for 1 min and extension at 72°C for 1

min, final extension of 72°C for 4 min. After the final extension of 72°C, reactions were

held at 4°C. PCR products were purified by the QIAquick PCR Purification Kit (Qiagen)

according to the manufacturer's protocol and submitted to sequencing. Samples to be

sequenced were processed by the DNA Sequence Facility at the Bio Molecular Research

(BMR), Servizio di Sequenziamento —- CRIBI, University of Padova (Italy).

282 ... Doveri, Sarrocco & Vannacci

Results

Cultural studies

Equations resulting from regression analysis obtained for each medium were

all highly significant (R*>0.880, P<0.001). From slopes comparison, growth rate

of Cleistothelebolus nipigonensis on YpSs (a=1.7 mm d") was statistically higher

than on all the other media (P<0.001) (data not shown).

Molecular study

Amplifications produced sequence lengths of about 600 bp (ITS) and 650

bp (LSU). Comparison of our ITS sequence (KC492060) with those deposited

in GenBank resulted in high similarity percentages (96%) with Antarctomyces

Stchigel & Guarro and other uncultured thelebolaceous fungi. Comparison

of the LSU sequence (KC492061) within the same database confirmed the

close relationship between C. nipigonensis and two species of Thelebolus

(T! ellipsoideus and T. globosus, 97% similarity).

Taxonomy and discussion

Emericella quadrilineata (Thom & Raper) C.R. Benj., Mycologia 47: 680, 1955 PL. 1

AscomatTA cleistothecioid, 300-350 um diam., globose, non-ostiolate,

smooth, purple-red but appearing straw coloured and slightly granulose-

reticulate at first sight, as surrounded and obscured by dense yellowish masses

of Hiille cells and sparse hyphae.

HULLE CELLS terminal, globose, exceptionally ellipsoidal, 15-25 um

diam., hyaline in the middle, with yellowish, up to 5 um thick walls, arising

out of hyaline, branched, septate, slightly encrusted hyphae, not forming a

pseudostalk at the peridial base; PERrp1um thin-walled, purple-red, a layer of

firmly pressed, narrow (about 1 um diam.), purple-red hyphae; PARAPHYSES

absent; Asci irregularly disposed, 8-spored, globose to broadly ellipsoidal,

8.5-11 x 8-9 um, thin-walled, lacking an apical apparatus, evanescent;

ASCOSPORES Oblate, globose in frontal view, ellipsoidal in side view, 4-5(-5.5)

x 3-3.5 um, bivalved, hyaline at first, becoming purple-red, smooth, with four

slightly raised equatorial crests, less than 0.5 um wide, seemingly with an entire

margin; ASEXUAL STATE only some hyaline, globose, echinulate conidia, about

3 um diam., observed in association with ascomata.

EcCOLoGY & DISTRIBUTION — Three specimens collected from sheep (Ovis

aries) dung in a damp chamber culture. April. Rare. Known from one island

coastal site in Italy, possibly cosmopolitan. Usually isolated from soil.

SPECIMEN EXAMINED — ITALY, Tuscany, Livorno, Capraia island, 9°49'13"N

43°01'29"E, 50 ma.s.L., 11.IV.2011, leg. L. Levorato (Herbarium F. Doveri 007.12).

Most plectomycetes are distributed in Eurotiomycetidae Doweld, particularly

in Eurotiales G.W. Martin ex Benny & Kimbr. and Onygenales Cif. ex Benny

Emericella, Lasiobolidium, & Cleistothelebolus spp. (Italy) ... 283

PLaTE 1. Emericella quadrilineata (Herbarium F. Doveri 007.12): A-B, clusters of Hiille cells

(arrows) around the peridial wall; C, asci with conglobate ascospores; D, asci in different stages of

maturity and some free ascospores; E-F, ascospores. Scale bars: A = 40 um; B-D = 10 um; E = 12

um; F = 8 um.

& Kimbr. Besides the cleistothecial ascomata and evanescent asci, these two

orders share a thin, variously structured, usually brightly coloured or pale

peridium, absence of an hamathecium, and small, usually brightly coloured,

284 ... Doveri, Sarrocco & Vannacci

one-celled, oblate ascospores often with equatorial ornamentations (Kirk et

al. 2008). Blastic phialoconidiogenesis distinguishes Eurotiales (von Arx 1987)

from Onygenales, where a thallic conidiogenesis produces both arthro- and

aleurioconidia (Benny & Kimbrough 1980, Kirk et al. 2008).

In Eurotiales, Trichocomaceae E. Fisch. (= Eurotiaceae Clem. & Shear)

encompasses several genera (including Emericella Berk.) characterised by

brightly coloured ascomata with a cellular or hyphal peridium, often catenulate

asci, usually saturnine (with equatorial crests) ascospores (Benny & Kimbrough

1980, Malloch 1985), and Aspergillus P. Micheli ex Link, Paecilomyces Bainier,

or Penicillium Link asexual states (Malloch & Cain 1972, Kirk et al. 2008). They

are cosmopolitan and usually soil inhabitants or saprotrophs on decaying plants;

some are medically or industrially important as producers of antibiotics or

toxins, sometimes behaving as opportunistic pathogens (Cannon & Kirk 2007)

or agents of food spoilage (Geiser et al. 2006). Molecular phylogenetic studies

(Berbee et al. 1995, Ogawa et al. 1997, Sugiyama 1998) have demonstrated that

Trichocomaceae is monophyletic.

Berkeley (1857) erected Emericella for E. variecolor Berk. & Broome, a

species with purplish long-spiny bordered ascospores and small cleistothecial

ascomata supported by a spongy column, from which numerous threads extend,

terminating in large globose cells. These stromatic (Malloch & Cain 1972),

thick-walled, intercalary or end cells surrounding cleistothecia (Ulloa & Hanlin

2000, Geiser et al. 2006, Kirk et al. 2008) are called “Hiille cells” (Malloch 1985).

In Trichocomaceae they are typical of Fennellia B.J. Wiley & E.G. Simmons and

Emericella; Fennellia has yellow or green, often confluent ascomata, hyaline

or pale ascospores, and often elongated Hiille cells (Wiley & Simmons 1973,

Locquin-Linard 1990, Guarro et al. 2012), and Emericella has usually discrete,

red or purplish to dark brown cleistothecia with a peridium of compressed

hyphae, and oblate, purple-red, orange or violet, rarely pale ascospores usually

ornamented with equatorial ridges. Emericella also has globose Hiille cells and

an Aspergillus asexual state (von Arx 1974, Hanlin 1998, Guarro et al. 2012) of

the so called “A. nidulans group” (Benjamin 1955, Christensen & Raper 1978,

Christensen and States 1982, Pitt & Samson 2007), which is the equivalent

of Aspergillus sect. Nidulantes (Gams et al. 1985). About forty species are

recognised at the present (Guarro et al. 2012) in Emericella; they are airborne

soil inhabitants (Stchigel & Guarro 1997, Samson et al. 2002), rarely growing

elsewhere (Pitt and Samson 2007), thermotolerant, but not thermophilic (Zalar

et al. 2008), and prefer warm climates and dry substrata (Samson & Mouchacca

1974). Some are opportunistic pathogens, uncommon agents of animal and

human infections (Balajee et al. 2007, Verweij et al. 2008).

Emericella quadrilineata is characterised by rapid growth in culture,

comparatively large ascomata, 8-spored asci, and smooth purple-red ascospores

Emericella, Lasiobolidium, & Cleistothelebolus spp. (Italy) ... 285

with four low equatorial crests (Thom & Raper 1939). According to Balajee et

al. (2007) and Verweij et al. (2008) the number of these inconspicuous crests

can be detected only by electron microscopy, but we think it can be detected

also by careful focusing under a light microscope.

Emericella acristata (Fennell & Raper) Y. Horie, E. miyajii Y. Horie, and

E. parvathecia (Raper & Fennell) Malloch & Cain are morphologically

indistinguishable from E. quadrilineata (Guarro et al. 2012) and possibly the

same species: E. acristata was originally described without equatorial crests

(Fennell & Raper 1955) but others have cited two (Ismail et al. 1995) or four low

crests (Horie et al. 1996, Guarro et al. 2012); E. miyajii has four inconspicuous

crests, and E. parvathecia has two (Raper & Fennel 1965).

The closely related E. nidulans (Eidam) Vuill. var. nidulans is also

morphologically indistinguishable from E. quadrilineata except for having

ascospores with two equatorial crests. Molecular studies that support

microscopic examinations have helped separate the two species (Verweij et al.

2008, Matsuzawa et al. 2012). Emericella quadrilineata, usually isolated from

soil (Thom & Raper 1939), has been also isolated sometimes from herbal drugs

(Horie 1979), once from white wine (Stchigel et al. 1999), and now from dung.

It is an opportunistic pathogen (Balajee et al. 2007), agent of onychomycosis

(Gugnaniet al. 2004) and rarely causes invasive aspergillosis (Drakos et al. 1993,

Verweij et al. 2008). Emericella nidulans, which is thermotolerant (Chen and

Chen 1991), cellulolytic (Reese & Downing 1951), possibly ligninolytic (Bull &

Carter 1973), and opportunistically pathogenic (Balajee et al. 2007, Verweij et

al. 2008) has been isolated not only from plants (Benjamin 1955), rotting wood

(Eidam, 1883, Lumley et. al. 2000), and soil (Chen & Chen 1991) but also from

rabbit (Benjamin 1955) and barking deer (Piasai & Manoch 2009) dung, goat

droppings, pellets of free-living birds, and fresh human faeces (Domsch et al.

1993).

We know very few records of other Emericella species from dung, apart from

E. foveolata Y. Horie from armadillo (Horie et al. 1996) and E. rugulosa (Thom

& Raper) C.R. Benj. from rat (Jeamjitt et al. 2007). Aspergillus recurvatus Raper

& Fennell, lacking a sexual state but related to Emericella as belonging to the

nidulans-group (Raper & Fennell 1965), was isolated from lizard dung.

Lasiobolidium orbiculoides Malloch & Benny, Mycologia 65: 655, 1973 PL. 2-3

Ascomara cleistothecioid, 200-500 um diam., globose to broadly ellipsoidal,

semi-membranous, yellowish at first, becoming yellow-ochraceous, finally

brownish, granulose due to protruding groups of peridial cells, fully covered by

dense flexuous, <4 mm long hairs.

PERIDIUM two-layered, pseudoparenchymatous: endostratum with textura

angularis composed of pale, thin-walled, polygonal cells, 5-8 um diam.,

286 ... Doveri, Sarrocco & Vannacci

exostratum thin, weakly dextrinoid, with textura globulosa—angularis composed

of thick-walled, yellowish brown cells, 12-28 x 11-22 um, interspaced with

some irregularly lobed cells; Hairs arising from the outermost exoperidial cells,

6-7.5 um diam., yellowish, smooth, thick-walled (up to 3 um), wavy, sometimes

slightly coiled, sparsely septate, more densely septate at their base, with rounded

tips and an usually swollen, bulbous, sometimes lobed base, 10-20 um diam;

PARAPHYSES apparently scarce and short, soon vanishing, mixed with asci, 2-3

um diam., not enlarged at the apex, septate, often branched in the lower portion,

filled with a few pigments; Ascr irregularly disposed, ephemeral, unitunicate,

non-amyloid, inoperculate, 8-spored, cylindrical, 80-90 x 10-11 um, thin-

walled, rounded at the apex, without an apical apparatus, with a very short,

slightly lobate stalk; ascospores regularly uniseriate, oblate, 11-12.5 x 9.5-11

x (7.5)8.5-10 um, globose to subglobose in frontal view, broadly ellipsoidal in

side view, hyaline to pale yellowish, smooth, thick-walled, uninucleate, with a

de Bary bubble in aqueous media, forming a powdery mass at maturity, when

asci disintegrate; ASEXUAL STATE unknown.

EcoLocy & DIsTRIBUTION — Hundreds of gregarious, superficial specimens

collected from blotting paper at the base of goat (Capra hircus) dung in a damp

chamber culture. April. Rare. Known from one island coastal site in Italy,

possibly cosmopolitan, also known from other herbivore dung, exceptionally

from soil.

SPECIMEN EXAMINED — ITALY, Tuscany, Livorno, Capraia island, 9°49'13"N

43°01'29"E, 50 ma.s.L., 11.IV.2011, leg. L. Levorato (Herbarium F. Doveri 006.12).

Malloch & Cain (1971) erected the monospecific genus Lasiobolidium,

describing L. spirale as a non-stromatic plectomycete with cleistothecial

ascomata, a differentiated peridium with helically coiled appendages, ephemeral

clavate asci, and hyaline, ellipsoidal ascospores. Malloch & Benny (1973) later

described a second species, L. orbiculoides, so called for its strong resemblance

to the monospecific genus Orbicula Cooke.

Lasiobolidium and Orbicula were initially placed (Malloch & Cain 1971,

Malloch & Benny 1973, Benny & Kimbrough 1980) in Eoterfeziaceae G.F.

Atk., now regarded as a plectomycete family with an uncertain position in

Pezizomycotina O.E. Erikss. & Winka (Cannon & Kirk 2007, Kirk et al. 2008,

Lumbsch & Huhndorf 2010). Jeng & Krug (1976) later transferred the genera

to the tribe Theleboleae Korf (= Thelebolaceae) of Pyronemataceae Corda

(Pezizales J. Schrot.), but phylogenetic studies (Hansen et al. 2005, Hansen

PLATE 2. Lasiobolidium orbiculoides (Herbarium FE. Doveri 006.12): A-B, ascomata (arrows) in

culture; C-D, details of exoperidium; E, free ascospores between paraphyses (black arrows) and

an immature ascus (white arrow); FE uniseriate ascospores inside a thin-walled (arrows) 8-spored

ascus. Scale bars: A = 100 um; B = 500 um; C, E= 10 um; D = 15 um; F= 8 um.

Emericella, Lasiobolidium, & Cleistothelebolus spp. (Italy) ... 287

288 ... Doveri, Sarrocco & Vannacci

PLatE 3. Lasiobolidium orbiculoides (Herbarium FE. Doveri 006.12): A-D, details of hairs

and some free ascospores; E, ascospores containing de Bary bubbles in aqueous medium;

F-G, ascospores. Scale bars: A = 10 um; B—D = 15 um; E = 50 um; F-G = 12 um.

Emericella, Lasiobolidium, & Cleistothelebolus spp. (Italy) ... 289

& Pfister 2006, Perry et al. 2007) place them in Pyronemataceae, a family of

apothecioid discomycetes, with a minority of cleistothecial fungi evolved from

an apothecial ancestor (Hansen et al. 2005).

Lasiobolidium orbiculoides is characterised by fast growth in culture,

wavy peridial hairs, cylindrical short-stalked asci, and oblate medium-sized

ascospores. The morphological characteristics of our collection fully match the

protologue, and its ascospores contain a large de Bary bubble when mounted

in aqueous media, a feature also reported by others (Janex-Favre & Locquin-

Linard 1979, Bell 2005).

The main differences between L. orbiculoides, six other recognised Lasiobolidium

species (Kirk et al. 2008), and Orbicula parietina (Schrad.) S. Hughes are

reported in the following key, partly based on Yaguchi et al. (1996) and Guarro

et al. (2012):

Worldwide key to Lasiobolidium and Orbicula

la Asctcylindrical aAscosp ones -Uniseriate ia. sae aga yang Haga eg hae Haas Haale 2

1b. Asci subglobose to claviform. Ascospores biseriate to conglobate, ellipsoidal ..... 4

2a. Ascospores broadly ellipsoidal, 11-12 x 8-9 um (Moustafa & Ezz-Eldin

NSD sou a8 saeas trash Sere san beres fe ste, Meas Soaca aa, AEE auleseTace lest lios Sree Rates L. aegyptiacum

PB ASCOUSDOPES OD EIEG oon enw tsce a cob ximsnee Mapiavttong # wots» Mote bead sattgenst Lane detent dale 3

3a. Cleistothecia usually not flattened at their bases. Peridium thin, appearing

one-layered, covered all over with hairy, wavy or sometimes irregularly coiled

appendages. Asci short- or non-stalked. Ascospores 11-12.5 x 9.5-11 x

8.5-10 um (9.8-14 x 9-12 um; Malloch & Benny 1973). From dung,

rarely from soil and vegetable material ....................044. L. orbiculoides

3b. Cleistothecia often with a flattened base. Peridium thicker, two-layered. Hairy

appendages hyphoid, present at the base only. Asci long-stalked. Ascospores

8-10.5 x 8-10.5 x 7-9 um (7-13 x 8-10 um; Hughes 1951). From several kinds

obduirs omveretabbleaiate nial yt aaa eu wah wi Og ow oes od gn ase O. parietina

4a, Ascospores. longer than TO prince yx, gave 5 we eek 9 ware ongdcye Pccege Pega Vkaeae Baa Een 5

AG AScosporessshOrters 2.720. <0 acetate ee mee wR ae OR, ee A, RR RN Re AN SB RRB, 6

5a. Hairs straight or somewhat flexuous, nodulose, septate, 2.5-3 um diam. Asci

subglobose or broadly clavate. Ascospores slightly warted, 18-24 x 12-18 um

(Yaouchivet dl -1996) SRTOnY SOU. ve. 2a «wad ket dig eh ict he nt sae L. gracile

5b. Hairs helically coiled, non-septate, 5-10.5 um diam. Asci clavate. Ascospores

smooth, 12-17 x 9-12 um (Malloch & Cain 1971). From dung, once from

wesetablest of ir rcs 0. aan nf ake clack atk ee ene ae ow bs L. spirale

6a. Hairs helical, non-septate. Ascospores 8-9 x 5-6 um (Locquin-Linard 1983). From

Poatland Shee prune Fe ks eee oa «lat Mes le oe Eo pa L. helicoideum

Ob Tarts-diflerentisrshapes cr. bbe. ew ke, Sos hey eee e ee A ey eh they eh ahs thee Ae dite hea ag 7.

290 ... Doveri, Sarrocco & Vannacci

7a. Hairs uncinate, not septate. Ascospores 8-10 x 5.5-7 um (Locquin-Linard 1983).

Fromrgazelleccit GAs em oR, Oc el 0A. ti Secdars ule trite tags we te L. recurvatum

7b. Hairs wavy, septate. Ascospores 9-10 x 5.5-7 um (Locquin-Linard 1983). From

camel-aird. other ting ulate dune ioe ie. Me eee Wei meena nie oe L. fallax

Guarro et al. (2012) regard L. aegyptiacum Mustafa & Ezz-Eldin is ‘practically

indistinguishable from L. orbiculoides, but Mustafa & Ezz-Eldin (1989)

originally described L. aegyptiacum with broadly ellipsoidal ascospores, quite

different from the oblate ascospores of the latter.

Although Lasiobolidium is regarded as coprophilous (Kirk et al. 2008) only

L. fallax Locgq.-Lin., L. helicoideum Locq.-Lin., and L. recurvatum Locq.-Lin.

have been isolated solely from dung (Locquin-Linard 1983, Abdullah & Alutby

1999). Lasiobolidium orbiculoides has been recorded from deer (Malloch &

Benny 1973), gazelle (Janex-Favre & Locquin-Linard 1979), donkey (Abdullah

& Alutby 1999), goat (Elshafie 2005), and possibly marsupial dung (Bell 2005)

as well as from cultivated soil (Horie et al. 1992). Moustafa & Sharkas (1982)

proved the cellulolytic activity of L. orbiculoides isolated from soil and grown

in culture on filter paper. Its growth in our culture on blotting paper around

the dung base confirms this activity. Simple or compound cellulose is found in

plants and their derivatives, soil, and dung, so it is understandable that some

cellulolytic fungi can develop on all these substrata without distinction, and it

is possible that all other Lasiobolidium species are cellulolytic.

Cleistothelebolus nipigonensis Malloch & Cain, Can. J. Bot. 49: 851,1971 PL. 4-5

COLONIES ON EMERSON YPSs medium attaining 60 mm diam. at 18 days,

doughy-waxy, pink, with thin and soft concentric rings, which are more patent

and brown shaded in the centre, margin even, reverse deeper pink, with clearly

outlined central rings due to the presence of immersed, brown ascomata (YpSs

medium resulted the best medium, suggesting complex nutrient requirements);

COLONIES ON PDA and V8 juice morphologically similar to colonies on YpSs;

CoLoniges ON MA with radial soft grooves and with a wavy and sublobate

margin, reverse deeper pink with more patent concentric rings and radial

grooves; COLONIES ON NA very pale pink, with an indented margin; ASCOMATA

cleistothecioid, globose, pale to dark brown at maturity, smooth, membranous,

60-80 um diam.

PLATE 4. Cleistothelebolus nipigonensis (DS 26817): A, ascomata on dung in a damp chamber;

B, ascoma in water; C-L, colonies at room temperature 18 days incubation in Petri dishes (C-D, in

Emerson YpSs agar; E-F, in PDA; G-H, in MA; I-J, in V8 agar; K-L, in NA; D, F, H, J, L, reverses).

Scale bars: A = 40 um; B = 30 um; C-F, K-L = 20 mm; G-J = 15 mm.

Emericella, Lasiobolidium, & Cleistothelebolus spp. (Italy) ... 291

292 ... Doveri, Sarrocco & Vannacci

PERIDIUM two-layered, pseudoparenchymatous, the inner layer hyaline,

the outer brown, both a textura epidermoidea of irregularly lobed, thin-walled

cells, 4-10 x 3-8 um, sometimes interspaced with spots of textura angularis;

INTERASCAL TISSUE absent; Ascr not born from croziers, irregularly disposed,

evanescent, unitunicate, inoperculate, non—amyloid, 8-spored, 10-14 x 9.5-10

um, globose or broadly ellipsoidal or even broadly clavate, then short-stipitate;

ASCOSPORES conglobate, 5-5.5 x 3.5-4 um, usually broadly ellipsoidal with

rounded ends, but also broadly ovoidal (Q = 1.37-1.42; Q average = 1.39),

sometimes hardly inequilateral, hyaline to pale yellow, thin-walled, lacking both

germ pores and de Bary bubbles; AsEXxuAL sTaTE filamentous, lacking phialides:

HYPHAE 1-3 um diam., thin-walled, hyaline, wavy, densely septate, splitting at

septa into swollen arthrospore-like cells in older colonies, branched, with long

or even short branches at right angles, sometimes diverticulate, often swollen

at the septa up to 5 um diam., abundantly vacuolate, with vacuoles particularly

observable in Congo red; conrp1A 3.5-7 x 2-3 um, 1-celled, smooth, hyaline,

thin-walled, cylindrical to ellipsoidal, with a truncate pedicel, born from very

small, cylindrical conidiophores along the upper hyphal side or at the apex of

short branches, usually budding.

EcoLocy & DIsTRIBUTION — More than fifty, gregarious or often crowded

and confluent, superficial specimens, collected from canid (possibly fox) dung

in a damp chamber culture. July. Very rare, known from one mountain site

(1300 m a.s.l.) in Italy, and from wolf (Canis lupus) dung of the type locality in

Ontario (Canada).

SPECIMEN EXAMINED — ITALY, VENETO, Vicenza, Roana, Cesuna, 11°27'29"N

45°50'21"E, 6.VII.2008, leg. R. Cerello and G. Robich (DS 26817; GenBank KC492060,

KC492061).

The name Cleistothelebolus means “Thelebolus developing cleistothecia and

emphasises the resemblance between these two coprophilous genera, which

usually share ascoma and ascospore shape, size, and colour. Sharp differences,

however, are noticeable, such as the presence of blastoconidia, production of

true cleistothecia, irregularly disposed ephemeral asci, lack of an interascal

tissue, and mesophily in Cleistothelebolus Malloch & Cain (Malloch & Cain

1971) versus no or a simple asexual state with production of arthrospores,

cleistohymenial apothecioid ascomata, persistent asci, presence of paraphyses,

and psychrophily (de Hoog et al. 2005) in Thelebolus Tode.

Coprotiella Jeng & J.C. Krug (Thelebolaceae) is similar to Cleistothelebolus

but differs in having larger and light coloured ascomata, a peridium ofa textura

PLATE 5 (right). Cleistothelebolus nipigonensis (DS 26817): A, free clustered conidia (white arrow)

and anamorph hyphae with short conidiophores bearing conidia (black arrow); B, detail of the

anamorph with some budding conidia (arrows); C, detail of exoperidial textura epidermoidea;

D, exoperidial pocket of textura angularis (white arrow) beneath some free ascospores and pale

Emericella, Lasiobolidium, & Cleistothelebolus spp. (Italy) ... 293

7 i

PONE Pr VeRy OLA

P we *

“Sars, co No Ne

Z hale alee ee

a 2

endoperidial cells (black arrow); E, ascospores; F, an empty ascus (arrow), asci with ascospores,

and free ascospores; G, 8-spored asci. Scale bars: A = 25 um; B, F = 15 um; C, G = 10 um; D = 20

um; E = 12 um.

294 ... Doveri, Sarrocco & Vannacci

angularis to globulosa, globose ascospores with a conspicuous de Bary bubble,

and in lacking an asexual state (Jeng & Krug 1976).

We agree with Malloch & Cain (1971) that Cleistothelebolus has a striking

morphological resemblance to Pseudeurotium J.EH. Beyma, the type genus

of Pseudeurotiaceae Malloch & Cain. This family is characterised by non-

stromatic, usually dark cleistothecial ascomata with a pseudoparenchymatous

peridium, (sub)globose, thin-walled, ephemeral asci, lacking apical apparatus

and irregularly arranged in the centrum, absence of interascal tissue, and small

usually smooth one-celled ascospores lacking germ pores. Pseudeurotiaceae,

which have a hyphomycetous, phialidic (Acremonium- or Stilbella-like) or

sympodial (Sporothrix- or Teberdinia-like) asexual state, can be isolated from

wood, soil, or dung (Malloch & Cain 1970, Kirk et al. 2001, Cannon & Kirk

2007, Stchigel & Guarro 2007). Pseudeurotium, with its superficial, dark, globose

ascomata, a peridium with a textura angularis, brown ascospores at maturity,

and a sympodial asexual state, differs from Connersia Malloch, Leuconeurospora

Malloch & Cain, and Pleuroascus Massee & E.S. Salmon. Connersia has

hyaline, ellipsoid-reniform, inaequilateral ascospores (Booth 1961, von Arx

1987) and lacks conidia (Malloch 1974); the psychrophilic, cephalothecoid

Leuconeurospora has ridged to reticulate ascospores (Malloch & Cain 1970, von

Arx et al. 1988), and Pleuroascus has ascomata with helical appendages and

hyaline ascospores (Massee & Salmon 1901, Malloch & Benny 1973, Barrasa &

Moreno 1984). Pseudogymnoascus Raillo, included in Pseudeurotiaceae by Wang

et al. (2006), is easily differentiated from Pseudeurotium by its gymnothecial

ascomata, fusoid ascospores, and a Geomyces Traaen asexual state (Rice &

Currah 2006).

Pseudeurotium ovale, with ovoidal-broadly ellipsoidal ascospores (Stolk

1955), differs from the majority of recognised Pseudeurotium species (Sogonov

et al. 2005) such as P. bakeri C. Booth, P. desertorum Mouch., P. macroglobosum

Matsush., P luteolum Matsush., and P. zonatum J.F.H. Beyma, which have

spherical ascospores (Beyma 1937; Routien 1957; Booth 1961; Mouchacca 1971;

Matsushima 1975, 1996). Pseudeurotium ovale differs also from P. irregulare

Lodha and P. jaipurense Lodha, which have irregularly ovoidal or subglobose

ascospores (Lodha 1971). The ascospore shape and size in P. ovale are similar

to those of Cleistothelebolus nipigonensis, which, however, can be distinguished

by a “lighter colored peridium, less complex ascogenous system, and lack of

phialides” (Malloch & Cain 1971). Based on our collection C. nipigonensis and

numerous reports on P. ovale (Stolk 1955, Booth 1961, Udagawa 1965, Ahmad

& Sultana 1973, Dennis 1981, Eriksson 1992, de Hoog et al. 2000), we will note

some additional differences between these two taxa. Cleistothelebolus ascomata

are smaller (15-80 versus 90-250 um diam.), asci are larger (9-14 x 8-10.5

versus 7-9 x 6.5-8 tm) and often short-stipitate (rather than globose to broadly

Emericella, Lasiobolidium, & Cleistothelebolus spp. (Italy) ... 295

ovoid or ellipsoid), and the peridium is two- instead of one-layered. The nature

of the Cleistothelebolus peridium is particularly noticeable: in our collection it is

formed of a TEXTURA EPIDERMOIDEA, not unmentioned but clearly depicted in

the protologue (Malloch and Cain 1971). A pseudoparenchymatous peridium

was also described in P ovale but depicted as a TEXTURA ANGULARIS (Stolk 1955,

Udagawa 1965, de Hoog et al. 2000). The habitat of the two species also differs:

originally isolated from nematode cysts (Stolk 1955), P ovale has been isolated

from soil (Udagawa 1965), grass, wood chips, and decaying algae (Richardson

2004), but only rarely from dung (Ahmad & Sultana 1973, Eriksson 1992),

whereas the only two C. nipigonensis collections come from wolf (the holotype)

and fox (ours) dung.

Based on morphological (Kimbrough & Korf 1967, Eckblad 1968)

and ultrastructural studies (Brummelen 1998), the Thelebolaceae were

accommodated in Pezizales, but the placement was later questioned by rDNA

sequence data (Momol & Kimbrough 1994, Momol et al. 1996). Thelebolus

and related genera were excluded from Pezizales based on morphological

and ultrastructural (Samuelson & Kimbrough 1978, Kimbrough 1981) and

molecular phylogenetic studies (Landvik et al. 1998, Mori et al. 2000, Gernandt

etal. 2001, Hansen et al. 2005). Molecular analysis of the nuclear SSU rDNA gene

(de Hoog et al. 2005) confirmed the placement of Thelebolus, Caccobius Kimbr.,

and Ascozonus (Renny) E.C. Hansen within the separate family Thelebolaceae

(Thelebolales PF. Cannon), near the leotialean fungi and far from those orders

to which they were assigned previously. Thelebolaceae now are considered allied

with the Leotiomycetes, as stated in a recent study by Hansen & Pfister (2006)

on the molecular systematics of Pezizomycetes O.E. Erikss. & Winka.

Cleistothelebolus was originally placed with Lasiobolidium, Orbicula, and

other cleistothecial fungi, in Eoterfeziaceae (Malloch & Cain 1971, Benny

& Kimbrough 1980), a family with an uncertain position in Ascomycota

morphologically similar to Pseudeurotiaceae but with an unknown asexual

state (Kirk et al. 2001, Cannon & Kirk 2007).

Cleistothelebolus was later transferred (Jeng & Krug 1976) to Pyronemataceae

trib. Theleboleae, which on the whole is the equivalent of the current

Thelebolaceae.

Landvik et al. (1997) suggested that genera other than Thelebolus belonging

to small pezizalean families with uncertain affinities (like Cleistothelebolus)

might be related to Leotiales and noted that further phylogenetic analyses

are needed to explain the relationships of Cleistothelebolus, which Malloch

& Cain (1971) originally regarded as probably evolved from Thelebolaceae.

Lumbsch & Huhndorf (2007, 2010) suggested placing Cleistothelebolus within

Pyronemataceae, but further phylogenetic studies are needed there as well.

Our ITS and LSU sequence analyses support Cleistothelebolus nipigonensis in

Thelebolaceae.

296 ... Doveri, Sarrocco & Vannacci

Acknowledgements

The authors wish to thank Prof. J. Guarro, Prof. Kevin Hyde, and the MycoTaxon

editors for critical revision of the manuscript, Gianni Robich and Lucia Levorato for

providing part of the material studied, and Maurizio Forti for technical support.

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MY COTAXON

http://dx.doi.org/10.5248/124.301

Volume 124, pp. 301-308 April-June 2013

Morphological and genetic characterisation of

Beauveria sinensis sp. nov. from China

MING-JUN CHEN’, Bo HUANG", ZENG-ZHI LI’ & JOSEPH W. SPATAFORA ?

"Anhui Provincial Key Laboratory of Microbial Control, Anhui Agricultural University,

West Changjiang Road 130, Hefei, Anhui 230036, China

? Department of Botany and Plant Pathology, Oregon State University,

Corvallis, Oregon 97331, U.S.A

* CORRESPONDENCE TO: bhuang@ahau.edu.cn

ABSTRACT —Beauveria sinensis sp. nov. was isolated from a larva of Geometridae

(Lepidoptera) collected from Tiantangzhai, Anhui province, China. It is characterized by

elongated ellipsoidal to cylindrical conidia, solitary conidiogenous cells that are cylindrical

or with slightly swollen bases, white to pale pink colony in culture, and small mycelial pellets

consisting of curved hyphae. Phylogenetic analyses of translation elongation factor-1 alpha

(TEF), RNA polymerase II largest subunit (RPB1), and RNA polymerase II second largest

subunit (RPB2) sequence data support it as a unique species and sister to B. sungii. It does not,

however, form a monophyletic group with other elongated ellipsoidal to cylindrical conidia-

producing species in Beauveria. Type isolates and holotype are deposited in the Research

Center for Entomogenous Fungi of Anhui Agricultural University (RCEF).

KEY worpDs —Ascomycetes, taxonomy, entomopathogenic fungi

Introduction

Beauveria Vuill. is a globally distributed genus of soil-borne and

entomopathogenic hyphomycetes. It is of particular interest as a model

system for the study of entomopathogenesis and the biocontrol of pest insects

(Rehner & Buckley 2005). Beauveria species are characterized by whorled

conidiophores and dense clusters of sympodial and globose or flask-shaped

short conidiogenous cells with apical denticulate rachi that give rise to one-

celled hyaline conidia. Conidial shape in Beauveria can be globose, ellipsoidal,

cylindrical, or comma-shaped.

Recent phylogenetic analyses of Beauveria inferred from partial sequences

of RNA polymerase II largest subunit (RPB1), RNA polymerase II second

largest subunit (RPB2), translation elongation factor-1 alpha (TEF) genes, and

the Bloc nuclear intergenic region (Bloc) resolved 12 clades, each of which

302 ... Chen &al.

includes one recognized species (Rehner et al. 2011). Since then Zhang et al.

(2013) have described B. lii Sheng L. Zhang & B. Huang from China, a species

with large cylindrical to ellipsoidal conidia. In Beauveria, there are eight species

with ellipsoidal, cylindrical, or comma-shaped conidia: B. amorpha (Hohn.)

Minnis et al., B. asiatica S.A. Rehner & Humber, B. brongniartii (Sacc.) Petch,

B. caledonica Bissett & Widden, B. lii, B. malawiensis S.A. Rehner & Aquino

de Muro, B. sungii S.A. Rehner & Humber, and B. vermiconia de Hoog & V.

Rao (Bissett & Widden 1988, de Hoog & Rao 1975, Rehner et al. 2006, 2011;

Samson & Evans 1982, Shimazu & Hashimota 1988, Zhang et al. (2013). Five

species with globose to subglobose conidia include the type of the genus,

B. bassiana (Bals.-Criv.) Vuill., and B. australis S.A. Rehner & Humber,

B. kipukae S.A. Rehner & Humber, B. pseudobassiana S.A. Rehner & Humber,

and B. varroae S.A. Rehner & Humber (Rehner et al. 2011).

Recently, we isolated a Beauveria with elongated ellipsoidal to cylindrical

conidia from Anhui province, China, infecting Geometridae larvae. ‘The isolate

possessed unique cultural and morphological characteristics and did not form

a monophyletic group with other Beauveria species that produce cylindrical

conidia. We concluded that it represents a new species and describe it here as

B. sinensis.

Materials & methods

Collection of specimens and isolation

An entomogenous specimen (TTZ070716-26) was collected from Tiantang zhai,

Jinzhai county, Anhui province, China, in July 2007. The host was identified as belonging

to Geometridae (Lepidoptera). Strain RCEF3903 was isolated from this collected

specimen on SDAY (1% w/v peptone, 4% w/v dextrose, 0.2% w/v yeast, 1.5% w/v agar)

medium.

Strain identification

Morphological characteristics are based on cultures incubated on quarter-strength

SDAY (SDAY/4) at 25 °C for 14 d. The isolated fungus was examined using classical

mycological techniques based on growth rate as well as macroscopic and microscopic

characteristics. The ex-type culture and a dried-culture holotype specimen has been

deposited in the Research Center for Entomogenous Fungi, Anhui Agriculture

University, Hefei, Anhui, China (RCEF).

DNA extraction, PCR and sequencing

Conidia were inoculated onto SDAY medium overlaid with a disc of sterilized

cellophane. After incubating at 25 °C for approximately 7 days, genomic DNA was

extracted from the mycelia scraped from the cellophane using modified CTAB method

(Gardes & Bruns 1993, Spatafora et al. 1998). The extracted DNA was stored in sterile

distilled water to a final concentration of 1-2 ng ul’ and stored at -20 °C.

To perform phylogenetic analysis of the isolated Beauveria strains (Zhang et al. 2013;

Rehner et al. 2011), the partial sequences of three nuclear loci, including TEF, RPB1,

Beauveria sinensis sp. nov. (China) ... 303

and RPB2, were amplified as described by Rehner & Buckley (2005) and Rehner et al.

(2011). After purifying PCR product using EasyPure quick gel extraction kit (TransGen

Biotech), DNA sequencing was performed at Sangon Company (Shanghai, China) and

the resulting sequences of RCEF3903 were submitted to GenBank.

Sequence alignment and phylogenetic analyses

DNA sequences generated in this study were assembled and edited using Codoncode

Aligner (version 3.6.1). Sequences of TEE, RPB1, and RPB2 from 69 taxa ( 68 Beauveria

isolates and one Isaria tenuipes strain as outgroup ) based on the results of Zhang et

al. (2013), Rehner & Buckley (2005), and Rehner et al. (2006) were downloaded form

GenBank. Multiple sequence alignments for TEF, RPB1, and RPB2 constructed using

Clustal X 1.81 (Thompson et al. 1997) were concatenated into a single file using the

program Mafft (Katoh et al. 2002), and MEGA was used to output a Nexus file for

phylogenetic analysis. The datasets were analysed using both maximum parsimony

(MP) and Bayesian algorithms.

PAUP* 4.0b10 (Swofford 2002) was used to perform maximum parsimony analyses

on the combined dataset of TEF, RPB1, and RPB2 with 1000 replicates of heuristic

search of random sequence additions, branch swapping algorithm by tree bisection-

reconnection (TBR) and MulTrees on. Insertions and deletions were minimized by

direct examination and treated as missing data. Ambiguously aligned sequence regions

were excluded from the data matrix before analysis. Branch support was estimated by

bootstrapping using 1000 replicates with full heuristic search (Felsenstein 1985). Clades

with bootstrap values =70% were considered strongly supported by the data.

Bayesian analyses were conducted with an online version of MrBayes 3.0b4

(Huelsenbeck et al. 2001) through the CIPRIS web portal. 1,000,000 Markov Chain

Monte Carlo (MCMC) generations were performed with four chains (three cold, one

heated) and trees were saved every 100 generations for a total of 100,000 trees. Analyses

were repeated two times and converged close to the same value (the standard deviation

of split frequencies about 0.005) and first 25% of trees were discarded as burn-in. The

consensus tree with the support values for each branch constituting their posterior

probability (=95% were considered as significantly supported by the data) was combined

into a single tree file. Posterior probabilities for branches receiving =95% support are

reported below the respective branches on the single best tree from the Bayesian analysis

(Fic. 1). The bootstrap values for branches supported in =70% are listed above branches

iBiGs

Results

For RCEF3903, PCR amplification yielded TEF, RPB1, and RPB2 amplicons

of 1714 bp, 2866 bp, and 2118 bp. The combined alignment of the above

genes included a total of 6698 characters. After ambiguously aligned positions

excluded, the final alignment comprised 6028 bp (TEF: 1004; RPB1: 2860;

RPB2: 2164), of which 771 were parsimony informative. Maximum parsimony

analysis of 70 taxa dataset yielded 48 equally most-parsimonious trees of 2018

tree length with consistency indices (CI) of 0.7096 and retention indices (RI)

of 0.9091. One of nine equally most-parsimonious trees is shown in Fic. 1 with

304 ... Chen &al.

bootstrap values =70% above the relevant branches. Bayesian analysis resulted

in the same topology as the MP analysis shown in Fie. 1.

The phylogenetic analysis of combined data set well supported RCEF3903

as a sister group to the B. sungii clade containing seven individuals. All isolates

from this clade produced ellipsoidal or oblong conidia in culture (Rehner et

al. 2011), and RCEF3903 differs from these seven isolates in conidial size. The

molecular data and analyses also suggest a wide relationship of RCEF3903 with

B. caledonica group, B. amorpha group, B. malawiensis group and B. lii.

Taxonomy

Beauveria sinensis Ming J. Chen, Z.Z. Li & B. Huang, sp. nov. PLATES 2-7

MycoBank MB 801325

Differs from all other Beauveria species by its pale pink colony and production of

mycelial pellets consisting of curved hyphae.

Type: China, Anhui Province, Jinzhai County, isolated from a larva of Geometridae, July

2007, coll. Mingjun Chen (Holotype, RCEF3903-DAC1; ex-type culture RCEF3903;

GenBank HQ270151, JX524283, JX524284).

EryMo_oey: sinensis, for the country of origin, China.

Colony on SDAY/4 attaining a diameter of 23-31 mm after 14 days at 25 °C,

aerial mycelium white, dense and cottony at first, slowly becoming pale pink and

producing considerable amounts of mycelial pellets on older portions, regular

in the margin, reverse light yellow-brown, odor indistinct. The mycelial pellets

attaining 95.0-150.0 um in diameter after 14 days at 25 °C, and consisting of

curved hyphae. Typical hyphae hyaline, smooth-walled, septate, branched,

1.8-2.5 um diam., curved mycelium a little slighter. Conidiophores arising on

aerial hyphae, similar to aerial hyphae, mostly simple. Conidiogenous cells

mostly borne on conidiophores, occasionally on aerial mycelium, solitary,

flask-shaped to cylindrical, 8.5-20.0 x 1.6-3.0 um, tapering into a long slender

denticulate rachis, geniculate or irregularly bent, 5.0-10.0 x 0.5-0.6 um.

Conidia elongate ellipsoidal to cylindrical, one-celled, hyaline, smooth, 3.0-5.0

x 1.5-2.0 um (L™ = 4.1 um, W™ = 1.7 um, Q™ = 2.4), chlamydospores absent.

Teleomorph not observed.

Discussion

We tried to amplify the Bloc loci of B. sinensis several times with Bloc

primers described by Rehner et al. (2011), but failed to get its PCR production.

The phylogenetic relationship among the Beauveria inferred from three nuclear

loci was mainly consistent with those inferred from four nuclear loci (Rehner

et al. 2011). Furthermore, Rehner et al. (2011) emphasized that each of the four

loci used to reconstruct the phylogeny of Beauveria was effective for accurate

diagnosis of all 12 species based on multiple species-specific phylogenetically

Beauveria sinensis sp. nov. (China) ... 305

RCEF3903 =~ 8B. sinensis

98/100 ARSEF1685

ARSEF5689

| ARSEF7043 ssh

100°TOO"| ARSEF7044 B. sungit

ARSEF7280

-/100 ARSEF7281

ARSEF7279

ARSEF1567

| ARSEF 2567

100/100 | aRSEF8024 B. caledonica

00/100 ARSEF2251

ARSEF7117

ARSEF 4302

ARSEF2922 —» JB. vermiconia

ARSEF1969

ARSEF4149

100/100 ARSEFBS518a B. amorpha

ARSEF7542

100) ABSEF2641

/ ARSEF4384 B. asiatica

ARSEF4850

00/100_ ARSEF4580

ARSEF4598 B. australis

ARSEF4622

99/100 ARSEF617

ARSEF979

00/100 ARSEFJe276

ARSEF985

ARSEF4362

ARSEF4363

ARSEF7516

ARSEF7517

oO ARSEF2271

ARSEF 2831 B. brongniartii

ARSEF7376

| ARSEF6213

| ARSEF6214

100/160 k ARSEF6215

| ARSEF7058

| ARSEF10277

| ARSEF10280

ARSEF7268

ARSEF10278

~ ARSEF300

100/106 ARSEF1811

ARSEF1848

100/100 ARSEF1040 B. bassiana

ARSEF 1478

ARSEF751

ARSEF 1564

saoeret ARSEF7518 i

ARSEF7032 —> B. kipukae

ARSEF2694

Ais ARSEF8257 B. varroae

100/1 ARSEF8259

RCEF5500 ——» 2 Ji

ARSEF1855

ARSEF3216

ARSEF3529

ARSEF4933 4,

100/100 ARSEF3220 B. pseudobassiana

ARSEF3405

ARSEF7242

ARSEF2997

ARSEF6229

100/100 | ARSEF4755 . .

ARSEF7760 B. malawiensis TIsaria tenuipes

ARSEF17613

ARSEF7260

¢ 4 250 changes

Fic. 1. A single tree from the maximum parsimony analysis showing phylogenetic relationships

among species of Beauveria inferred from a combined dataset of TEF, RPB1, and RPB2 data.

Bootstrap values =70% and posterior probabilities =95% are labeled above and below the

appropriate branches.

306 ... Chen & al.

Fics 2-7. Beauveria sinensis. 2. Colony on SDAY/4, showing mycelial pellets. 3. Typical hyphae.

4, Mycelial pellets. 5. Conidiophores, conidiogenous cells, and conidia. 6. Curved hyphae.

7. Phialide and conidia. Scale bars: 2 = 50 mm; 3, 5, 6, 7 = 10 um; 4 = 30 um.

informative nucleotide characters. Therefore, the position of B. sinensis in the

tree from the combined analysis of RPB1, RPB2, and TEF data is reasonable

and reliable.

In culture, B. sinensis is distinguishable by a pale pink colony and production

of mycelial pellets consisting of curved hyphae. Most species of Beauveria are

white to buff on solid media except B. malawiensis, whose colonies become

light pink in older portions. Mycelial pellets consisting of curved hyphae are

formed by B. sinensis isolates but are unknown in any other Beauveria species.

Subcultured for five generations, the isolates still produced significant amounts

of mycelial pellets. The unique stable growth behavior may prove to be of

taxonomic value in Beauveria, with additional isolates discovered.

In Beauveria, there were three species with ellipsoidal conidia (B. brongniartii,

B. asiatica, and B. sungii) and four species with elongate ellipsoidal to cylindrical

conidia similar to the new species (B. amorpha, B. caledonica, B. malawiensis,

and B. lii). TABLE 1 provides a comparative summary of the main characters

of B. sinensis and the other four species with elongate ellipsoidal to cylindrical

conidia. Microscopically, B. sinensis is distinguished by conidiogenous cell

shape from B. amorpha, B. caledonica, and B. malawiensis, all of which produce

whorls of conidiogenous cells with globose to subglobose bases. Beauveria

malawiensis is further distinguished by its short and thick rachis, B. amorpha

Beauveria sinensis sp. nov. (China) ... 307

TABLE 1. Morphological comparisons of Beauveria spp. with

elongated ellipsoidal to cylindrical conidia.

COLONY CONIDIA CONIDIAL SHAPE CONIDIOGENOUS CELL

SPECIES

(um)

B. amorpha White to yellow 3.5-5 x Cylindrical, Whorled, globose to

1.5-2 often flattened subglobose base,

on one side or basal part 2-4 um

slightly curved

B. caledonica White to cream 3-5 x Ellipsoidal to Simple to whorled, swollen

to dull buff 1-1.8 cylindrical base, 2.6-4.2 x 1.6-3.3 um

B. lii White to cream 3.1-10.1x — Ellipsoidal to Solitary, occasionally

1.4-3.6 cylindrical, tight clusters of

occasionally 2-3, base ellipsoidal to

obovoid cylindrical 4.8-9.0 pm

B. malawiensis White to pale 3.7-4.5 x Cylindrical Whorled, base globose to

pink, slightly 1.3-1.9 obpyriform, 2.0-6.6 um

zonate

B. sinensis White to pale 3-5 x Elongate Solitary, slightly or no

pink, curved 1.5-2 ellipsoidal to swollen base, 8.2-20

mycelium in cylindrical x 1.6-3.0 um

mycelial pellet

has a characteristically shaped conidium that is often slightly curved or flattened

on one side (Samson & Evans 1982), and B. lii produces the largest conidia

(Zhang et al. 2013).

In the Bayesian analysis and maximum parsimony of TEF, RPB1, and RPB2

sequence data, Beauveria sinensis was the sister species to B. sungii group

according to Rehner et al. (2011), and was distinct from other Beauveria

species with cylindrical conidia. Thus, the phylogenetic studies also supported

the recognition of Beauveria sinensis as a distinct species.

Acknowledgments

We are grateful to Prof. Z.Q. Liang and Dr. L. Wang for reviewing the manuscript.

We also thank Drs. Shengli Zhang and Yue Cai for their kind help with molecular work.

This work was supported by the Natural Science Foundation of China (No.31200114

and 31070009).

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Canadian Journal of Botany 66(2): 361-362. http://dx.doi.org/10.1139/b88-057

de Hoog GS, Rao V. 1975. Some new hyphomycetes. Persoonia 8: 207-212.

Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution

38: 783-791. http://dx.doi.org/10.2307/2408678

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

Huelsenbeck JP, Ronquist F. 2001. Mr Bayes: Bayesian inference of phylogeny. Bioinformatics 17:

754-755. http://dx.doi.org/10.1093/bioinformatics/17.8.754

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Katoh K, Misawa K, Kuma K, Miyata T. 2002. MAFFT: a novel method for rapid multiple sequence

alignment based on fast Fourier transform. Nucleic Acids Research 30(14): 3059-3066.

http://dx.doi.org/10.1093/nar/gkf436

Rehner SA, Buckley E. 2005. A Beauveria phylogeny inferred from nuclear ITS and EF1-alpha

sequences: evidence for cryptic diversification and links to Cordyceps teleomorphs. Mycologia

97(1): 84-98. http://dx.doi.org/10.3852/mycologia.97.1.84

Rehner SA, Aquino de Muro M, Bischoff JE 2006. Description and phylogenetic placement of

Beauveria malawiensis sp. nov. (Clavicipitaceae, Hypocreales). Mycotaxon 98: 137-145.

Rehner SA, Minnis AM, Sung G-H, Luangsa-Ard JJ, Devotto L, Humber RA. 2011. Phylogeny

and systematics of the anamorphic, entomopathogenic genus Beauveria. Mycologia 103(5):

1055-1073. http://dx.doi.org/10.3852/10-302

Samson RA, Evans HC. 1982. Two new Beauveria spp. from South America. Journal of Invertebrate

Pathology 39(1): 93-97. http://dx.doi.org/10.1016/0022-2011(82)90162-8

Spatafora JW, Kohlmeyer BV, Kohlmeyer J. 1998. Independent terrestrial origins of the

Halosphaeriales (marine Ascomycota). American Journal of Botany 85: 1569-1580.

http://dx.doi.org/10.2307/2446483

Shimazu M, Mitsuhashi W, Hashimoto H. 1988. Cordyceps brongniartii sp. nov., the teleomorph of

Beauveria brongniartii. Transactions of the Mycological Society of Japan 29: 323-330.

Swofford DL. 2003. PAUP”: phylogenetic analysis using parsimony (*and other methods). Version

4. Sunderland, Massachusetts: Sinauer Associates.

Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997. The Clustal X windows

interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.

Nucleic Acid Research 24: 4876-4882. http://dx.doi.org/10.1093/nar/25.24.4876

Zhang SL, He LM, Chen X, Huang B. 2013 [“2012”]. Beauverialiisp. nov. isolated from Henosepilachna

vigintioctopunctata. Mycotaxon 121: 199-206. http://dx.doi.org/10.54248/121.199

MY COTAXON

http://dx.doi.org/10.5248/124.309

Volume 124, pp. 309-321 April-June 2013

The lichen genus Fissurina (Graphidaceae) in Vietnam

SANTOSH JOSHI’, THI THUY NGUYEN’, NGUYEN ANH DZUNG?’,

UDENI JAYALAL’, SOON-OK OH!’ & JAE-SEOUN HurR*™*

‘Korean Lichen Research Institute, Sunchon National University, Suncheon-540 742, Korea

Biotechnology Center, Tay Nguyen University, 567 Le Duan, Buon Ma Thuot City, Vietnam

CORRESPONDENCE TO *: jshur1@sunchon.ac.kr

ABSTRACT — Nine species of Fissurina from Vietnam are briefly commented on. Fissurina

dumastii, FE dumastioides, F. instabilis, E rubiginosa, and F. undulata are recorded as new

for the Vietnam lichen biota. Characteristic morpho-anatomical and chemical features are

described and summarized in an artificial key to all known taxa of Fissurina from Vietnam.

Key worps — Chu Yang Sin National Park, corticolous, graphidoid, taxonomy

Introduction

Fissurina Fée (Ascomycota: Ostropales) accommodates species with mostly

slit-like lirellae (Staiger 2002; Archer 2009). The diagnostic characters of the

genus includes a pale to yellow-brown to olive green (rarely whitish), mostly

smooth and glossy thallus, fissurine, simple to branched, immersed to prominent

lirellae, uncarbonized or rarely carbonized proper exciple, clear hyaline non-

amyloid hymenium, 1-8-spored asci, and hyaline, oval or narrowly to broadly

ellipsoid, trans-septate to muriform, amyloid to non-amyloid, thick-walled,

mostly halonate ascospores (Archer 2009; Sharma et al. 2012). Fissurina occurs

in the tropics and presently comprises more than 100 species worldwide. Many

species have recently been added to the genus as a consequence of a molecular

and phylogenetic revision of the Graphidaceae (Rivas Plata et al. 2012; Sharma

et al. 2012).

Aptroot & Sparrius (2006) recorded four Fissurina species — F. marginata

(=F elaiocarpa), F. egena, EF incrustans, and E radiata —from different Botanical

Gardens and National Parks of Vietnam. During a recent field excursion to

Chu Yang Sin National Park (Vietnam), several interesting graphidoid taxa

were collected, including five new records of Fissurina for Vietnam: F. dumastii,

F. dumastioides, F. instabilis, F. rubiginosa, and F. undulata. Among the Fissurina

310 ... Joshi & al.

species in the country, F dumastioides and F. rubiginosa produce stictic acid,

whereas the others lack secondary metabolites.

This report is a preliminary attempt to assess the taxonomy of fissurine

lichen species known so far from Vietnam. The synopsis clarifies the current

status of the genus within the country and confirms the high diversity of

graphidoid taxa in tropical rain forests. Diagnostic characters are studied from

the available material. Unfortunately, many of the samples were either immature

or collected in low frequency and many others that could increase the diversity

considerably were sterile. In the following account nine reports of Fissurina are

briefly described and summarized in an artificial key.

Materials & methods

Material deposited in the herbarium of the Korean Lichen Research Institute

(KoLRI) was examined morphologically, anatomically, and chemically. The morphology

of thallus and the ascomata were observed using a SMZ-168 dissecting microscope.

Thin hand-cut sections mounted in tap water, in lactophenol cotton blue, in 5% KOH,

or in Lugol’s solution were observed under a Olympus BX50 compound microscope.

Chemical spot tests and TLC (using solvent system C & A) were performed according

to Orange et al. (2010).

Taxonomic descriptions

Fissurina dumastii Fée, Essai Crypt. Ecorc.: xc, 1825. Piya

Thallus corticolous, epiperidermal, corticate, to ca. 250 um thick, glossy,

smooth, continuous to uneven, greenish grey, pale green or olivaceous to dull

green; cortex 15-30 um thick; algal layer well developed, continuous, 20-35 um

thick; medulla indistinct, mostly endophloeodal, studded with crystals; calcium

oxalate crystals small to moderately large, clustered or scattered; prothallus

distinct, white. Vegetative propagules not seen.

Ascocarps numerous, lirelliform, + conspicuous, immersed to erumpent,

indicated by a thin line between two lips, sometimes slightly open, slightly

raised and paler than the thallus, straight, curved or sinuous, often branched,

0.6-2 x 0.2-0.25 mm, dumastii-type (Staiger 2002); disc slit-like, narrow

to sometimes open in mature apothecia, flesh-colored. Thalline margin

concolorous with the thallus or slightly paler, thin to thick, + incurved or

straight, 10-40 um thick. Proper exciple brownish to dark brown, ca. 15-50

um thick. Epihymenium hyaline, indistinct, with greyish or brownish granules,

<10 um high. Hymenium hyaline, clear, to ca. 50-95 um high, I-. Paraphyses

straight to bent, + interwoven, unbranched, with slightly thickened tips,

moderately to distinctly conglutinated, 1-2 um thick. Subhymenium indistinct

to hyaline, ca. 20 um high. Asci clavate, 8-spored, ca. 65-80 um, I-. Ascospores

uniseriate, hyaline, becoming mottled or brown in late maturity, transversely

Fissurina in Vietnam ... 311

a a ea SS *

PLaTE 1. Fissurina dumastii. A. Habit. B. Ascus with ascospores. C. Ascospores.

Scale bars: A = 2 mm; B = 25 um; C = 10 um.

septate, oblong to ellipsoidal, with + rounded to narrowly rounded to subacute

ends, thick walled, halonate, 15-18 x 4-9 um, with 4 lenticular to + round

locules, I+ blue violet.

CHEMISTRY: K-, PD-, C-; no lichen compound detected in TLC.

DISTRIBUTION & ECOLOGY: This species has a pantropical distribution

and was previously reported from Queensland, New South Wales, Indonesia,

312 ... Joshi & al.

Solomon Islands, and Brazil (Staiger 2002; Archer 2006, 2009). In Vietnam

it was collected from tree trunks of an evergreen forest in the Chu Yang Sin

National Park at an altitude of ca. 600 m.

SPECIMEN EXAMINED: VIETNAM. Dak LAK PROVINCE: Chu Yang Sin National Park,

12°28'04.4"N 108°20'39.0"E, alt. ca. 622 m, on tree trunk, 20 April 2012, Hur, Oh &

Nguyen 120282 (KoLRI).

REMARKS: Fissurina dumastii seems close to E insidiosa C. Knight & Mitt. in

having trans-septate ascospores and thallus lacking chemical substances, but

differs in thallus and apothecial morphology. Fissurina insidiosa has distinct

verrucose-bullate thallus and labiate lirellae. Moreover, the ascospores of

F. dumastii show distinct amyloidity, absent in E insidiosa. Another species

belonging to the dumastii species complex is F. analphabetica Common &

Licking, but it has very small delicate lirellae (0.3-0.5 mm) and comparatively

smaller ascospores of 15 x 7 um (Licking et al. 2011). Fissurina egena also has

roof-like apothecial margins but differs in its muriform ascospores.

Fissurina dumastioides (Fink) Staiger, Biblioth. Lichenol. 85: 161, 2002. PL. 2

Thallus corticolous, epiperidermal, corticate, to ca. 250 um thick, glossy,

smooth, continuous to uneven, green to olive-green; cortex 15-25 um thick;

algal layer well developed, continuous, 20-30 um thick; medulla indistinct,

mostly endophloeodal, studded with crystals; calcium oxalate crystals small to

moderately large, clustered or scattered; prothallus distinct, white. Vegetative

propagules not seen.

Ascocarps numerous, lirelliform, + conspicuous, immersed to erumpent,

indicated by a thin line between two lips, sometimes broadly open, slightly

raised and paler than the thallus, straight, curved or sinuous, often branched,

<5 mm long and 0.2-0.3 mm wide with recurved margins, dumastii-type

(Staiger 2002); disc narrow, open in mature apothecia, greyish to flesh-colored.

Thalline margin concolorous with the thallus or slightly paler, thin to thick, +

incurved or straight, 10-40 um thick. Proper exciple brownish to dark brown,

ca. 15-50 um thick. Epihymenium hyaline, indistinct, with greyish or brownish

granules, <10 um high. Hymenium hyaline, clear, to ca. 50-90 um high, I-.

Paraphyses straight to bent, + interwoven, unbranched, with slightly thickened

tips, moderately to distinctly conglutinated, 1-2 um thick. Subhymenium

indistinct to hyaline, ca. 20 um high. Asci clavate, 8-spored, ca. 60-80 um, I-.

Ascospores uniseriate, hyaline, becoming mottled or brown in late maturity,

transversely septate, oblong to ellipsoidal, with + rounded to narrowly rounded

to subacute ends, thick walled, halonate, 15-20 x 4-9 um, with 4 lenticular to

+ round locules, I+ blue violet.

Fissurina in Vietnam ... 313

PLATE.2. Fissurina dumastioides. Habit. Scale bar: 2 mm

CHEMISTRY: K+ yellow, PD+ yellow-orange, C-; stictic acid detected in

EGE

DISTRIBUTION & ECOLOGY: The species is known to occur in Puerto Rico

and India (Fink 1927, Sharma et al. 2012), and has now been identified from

Vietnam. It was collected from trees in an evergreen tropical forest of the Chu

Yang Sin National Park at an altitude between 700-800 m.

SPECIMEN EXAMINED: VIETNAM. Dak LAK PROVINCE: Chu Yang Sin National Park,

12°27'57.0"N 108°20'34.9"E, alt. ca. 780 m, on tree trunk, 21 April 2012, Hur, Oh &

Nguyen 120267-1 (KoLRI).

REMARKS: Fissurina dumastioides resembles FE dumastii morphologically and

anatomically, but differs in containing stictic acid as thallus compound.

Fissurina egena (Nyl.) Nyl., Sert. Lich. Trop.: 13, 1891

This taxon is morphologically similar to FE cingalina (Nyl.) Staiger, with a

ochre, yellow-grey, yellowish green, slightly rough or warty thallus (Staiger

2002). However, F. egena can be distinguished from E cingalina in its immersed

lirellae with roof-like margins and concealed to slightly exposed disc, its

8-spored asci, its muriform, amyloid, 15-23 x 7-10 um ascospores and the lack

of secondary metabolites.

It has been reported from Singapore, North and South America, and Vietnam

(Staiger 2002; Aptroot & Sparrius 2006).

314 ... Joshi & al.

Fissurina elaiocarpa (A.W. Archer) A.W. Archer, Telopea 11:71, 2005.

= Fissurina marginata Staiger, Bibliotheca Lichenologica 85: 144, 2002.

This species is characterized by a olive green, slightly subtuberculate, glossy

thallus, conspicuous, sessile, straight, curved or sinuous, unbranched, ca. 1-8

x 4 mm ascomata, scarcely open lip, reddish brown proper exciple, 150-200

um high hymenium, 4-5 x 3-4 locular, 21-28 x 13-18 um large, amyloid

ascospores and absence of secondary metabolites (Archer 2009).

Aptroot & Sparrius (2006) reported this species from Vietnam as

F marginata and collected it from a secondary mountain forest of Ba Vi National

Park (Hanoi Province), at a 1200-1300 m altitude. The species has a worldwide

distribution and is found in Africa, Australia, South and Western America, and

Western Indian Ocean (Staiger 2002).

Fissurina incrustans Fée, Essai Crypt. Ecorc.: 60, 1825.

This species is characterized by a beige green, smooth to warty thallus,

elongate to short (1-4 x 0.3-0.4 mm), sinuous, branched ascomata, erumpent

labia, gaping disc, poorly developed proper exciple, usually intermingled with

bark substratum, 8-spored asci, muriform, amyloid ascospores of 15-28 x

7-13 um and absence of secondary compounds. It closely resembles F. egena

in having similar-sized muriform amyloid ascospores and a thallus lacking

chemical compounds, but F. incrustans has erumpent lirellae more similar to

the ones found in Platythecium than the ones found in Fissurina (Staiger 2002;

Licking et al. 2011).

The species has been reported in Vietnam from a secondary mountain forest

in Ba Vi National Park by Aptroot & Sparrius (2006). It is also found in South

America (Staiger 2002).

Fissurina instabilis (Nyl.) Nyl., Sert. Lich. Trop.: 36, 1891. Pig3

Thallus corticolous, epiphloeodal, corticate, <300 um thick, glossy, smooth,

continuous to + verrucose (due to bark texture), non-rimose, green, olive green

to pale green; cortex 35-45 um thick; algal layer well developed, continuous,

densely integrated with crystals, <215 um thick; medulla indistinct, mostly

endophloeodal, integrated with crystals; calcium oxalate crystals small to

moderately large, clustered or scattered; prothallus indistinctly black. Vegetative

propagules not seen.

Ascocarps numerous, lirelliform, inconspicuous, immersed to + erumpent,

indicated by a thin line between two lips (labia), sometimes slightly open,

slightly raised, curved or sinuous, often branched, <10 x 0.6 mm, globulifica-

type (Staiger 2002); labia entire, paler than the thallus; disc slit-like, narrow,

scarcely open in mature apothecia, flesh-colored. Thalline margin concolorous

with the thallus, thick, tincurved or straight, 180-215 um thick. Proper

Fissurina in Vietnam ... 315

PLATE 3. Fissurina instabilis A. Habit. B. Cross section of apothecia. C. Ascospores.

Scale bars: A = 2 mm; B = 100 um; C = 20 um.

exciple well developed, brownish to reddish brown, superficially exposed as a

red-brown lining, ca. 30-55 um thick. Epihymenium hyaline, indistinct, with

greyish or brownish granules, 3-6 um high. Hymenium hyaline, clear, to ca.

75-100 um high, I-. Paraphyses straight to bent, +interwoven, unbranched,

316 ... Joshi & al.

with slightly thickened tips, moderately to distinctly conglutinated, 1-2

um thick. Subhymenium distinct, hyaline, ca. 40-50 um high. Asci clavate,

8-spored, ca. 65-80 um, I-. Ascospores uni- or biseriate, hyaline, muriform,

oblong to ellipsoidal, with + rounded ends, thick walled, 13-20 x 7-11 um,

with 3-4(-5) x 1-2 tround locules, I+ blue violet.

CHEMISTRY: K-, PD-, C-; no lichen compound detected in TLC.

DISTRIBUTION & ECOLOGY: This species has a Pacific distribution (Staiger

2002). In Vietnam it was collected from tree trunks in an evergreen forest of the

Chu Yang Sin National Park at an altitude between 700-800 m.

SPECIMENS EXAMINED: VIETNAM. Dak LAK PROVINCE: Chu Yang Sin National Park,

12°27'57.0"N 108°20'34.9"E, alt. ca. 780 m, on tree trunk, 21 April 2012, Hur, Oh &

Nguyen 120132 (KoLRD); 12°28'12.3"N 108°20'59.9"E, alt. ca. 763 m, on tree trunk, 20

April 2012, Hur, Oh & Nguyen 120087 (KoLRI).

REMARKS: Fissurina instabilis resembles EF confusa Common & Licking and

EF. globulifica (Nyl.) Staiger in having the same thallus morphology, globulifica-

type lirellae, and muriform amyloid ascospores but F. confusa and E globulifica

produce psoromic acid and EF globulifica has smaller globose (10 x 10 um)

ascospores (Liicking et al. 2011).

Fissurina radiata Mont., Annls Sci. Nat., Bot., sér. 2, 18: 280, 1842.

This species differs from other Fissurina species in having aggregated, mostly

radiate ascomata forming a pseudostroma, inconspicuous and gaping labia,

amyloid, 4-locular ascospores of 15 x 7 um, and a thallus lacking secondary

metabolites (Liicking et al. 2011). It is comparable to material described from

Florida as Fissurina aggregatula Common & Liicking (Licking et al. 2011),

which differs in having conspicuous labia and a concealed disc, sparsely

branched aggregated lirellae, and non-amyloid ascospores.

Fissurina radiata has been reported from the Botanical Garden (Bach Thao

Park) in Hanoi City in Vietnam at an altitude between 50-60 m (Aptroot &

Sparrius 2006). It has a distribution in tropical regions of America (Licking et

al. 2011), Malaysia, Singapore, and Taiwan.

Fissurina rubiginosa (Fée) Staiger, Biblioth. Lichenol. 85: 148, 2002. PL. 4

Thallus corticolous, epiphloeodal, corticate, <350 um thick, glossy, smooth,

continuous, non-rimose, green, olive green to pale green; cortex 30-50 um thick;

algal layer well developed, continuous, densely integrated with crystals, <200

um thick; medulla indistinct, mostly endophloeodal, studded with crystals;

calcium oxalate crystals small to moderately large, clustered or scattered;

prothallus indistinctly black. Vegetative propagules not seen.

Ascocarps numerous, scattered, lirelliform, inconspicuous, immersed,

indicated by a thin line, sometimes slightly open, slightly raised and paler than

Fissurina in Vietnam ... 317

el >

* in -

2 EE a5 Y

> ee (Seq

aS! .

Pate 4. Fissurina rubiginosa. A. Habit. B. Cross section of apothecia. C. Ascospores.

Scale bars: A = 2 mm; B = 100 um; C = 30 um.

the thallus, straight, curved or sinuous, often branched, <2 x 0.25 mm, dumastii-

type; disc slit-like, narrow, scarcely open in mature apothecia, flesh-colored.

Thalline margin concolorous with thallus, thick, +incurved or straight, 75-150

um thick. Proper exciple well developed, pale to reddish brown, intermingled

with bark cells, <75 um thick. Epihymenium hyaline, indistinct, with greyish

318 ... Joshi & al.

or brownish granules, 10-15 um high. Hymenium hyaline, clear, <120-150

um high, I-. Paraphyses straight to bent, tinterwoven, unbranched, with

slightly thickened tips, moderately to distinctly conglutinated, 1-2 um thick.

Subhymenium indistinct, hyaline, ca. 25-30 um high. Asci clavate, 8-spored,

ca. 65-80 um, I-. Ascospores uni- or biseriate, hyaline, muriform, oblong to

ellipsoidal, with trounded ends, thick walled, 20-27 x 10-12 um, with 6-8 x

2-3 + round locules, I-.

CHEMIstTRY: K-, PD-, C-; no lichen compound detected in TLC.

DISTRIBUTION & ECOLOGY: The species is found in French Guiana, USA

and India (Sharma et al. 2012), but is now also reported from Vietnam where it

was and collected from tree trunks in an evergreen forest of the Chu Yang Sin

National Park at an altitude between 700-800 m.

SPECIMEN EXAMINED: VIETNAM. Dak LAK PROVINCE: Chu Yang Sin National Park,

12°28'12.3"N 108°20'59.9"E, alt. ca. 763 m, on tree trunk, 20 April 2012, Hur, Oh &

Nguyen 120246 (KoLRI).

REMARKS: This species is close to F elaiocarpa. Their thallus morphology is

similar and they both have muriform ascospores, although the spores are

comparatively larger (18-32 x 13-19 um) and I+ blue in F elaiocarpa. Another

closely related taxon, Fissurina aff. elaiocarpa, resembles F. rubiginosa in

having muriform, I- ascospores of similar size (25 x 15 um), but differs in

labiate ascomata and verrucose thallus. The description above does not agree

with that of E rubiginosa provided by Sharma et al. (2012), which describes

a brown, cracked and rough thallus and much smaller lirellae (0.4-0.6 mm).

However, the investigated sample shares some main characteristics with

F. rubiginosa, such as dumastii-type lirellae and non-amyloid ascospores.

Because of insufficient material, we provisionally identify our specimen from

Vietnam as F rubiginosa.

Fissurina undulata (Mill. Arg.) M. Nakan. & Kashiw., Bull. natn. Sci. Mus.,

Tokyo, B 29: 87, 2003. PL. 5

Thallus corticolous, epiphloeodal, corticate, to ca. 400 um thick, glossy,

verrucose to smooth, continuous, non-rimose or slightly cracked due to bark

texture, green, olive green to pale green or dull green; cortex 25-45 um thick;

algal layer well developed, continuous, densely integrated with crystals, <100

um thick; calcium oxalate crystals small to moderately large, clustered or

scattered; medulla indistinct, mostly endophloeodal, studded with crystals;

prothallus not seen. Vegetative propagules not seen.

Ascocarps numerous, scattered, lirelliform, inconspicuous, immersed,

indicated by a thin line, sometimes slightly open, slightly raised and paler

than the thallus, straight, curved or sinuous, often branched, <2 x 0.3 mm,

dumastii-type; disc slit-like, narrow, scarcely open in mature apothecia, flesh-

Fissurina in Vietnam ... 319

PiaTE 5. Fissurina undulata. A. Habit. B. Cross section of apothecia. C. Ascospores.

Scale bars: A = 2 mm; B = 200 um; C = 30 um.

colored. Thalline margin concolorous with thallus, thick, +incurved or straight,

50-100 um thick. Proper exciple poorly to well developed in mature fruits,

hyaline to pale, reddish brown when intermingled with bark cells, <30 um

thick. Epihymenium hyaline, indistinct, with greyish or brownish granules.

Hymenium hyaline, clear, to ca. 60-100 um high, I-. Paraphyses straight to

bent, +interwoven, unbranched, with slightly thickened tips, moderately to

320 ... Joshi & al.

distinctly conglutinated, 1-2 um thick. Subhymenium indistinct, hyaline, ca.

20-30 um high. Asci clavate, 8-spored, ca. 70-95 x 12-15 um, I-. Ascospores

uni- or biseriate, hyaline, muriform, oblong to ellipsoidal, with trounded ends,

thick walled, gelatinous sheath present, 22-32(-35) x 10-13 um, with 6-12 x

2-4 locules, I-; locules tround to angular.

CHEMIstTRY: K-, PD-, C-; no lichen compounds detected in TLC.

DISTRIBUTION & ECOLOGY: The species is found in Japan (Nakanishi 1966),

but is now also reported from Vietnam where it was collected from tree trunks

in an evergreen forest in Chu Yang Sin National Park at an altitude between

700-800 m.

SPECIMEN EXAMINED: VIETNAM. Dak LAK PROVINCE: Chu Yang Sin National Park,

12°28'12.3"N 108°20'59.9"E, alt. ca. 763 m, on tree trunk, 20 April 2012, Hur, Oh &

Nguyen 120247 (KoLRI).

REMARKS: The above description is based on a specimen with mostly immature

fruits, which appear more similar to E undulata than to F. inabensis (Vain.)

M. Nakan. & Kashiw. (erroneously cited as F fujisanensis (Kashiw. & M.

Nakan.) M. Nakan. & Kashiw. in Nakanishi et al. 2003). Both FE undulata and

F. inabensis produce stictic acid and have similar-sized ascospores (21-30 x

10-14 um for E inabensis). However, a thin thallus, a well developed proper

exciple, crenate labia, a medulla with gonidia and 2-3-celled ascospores

clearly separate FE inabensis from F undulata (Nakanishi 1966). Although the

descriptions of F. undulata by Nakanishi (1966), Nakanishi et al. (2003), and

Sharma et al. (2012) differ in the number of ascospores per ascus (8-spored asci

sensu Nakanishi vs. 1-spored asci sensu Sharma), our material agrees well with

a species with 8-spored asci. Moreover, the ascospore size (30-38 x 15-18 um)

of FE. undulata is in conflict with the ascospore size of species with 1-spored asci.

A further revision of E undulata would be necessary, as it seems closely related

to EF inabensis, particularly in immature stages (Nakanishi 1966). Pending some

more collections, the Vietnamese material is placed in F undulata.

Key to Fissurina species recorded from Vietnam

1A VPA ES COMEATINITIG SECU ACIG Ys as nets Sanaa ntiy Shane NOY es nae iced ees co Peta ash Rena dat 2,

Ib. Thallus lacking secondary metabolites. on... seu .eic state alan ie ples ole od ee On ae :

2a. Ascospores trahsversely septate § 0... saw Hee eee eee cea F. dumastioides

20, ASCOSPORES TIUTTOTI, 9 kM nie Uh taalg oh eee em bile ORR OR ee Oh ces F. rubiginosa

BdwASCOSpOTes transversely: Septaie: ety Lote Maile pele anita da tal denial eos abulkhi Ne weet Be abate 4

SRA SCOS POET ORTISA 2.2, sundae tte, Wide anaes eekly Weg ht AE Ga Ae ER, oer 5

4a. Lirellae mostly aggregated, radiate, forming a pseudostroma ........... F, radiata

4b. Lirellae disperse, solitary, branched, non-radiate..................... F, dumastii

5a. Ascospores non-amyloid, 22-32(-35) x 10-13 um................0. F. undulata

SDs NSCOSPOTES CHSUITICE Vea TTI ON Ge ol bay la ela clits tan dle a la dle glial eabandl a eacch eo Seater 6

Fissurina in Vietnam ... 321

6a. Lirellae immersed, fissurine, roof like margins,

ASCOSPOLES DADS F IOs ITN rhe stey WAS00h Achy eins heh er tech etek ples hp F egena

6b. Lirellae immersed to erumpent or prominent, labiate......................202. 7

7a. Lirellae immersed to + erumpent, ascospores 13-20 x 7-ll um ...... F. instabilis

7D; Lireliae. erunip enitiato:proindn Cite Ai). 0 0! occleedaind ore taind adler -« geen ne cesne Meee a Mae 8

8a. Thallus + smooth, proper exciple poorly developed,

ascospores 5 =28 9% FT 5: Wins ou chy a6: sdeg Sp tea yep tee keg tee hg F. incrustans

8b. Thallus verrucose, proper exciple well developed,

ASCOSPOTES ALS oT S= ISNT ine es ves Meaty Noahs oat, Headly ve F. elaiocarpa

Acknowledgments

This work was supported by a grant from the National Research Foundation of Korea

(#2011-0031494) and the Korea National Research Resource Center Program. Authors

are grateful to Dr. Robert Licking, Dr. Laszlé L6kGs, and Dr. Cecile Gueidan for their

valuable comments on the manuscript. Santosh Joshi also thanks Dr André Aptroot for

providing the lichen checklist of Vietnam, and helpful suggestions.

Literature cited

Aptroot A, Sparrius LB. 2006. Addition to the lichen flora of Vietnam, with an annotated checklist

and bibliography. Bryologist 109(3): 358-371.

http://dx.doi.org/10.1639/0007-2745(2006)109[358:ATTLFO]2.0.CO;2

Archer AW. 2006. The lichen family Graphidaceae in Australia. Bibliotheca Lichenologica 94:

1-191.Archer AW. 2009. Graphidaceae. 84-194, in: PM McCarthy, B Kuchlmayr (eds). Flora of

Australia, Vol. 57 (Lichens 5). Canberra & Melbourne: ABRS/CSIRO.

Archer AW. 2009. Graphidaceae. 84-194, in: PM McCarthy, B Kuchlmayr (eds). Flora of Australia,

Vol. 57 (Lichens 5). Canberra & Melbourne: ABRS/CSIRO.

Fink B.1927. New species of lichens from Porto Rico, I. Graphidaceae. Mycologia 19(4): 206-221.

http://dx.doi.org/10.2307/3753982

Liicking R, Seavey F. Common RS, Beeching SQ, Breuss O, Buck WR, Crane L, Hodges M,

Hodkinson BP, Lay E, et al. 2011. The lichens of Fakahatchee Strand Preserve State Park,

Florida: Proceedings from the 18th Tuckerman workshop. Bulletin of the Florida Museum of

Natural History 49(4):127-186.

Nakanishi M. 1966. Taxonomical studies on the family Graphidaceae of Japan. Journal of Science of

the Hiroshima University, Series B, 11(2): 51-126.

Nakanishi M, Kashiwadani H, Moon KH. 2003. Taxonomical notes on Japanese Graphidaceae

(Ascomycotina), including some new combinations. Bull. natn. Sci. Mus., Tokyo, B 29(2):

83-90.

Orange A, James PW, White FJ. 2010. Microchemical methods for the identification of lichens.

British Lichen Society, London.

Rivas Plata E, Licking R, Lumbsch TH. 2012. A new classification of the family Graphidaceae

(Ascomycota: Lecanoromycetes: Ostropales). Fungal Diversity 52: 107-121. http://dx.doi.

org/10.1007/s132225-011-0135-8

Sharma BO, Khadilkar P, Makhija U. 2012. New species and new combinations in the

lichen genera Fissurina and Hemithecium from India. Lichenologist 44(3): 339-362.

http://dx.doi.org/10.1017/S0024282911000752

Staiger B. 2002. Die Flechtenfamilie Graphidaceae. Studien in Richtung einer natiirlicheren

Gliederung. Bibliotheca Lichenologica 85: 1-526.

MY COTAXON

http://dx.doi.org/10.5248/124.323

Volume 124, pp. 323-332 April-June 2013

Studies of North American macrofungi, 1.

Validation of Lactarius rubidus comb. nov.

and Leccinellum quercophilum sp. nov.

MICHAEL Kuo,’* ANDREW S. METHVEN,”

ANDREW M. MINNIS,? & Roy E. HALLING4

‘Department of English & *Department of Biological Sciences, Eastern Illinois University,

600 Lincoln Avenue, Charleston IL 61920 USA

>Center for Forest Mycology Research, Northern Research Station, USDA-U.S. Forest Service,

One Gifford Pinchot Dr., Madison WI 53726 USA

‘Institute of Systematic Botany, The New York Botanical Garden,

2900 Southern Boulevard, Bronx NY 10458 USA

* CORRESPONDENCE TO: michael@mushroomexpert.com

ABSTRACT — Two mycorrhizal taxa are described: Lactarius rubidus from the west coast

and Leccinellum quercophilum from eastern North America. Lactarius rubidus is a new

combination for Lactarius fragilis var. rubidus, here validated after a previous invalid

publication. Leccinellum quercophilum is a new species. Detailed macro- and microscopic

descriptions, ITS and LSU sequences, illustrations of microscopic features, and plates are

presented for each species; a type study of Lactarius fragilis var. rubidus is also provided.

Key worps — Boletaceae, Boletales, Russulaceae, Russulales, taxonomy

Introduction

The North American macrofungi known as Lactarius fragilis var. rubidus and

Leccinum griseum (also sometimes known as Leccinum carpini) are commonly

collected fungi for which currently applied names are unsatisfactory. The west

coast fungus Lactarius fragilis var. rubidus, which differs substantially from the

eastern North American type variety in morphology, ecology, and geographic

distribution, merits taxonomic rank as a species. Leccinum griseum is a

European fungus clearly distinct from the North American entity frequently

bearing its name in herbaria. Here we describe Leccinellum quercophilum, a

North American bolete sometimes identified as Leccinum griseum, as a new

species and validate the combination Lactarius rubidus to apply to Lactarius

fragilis var. rubidus.

324 ... Kuo & al.

Materials & methods

Both fresh basidiomes and herbarium specimens were studied. Colors were recorded

and codified following Kornerup & Wanscher (1967), or the Online Auction Color

Chart (www.onlineauctioncolorchart.com — abbreviated “OAC” herein). Microscopic

features were studied using hand sections of fresh material, and of dried specimens

rehydrated in water after immersion in 90% alcohol. Sections were mounted in 2% KOH

and Melzer’s reagent, and viewed using an Olympus BX51 microscope. Specimens are

deposited in herbaria and cited according to Thiers (2012).

DNA from each taxon was extracted from small hymenium-bearing pieces of

dried basidiocarp following the methods of Lindner & Banik (2009). Protocols for ITS

amplification, sequencing, sequence editing, and other related procedures followed

Lorch et al. (2013) with the following modifications: denaturing for 40 s, annealing at 53

°C for 40 s, and extension for 90 s in the 30 PCR cycles. LSU sequences were obtained

with the same protocols using the primers LROR (Moncalvo et al. 2000) and either LR5

or LR7 (Vilgalys & Hester 1990). Newly generated DNA sequences were deposited in

GenBank and compared with available sequences via GenBank BLAST searches.

Taxonomy

Lactarius rubidus (Hesler & A.H. Sm.) Methven, comb. & stat. nov. PLATE 1

MycoBank MB803100

= Lactarius fragilis var. rubidus Hesler & A.H. Sm., N. Amer. Sp. Lactarius: 505. 1979.

“Lactarius rubidus” Methven, Agaricales (Gilled Fungi) of California,

10, I: 67. 1997, nom. inval. [provisional name].

PiLEus 2-8 cm broad, convex to plane, disc shallowly depressed, occasionally

umbonate; margin incurved to decurved, glabrous; surface glabrous, dry to

moist, rugose to rugulose, azonate, light brown (6D7-5) to brown (7D8-5), or

reddish brown (8D8-6), negative with the application of 15% KOH; context up

to 5 mm thick at the disc, light orange (5A4-2), unstaining on exposure; odor of

maple syrup or fenugreek when dried; taste not distinctive. LAMELLAE adnate

to subdecurrent, close to distant, narrow, rarely forked, light orange (5A4-2,

6A4-3), not marginate, unstaining where cut. STIPE 2-7 cm x 4-10(-15) mm,

terete, equal; surface glabrous, dry, not scrobiculate, brownish orange (6C7-5)

to light brown (7D7-5); white to light orange tomentum at the base; context

hollow, concolorous with the pileal context, unstaining on exposure. LATEX

whey-like, not copious, unstaining, unchanging, taste not distinctive.

BASIDIOSPORES white to pale yellow (4A3-2) in mass, 6-8(-8.5) x 6-7.5

um, globose to subglobose; amyloid ornamentation a broken to partial or

nearly complete reticulum 0.5-1 um high. Basip1a 35-55 x 7.5-12.5 um,

tetrasterigmate. CHEILOCYSTIDIA 25-35 x 6-9 um, clavate. MACROCYSTIDIA

not observed. PILEIPELLIs an epithelium composed of clavate to vesiculose cells

in short chains, tangled in age, dry. PILEUS TRAMA heteromerous, sphaerocysts

in rosettes, lactiferous hyphae inconspicuous. STIPITIPELLIS a simple cutis with

Lactarius rubidus validated & Leccinellum quercophilum sp. nov. ... 325

PiaTE 1: Lactarius rubidus, M. Kuo 01131106 (NY). Basidiocarps (top), basidiospores (middle),

and pileipellis (bottom). Scale bars = 10 um. [For additional illustrations see Hesler & Smith (1979:

Fig. 247, basidiospores) and Methven (1997: Pl. 2, basidiocarps).]

scattered projecting hyphal tips, stipe cortex heteromerous, sphaerocysts in

rosettes.

ECOLOGY & DISTRIBUTION — Scattered, gregarious, or caespitose in duff,

in coastal coniferous-deciduous forests in apparent association with Quercus

agrifolia, Notholithocarpus densiflorus, and Pseudotsuga menziesii. Common.

September through February. California, Oregon, and Washington.

COLLECTIONS EXAMINED — UNITED STATES. CALIFORNIA: MARIN County, 10

Dec 1982, ASM 2261 (EIU); 19 Dec 1984, ASM 3523 (EIU); 27 Dec 1984, ASM 3632

326 ... Kuo & al.

(EIU). MENDOCINO County, 30 Apr 1983, CM Bern, ASM 2456 (EIU); 13 Dec 1990,

ASM 6523 (EIU). SAN MaTEO County, 17 Dec 1984, ASM 3600 (EIU); 12 Jan 1985, H

Saylor 2297 (EIU); 13 Jan 2005, M Kuo 01130522 (NY); 13 Jan 2011, M Kuo 01131106

(NY, GenBank ITS KC691205, LSU KC691206). SonoMA Counry, 02 Feb 2003, M Kuo

02200309 (NY). OREGON: POLK County, 14 Nov 1970, AH Smith 79939 (MICH 23242,

paratype); 14 Nov 1970, AH Smith 79942 (MICH 11133, holotype).

ComMENTs — Lactarius rubidus is characterized by the reddish brown, azonate,

dry to moist pileus, whey-like latex, the maple syrup or fenugreek odor when

dried, and globose basidiospores with a broken to partial or nearly complete

amyloid reticulum. Lactarius fragilis (Burl.) Hesler & A.H. Sm. var. fragilis,

known from eastern North America, differs in having a snuff brown to burnt

umber pileus, subdistant yellowish lamellae, and globose basidiospores with

heavier deposits of amyloid ornamentation that form a partial to complete

reticulum. Lactarius rubidus has frequently been misidentified as L. camphoratus

(Bull.) Fr. However, L. camphoratus is a European and eastern North American

species featuring a red brown pileus, close to crowded pale pinkish cinnamon

lamellae, milk-white latex, and broadly ellipsoid basidiospores with amyloid

ornamentation of isolated spines and nodulose ridges forming at most a

broken reticulum. Lactarius rubidus is a popular edible commonly called the

“candy cap” by collectors (Kuo 2007). Wood et al. (2012) recently determined

quabalactone III to be the source of the maple syrup odor of L. rubidus.

ITS sequence comparisons via GenBank BLAST searches indicate L. rubidus

is not conspecific with European and North American L. camphoratus. Lactarius

cf. rubidus (GenBank DQ822820.1 from a basidiocarp and GU180303.1

from a Pinus muricata root tip; Peay et al. 2007) and unidentified samples

GenBank AF323116.1 from a Pinus muricata root (Wurzberger et al. 2001)

and DQ273391.1 from a Notholithocarpus densiflorus root (Bergemann &

Garbelotto 2006), all from California, appear to represent L. rubidus at 99%

sequence identity.

In Methven (1997: 67), the combination “Lactarius rubidus (Hesler & A.H.

Sm.) Methven” appeared with a citation of its forthcoming publication as

“Mycotaxon (in press). 1998” but was not validly published since it was merely

proposed in anticipation of future acceptance of this taxon at the species rank

(ICN Art. 36.1, McNeill et al. 2012). The subsequent Mycotaxon publication

did not occur, and the combination Lactarius rubidus has not been validly

published elsewhere since. A record for “Lactarius rubidus Arora 1991” appears

in Index Fungorum (www.indexfungorum.org) but no further information is

provided; a similar record appears in the MycoBank database (www.mycobank.

org), again without any supporting publication information. David Arora’s

(1991) field guide, All That the Rain Promises and More: a Hip Pocket Guide to

Western Mushrooms is the only plausible basis we have been able to discover for

these website entries. Since Arora treats the species as “Lactarius fragilis, listing

Lactarius rubidus validated & Leccinellum quercophilum sp. nov. ... 327

“other names” as “Lactarius fragilis var. rubidus, L. rubidus, without citing

taxon authorities or providing full and direct references to any of the names as

required for publishing a new combination, his book cannot be considered a

valid publication of Lactarius rubidus. We validate the combination here.

STUDY OF THE HoLotype — The holotype of Lactarius fragilis var. rubidus

is held in the University of Michigan Herbarium (MICH 11133) and was

collected by A.H. Smith (79942) on “rotten wood” in the Van Duzer Corridor,

Polk County, OR; it consists of several well preserved basidiomes with a strong

odor of fenugreek. BASIDIOSPORES 6.5-8.5 x 6-7.5 um, globose to subglobose,

ornamentation a partial to more or less complete reticulum 0.5-1 um high,

with scattered free ends and rare isolated elements, plage conspicuous and

lacking amyloid debris. Bastp1a 35-50 x 7.5-12.5 um, clavate, tetrasterigmate.

CHEILOCYSTIDIA not clearly differentiated. MACROCysTIDIA not observed.

PILEIPELLIS an epithelium composed of vesiculose or irregularly shaped

inflated cells in short chains, at times aggregated into mounds, especially in age.

PiLEus TRAMA heteromerous. STIPITIPELLIS a dry, simple cutis of interwoven

hyphae with scattered, projecting, pyriform to subfusiform hyphal tips 25-35

x 7.5-15 um.

Leccinellum quercophilum M. Kuo, sp. nov. PLATE 2

MycoBank MB802407

Differs from Leccinum griseum by its bluish green staining on the pileus and stipe, its

creamy whitish tubes, its context staining gray when sliced, and its association with

Quercus in eastern North America.

TyPeE: United States, Illinois, Coles County, Charleston, under Quercus alba on a lawn,

12 July 2008, coll. M Kuo 07120801 (Holotype, NY; GenBank ITS KC691207, LSU

KC691208).

ErymMo oey: from a combination of the Latin Quercus = oak, and the Greek otAoc =

friend; “oak-loving”

PILEus 3-9 cm broad; convex; dry; glabrous; dull orangish brown (OAC 749) to

medium yellow-brown (OAC 707, 715); rugulose-pitted when young, becoming

conspicuously areolate; without an overlapping sterile margin; discoloring bluish

green marginally with age; surface negative with the application of ammonia

or FeSO,, negative to yellowish with 15% KOH. HyMENopPHORE tubulose;

depressed at the stipe. TUBES 1-2 cm deep, creamy whitish (contrasting with

the pore surface), discoloring slowly blackish when sliced; pores 1-3 per mm at

maturity, round becoming angular with maturity, whitish or medium grayish

brown (OAC 736) when young, becoming yellowish brown to brownish;

discoloring greenish in places with age; bruising slowly dark brown, with or

without a bluish stage. StrpE 5-9 cm x 8-20 mm; equal, or tapered slightly to

apex, or slightly ventricose; dry; whitish underneath fine, tiny scabers that are

whitish apically and brown below; the scabers arranged in vague longitudinal

328 ... Kuo & al.

lines, darkening somewhat with age but not blackening; bruising or staining

greenish to bluish basally; basal mycelium whitish. CONTEXT whitish, staining

grayish to gray within 15-30 minutes of being sliced, with or without a faintly

pinkish stage; sometimes staining bluish in the stem base when sliced; pinkish

to negative with the application of ammonia, negative to greenish with FeSO,,

gray to greenish gray with 15% KOH. Opor AND TASTE not distinctive.

BASIDIOSPORES 15-18(-28) x 5-7.5 um; fusiform; ochraceous in 2%

KOH; inamyloid; aseptate; smooth. Basip1a 25-30 x 9-12 um; clavate;

tetrasterigmate. PLEUROCYSTIDIA 25-40 x 8-12 um; occasional or frequent;

fusoid-ventricose; thin-walled; smooth; hyaline to ochraceous or golden in 2%

KOH. CHEILocystTipia 35-50 x 5-10 um; fusoid-ventricose to mucronate,

subclavate, or irregular; thin-walled; smooth or roughened; ochraceous to golden

in 2% KOH. CauLocystTIDIA 30-50 x 10-15 um; mostly fusoid-ventricose but

occasionally ventricose-rostrate, or lageniform; apices occasionally mucronate;

thin-walled; smooth; hyaline to ochraceous in 2% KOH. PILEIPELLIS a

trichodermium of septate hyphae; hyaline to ochraceous or brownish in 2%

KOH; terminal elements subglobose to clavate or irregular, 25-50 um wide.

ECOLOGY & DISTRIBUTION — Scattered to gregarious under oaks; appearing

in hardwood forests and in urban settings (lawns, cemeteries, etc.); summer

and early fall; currently known from Illinois and Michigan.

ADDITIONAL COLLECTIONS EXAMINED — UNITED STATES. ILiinots: Cotes County,

20 Jun 2010, M Kuo 06201003 (NY). MicHIGAN: LIVINGSTON County, Aug 1972, AH

Smith 84011 (MICH 35770, as Leccinum griseum); WASHTENAW COUNTY, 16 Sep 1940,

AH Smith 15410 (MICH 35774, as Leccinum griseum).

ComMENts — Leccinellum quercophilum can be identified by its association

with oaks, the dull brown or yellowish brown rugulose-pitted cap that becomes

conspicuously areolate, the fine brown scabers, the green and blue staining,

and the trichoderm pileipellis. It has been thought to represent the European

species Leccinum griseum (Quél.) Singer (=Gyroporus griseus Quél.) by various

North American authors, including Smith & Thiers (1971: 214). However, the

name Leccinum griseum is problematic (Lannoy & Estades 1995; den Bakker

& Noordeloos 2005). Regardless of its correct name, this European species,

sometimes listed as Leccinum carpini (R. Schulz) D.A. Reid (Lannoy & Estades

1995) or Leccinum pseudoscabrum (Kallenb.) Sutara (den Bakker & Noordeloos

2005), is primarily associated with Carpinus and does not stain blue or green

(except very rarely in the base of the stem). Also, the European species has

yellowish tubes when young and flesh that stains deep violet when sliced

(Lannoy & Estades 1995; den Bakker & Noordeloos 2005)—a difference noted

by Smith & Thiers (1971). On the basis of these morphological differences,

we recognize the North American species as new and have selected an epithet

reflecting its association with oaks.

Lactarius rubidus validated & Leccinellum quercophilum sp. nov. ... 329

PLATE 2: Leccinellum quercophilum, M. Kuo 07120801 (NY; holotype). Basidiocarps (top),

pileipellis elements (bottom left), cheilocystidia (middle right), and basidiospores (bottom right).

Scale bars = 10 um.

Leccinellum Bresinsky & Manfr. Binder was erected in 2003 (Bresinsky &

Besl 2003) to accommodate Leccinum species with a yellow hymenophore and

a trichoderm-like pileipellis. Boletus nigrescens Richon & Roze (nom. illegit.;

= Leccinellum nigrescens (Singer) Bresinsky & Manfr. Binder) was designated

as the type of the genus. Bresinsky & Besl (2003) included Leccinum carpini and

L. griseum among eight existing species they accepted in Leccinellum, but the

nomenclature and taxonomy warrant review. Although the hymenophore of

Leccinellum quercophilum is not yellow, it falls within Leccinellum as defined in

Bresinsky & Besl (2003) on the basis of its pileipellis.

330 ... Kuo & al.

GenBank BLAST searches with an ITS sequence of Leccinellum quercophilum

reveal that the closest matches include Leccinum carpini |AF454588.1 (The

Netherlands)], Leccinum crocipodium (Letell.) Watling [AF454589.1 (Belgium),

JN021053.1 (France), JF908325.1 unpublished (Italy), and AF454590.1 (The

Netherlands)], and Leccinum talamancae Halling et al. [AY544779.1 (Costa

Rica)] as labeled and numbered in GenBank with published citations (den

Bakker et al. 2004a,b; Dentinger et al. 2011). The >3% difference in sequence

identity supports Leccinellum quercophilum as a distinct species. Morphological

differences also separate these taxa. As noted above, the European species

associated with Carpinus has yellowish young tubes, flesh that turns violet

when sliced, and does not stain blue or green; Leccinum crocipodium has a

yellow hymenophore and does not stain green (den Bakker & Noordeloos

2005); Leccinum talamancae is less areolate and features flesh that turns slowly

pink to reddish orange in the pileus and stipe apex when exposed (Halling

1999). Our own additional unpublished alignments and phylogenetic analyses,

along with the ITS tree in Lebel et al. (2012a,b), support generic placement in

Leccinellum. Further research will determine whether Leccinellum as presently

circumscribed is or is not supported phylogenetically.

Acknowledgments

We are grateful to Timothy J. Baroni and Patrick R. Leacock for serving as pre-

submission reviewers and for their helpful comments on the manuscript; Darvin

DeShazer also provided helpful commentary. We thank the University of Michigan

Herbarium (MICH) for kindly lending specimens, and the William and Linda Steere

Herbarium of the New York Botanical Garden (NY) for processing deposits. We also

thank Henk den Bakker, Dana Ringuette, Barbara Thiers, Jean Toothman, and Sue

Yocum for invaluable assistance in various aspects of this study. The U.S. Forest Service,

Northern Research Station, Center for Forest Mycology Research provided funds for

DNA sequencing. We thank Kyah Norton of the CFMR for providing expert technical

support. Support to REH from the National Science Foundation, via grants DEB

#9972018, DEB #0414665, and DEB #1020421, is gratefully acknowledged.

Literature cited

Arora D. 1991. All that the rain promises and more: a hip pocket guide to western mushrooms.

Berkeley: Ten Speed Press.

Bergemann SE, Garbelotto M. 2006. High diversity of fungi recovered from the roots of mature

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MYCOTAXON

http://dx.doi.org/10.5248/124.333

Volume 124, pp. 333-340 April-June 2013

Cladonia dunensis sp. nov. from southern Brazil,

with notes on the genus in beach dune environments

EMERSON LUIZ GUMBOSKI'*, FLAVIO BEILKE” & SIONARA ELIASARO?

' Programa de Pés-Graduacdo em Botanica, Universidade Federal do Rio Grande do Sul,

Av. Bento Goncalves, 9500, Agronomia, Porto Alegre, RS, 91501-970, Brazil

? Rua Roberto Ponick, 292, Bairro Bom Retiro, Joinville, SC, 89223-250, Brazil

* Departamento de Botanica, Universidade Federal do Parana,

Setor de Ciéncias Biologicas, Curitiba, PR, 81531-980, Brazil

* CORRESPONDENCE TO: emersongumboski@gmail.com

ABSTRACT — Cladonia dunensis is described. The new species resembles C. celata in the

corticate and scyphose podetia that are simple at first before branching fastigiately but

differs in forming squamulose podetia and producing psoromic acid. Seven Cladonia species

are reported for the first time from beach dune environments in the coastal plain of Santa

Catarina State, southern Brazil.

Key worps — Ascomycota, Cladoniaceae, dimorphic thallus, lichens, taxonomy

Introduction

In the Brazilian coastal areas, sand dune habitats are covered by herbaceous,

arbustive and arboreal vegetation types named restinga vegetation (Araujo

1992). The lichenized restinga mycota has been intensively studied in the last

decade, primarily within arbustive-arboreal vegetation (e.g., Benatti & Marcelli

2008, 2009; Dal-Forno & Eliasaro 2010 a,b; Donha & Eliasaro 2006; Marcelli

& Benatti 2011; Martins & Marcelli 2011). However, there is no information

about species that colonize the beach dune environments in Brazil (Marcelli

1998).

Cladonia P. Browne, the largest genus in Cladoniaceae with c. 400 species

(Stenroos et al. 2002; Burgaz & Ahti 2009), is frequent and abundant in

arbustive restinga vegetation (Ahti 2000; Gumboski & Eliasaro 2012a,b), and

some species can colonize other adjacent environments.

As a part of a survey of the Cladoniaceae in the coastal region of southern

Brazil (Gumboski & Eliasaro 2012a,b) we present a first report on Cladonia

species found in the beach dune environment in Brazil, one of which is new to

science. Descriptions, comments and illustrations are presented.

334 ... Gumboski, Beilke & Eliasaro

Materials & methods

The specimens were collected in depressions between beach dunes in the Municipality

of Imbituba, Santa Catarina State, Brazil (Fic. 1). Specimens were examined using

standard stereoscopic (20-50x) and light microscopic (400-1000x) techniques. Sections

of primary thallus, podetia, and pycnidia were mounted in water. Chemical constituents

were identified by spot tests, under UV light, and thin layer chromatography using

solvent system C (Orange et al. 2001) and by comparison with authentic samples.

Taxonomy

Cladonia dunensis Gumboski, Beilke & Eliasaro, sp. nov. Fic. 2

MycoBank MB 804907

Differs from Cladonia celata by its squamulose podetia and by its production of psoromic

acid.

Type: Brazil. Santa Catarina State: Municipality of Imbituba, 28°17'57"S 48°42'14"W,

sea level, on acid soil in depressions between dunes, 19 Feb 2010, E. Gumboski & FE

Beilke 1750 (Holotype, UPCB; isotypes, ICN, JOI).

Erymo.oey: The epithet refers to the habitat of this species.

Primary thallus evanescent, lobate squamules, 1.0-3.0 mm long, 0.5-1.2 mm

wide, planes, not divided, smooth margins, without rhizines, esorediate; upper

surface greenish to whitish, corticate, smooth, not pruinose, without pycnidia,

lower surface white, ecorticate, smoothly arachnoid; cortex 40-70 um, medulla

110-420 um. Podetia 0.8-2.0 cm tall, 0.4-0.9 mm thick, greenish to brownish,

scyphose, irregularly branching, at first simple then finally fastigiate mainly

dichotomously, but with irregular branches common on podetial sides; scyphi

sometimes deformed at margin, <1.2 mm wide; surface corticate and verrucose,

sometimes cracked (some cracks can reveal the white medulla below), without

soredia or granules, schizidia and squamules present, lobate squamules <0.5

mm long; cortex 20-50(-80) um, medulla 50-120 um, hyaline stereome, 130-

190 um, papillate central canal, 270-500 um wide. Hymenial disks common,

globose, apical on podetia, pale brown to brownish, immature, ascospores not

seen. Pycnidia rare, brow to blackish, to 0.2 mm wide, slime and conidia not

seen.

CuHemistry: Thallus K-, C-, KC-, UV-. Psoromic acid [major] and 2’-O-

demethylpsoromic acid [minor] detected by TLC.

ECOLOGY AND DISTRIBUTION: The new species is known only on sandy soil

in depressions between dunes, where it usually grows intermixed with other

Cladonia species, such as C. ramulosa, C. didyma, and C. merochlorophaea.

This area, characterized by acidity and high humidity, develops an herbaceous

vegetation that is often used for cattle grazing and is periodically burned, as

observed in November 2011; thus the species is endangered.

ADDITIONAL SPECIMENS EXAMINED: BRAZIL. SANTA CATARINA STATE: Municipality

of Imbituba, 28°17'57"S 48°42'14"W, sea level, 19 Feb 2010, E. Gumboski & F. Beilke

1754b (JOD).

Cladonia dunensis sp. nov. (Brazil) ... 335

FiGure 1. The studied area: dunes from Municipality of Imbituba, Santa Catarina State, Brazil.

FiGuRE 2. Cladonia dunensis (holotype). Scale bar = 1 cm.

336 ... Gumboski, Beilke & Eliasaro

REMARKS: The new species is characterized by the evanescent primary

squamules, short, corticate, scyphose and squamulose podetia, at first simple

then branching fastigiately, by the brownish ascomata, and the psoromic acid

production.

According to the infrageneric classification suggested by Stenroos et al.

(2002), Cladonia dunensis probably belongs in supergroup Cladonia based on its

squamulose primary thallus, sparsely branched corticate podetia, unperforated

axils, and brownish ascomata.

Cladonia dunensis resembles C. celata A.W. Archer in similar podetia (Archer

1984, 1992). However, C. dunensis has squamulose podetia with schizidia and

contains psoromic acid, while C. celata has podetia without schizidia and

squamules and produces fumarprotocetraric acid. These two species also

exhibit disjunct geographic distributions, with C. celata occurring at 450-1100

m in Australia (Archer 1984), whereas C. dunensis is known only from the type

locality at sea level in dune environments in southern Brazil.

Cladonia fruticulosa Kremp. differs morphologically in having a persistent

primary thallus up to 5(-10) mm long and 2(-3) mm wide with crenate-incised

to deeply divided squamules, moderately branching podetia with variably

smooth or roughly corticate to partly or almost totally sorediate surfaces, and

is found at 440-3800 m (Swinscow & Krog 1988; Stenroos 1988).

The specimens of Cladonia ochracea found in the studied area are quite

similar to C. dunensis, but C. ochracea has scyphose to capitate podetia,

produces fumarprotocetraric acid and grows up to 3.5 cm tall.

Psoromic acid is very rare in Cladonia species from Brazil. Gumboski &

Eliasaro (2012a,b) recorded 23 species from restinga vegetation and rocky

shores from southern Brazil, and none of them produced this compound. Ahti

(2000) recorded 180 species from Brazil and psoromic acid was detected in

only some specimens of C. acuminata (Ach.) Norrl., C. cartilaginea Mill. Arg.,

and C. dactylota Tuck. Both C. acuminata and C. cartilaginea have ascyphose

podetia that have a granulose-sorediate surface in C. acuminata (Ahti 2000)

and are decorticate in C. cartilaginea (Herb. ICN, M. Fleig 3416, 6041). In

Cladonia dactylota the primary thallus is persistent, sometimes sorediate, and

the podetia, although scyphose, have sorediate surface with tuberculose soralia

below the scyphi or on podetial stalks (Ahti 2000; Herb. ICN, M. Fleig 5191,

5969).

The six other species found in beach dune environments are:

Cladonia crispatula (Nyl.) Ahti, Lichenologist 9: 14. 1977.

ECOLOGICAL NOTES: Specimens were found mainly between herbs and

isolated from other species on sandy soil. The specimens are smaller and

Cladonia dunensis sp. nov. (Brazil) ... 337

less frequent when compared to those seen in arbustive restinga vegetation

(Gumboski & Eliasaro 2012b).

SPECIMENS EXAMINED: BRAZIL. SANTA CATARINA STATE: Municipality of Imbituba,

28°17'S 48°42’W, sea level, 19 Feb 2010, E. Gumboski & FE. Beilke 1754a, 1755 (JOI); 10

Nov 2011, E. Gumboski, F. Beilke & S. Eliasaro 3020, 3027 (JOI).

DISTRIBUTION: Occurs from sea level to 2000 m. Recorded only from South

America: Brazil, Paraguay and Uruguay (Ahti 2000).

See Ahti (2000), Gumboski & Eliasaro (2012b) for descriptions.

Cladonia didyma (Fée) Vain., Acta Soc. Fauna Fl. Fenn. 4: 137. 1887.

ECOLOGICAL NOTES: The most frequent species found in the beach dunes,

growing on sandy soil in open sites or between herbs. Usually intermixed with

C. ochracea and C. ramulosa specimens.

SPECIMENS EXAMINED: BRAZIL. SANTA CATARINA STATE: Municipality of Imbituba,

28°17'S 48°42'W, sea level, 19 Feb 2010, E. Gumboski & EF. Beilke 1751 (JOI); 10 Nov

2011, E. Gumboski, F. Beilke & S. Eliasaro 3017, 3022 (JOI).

DISTRIBUTION: Cosmopolitan, from sea level to 3000 m (Ahti 2000). Recorded

from Oceania, Asia (Vainio 1887), Africa (Doidge 1950), North America

(Fulford 1937), Central America (Vainio 1887), and South America in Argentina

(Calvelo & Liberatore 2002), Bolivia (Ahti 2000), Brazil (Krempelhuber 1876,

as C. muscigena), Chile (Crombie 1876, as C. melanodes), Colombia, Ecuador

(Ahti 2000), Guyana (Sipman 1990), and Paraguay, Peru, Suriname, Trinidad &

Tobago, Uruguay, Venezuela (Ahti 2000).

See Ahti (2000), Ahti & Hammer (2002), Gumboski & Eliasaro (2012b) for

descriptions.

Cladonia latiloba Ahti & Marcelli, Fl. Neotrop., Monogr. 78: 249. 2000.

ECOLOGICAL NOTES: Only little thalli of this species were found. The

specimens were growing on sandy soil in open sites without vegetation cover

around them and usually isolated from other species of Cladonia.

SPECIMENS EXAMINED: BRAZIL. SANTA CATARINA STATE: Municipality of Imbituba

28°17'S 48°42'W, sea level, 19 Feb 2010, E. Gumboski & F. Beilke 1756, 1755 (JOI); 10

Nov 2011, E. Gumboski, FE. Beilke & S. Eliasaro 3018, 3019, 3026 (JOI).

DISTRIBUTION: Recorded only from Brazil, at sea level (Fleig et al. 1995; Ahti

2000).

See Ahti (2000), Gumboski & Eliasaro (2012a) for descriptions.

Cladonia merochlorophaea Asahina, J. Jap. Bot. 16: 713. 1940.

ECOLOGICAL NOTES: The specimens were represented by scattered podetia,

almost always intermixed with C. ochracea and C. ramulosa specimens on

sandy soil in open places.

338 ... Gumboski, Beilke & Eliasaro

SPECIMENS EXAMINED: BRAZIL. SANTA CATARINA STATE: Municipality of Imbituba,

28°17'S 48°42’'W, sea level, 19 Feb 2010, E. Gumboski & EF. Beilke 1749 (JOI); 10 Nov

2011, E. Gumboski, F. Beilke & S. Eliasaro 3021, 3025 (JOI).

DISTRIBUTION: According to Ahti (2000), this species usually occurs in high

altitudes from 900 to 4100 m. Recorded from Oceania (Galloway 1985), Asia

(Ahti 1976), Europe (Asahina 1940), North America (Archer 1992), and South

America in Brazil (Fleig et al. 1995), Colombia (Flakus et al. 2008), Peru, and

Venezuela (Ahti 2000).

See Ahti (2000), Burgaz & Ahti (2009), Gumboski & Eliasaro (2012a) for

descriptions.

Cladonia ochracea L. Scriba, Cladon. Exs.: no. 1006. 1923.

ECOLOGICAL NOTES: Usually growing intermixed with C. didyma and

C. ramulosa specimens on sandy soil in open places. A few specimens were

found growing on decaying logs and on an anthill between herbs.

SPECIMENS EXAMINED: BRAZIL. SANTA CATARINA STATE: Municipality of Imbituba,

28°17'S 48°42’'W, sea level, 19 Feb 2010, E. Gumboski & EF. Beilke 1752 (JOI); 10 Nov

2011, E. Gumboski, F. Beilke & S. Eliasaro 3023, 3028 (JOI).

DISTRIBUTION: Occurs from sea level to 1900 m (Ahti 2000). Recorded only

from South America in Argentina (Ahti 2000), Brazil (Osorio & Fleig 1991),

and Paraguay and Uruguay (Ahti 2000).

See Fleig et al. (1995), Ahti (2000), Gumboski & Eliasaro (2012a) for

descriptions.

Cladonia ramulosa (With.) J.R. Laundon, Lichenologist 16: 225. 1984.

ECOLOGICAL NOTES: The specimens were mainly found as scattered podetia

usually growing intermixed with C. didyma and C. ochracea specimens on

sandy soil in open places.

SPECIMENS EXAMINED: BRAZIL. SANTA CATARINA STATE: Municipality of Imbituba,

28°17'S 48°42'W, sea level, 19 Feb 2010, E. Gumboski & F. Beilke 1748, 1753 (JOI); 10

Nov 2011, E. Gumboski, F. Beilke & S. Eliasaro 3024 (JOI).

DISTRIBUTION: Cosmopolitan (Burgaz & Ahti 2009), occurring from sea level

to 2000 m (Ahti 2000). Recorded from Oceania, Asia, Europe, Africa, North

America, Central America (Vainio 1894, as C. pityrea), and South America

in Argentina (Ferraro & Ahti 1987), Brazil (Osorio & Fleig 1988), Colombia,

Paraguay (Ahti 2000), Peru (Vainio 1894, as C. pityrea), and Uruguay and

Venezuela (Ahti 2000).

See Ahti (2000), Burgaz & Ahti (2009), Gumboski & Eliasaro (2012a) for

descriptions.

Cladonia dunensis sp. nov. (Brazil) ... 339

Acknowledgments

We are grateful to Dr. Alan W. Archer, Dr. Lidia I. Ferraro, Dr. Soili Stenroos, and

Msc. Celso Voos Vieira for providing a valuable bibliography and for discussions with

the first author and to the reviewers Dr. Marcelo Pinto Marcelli, Dr. Samuel Hammer,

and Dr. Shaun R. Pennycook for all the comments and suggestions. The authors also wish

to thank CAPES (Coordenadoria de Aperfeigoamento do Pessoal do Ensino Superior)

for granting a Mastership to Gumboski and to Beilke.

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MY COTAXON

http://dx.doi.org/10.5248/124.341

Volume 124, pp. 341-347 April-June 2013

Coniolepiota spongodes (Agaricaceae, Basidiomycota)

in Bangladesh and China

Mb. IQBAL HOSEN”? & ZHU L. YANG?

'Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany,

Chinese Academy of Sciences, Kunming 650201, Yunnan, P. R. China

*University of Chinese Academy of Sciences, Beijing 100049, P. R. China

CORRESPONDENCE TO: ‘fungi@mail.kib.ac.cn; '’iqbalpatho@gmail.com

AsBsTRACT — The genus Coniolepiota is recorded for the first time from Bangladesh and

China. Coniolepiota spongodes is characterized by its gray lilac to purple floccose basidiomata

and relatively small smooth ellipsoid to oblong basidiospores. A detailed morphological

description, photographs and line drawings, with confirmation of the phylogenetic position

of newly reported taxon are provided herein.

Key worps — Agaricales, Asian fungus, ITS-nrLSU, morphology, new record

Introduction

Coniolepiota Vellinga, a genus of family Agaricaceae recently described

from tropical Asia, harbors a single relatively well-known species, C. spongodes

(Vellinga et al. 2011). During studies of the higher fungi (Hosen & Ge 2011;

Hosen et al. 2013), collections of C. spongodes were made for the first time

from Bangladesh. A study of the Bangladesh and Chinese materials is provided

here.

Materials & methods

Morphological studies

Collections were made along roadsides in forests of Shorea robusta Gaertn.

(Dipterocarpaceae) in Bangladesh, and from tropical China. Fresh specimens were

described and photographed in the field. Dried specimens were deposited in the

Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences, Yunnan,

China (KUN-HKAS), and in the Herbarium of Agarics Flora, Department of Plant

Pathology, Faculty of Agriculture, Sher-e-Bangla Agricultural University, Dhaka,

Bangladesh (SHAF).

342 ... Hosen & Yang

Microscopic observations were made in 5% aqueous KOH, and structures drawn

by free hand. The notations “(n/m/p)’ indicate that the measurements were made on ‘n’

basidiospores from ‘m basidiomata of ‘p’ collections with a minimum of 20 basidiospores

per basidioma. Dimensions of basidiospores are presented in the following form

Coniolepiota spongodes HM488756, HM488774; Thailand

Coniolepiota spongodes -, KC871015; China

100 } Coniolepiota spongodes KC625531, KC625530; Bangladesh

97 Coniolepiota aff. spongodes HM488757, -; Thailand

Heinemannomyces splendidissima HM488760, HM488769

Agaricaceae sp. HM488761, HM488770

100

65 Eriocybe chionea HM488752, HM488773

Clarkeinda trachodes HM478750, HM488771

Chlorophyllum brunneum AY083205, AF482886

100

100 Chlorophyllum rachodes AY081236, AY176345

Chlorophyllum molybdites AY243617, U85303

Leucoagaricus americanus AY 176407, AF482891

Leucoagaricus meleagris AF482867, AF482890

100

Micropsalliota sp. HM488762, HM488768

100

Allopsalliota geesterani AF482857, AF482888

Macrolepiota dolichaula HM125522, JN940269

100

Macrolepiota procera JQ683110, AM946456

Lycoperdon pyriforme AY854075, NG0O27553

0.1

Tubaria vinicolor DQ536417, DQ536415

FicureE 1. A phylogenetic tree generated from the combined ITS-nrLSU dataset of Agaricaceae

using maximum likelihood (ML) method. Posterior probabilities from Bayesian inference (BI)

(>0.97) are indicated as thick branches and bootstrap values derived from ML (BS>60%) are shown

above the branches at nodes. Newly generated sequences for Coniolepiota spongodes are highlighted

in boldface with red color. GenBank accessions ITS and nrLSU are provided after the species name

and unavailable sequence is marked as hyphen sign.

Coniolepiota spongodes in Bangladesh & China ... 343

(a—)b-c(—d); in which ‘b-c’ contains at least 90% of the measured values and extreme

values ‘a and ‘d’ are setin parentheses.Q_ =Q+SD;Q refers to the ratio of individual spore,

Q__ refers to the average Q of basidiospores, and SD is the standard deviation.

Molecular procedures and phylogenetic analysis

The protocol for DNA extraction followed those of Doyle & Doyle (1987). ITS1/

ITS4 (White et al. 1090) and LROR/LRS (Vilgalys & Hester 1990) primer pairs were

used for the amplification of internal transcribed spacer (ITS) and the large subunit

nuclear ribosomal RNA (nrLSU) regions, respectively. PCR amplification, sequencing

and sequence alignment, Maximum Likelihood (ML) and Bayesian Inference (BI)

methods followed those in Hosen et al. (2013). Phylogenetic analysis was conducted on

the combined dataset of the ITS and the nrLSU.

Molecular results

Two sequences were newly generated in this study and are deposited in

GenBank. The combined ITS-nrLSU dataset consisted of 1689 nucleotide

sites (including gaps). In this dataset, 1116 characters were constant, while

573 characters were variable, of which 211 were parsimony uninformative

and 362 were parsimony informative. Parsimony analysis resulted in one

most parsimonious tree of 1329 steps, with Consistency Index (CI) = 0.634,

Retention Index (RI) = 0.590 and Rescaled Consistency Index (RC) = 0.374.

Tree topologies obtained from both ML and BI methods of phylogenetic

analysis were almost identical. Sequences from collection of C. spongodes made

from Bangladesh fall in a highly supported clade with those described from

northern Thailand (Fic. 1).

Taxonomy

Coniolepiota spongodes (Berk. & Broome) Vellinga, Mycologia 103: 502, 2011.

Figs 2, 3

BASIDIOMATA solitary, medium-sized. PILEus 40-80 mm broad, plano-

convex to applanate with slightly inflexed margin, covered with lilac to purple-

gray powdery velar warts at young stage which remain in some areas especially

in the center, but in outer zone sometimes disappearing, showing the pallid

to whitish background; warts thick at center, becoming spread out towards

margin, sometimes with powdery warts hanging over the margin. LAMELLAE

14 x 4 mm, free and remote from the stipe, crowded with lamellulae, pallid

cream to pale yellowish. StrpE 25-50 x 6-10 mm, central, cylindrical, usually

slightly tapering upward, glabrous and pallid white at apex but the remaining

parts covered with powdery lilac warts as on pileus, base slightly swollen and

covered with whitish mycelium, hollow. ANNULUs present, thin membranous

and easily detachable. CONTEXT 7 mm thick at the center of the pileus, dull

white to slightly pale purplish.

344 ... Hosen & Yang

FIGURE 2. Coniolepiota spongodes. a—b. Basidiomata show gray lilac velar coverings on the surface

of pileus and stipe. c. Hymenophore showing free, crowded, pallid to cream-white lamellae with

powdery warts hanging over the margin. d. Gray lilac velar coverings disappearing from the

surface of the pileus and stipe at maturity and/or handing. (a = HKAS 77576; b = HKAS 60246;

c = SHAF 5; d= HKAS 77574). Photos: a, c, d, Md. Iqbal Hosen; b, Zai- Wei Ge.

BasIpiospores [80/4/4] 4-5(-6) x (2.8-)3-3.2(-3.5) um [Q = 1.43-1.57,

Q_ = 1.52 + 0.127], ellipsoid to somewhat oblong, hyaline to pale yellowish,

dextrinoid, inamyloid, nearly rounded apex, smooth, slightly thick-walled,

without germ pore. BAsip1a 12-20 x 5-7 um, cylindro-clavate to clavate,

hyaline to pale yellowish in H,O, thin-walled, tetrasporic but occasionally 2- or

3-spored. CHEILOCYSTIDIA 20-26 x 6-9 um, clavate to clavulate, some cylindro-

clavate, hyaline, thin-walled, smooth, without encrustations, not abundant,

scattered and sometimes hard to find. PLEUROCysTIDIA not observed. PILEUS

COVERING a powdery layer made up of irregularly shaped cylindrical cells,

three dimensional (T-shaped), some anastomosing (H-shaped), individual

elements 20-50 x 4-10 um, hyaline, smooth, without incrusting pigments,

refractive connecting points often present at the septate portion or near to the

ending points of the pileal hyphae. STIPE COVERING similar to those of pileus

but refractive connecting points is less common. CLAMP CONNECTIONS absent

in all tissues.

Coniolepiota spongodes in Bangladesh & China... 345

10 pm

—

——

FIGURE 3. Coniolepiota spongodes. a. Basidiospores. b. Basidia at different stages of development;

c. Stipe covering elements; d. Pileus covering elements; e. Cheilocystidia. (a = HKAS 77575;

b-e = HKAS 77576).

346 ... Hosen & Yang

Hasit & DIsTRIBUTION — Solitary or in small groups of two along roadsides

in forests of S. robusta, in Bangladesh. Known also from Sri Lanka, Thailand,

Singapore, Malaysia, tropical China.

SPECIMENS EXAMINED — BANGLADESH. RanGpvur Division: Thakurgaon, Pirganj,

Thumnia Sal Baghan, 70 ma.s.l., 18 July 2011, M.I. Hosen 239a (HKAS 77574; GenBank

KC625530, KC625531); 18 June 2012, M.I. Hosen 373 (HKAS 77575); Dinajpur, Birganj,

Singra Forest, 60 m a.s.l., 1 July 2013, M.I. Hosen 708 (SHAF 5); DHaka DIVISION:

Tangail, Madhupur, Madupur National Park, 20 m a.s.l, 3 August 2012, M.I. Hosen 561

(HKAS 77576). CHINA. YUNNAN PROVINCE: Luxi, Santai Mountain, 1200 m a.s.1., 29

July 1979, Zheng Wen Kang 79078 (HKAS 4848); Menhai, Mangao Nature Reserve,

1250 m a.s.l., 16 August 1991, Zhu L. Yang 1590 (HKAS 24071); Ruili, Nongdao, 1130

m a.s.l., 31 July 2003, Luo Hong 92 (HKAS 43631); HAINAN PROVINCE: Wuzhishan,

Wuzhishan Nature Reserve, 1867 m a.s.l., 1 August 2010, Z.W. Ge 2570 (HKAS 60246;

GenBank KC871015).

COMMENTS — Coniolepiota spongodes is restricted to southeast and south Asia.

It is an attractive species for mushroom pickers as it can be readily recognized by

the purplish to gray lilac coverings of the pileus and stipe surface. Distinguishing

microscopic characters include the absence of abundant cheilocystidia, small

smooth ellipsoid to oblong basidiospores, cylindrical elements in the pileal

and stipe coverings, and absence of clamp connections in all tissues. Vellinga,

who established Coniolepiota as an independent genus in Agaricaceae based

on morphological and molecular evidence, presented the full synonymy of

C. spongodes (Vellingaetal. 2011). According to Pegler (1972, 1986) basidiospores

of C. spongodes are dextrinoid, which was confirmed by our study. The Chinese

materials of C. spongodes also bear the same features that were observed in

Bangladesh specimens: pileus and stipe coverings gray purple, basidiospores

measuring [4/4/80] 4—5.5 x (2.5-)3-3.5(-4.0) um [Q = 1.41-1.67, Q. = 1.52

+ 0.118], ellipsoid, colorless, dextrinoid, inamyloid, slightly thick-walled, and

without germ pore. Vellinga et al. (2011) reported that the basidiospores from

Thai collections are pale or barely weakly dextrinoid in Melzer’s reagent.

Phylogenetically, C. spongodes nests within the Agaricus s.l. clade based

on multi-gene analysis with four different loci (ITS, nrLSU, tefl-a, and rpb2;

Vellinga et al. 2011). Coniolepiota spongodes is closely related to the colored-

spored genera Agaricus L., Heinemannomyces Watling, and Clarkeinda Kuntze

(Vellinga et al. 2011). In our molecular analysis H. splendidissima Watling

is strongly supported to be sister group to Coniolepiota with ML (96%) and

BI (1.0) values, followed by Agaricaceae sp., Eriocybe chionea Vellinga and

Clarkeinda trachodes (Berk.) Singer (Fic. 1). Vellinga et al. (2011) described

the genus Coniolepiota to include collections of C. spongodes from Malaysia,

Singapore, and Thailand. A collection from Thailand (ECV 3613), described as

having pink-tinges to the pileus and stipe, was designated as C. aff. spongodes

but not formally named. Minor differences in cheilocystidia shape and color

of basidiomata were possibly due to environmental variation. Inclusion of our

Coniolepiota spongodes in Bangladesh & China ... 347

Bangladesh and Chinese collections in phylogenetic analysis of ITS-nrLSU

support the recognition of a single species in the genus rather than two distinct,

sister species.

Acknowledgments

This study was supported by the Knowledge Innovation Program of the Chinese

Academy of Sciences (No. KSCX2-EW-Z-9). Dr. Zai-Wei Ge (China) is gratefully

acknowledged for providing photo of C. spongodes. The authors are grateful and sincere

thanks to Dr. Teresa Lebel (Australia) and Dr. Takamichi Orihara (Japan) for their

critical revision, comments, and reviewing this manuscript prior to submission.

Literature cited

Doyle JJ, Doyle JL. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue.

Phytochem. Bull. 19: 11-15.

Hosen MI, Feng B, Wu G, Zhu XT, Li YC, Yang ZL. 2013. Borofutus, a new genus of Boletaceae

from tropical Asia: phylogeny, morphology and taxonomy. Fungal Divers. 58(1): 215-226.

http://dx.doi.org/10.1007/s13225-012-0211-8

Hosen MI, Ge ZW. 2011. Clarkeinda trachodes (Agaricales, Basidiomycetes), first record from

Bangladesh. Mycotaxon 118: 331-336. http://dx.doi.org/10.5248/118.331

Pegler DN. 1972. A revision of the genus Lepiota from Ceylon. Kew Bull. 27: 155-202.

http://dx.doi.org/10.2307/4117880

Pegler DN. 1986. Agaric flora of Sri Lanka. Kew Bull. add. Series 12: 1-519.

Vellinga EC, Sysouphanthong P, Hyde KD. 2011. The family Agaricaceae: phylogenies and two new

white-spored genera. Mycologia 103(3): 494-509. http://dx.doi.org/10.3852/10-204

Vilgalys R, Hester M. 1990. Rapid genetic identification and mapping of enzymatically amplified

ribosomal DNA from several Cryptococcus species. J. Bacteriol. 172: 4238-4246.

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, San Diego.

MY COTAXON

http://dx.doi.org/10.5248/124.349

Volume 124, pp. 349-352 April-June 2013

Pertusaria albiglobosa, a new lichen from China

QIANG REN

College of Life Science, Shandong Normal University, Jinan, 250014, China

CORRESPONDENCE TO: rendaqiang@hotmail.com

ABSTRACT — Pertusaria albiglobosa is described as new, based on collections from Shaanxi

province in China. The species is characterized by the white thallus, spherical verrucae,

asci with 2 smooth-walled ascospores, and the presence of 2'-O-methylperlatolic acid and

4,5-dichlorolichexanthone. It grows on rotten mosses in alpine meadow at elevations of

3570-3760 m.

Key worps — Pertusariaceae, Pertusariales, lichenized fungi, western China

Introduction

Species of Pertusaria DC. colonize almost all habitats. The apothecial

structure, the number and structure of the ascospores and the chemistry are

the diagnostic characters in identification of Pertusaria species (Oshio 1968,

Dibben 1980, Archer 1997, Schmitt & Lumbsch 2004). In China, the genus

received little attention until Zhao et al. (2004) published an annotated key to

the revised taxa of Pertusaria from China in which 41 species and 4 varieties

were accepted. A recent study of the Pertusaria specimens collected from

Qinling Mountains in western China revealed a new species, described here.

Materials & methods

All specimens examined are preserved in SDNU (the Lichen Section of Botanical

Herbarium, Shandong Normal University). An Olympus SZ 51stereomicroscope and

Olympus CX 21 compound microscope were routinely used for the morphological

and anatomical examinations. Colour reactions (spot tests) were made using standard

methods (Orange et al. 2001). The chemical constituents were identified using thin layer

chromatography (TLC) (Culberson 1972) and gradient-elution high performance liquid

chromatography (HPLC) (Lumbsch 2002).

Pertusaria albiglobosa Q. Ren, sp. nov. PLATE 1

MycoBank MB 804971

Differs from Pertusaria trochiscea by its 2-spored asci, its production of 2'-O-

methylperlatolic acid, and its lack of stictic acid.

350 ... Ren

Pate 1. Pertusaria albiglobosa (J.Zh. Zhao 899, SDNU). A. Thallus; B. Apothecium, cross-section;

C-D. Asci with 2 ascospores. Bars: A = 1 mm; B & C= 100 um; D = 50 um.

Type: China. Shaanxi: Taibai Co., Mt. Qinling, Baxiantai, 33°67'N 107°58’E, alt. 3760 m,

5 Aug. 2005, J.Zh. Zhao 899 (Holotype, SDNU).

Erymo oey: Latin albiglobosa, referring to the colour of the thallus and the shape of

the fertile verrucae.

THALLUS white or whitish-gray, episubstratal, thin or fairly thick, the margin

entire and unzoned; suRFACE tuberculate, matt and continuous; IsIDIA AND

SOREDIA absent. VERRUCAE numerous, crowded or well dispersed, but

occasionally fused, (0.5-)0.7-1.0(-1.2) mm in diameter, concolorous with

the thallus, persistently closed and appearing as a spherical protuberance;

Pertusaria albiglobosa sp. nov. (China) ... 351

OSTIOLES single, black. APOTHECIA 1 (occasionally 2-3) per verruca, the fruit

center hyaline; EPITHECIUM brown, K+ violet, hypothecium hyaline, hymenium

colorless, 325-450 um thick. Asci clavate or cylindric; AscosPORES 2 per ascus,

uniseriate, ellipsoid to fusiform (at times deformed), 63-135(-163) x 30-55

um, the spore wall double, smooth, 7.5-10 um thick, and usually trimmed, the

end wall 12.5-17.5 um thick. Pycnrp1a unknown.

SPOT TESTS — cortex: all chemical tests negative, UV-; medulla: all chemical

tests negative.

SECONDARY METABOLITES — 4,5-dichlorolichexanthone and 2’-O-

methylperlatolic acid (HPLC).

EcoLocy — Pertusaria albiglobosa is a muscicolous species occurring in

alpine meadows. It is known only from Shaanxi province (western China) at

elevations of 3570-3760 m.

ADDITIONAL SPECIMENS EXAMINED — CHINA. SHAANXI: Taibai Co., Mt. Qinling,

Wengong Temple, alt. 3570 m, 5 Aug. 2005, Ch.L Wang & F. Yang TBW052 (SDNU); alt.

3600 m, 5 Aug. 2005, Sh.X. Guo & YJ. Li QL004 (SDNU)); Baxiantai, alt. 3767 m, 5 Aug.

2005, Sh.X. Guo & X.L. Shi QL191 (SDNU).

Comments — Pertusaria albiglobosa is characterised by the matte white thallus,

spherical verrucae, 2-spored asci, smooth-walled spores, and chemistry. In

morphology and habitat, this species is very similar to P trochiscea Norman and

Megaspora verrucosa (Ach.) L. Arcadia & A. Nordin, which differ in number

of ascospores per ascus and the chemistry: P trochiscea produces 4 spores

per ascus and 4,5-dichlorolichexanthone and stictic acid, and M. verrucosa

produces 8 spores per ascus and no lichen products. Pertusaria saximontana

Wetmore also has a similar anatomy and chemistry but is readily distinguished

by the shiny gray thallus, wide blackish discs usually surrounded by an eroded

inner verrucal margin, smaller ascospores, and its habitats. Pertusaria signyae

@Ovstedal, which might be confused with the new species, is also a muscicolous

(also saxicolous) species with 2 smooth spores per ascus, but it has isidia and

contains 2’-O-methylperlatolic acid only.

Acknowledgements

The author is grateful to Prof. Jack Elix (Australian National University) for his help in

confirming the chemistry and to Dr. Alan W. Archer (Royal Botanic Gardens, Australia)

for professional advice. The author thanks Drs. André Aptroot (ABL Herbarium, Soest,

The Netherlands) and Shouyu Guo (Institute of Microbiology, CAS) for presubmission

review. This study was supported by the National Natural Science Foundation (nos.

31100011 & 31093440).

Literature cited

Archer AW. 1997. The lichen genus Pertusaria in Australia. Bibliotheca Lichenologica 69: 1-249.

Culberson CE. 1972. Improved conditions and new data for the identification of lichen products

by a standardized thin-layer chromatographic method. Journal of Chromatography A 72:

113-125. http://dx.doi.org/10.1016/0021-9673(72)80013-X

352... Ren

Dibben MJ. 1980. The chemosystematics of the lichen genus Pertusaria in North America North of

Mexico. Publications in Biology and Geology, Milwaukee Public Museum 5: 1-162.

Lumbsch HT. 2002. Analysis of phenolic products in lichens for identification and taxonomy.

281-295, in: I Kranner et al. (eds). Protocols in Lichenology. Berlin: Springer Verlag.

Orange A, James PW, White FJ. 2001. Microchemical methods for the identification of lichens.

London: British Lichen Society.

Oshio M. 1968. Taxonomical studies on the family Pertusariaceae of Japan. Journal of Science of

the Hiroshima University, Series B, Div. 2 (Botany) 12: 81-151.

Schmitt I, Lumbsch HT. 2004. Molecular phylogeny of the Pertusariaceae supports secondary

chemistry as an important systematic character set in lichen-forming ascomycetes. Molecular

Phylogenetics and Evolution 33: 43-55. http://dx.doi.org/10.1016/j.ympev.2004.04.014

Zhao ZT, Ren Q, Aptroot A. 2004. Anannotated key to the lichen genus Pertusariain China. Bryologist

107(4): 531-541. http://dx.doi.org/10.1639/0007-2745(2004)107[531:AAKTTL]2.0.CO;2

Zhurbenko MP, Lumbsch HT. 2006. Pertusaria christae is a synonym of P. saximontana.

Lichenologist 38(5): 487-490. http://dx.doi.org/10.1017/S0024282906005767

MYCOTAXON

http://dx.doi.org/10.5248/124.353

Volume 124, pp. 353-359 April-June 2013

Lichenological notes 6: nomenclatural acts

KERRY KNUDSEN” & JANA KOCOURKOVA?’

'The Herbarium, Department of Botany and Plant Sciences, University of California,

Riverside, CA 92521-0124, U.S.A.

*Department of Ecology, Faculty of Environmental Sciences, Czech University of Life Sciences,

Prague, Kamycka 129, Praha 6 - Suchdol, CZ-165 21, Czech Republic

CORRESPONDENCE TO: '’Knudsen@ucr.edu & kocourkovaj@fzp.czu.cz ”

ABSTRACT — Lecanora peltastictoides is transferred to Aspicilia. Thelenella americana is

treated as Melanophloea americana and T: montana as M. montana. Sarcogyne athroocarpa

is treated as a synonym of Acarospora badiofusca and a lectotype is designated. Pleopsidium

stenosporum is treated as a synonym of P. flavum.

Key worps — Acarosporaceae, Joshua Tree National Park, Thelocarpaceae

The species

Acarospora badiofusca (Nyl.) Th. Fr., Nova Acta Regiae Soc. Sci. Upsal.,

Ser. 3: 3 190 (1861).

= Lecanora badiofusca Nyl., Herb. Mus. Fenn.: 110 (1859).

Type: Ad saxa granitica in Lapponia, Nylander s.n. (H-NYL! holotype).

= Sarcogyne athroocarpa H. Magn., Ann. Crypt. Exot. 7: 137 (1935) [“1934”], syn. nov.

Tye: U.S.A., Colorado, Boulder Co., Sugar Loaf, 2743 m, on granite, Aug. 1898, Morgan

s.n., Lichenes Boreali-Americani 227 (FH! holotype, destroyed by sampling?; COLO!

L-79776, lectotype designated here).

The holotype collection of Sarcogyne athroocarpa has only a Lecidea

species remaining, and the Sarcogyne is presumed to have been destroyed by

sampling. In COLO we have discovered a syntype of S. athroocarpa, originally

identified by W.A. Weber, which we designate as a lectotype. Unfamiliar with

extremely reduced forms of A. badiofusca in North America at high elevations,

H. Magnusson, who described the reduced dispersed apothecia of A. badiofusca

as an endolithic Sarcogyne, suggested that its sterile areoles represented another

“Acarospora” species (Magnusson 1935). As with A. badiofusca, the hymenium

is low (60-70 um) and the perithecial crown (exciple) is brown (it often becomes

354 ... Knudsen & Kocourkova

blackened in many specimens). Therefore, we place Sarcogyne athroocarpa in

synonymy with A. badiofusca.

"ty

PLATE 1. Aspicilia peltastictoides (Knudsen et al. 14420, UCR),

Coxcomb Mountains, Joshua Tree National Park. (Tim Wheeler, photo). Scale bar = 1 mm.

Aspicilia peltastictoides (Hasse) K. Knudsen & Kocourk., comb. nov. PLATE 1

MycoBank MB 803946

= Lecanora peltastictoides Hasse, Bryologist 17: 63 (1914).

Type: U.S.A., California, Palm Springs, on granite, 1901, Hasse 861 (FH!, holotype)

In 1914, the pioneer California lichenologist H.E. Hasse (1836-1915)

described Lecanora peltastictoides from granite in the Sonoran Desert in Palm

Springs, California. During the revision of Lecanora for the Sonoran Lichen

Flora, no specimens besides the type were seen, and the species remained

unrevised but recognized as an Aspicilia (Knudsen 2003; Ryan et al. 2004).

During our inventory of Joshua Tree National Park, we collected five specimens

of Lecanora peltastictoides in the Mojave and Sonoran Deserts.

Lecanora peltastictoides is an Aspicilia with small ascospores (mostly 12-13

x 9-10 um), eight per ascus, a white striated thallus, black pruinose apothecia

with elevated crenulate margins, and no secondary metabolites. No conidia have

been observed. It occurs on granite, usually on softer decaying rock at Joshua

Tree National Park. There are only two other Aspicilia species in California

with similar sized ascospores. Both species, A. brucei Owe.-Larss. & A. Nordin

and A. cinerea (L.) K6rb., can easily be distinguished from A. peltastictoides

Lichenological notes 6... 355

by their production of norstictic acid. Aspicilia peltastictoides looks similar

to two other lichen species in southern California, Acarospora strigata (Nyl.)

Jatta and Lecanora utahensis H. Magn. The polyspored asci easily distinguish

A. strigata from A. peltastictoides. An older name for A. strigatain North America

is A. peltasticta Zahlbr., which explains the etymology of the specific name of

peltastictoides. Lecanora utahensis has been confused with A. peltastictoides

not only because they look similar, but because they both have similar sized

ascospores (Knudsen & Lendemer 2006; Knudsen 2012). Lecanora utahensis,

a member of the Lecanora dispersa group (Sliwa 2007), differs especially in the

production of isousnic acid and the Lecanora-type ascus stain. We regard both

A. peltastictoides and L. utahensis as rare in California.

ADDITIONAL SPECIMENS EXAMINED — U.S.A., California, Riverside Co., Little San

Bernardino Mountains, Berdoo Canyon, 1025 m, rare on granite along wash, 7 Dec.

2010, Knudsen 12869.2 & Harding (UCR); upper Covington Flats, 1426 m, on granite,

9 Nov. 2011, Knudsen 14043 & Kocourkova (UCR); SAN BERNARDINO Co., 49 Palms

Canyon, 844 m, on granite boulder in palm oasis, 29 Dec. 2010, Knudsen 13469 (UCR);

Coxscomb Mountains, 635 m, on decaying granite boulder, 7 Dec. 2011, Knudsen et al.

14420 (UCR).

Melanophloea americana K. Knudsen & Lendemer, Opusc. Philolich. 9: 61 (2011).

= Thelenella americana (K. Knudsen & Lendemer) Aptroot, Lichenologist 44: 504 (2012).

Type: U.S.A., Pennsylvania, Montgomery Co., Pennypack Watershed, on silicate rock,

Nov. 1987, A. Aptroot 21376 (NY! holotype; ABL[n.v.], BR[n.v.], isotypes).

Melanophloea montana P.M. McCarthy, Australas. Lichenol. 62: 26 (2008).

= Thelenella montana (P.M. McCarthy) Aptroot, Lichenologist 44: 506 (2012).

Type: Australia, Queensland, Atherton Tableland, Tully Falls, on siliceous rock, 7 August

2006, McCarthy 2520 (CANB, holotype, n.v.).

Melanophloea (Thelocarpaceae) contains three species. The type of the genus

is M. pacifica P. James & Vézda, described from the Solomon Islands (James

& Vézda 1971). The saxicolous M. montana was described from Australia

(McCarthy 2008), and the saxicolous M. americana was described from

eastern North America (Knudsen et al. 2011). Recently Aptroot & Schumm

(2012) transferred M. americana and M. montana from the Thelocarpaceae

to Thelenella in the Thelenellaceae. Melanophloea americana and M. montana

differ from Thelenella species in the following important characters that were

overlooked by these authors:

1. Melanophloea americana and M. montana have Thelocarpon-like ascomata

that are apothecioid with an inner wall that is an incurving exciple, reducing

the size of the hymenial disc (clearly visible when wetted), and an outer wall

that is basically a thalline margin (Salisbury 1966; Horakova 1998; Knudsen

& Lumbsch 2007; McCarthy 2008; Knudsen et al. 2011). In M. americana and

356 ... Knudsen & Kocourkova

M. pacifica, algae are scattered between the outer and inner walls, extending

up from the algal layer in the thallus. This apothecioid fruiting body differs

fundamentally from the single-walled perithecia of Thelenella species

(Mayrhofer & Poelt 1985; Mayrhofer 2002; Orange et al. 2009).

2. Melanophloea americana and M. montana differ from Thelenella species in

having apothecioid ascomata with distinct outer and inner walls. The outer or

thalline wall of the ascomata in the Melanophloea species is melanized, with the

melanization of the outer thalline wall possibly protecting the algae beneath.

The inner wall (an incurving exciple) is hyaline. Aptroot & Schumm (2012)

misinterpreted the outer wall of M. americana as a clypeus, which we define as

a wall in corticolous species formed of fungal and bark cells, not the melanized

wall of a saxicolous fungus containing no distinct substrate particles and which

is basically thalline (Nash et al. 2002). Thelenella has single-walled perithecia,

also without a clypeus using the definition we accept (Mayrhofer & Poelt 1985;

Mayrhofer 2002; Orange et al. 2009).

3. Melanophloea americana and M. montana have hyaline simple ascospores

in polyspored asci with a K/I+ blue stain, while all Thelenella taxa have

2-8-spored asci with a K/I- stain and large muriform or submuriform spores

(Mayrhofer & Poelt 1985; Mayrhofer 2002; Orange et al. 2009).

Based on these characters we reject the emendation of Thelenellaceae proposed

by Aptroot & Schumm (2012). The speculations of Aptroot & Schumm (2012)

about the evolutionary development of polyspory are not relevant in this case

because M. americana and M. montana are not Thelenella species.

The radical emendation of Thelenella by Aptroot & Schumm (2012) lacks

not only convincing morphological evidence but molecular support. While

we are open to the possibility that M. americana and M. montana are not

congeneric with M. pacifica, to radically emend another genus and family

lacking molecular data and strong morphological support is speculative

and undermines nomenclatural stability. That molecular data are required

to confirm their hypothesis is reinforced by the fact that M. americana and

M. montana differ from Thelenella in significant morphological characters.

Until these further results are obtained, we treat T. americana and T: montana

as members of Melanophloea using the names M. americana and M. montana.

It should be noted that Aptroot & Schumm (2012) cited inaccurate type

collections for Melanophloea americana. When the name M. americana was

introduced, the type collection was clearly stated to be Aptroot 21376 (with

the holotype deposited at NY). A second collection from the same site as the

holotype, Aptroot 21389, was used by NY curator R.C. Harris to produce

the microscopic images for the protologue to preserve the holotype material.

Although Aptroot 21389 was cited in the figure caption in the protologue, it

was inadvertently omitted from list of additional specimens examined. This

Lichenological notes 6 ... 357

collection (Aptroot 21389) is a paratype or a topotype of M. americana, but it is

not an isotype as stated by Aptroot & Schumm (2012: 504), because a separate

collection number clearly indicates it does not represent part of the same

gathering as the type. The holotype of M. americana consists of a large rock,

with abundant and well developed thalli and ascomata. Although the quality

of a specimen is irrelevant from a nomenclatural standpoint, this particular

collection was selected as the type because of its exemplary condition compared

with the other available material.

PLATE 2. Pleopsidium flavum (Knudsen 12911, UCR), Joshua Tree National Park.

(Tim Wheeler, photo). Scale bar = 1 mm.

Pleopsidium flavum Korb., Syst. Lich. Germ.: 113 (1855). PLATE 2

TyPeE: Switzerland, Schaerer, Lich. Helvet. exs. No. 335, sub Parmelia flava a oxytona

(GZU, neotype, designated by Hafellner 1993, n.v.).

= Lecanora stenospora Stizenb. ex Hasse, Bull. Torrey Bot. Club 24: 447. 1897, syn. nov.

= Acarospora stenospora (Stizenb. ex Hasse) Hue, Nouv.

Arch. Mus. Hist. Nat., Sér. 5, 1: 161 (1909).

= Acarospora stenospora (Stizenb. ex Hasse) Zahlbr., Cat.

Lich. Univ. 5: 108 (1928), nom. illeg. superf.

= Pleopsidium stenosporum (Stizenb. ex Hasse) K.

Knudsen, Opusc. Philolich. 9: 79 (2011).

Type: U.S.A., California, [Los Angeles or San Bernardino Co.] San Gabriel Mountains,

above 800 m, on unknown rock type with some HCl+ inclusions, 1895, Hasse s.n. (NY!

[Hasse Herb. No. 3682], neotype designated Knudsen 2011).

358 ... Knudsen & Kocourkova

Lecanora stenospora was described from the San Gabriel Mountains in

southern California. The holotype was missing from ZT and a neotype

from NY was designated. The name was transferred to Pleopsidium based

on its distinctive Pleopsidium-type ascus stain (Knudsen 2011). Due to the

dispersed thallus, wandering lines of areoles, and a possible difference in the

production of secondary metabolites, P. stenosporum was tentatively treated as

a separate species from P. flavum. Field studies in Joshua Tree National Park

and Yosemite National Park by the first author, as well as in Montana by Tim

Wheeler, convinced us that the normally contiguous and effigurate thallus

of P. flavum will become dispersed or form long lines of areoles, especially

when growing in shaded overhangs or if infected with Lichenostigma saxicola

K. Knudsen & Kocourk. Michalova (2012), who investigated the neotype for

secondary metabolites, detected acaranoic acid, a substance typically present

with rhizocarpic acid in P flavum. Since P. stenosporum does not differ from

P. flavum in thallus morphology or secondary metabolites, it is treated as a

synonym of P. flavum, which is common in North America.

Acknowledgments

We thank our reviewers, J.C. Lendemer (NY) and P.M. McCarthy (Australia). The

work of Kerry Knudsen was supported by a co-operative agreement between Joshua

Tree National Park and the University of California at Riverside. The work of Jana

Kocourkova was supported financially by the Kontaxr H, Program of International

Cooperation in Research and Development for scientific cooperation between the

CR and USA, LH 11057 from Ministry of Education, Youth and Sports. We thank for

their help the curators of COLO, FH, and ZT. We thank Tim Wheeler (Montana) for

discussion about Pleopsidium and for the figures.

Literature cited

Aptroot A, Schumm F, 2012. The genus Melanophloea, an example of convergent evolution towards

polyspory. Lichenologist 44: 501-509. http://dx.doi.org/10.1017/S0024282912000035

Hafellner J. 1993. Acarospora und Pleopsidium - zwei lichenisierte Ascomycetengattungen

(Lecanorales) mit zahlreichen Konvergenzen. Nova Hedwigia 56: 281-305.

Horakova J. 1998. Distribution and ecology of the genus Thelocarpon (Lecanorales, Thelocarpaceae)

in the Czech Republic. Czech Mycology 50: 271-302.

James PW, Vézda A. 1971. Melanophloea P. James & Vézda, a new lichen genus. Lichenologist 5:

89-91. http://dx.doi.org/10.1017/S0024282971000112

Knudsen K. 2003. Type specimens: investigations and observations. Bulletin of the California

Lichen Society 10(2): 36-38.

Knudsen K. 2011. A preliminary study of Pleopsidium stenosporum (Stizenb. ex Hasse) K. Knudsen.

Opuscula Philolichenum 9: 77-83.

Knudsen K. 2012. Notes on the California Lichen Flora # 4. Bulletin of the California Lichen

Society 19(1): 4-7.

Knudsen K, Kocourkova J. 2012. Lichenological notes 3: Sarcogyne plicata in California. Mycotaxon

118: 423-431. http://dx.doi.org/10.5248/118.423

Lichenological notes 6 ... 359

Knudsen K, Kocourkova J. 2013 [“2012”]. Lichenological notes 5: neotypification of Sarcogyne

magnussonii (Acarosporaceae). Mycotaxon 121: 139-145. http://dx.doi.org/10.5248/121.139

Knudsen K, Lendemer JC. 2006. Changes and additions to the North American lichen mycota — V.

Mycotaxon 95: 309-313.

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Flora of the Greater Sonoran Desert Region. Volume 3. Lichens Unlimited, Arizona State

University, Tempe.

Knudsen K, Lendemer JC, Harris RC. 2011. Lichens and lichenicolous fungi - no. 15: miscellaneous

notes on species from eastern North America. Opuscula Philolichenum 9: 45-75.

Magnusson AH. 1935 [“1934”]. On the species of Biatorella and Sarcogyne in America. Annales de

Cryptogamie Exotique 7: 115-145.

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Sonoran Desert Region. Volume 1. Lichens Unlimited, Arizona State University, Tempe.

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Herzogia 7: 13-79.

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Australasian Lichenology 62: 26-28.

Michalova M. 2012. Chemistry of lichens of southwest Mojave Desert. 101 p., Ms [Dipl. Pr.; depon.

in: Faculty of Environmental Sciences, Czech University of Life Sciences, Praha]. [Duplicate

copy in library of New York Botanical Garden].

Nash III TH, Ryan BD, Gries C, Bungartz F. 2002. Lichen Flora of the Great Sonoran Desert Region.

Volume 1. Lichens Unlimited, Arizona State University, Tempe. 532 p.

Orange A, Purvis OW, James PW. 2009. Thelenella Nyl. (1855). 877-879, in: CW Smith et al. (eds).

The Lichens of Great Britain and Ireland. British Lichen Society, Natural History Museum Pub.,

UK.

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Journal 52(1): 1-70.

MYCOTAXON

http://dx.doi.org/10.5248/124.361

Volume 124, pp. 361-365 April-June 2013

Phylloporia tiliae sp. nov. from China

L1-WEI ZHOU

State Key Laboratory of Forest and Soil Ecology, Institute of Applied Ecology,

Chinese Academy of Sciences, Shenyang 110164, PB. R. China

CORRESPONDENCE TO: liwei_zhou1982@163.com

ABSTRACT — A new species, Phylloporia tiliae, is described and illustrated from Hunan

Province, China. It is distinguished from other Phylloporia species by its combination of a

perennial habit, pileate basidiocarps, a crusted pileal surface with tomentose margin, minute

pores, and a monomitic hyphal system.

Key worps — Hymenochaetaceae, Hymenochaetales, polypore, taxonomy

Introduction

Phylloporia Murrill was erected for P. parasitica Murrill, growing on living

leaves (Murrill 1904). Most species of this genus have been found on living

trees and also sometimes on dead wood (Zhou & Dai 2012). The genus has a

worldwide distribution (Ryvarden & Johansen 1980, Gilbertson & Ryvarden

1987, Ryvarden & Gilbertson 1994, Nufez & Ryvarden 2000, Dai 2010, 2012),

and includes a total of 24 species (Zhou & Dai 2012, Decock et al. 2013).

Phylloporia is a monophyletic genus within the Hymenochaetaceae

(Valenzuela et al. 2011, Zhou & Dai 2012). All Phylloporia species share

abundant thick-walled tiny colored basidiospores and lack setae (Wagner &

Ryvarden 2002, Ipulet & Ryvarden 2005, Douanla-Meli et al. 2007, Cui et al.

2010, Valenzuela et al. 2011, Zhou & Dai 2012); however, this genus is highly

diverse in morphology and life strategy (see Zhou & Dai 2012 for detail).

Cui et al. (2010) reported two new species of Phylloporia from South China.

Recently, Zhou & Dai (2012) described five new species from all over China

based on morphological and phylogenetic evidence and provided a key to

worldwide species thus far accepted in Phylloporia. However, there are still a

number of Chinese specimens that show generic characters but have not been

identified to species level. One of these is described and illustrated here as

Phylloporia tiliae.

362 ... Zhou

Materials & methods

The holotype specimen was deposited at the herbarium of Institute of Applied

Ecology, Chinese Academy of Sciences, Shenyang, P.R. China (IFP). The microscopic

procedure follows Zhao et al. (2013). The following abbreviations are used:

IKI = Melzer’s reagent, IKI- = inamyloid and non-dextrinoid, CB = Cotton Blue,

CB(+) = weakly cyanophilous, KOH = 5% potassium hydroxide, L = mean basidiospore

length (arithmetic average of all basidiospores), W = mean basidiospore width

(arithmetic average of all basidiospores), Q = variation in the L/W ratios between the

specimens, and n = number of basidiospores measured/number of specimens measured.

Sections prepared in IKI, CB and KOH solutions were studied using a Nikon Eclipse

80i microscope at magnifications up to x1000. When presenting the basidiospore size

variation, the upper and lower 5% of measurements are excluded from the range and the

extreme values are presented in parentheses. Line drawings were made with the aid of a

light tube. Special color terms follow Petersen (1996).

Taxonomy

Phylloporia tiliae L.W. Zhou, sp. nov. FIGURE 1

MycoBank MB 804993

Differs from Phylloporia pectinata in its monomitic hyphal system and narrower

basidiospores.

Type: China. Hunan Province, Zhangjiajie, Tianmenshan National Forest Park, on living

Tilia (Malvaceae), 19.V1II.2010, Yuan 5491 (holotype, IFP).

ErymMo_oey: tiliae (Lat.): refers to the host genus.

BASIDIOCARPS perennial, pileate, single, without odor or taste when fresh.

PrLEI dimidiate, projecting up to 7.5 cm, 5.5 cm wide, and 2 cm thick at base.

PILEAL SURFACE glabrous, crust, mouse-grey, azonate; MARGIN tomentose,

obtuse, curry-yellow to yellowish brown, a distinct groove between margin and

other part of the pileal surface. PORE SURFACE honey-yellow, glancing; STERILE

MARGIN distinct, curry-yellow, up to 1 mm wide; Porgs circular, 9-12 per mm;

DISSEPIMENTS thin, entire. CONTEXT duplex when juvenile, up to 6.5 mm thick,

with a black line, the lower context woody hard, yellowish brown, up to 2.5

mm thick, upper context as a tomentum, soft corky, cinnamon-buff, yellowish

brown, up to 4 mm thick, the black line becoming a crust with age. TUBEs

yellowish brown, woody, each layer up to 1 mm long.

HYPHAL SYSTEM monomitic; GENERATIVE HYPHAE simple septate; tissue

becoming reddish brown in KOH but otherwise unchanged. CONTEXTUAL

HYPHAE pale yellowish to yellowish, slightly thick- to thick-walled with a wide

lumen, rarely branched, frequently simple septate, straight, regularly arranged,

2.5-4.5 um in diam; HYPHAE IN TOMENTUM yellowish brown, slightly thick-

walled, occasionally branched, frequently simple septate, loosely interwoven,

2-5 um in diam; HYPHAE IN THE BLACK ZONE (CRUST) dark brown, distinctly

thick-walled with a narrow lumen, strongly agglutinate, interwoven. TRAMAL

Phylloporia tiliae sp. nov. (China) ... 363

oOo Cc CG G

Figure 1. Phylloporia tiliae (holotype).

a: Basidiospores. b: Basidia and basidioles.

c: Hyphae from trama. d: Hyphae from the lower context.

HYPHAE hyaline, thin-walled to yellowish, thick-walled with a wide to narrow

lumen, rarely branched, frequently simple septate, more or less straight,

subparallel along the tubes, 2-4 um in diam. SETAE absent; cysTrp1A absent;

CYSTIDIOLES absent. Basip1A barrel-shaped with four sterigmata and a simple

septum at the base, 4-6 x 3-4.5 um; BASIDIOLES dominant, similar to basidia

in shape, but slightly smaller. Basip1osporss ellipsoid, yellowish, thick-walled,

smooth, IKI-, CB(+), (2.8-)3-3.4(-3.5) x (1.9-)2-2.5(-2.6) um, L = 3.17 um,

W = 2.29 um, Q = 1.38 (n = 30/1).

REMARKS: Phylloporia tiliae has thick-walled tiny colored basidiospores

and lacks setae, fulfilling the morphological characters of Phylloporia. The

new species is characterized by the combination of a perennial habit, pileate

basidiocarps, a crusted pileal surface with tomentose margin, small pores, and

a monomitic hyphal system.

Macroscopically, Phylloporia crataegi L.W. Zhou & Y.C. Dai, P. ephedrae

(Woron.) Parmasto, P gutta L.W. Zhou & Y.C. Dai, P. pectinata (Klotzsch)

364 ... Zhou

Ryvarden, and P_ ribis (Schumach.) Ryvarden share perennial pileate

basidiocarps with P tiliae. However, P. crataegi, P. ephedrae, P. gutta, and P. ribis

have larger pores (<9 per mm; Wagner & Ryvarden 2002, Zhou & Dai 2012).

Phylloporia pectinata has pores (8-10 per mm) similar in size to those of

P. tiliae, and both species also share a black crust at the old part of pileal surface

and duplex context; however, P. pectinata has a zonate and sulcate pileal surface

with acute margin and (more importantly) a dimitic hyphal system and wider

basidiospores (3 um wide; Wagner & Ryvarden 2002).

Phylloporia fontanesiae L.W. Zhou & Y.C. Dai also has small pores (10-12 per

mm) and a monomitic hyphal system like P. tiliae, but its annual habit, zonate

sulcate pileal surface with acute margin, and shorter basidiospores (2.6-3 um

long; Zhou & Dai 2012) distinguish it from P. tiliae. In addition, P. fontanesiae

was found on living Fontanesia (Oleaceae) in temperate China in Henan (Zhou

& Dai 2012) and Shandong (data unpublished) provinces.

Phellinus minisporus B.K. Cui & Y.C. Dai, which was described from southern

China and has similar pores, differs from Phylloporia tiliae in its resupinate

habit, abundant hymenial setae, and smaller basidiospores (Cui et al. 2009).

Some Phylloporia species often inhabit living trees, affecting leaves, twigs,

branches, and trunk bases, and have an exclusive host-specificity (Zhou &

Dai 2012). This phenomenon exists in certain species of Inonotus linteus

complex as well (Tian et al. 2003). Therefore, it is important to record the host

information when collecting specimens of Phylloporia and even other genera

in Hymenochaetaceae.

Acknowledgements

Dr. Deepika Kumari (Xinjiang Institute of Ecology and Geography, Chinese Academy

of Sciences, China) is thanked for improving this manuscript. I express my gratitude

to Prof. Yu-Cheng Dai (Beijing Forestry University, China) and Dr. Viacheslav Spirin

(Helsinki University, Finland) who reviewed the manuscript before final submission.

The research was financed by the National Natural Science Foundation of China (Project

No. 31200015).

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MYCOTAXON

http://dx.doi.org/10.5248/124.367

Volume 124, pp. 367-368 April-June 2013

NOMENCLATURAL NOVELTIES AND TYPIFICATIONS

PROPOSED IN MYCOTAXON 124

Arenariomyces truncatellus Jorg. Koch, p. 70

Aspicilia peltastictoides (Hasse) K. Knudsen & Kocourk., p. 354

Beauveria sinensis Ming J. Chen, Z.Z. Li & B. Huang, p. 304

Cantharocybe virosa (Manim. & K.B. Vrinda) T.K.A. Kumar, p. 235

Chaetomium jatrophae Rohit Sharma, p. 120

Cladonia dunensis Gumboski, Beilke & Eliasaro, p. 334

Coleosporium tussilaginis f.sp. doronici S. Helfer, p. 92

Coleosporium tussilaginis f.sp. inulae S. Helfer, p. 92

Coleosporium tussilaginis f.sp. telekiae S. Helfer, p. 96

Entoloma shandongense T. Bau & J.R. Wang, p. 166

Glomus mume B.P. Cai, Jun Y. Chen, Q.X. Zhang & L.D. Guo, p. 264

Hypochnicium pini Y. Jang & J.J. Kim, p. 211

Inonotus niveomarginatus H.Y. Yu, C.L. Zhao & Y.C. Dai, p. 62

Inonotus tenuissimus H.Y. Yu, C.L. Zhao & Y.C. Dai, p. 64

Kuklospora spinosa B.P. Cai, Jun Y. Chen, Q.X. Zhang & L.D. Guo, p. 265

Lactarius rubidus (Hesler & A.H. Sm.) Methven, p. 324

Leccinellum quercophilum M. Kuo, p. 327

Lyromma coronatum Flakus & Farkas, p. 128

Lyromma multisetulatum Flakus & Farkas, p. 130

Myrmecridium obovoideum Jie, Y.L. Jiang, McKenzie & Yong Wang bis, p. 5

Pertusaria albiglobosa Q. Ren, p. 349

Phialophora avicenniae Yue L. Liu & Z.D. Jiang, p. 33

Phylloporia tiliae L.W. Zhou, p. 362

Pisolithus calongei M.P. Martin, Phosri & Watling, p. 151

Polycoccum anatolicum Halici & E. Akgiil, p. 46

Russula atroaeruginea G,J. Li, Q. Zhao & H.A. Wen, p. 175

Russula changbaiensis G.J. Li & H.A. Wen, p. 270

Russula sichuanensis G.J. Li & H.A. Wen, p. 179

368 ... MYCOTAXON 124

Rutstroemia coracina (Dur. & Lév.) Dennis 1964 (epitypified), p. 15

Sarcogyne athroocarpa H. Magn. 1935 (lectotypified), p. 353

Scolecobeltrania Iturr., R.F. Castafieda & Rob. Fernandez, p. 144

Scolecobeltrania vermispora Iturr., R.E Castafieda & Rob. Fernandez, p. 144

Septoglomus titan B.T. Goto & G.A. Silva, p. 105

Terfezia albida Ant. Rodr., Mufioz-Mohedano & Bordallo, p. 201

Terfezia eliocrocae Bordallo, Morte & Honrubia, p. 196

Terfezia extremadurensis Mufhioz-Mohedano, Ant. Rodr. & Bordallo, p. 195

Terfezia pini Bordallo, Ant. Rodr. & Mufioz-Mohedano, p. 198

Terfezia pseudoleptoderma Bordallo, Ant. Rodr. & Mufioz-Mohedano, p. 200

Tuber neoexcavatum L. Fan &Yu Li, p. 159

Xerocomus porophyllus T.H. Li, WJ. Yan & Ming Zhang, p. 257