IV ... MYCOTAXON 131(1)

MYCOTAXON

VOLUME ONE HUNDRED THIRTY-ONE (1) — TABLE OF CONTENTS

COVER SECTION

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EPPA 9 © aA Nees gS Renate ae tees coe AE De aisles, aig ee taet Aes Vili

EROMSEDEAULOI chy 10 BIL oR ten, fy alt Me ie OSG AL oc Satie MN se Behe ix

SUDITISSION PrOCCdUTCS: = A008 Sw lo newt) oat ae th ows Rawle oan aaed & xi

RESEARCH ARTICLES

New hosts for Bartalinia and Chaetopyrena in China YAN WANG,

LinG JIN, Li Lin, TIAN-TIAN ZHU, XIU-RONG CHEN & LI-PING CHAO

Aspergillus similanensis sp. nov. from a marine sponge in Thailand

TrpA DETHOUP, NELSON GONGALO MARTAGUA GOMES,

SRIMEK CHAOPONGPANG & ANAKE KIJJOA

First record of Mycena plumipes in Romania, with notes on

its presence on pine cones VASILICA CLAUDIU CHINAN & LUCIAN Fusu

Passalora caesalpiniicola sp. nov. from India on Caesalpinia bonduc

SHAMBHU KUMAR & RAGHVENDRA SINGH

First record of Russula anthracina and its ectomycorrhiza

associated with Himalayan cedar from South Asia

SANA JABEEN, ABDUL REHMAN NIAZI & ABDUL NASIR KHALID

Sympodiosynnema, a new genus of dematiaceous hyphomycetes

from southern China JI- WEN X14, YiING-Rur Ma,

JiaN-MEI GAo, ZHUANG LI & XIU-GUO ZHANG

New records from Lithuania of fungi alien to Europe

JURGA MOTIEJUNAITE, ERNESTAS KUTORGA,

JONAS KASPARAVICIUS, VAIDOTAS LYGIS & GODA NORKUTE

Some stromatic pyrenomycetous fungi from northern Thailand—2.

Annulohypoxylon and Ustulina

LARISSA N. VASILYEVA, STEVEN L. STEPHENSON & KEVIN D. HYDE

Taxonomic re-evaluation and phylogenetic position of

Hemibeltrania cinnamomi within Xylariales

KUNHIRAMAN C. RAJESHKUMAR, SAYALI D. MARATHE,

KARADKA MADHUSUDHANAN & RAFAEL F. CASTANEDA-RUIZ

Ramularia hydrangeicola sp. nov. with distinctive traits on

Hydrangea serrata f. acuminata in Korea

Ji-HyunN PARK & HYEON-DONG SHIN

JANUARY-MARCH 2016... V

Phylogenetic placement of the secotioid fungus Araneosa columellata

within Agaricus Scott T. BaTEs, ROBERT M. CHAPMAN,

Me Issa B. IsLaM, ANNA SCHWABE, ERIK C.P. WARDENAAR & VERA S. EVENSON 103

Morphological traits, DELTA system, and molecular analyses for

Gibellula clavispora sp. nov. from China

WaAN-HAo CHEN, YAN-FENG HAN, ZONG-Q] LIANG & X1A0 Zou 111

Olpitrichum sphaerosporum: a new USA record and

phylogenetic placement Dx-Wer Li, Nett P. SCHULTES & CHARLES VossBRINCK 123

Sarcogyne saphyniana sp. nov., a saxicolous lichen from

northwestern China Lazzat Nurtal, Kerry KNUDSEN & ABDULLA ABBAS 135

Mirandina uncinata sp. nov. from submerged leaves from Brazil

PATRICIA OLIVEIRA FIUZA, JOSIANE SANTANA MONTEIRO,

Luis FERNANDO PASCHOLATI GUSMAO, & RAFAEL F. CASTANEDA-Ruiz 141

Pleurothecium & Sporidesmiopsis spp. nov. from Brazil

JOSIANE SANTANA MONTEIRO, Luis FERNANDO PASCHOLATI GUSMAO

& RAFAEL FE. CASTANEDA-Ru1z 145

New leptonioid species from New South Wales and

northeastern Queensland, Australia Davip L. LARGENT,

KERRI L. KLUTING, NOELLE M. ANDERSON & SARAH E. BERGEMANN 153

Dictyoaquaphila appendiculata gen. & sp. nov. from

submerged wood from Brazil JOSIANE SANTANA MONTEIRO,

Lucas BARBOSA CONCEIGAO, MARCOS FABIO DE OLIVEIRA MARQUES,

Luis FERNANDO PASCHOLATI GUSMAO & RAFAEL F. CASTANEDA-RuIz 177

Anacoronospora diversiseptata gen. & sp. nov. from Brazil

JOSIANE SANTANA MONTEIRO, Luis FERNANDO PASCHOLATI GUSMAO

& RAFAEL E CASTANEDA-Rui1z 185

Two new polypore species from the southwestern USA:

Fomitiporia fissurata and F. deserticola JosEF VLASAK & JOSEF VLASAK Jr. 193

First report of Singerocybe in Thailand JATURONG KuMLA,

NAKARIN SUWANNARACH, SANTHITI VADTHANARAT,

OLIVIER RASPE & SAISAMORN LuMyonG 205

Acremonium camptosporum isolated as an endophyte of

Bursera simaruba from Yucatan Peninsula, Mexico Maria C. GONZALEZ,

ANTHONY E. GLENN, RICHARD T. HANLIN, MARTHA L. MAciAs RUBALCAVA,

BLANCA E. HERNANDEZ BAUTISTA & ANA Luisa ANAYA 211

Aspicilia, Lobothallia, and Rhizocarpon species new for

Turkey and Asia KENAN YAZICI & ALI ASLAN 227

New species of neotropical Rhodocollybia JuAN L. Mata,

CLARK L. OVREBO, TIMOTHY J. BARONI & KAREN W. HuGHES 235

VI ... MYCOTAXON 131(1)

Two new Puccinia species on Melica (Poaceae) from USA

MEHRDAD ABBASI & MARY CATHERINE AIME 247

REGIONAL MYCOBIOTAS NEW TO THE MYCOTAXON WEBSITE 255

Checklist of the Glomeromycota in the Brazilian Savanna

KHADIJA JOBIM, BRUNA IOHANNA SANTOS OLIVEIRA & BRUNO TOMIO GOTO

Macromycetes within Cistaceae-dominated ecosystems in Cyprus

MICHAEL LOIZIDES

Contributions to the macrofungal diversity of Mugla province (Turkey)

HALIL GUNGOR, MEHMET HALIL SOLAK, HAKAN ALLI,

MusTAFA ISILOGLU & ERBIL KALMIS

BOOK REVIEWS AND NOTICES ELsE C. VELLINGA & RIKKE REESE NAESBORG 257

NOMENCLATURAL NOVELTIES AND TYPIFICATIONS

PROPOSED IN VOLUME 131(1) 261

JANUARY-MARCH 2016...

REVIEWERS — VOLUME ONE HUNDRED THIRTY-ONE (1)

The Editors express their appreciation to the following individuals who have,

prior to acceptance for publication, reviewed one or more of the papers

prepared for this quarter.

Joe Ammirati

Vladimir Antonin

Boris Assyov

Charles W. Bacon

Timothy J. Baroni

Uwe Braun

R.E Castaneda-Ruiz

Yu-Cheng Dai

Victor Rafael M. Coimbra

Eduardo Furrazola

Anna Guttova

Mehmet Gokhan Halic1

Stephan Helfer

Fabien Charles Cossi Hountondji

Bo Huang

Richard A. Humber

Abdullah Kaya

Bryce Kendrick

Bradley R. Kropp

T.K. Arun Kumar

James C. Lendemer

De-Wei Li

Elaine Malosso

Eric H.C. McKenzie

Pierre-Arthur Moreau

Keiichi Motohashi

Chiharu Nakashima

Karen K. Nakasone

Lorelei L. Norvell

Lorenzo Pecoraro

Shaun R. Pennycook

Roger S. Peterson

Stephen Peterson

Liliane E. Petrini

Michele Piercey-Normore

Amy Y. Rossman

Irja Saar

Derek Schafer

Markus Scholler

Gladstone Alves da Silva

Narong Singburaudom

Steven L. Stephenson

Aziz Tirkoglu

Else C. Vellinga

Giuseppe Venturella

James Francis White

Andrew Wilson

Katherine E. Winsett

Alan R. Wood

Marta Wrzosek

Xiu-Guo Zhang

VII

vul ... MYCOTAXON 131(1)

ERRATA FROM PREVIOUS VOLUMES

VOLUME 130(3)

p.746, line 12 App: We also thank Dr. Alan Fryday (Michigan State University, USA)

for valuable comments and reviews on the manuscript.

VOLUME 130(4)

p-964, line 10 FOR: 1.5-4 um diameter READ: 1.5-4 um diam.

p.964, line 19 FOR: Pegler et. al. 1997 READ: Pegler et al. 1997

p.965, lines 17-18 DELETE:

Yao YJ, Spooner BM. 1995. Notes on British species of Lamprospora

and Ramsbottomia. Mycological Research 99(12): 1521-1524.

http://dx.doi.org/10.1016/S0953-7562(09)80804-6

p.1038, line 25 FoR: Zhuang WY, Liu XX. READ: Liu XX, Zhuang WY.

JANUARY-MARCH 2016 ... IX

FROM THE EDITOR-IN-CHIEF

GUIDE TO ETHICAL RESEARCH NETWORKING — Lucky indeed is the twenty-first

century scientist! The rise of academic research network sites (e.g., ResearchGate,

Academia.edu, LinkedIn) has recently flooded the Internet with research &

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Much of the information is open and free. Mycotaxon has long supported

open access and is proud of our affiliation with CyBERLIBER, the electronic

library for Mycology. Our long-standing agreement allows CYBERLIBER to

scan and post all MycoTaxon papers on its website two years after publication.

All mycologists would do well to check the website frequently to access the

wealth of mycological literature available there.

However, as Dr. H. Frederick Dylla noted in his letter to the WALL STREET

JOURNAL (April 18, Opinion | Letters), “Unless a raw manuscript placed online

somehow constitutes the finished product of scientific publishing, “free” can

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And as much as Mycotaxon would love to publish an entirely free & open

access journal, it is our modest page charges and subscription fees that make it

possible for us to publish well formatted and thoroughly reviewed mycological

research papers. Our dependence upon subscriptions means that unless our

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Nonetheless, Mycotaxon DOES recommend (perhaps even URGE) its authors

to announce new papers by submitting title, abstract, and por number to their

favorite research network as soon as their work has been published. Informing

these networks of your recent accomplishments only makes sense!

MYCOTAXON 131(1) covers 28 papers by 101 authors (representing 17 countries)

and revised by 51 expert reviewers.

Within its pages are three new genera (Anacoronospora and Dictyoaquaphila

from Brazil, and Sympodiosynnema from China) and 33 species new to science

representing Leptonia from Australia; Anacoronospora, Dictyoaquaphila,

Mirandina, Pleurothecium, and Sporidesmiopsis from Brazil; Gibellula,

Sarcogyne, and Sympodiosynnema from China; Rhodocollybia from Costa

Rica, Dominican Republic, and Puerto Rico; Passalora from India; Ramularia

from Korea; Annulohypoxylon, Aspergillus, and Ustulina from Thailand; and

Fomitiporia and Puccinia from the U.S.A.

x ... MYCOTAXON 131(1)

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

we also offer new combinations in Agaricus and Annulohypoxylon.

2016 brings additional changes: A full color photo now graces our front cover,

while an original drawing graces the back cover. Our nomenclatural novelties

and typifications page now also presents the nomenclatural identification

number after each new name.

Warm regards,

Lorelei Norvell (Editor-in-Chief)

20 April 2016

PUBLICATION DATE FOR VOLUME ONE HUNDRED THIRTY (4)

MYCOTAXON for OCTOBER-DECEMBER 2015, (I-XII + 929-1215)

was issued on January 11, 2016

JANUARY-MARCH 2016... XI

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MY COTAXON

January-March 2016—Volume 131, pp. 1-6

http://dx.doi.org/10.5248/131.1

New hosts for Bartalinia and Chaetopyrena in China

YAN WANG’, LING JIN™*, Li LIN’, TIAN-TIAN ZHU’,

XIU-RONG CHEN?’ & LI-PING CHAO?

"Gansu University of Traditional Chinese Medicine, Lanzhou 730000, China

? College of Pratacultural Science & Key Laboratory of Grassland Ecosystem,

Gansu Agricultural University, Ministry of Education

Sino-U.S. Center for Grazingland Ecosystem Sustainability, Lanzhou 730070, China

° Tianjin University of Traditional Chinese Medicine, Tianjin 300100, China

*CORRESPONDENCE TO: zyxyjl@163.com

ABSTRACT—Two anamorphic species, Bartalinia robillardoides and Chaetopyrena penicillata,

are reported on a new host in China. Both endophytic fungi were isolated from stems of

Ephedra intermedia and are described and illustrated.

Key worps—Amphisphaeriaceae, appendaged coelomycetes, Didymellaceae, endophytes,

taxonomy

Introduction

In the course of investigating the endophytic fungi of Ephedra intermedia

in Gansu province, China, healthy stems were surface-sterilized in 1% sodium

hypochlorite for 1 minute and then rinsed three times in sterilized distilled water,

and tissues were plated onto PDA to obtain pure cultures. Two coelomycetous

fungi were isolated. Based on their cultural characteristics, morphological

comparison, and analysis of the internal transcribed spacer region of ribosomal

DNA (rDNA-ITS), the fungi were determined as two coelomycetous species

on a new host in China. Chaetopyrena penicillata is reported for the first time

from China.

Materials & methods

Isolates and morphology

The morphology of the strains was examined on OA: oatmeal agar (30 g oatmeal,

1000 mL distilled water), MEA: malt extract agar (15 g malt extract, 1 g peptone, 20 g

2 ... Wang & al.

dextrose, 15 agar, 1000 mL distilled water) and PDA: potato dextrose agar (200 g potato,

20 g dextrose, 15 g agar, 1000 mL distilled water), incubated for 10 d at 25°C in the dark.

All structures were described from 1-month-old PDA cultures.

Specimens are deposited in the Herbarium of Gansu University of Traditional

Chinese Medicine, Lanzhou, China (HTCM).

DNA extraction and sequencing of (rDNA-ITS)

Mycelium was scraped from cultures growing on PDA, put into 100 mL PD, agitated

using an orbital shaker set at 45 rp.m. and incubated at room temperature under

constant illumination by fluorescent lights. Mycelium (100 mg) was removed after 7

days and transferred to a mortar and pestle for grinding with liquid nitrogen. DNA

extraction was then carried out using the FastDNAkit (SK1375, Sangon Biotech Co.

Ltd.) following the manufacturer's instructions. The presence of DNA was verified by

separation on a 0.8% agarose gel in 1xTAE (40 mM Tris-acetate, 1 mM EDTA, pH 8.0)

stained with ethidium bromide (0.5 mg/L) and visualized under UV light. The ITS region

was amplified using the universal primers, ITS1 and ITS4 (White et al. 1990). PCR was

performed in 50 uL reaction volumes containing 10-100 ng of genomic DNA, 2.5 uL of

each primer (10 uM), 0.25 uL dNTP (10 mM), 2.0 unit Supertaq DNA polymerase and

5 uL 10 x PCR buffer (Sangon Biotech Co. Ltd.). The amplification was performed in a

thermocycler (Biometra Tg PCR). The PCR thermal cycle program was as follows: 95°C

for 3 min, followed by 30 cycles of denaturation at 94°C for 3 min, annealing at 52°C

for 45 s, and elongation at 72°C for 1 min, with a final extension step of 72°C for 8 min.

The PCR products, spanning approximately 500 bp of ITS, were checked on 1% agarose

electrophoresis gels stained with ethidium bromide. The PCR product was purified and

sequenced using the above-mentioned primers by Sangon. Approximate phylogenetic

placements were determined with the Blastn search option of the National Center for

Biotechnology Information (NCBI) and the suggested identities are discussed in the

descriptive notes below.

Taxonomy

Bartalinia robillardoides Tassi, Bull. Lab. Orto Bot. Reale Univ. Siena 3: 5. 1900.

PLATE 1

Black conidiomata in PDA culture, stromatic, globose or subglobose,

immersed or semi-immersed, solitary, glabrous, 605-757 x 585-702 um

diam, black to brownish black, wall thick, composed of bright brown, thick-

walled cells, paler and thinner in inner layers. Ostiole absent, dehiscence

irregular by breakdown of upper wall. Conidiophores absent. Conidiogenous

cells cylindrical to subcylindrical, hyaline, 6-9 x 3 um. Conidia cylindrical

or fusiform, smooth-walled, straight to slightly curved, 4-septate, slightly

constricted at septa, hyaline, 25.6-33.3 x 2.6-3.1 um, length/width ratio

9.9. Apical cell hyaline, conic, thin-walled, 3.0 um long, bearing (2-)3(-4)

appendages from apex. Appendage branches 2-3, divergent, flexible, tapering

gradually towards apex, 11.5-19.8 um long. Median cells cylindrical, ochraceous

Bartalinia & Chaetopyrena—new hosts (China) ... 3

PiateE 1. Bartalinia robillardoides (HTCM 1009024). A-C: Ten-day-old colonies on PDA (A),

OA (B), and MEA (C); D, E: Conidiomata; F: Conidiogenous cells with developing conidia;

G-I: Conidia.

to pale brown; the cell next to basal cell longest, 7.7-11.5(-14.1) um (mean =

10.1 um), |: w = 3. Basal appendage straight, (5.1-)7.7-9.5(-10.3) um long

(mean = 8.5 um). Colony on PDA at 25°C grayish green, regular, with darker

concentric zones, aerial mycelium floccose, slightly grey black in reverse,

producing numerous black conidiomata, growth rate 7.3 mm/d. Colony on OA

grayish green, no concentric zones, the reverse grey, producing sparse brown

conidiomata, growth rate 6.5 mm/d. Colonies on MEA white to grayish white,

sparse conidiomata, growth rate 6.3 mm/d.

SPECIMEN EXAMINED: CHINA: GANSU PROVINCE, Lanzhou city, Renshou mountain,

isolated from stems of Ephedra intermedia Schrenk & C.A. Mey. (Ephedraceae), 20 Sep,

2009, Yan Wang. (HTCM 1009024; dried cultures, HTCM-F: 09122, 09123; GenBank,

KF656706).

ComMMENTS — Bartalinia robillardoides is reported from diverse hosts around

the world, including Hong Kong, China (Zhuang 2001, as Seimatosporium

robillardoides), but it has not previously been reported from Ephedra intermedia.

The identification of this isolate was made based on conidial morphology

as well as on molecular sequence data. The Blastn search results of the ITS

sequence of our Chinese isolate from Ephedra (GenBank KF656706) showed

99% similarity to B. robillardoides (GenBank EU552102), 100% similarity to

4 ... Wang & al.

Ellurema sp. (GenBank AY148442), 96 and 95% similarity to two isolates

of B. pondoensis (GenBank GU291796, AF405302), and 95% similarity to

B. laurina (GenBank AF405302).

Nag Raj (1993), who published a comprehensive account of Bartalinia,

accepted and keyed six species and listed another nine species as “unexamined

and excluded taxa.” Andrianova & Minter (2007) accepted and keyed ten species

were by, and Marincowitz et al. (2010) added a futher species. Based on Nag

Raj (1993), our Ephedra isolate keys to B. robillardoides based on its size and

4-septate conidia. Andrianova & Minter (2007) emphasized the importance

of even or uneven conidial septation, conidial size, and number of appendage

branches; in their key, our Ephedra isolate again keys to B. robillardoides

because of the large cell next to the base, conidial size, and generally 3-branched

apical appendage. The most recently described species, B. pondoensis, is unlike

B. robillardoides in having 3-septate conidia. The genus Ellurema is applied to

the sexual state of Hyalotiopsis, a genus that is distinguished from Bartalinia by

characteristics of the conidial appendages (Nag Raj 1993).

Chaetopyrena penicillata (Fuckel) Héhn., Hedwigia 60: 132, 1918. PLATE 2

Conidiomata superficial. Pycnidia single, solitary, globose to subglobose,

dark brown to black, ostiolate, 320-400 um diam, surrounded by setae, neck

100 x 50 um; setae brown, thick-walled, septate, unbranched, gradually

tapering upward, up to 150-180 um long; wall consisting of 3-4 layers of brown

textura angularis. Conidiophores hyaline, simple. Conidia hyaline, smooth,

1-celled, cylindrical to sub-cylindrical, straight, with rounded apex and

truncate base, 12.6-17.7 x 2.5-5.1 um (mean = 15.9 x 3.9 um). Colonies on

PDA 42 mm diam. after 7 days at 25°C in dark, smooth, black, margins grayish

white, regular, reverse black, aerial mycelium sparse. Colonies on OA 36 mm

diam after 7 days at 25°C in dark, substrate mycelium orange-green. Colonies

on MEA reaching 30 mm diam. after 7 days at 25°C in dark; aerial mycelium

grayish white; pycnidia not formed; chlamydospores not seen.

SPECIMEN EXAMINED: CHINA: GANSU PROVINCE, Lanzhou city, Renshou mountain,

isolated from stems of Ephedra intermedia , 1 Oct. 2009, Yan Wang. (HTCM 1009028;

dried cultures, HTCM-F: 09124, 09125; GenBank, KC492443).

CoMMENTS — A Blastn search of the ITS sequence of our Chinese isolate

from Ephedra (GenBank KC492443) showed 99% similarity to Chaetopyrena

penicillata (GenBank JQ663990). Our isolate agrees morphologically with this

species as described in Arzanlou & Khodaei (2012). This genus is currently

placed in the Didymellaceae (Gruyter et al. 2010). Although 19 species have

been placed in Chaetopyrena, most species are obscure and do not have living

cultures. Arzanlou & Khodaei (2012) reported C. penicillata as the cause of

Bartalinia & Chaetopyrena—new hosts (China) ... 5

PLATE 2. Chaetopyrena penicillata (HTCM 1009028). A-C: Ten-day-old colonies on PDA (A),

OA (B), and MEA (C); D: Conidiomata; E, F: Conidia.

dry rot on Russian olive (Elaeagnus angustifolia). It is also reported from soil

as well as dead twigs, fruit, and stubble in the Middle East, Russia, and South

Africa. This is the first report of C. penicillata from China, and the first report

from Ephedra.

Acknowledgements

The authors are grateful to Amy Rossman (Systematic Mycology & Microbiology

Laboratory, USDA-ARS), Keiichi Motohashi (Electron Microscope Center, Tokyo

University of Agriculture), and Lorelei Norvell (Mycotaxon Editor-in-Chief) for critical

comments, corrections, and pre-submission reviews. This study has been supported

by National Nature Science Foundation, project 31460013, Gansu Nature Science

Foundation, project 1212RJZA092, Gansu College Scientific Research, project 2013A-

089 and Gansu Herbal Medicine Industry Research Foundation, project GYC11-01.

Literature cited

Andrianova TV, Minter DW. 2007. New species of Bartalinia and Septoriella from the Altai

Mountains (Russia). Mycotaxon 101: 297-313.

Arzanlou M, Khodaei S. 2012. Phenotypic and molecular characterization of Chaetopyrena

penicillata from Iran with description of a hyphomycete synanamorph. Mycosphere 3(1):

73-77. http://dx.doi.org/10.5943/mycosphere/3/1/9

Aveskamp M, de Gruyter H, Woudenberg J, Verkley G, Crous PW. 2010. Highlights of the

Didymellaceae: a polyphasic approach to characterise Phoma and related pleosporalean genera.

Stud. Mycol. 65: 1-64. http://dx.doi-org/10.3114/sim.2010.65.01

6 ... Wang & al.

Gruyter J de, Woudenberg JHC, Aveskamp MM, Verkley GJM, Groenewald JZ, Crous PW. 2010.

Systematic reappraisal of species in Phoma section Paraphoma, Pyrenochaeta and Pleurophoma.

Mycologia 102: 1066-1081. http://dx.doi.org/10.3852/09-240

Marincowitz S, Gryzenhout M, Wingfield MJ. 2010. New and rare coelomycetes with

appendage-bearing conidia from Pondoland, South Africa. Mycotaxon 111: 309-322.

http://dx.doi.org/10.5248/111.309

Nag Raj TR. 1993. Coelomycetous anamorphs with appendage-bearing conidia. University of

Waterloo: Waterloo, Canada. 1101 p.

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

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

methods and applications. Academic, New York.

Zhuang WY. 2001. Higher fungi of tropical China. Mycotaxon Ltd., Ithaca, NY. 485 p.

MY COTAXON

January-March 2016—Volume 131, pp. 7-15

http://dx.doi.org/10.5248/131.7

Aspergillus similanensis sp. nov.

from a marine sponge in Thailand

TipA DETHOUP’ , NELSON GONGALO MoRTAGUA GOMES?,

SRIMEK CHAOPONGPANG’ & ANAKE KIJJOA?

"Department of Plant Pathology, Faculty of Agriculture, Kasetsart University,

50 Ngam Wong Wan Road, Chatuchak, Bangkok, 10900, Thailand

? ICBAS-Instituto de Ciéncias Biomédicas Abel Salazar and Interdisciplinary Centre of Marine &

Environmental Research (CIIMAR), Universidade do Porto,

Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal

" CORRESPONDENCE TO: agrtdd@ku.ac.th

ABSTRACT —A new species of Aspergillus sect. Fumigati, A. similanensis, isolated from the

marine sponge Rhabdermia sp. collected from a coral reef of the Similan Island, Phangnga

province, Thailand, is described and illustrated. Aspergillus similanensis is characterized by

production of finely reticulate ascospores with two low equatorial crests, a yellowish colony

on malt extract agar, and extremely restricted growth on Czapek agar. ‘The validation of this

new species is further supported by sequence analyses of the 6-tubulin, calmodulin, and actin

genes.

KEY WORDS — sponge-associated fungi, phylogeny, taxonomy

Introduction

Aspergillus sect. Fumigati W. Gams et al. [= Neosartorya Malloch & Cain]

is widely distributed, being reported mainly from soil, debris, and foods

(Samson et al. 2007, Matsuzawa et al. 2014a,b) but recently also from marine

environments, such as from marine sponges (Eamvijarn et al. 2013a). Despite

the reports on their pathogenicity to humans and animals (Farrell et al. 2014,

Howard 2014), Neosartorya species are also prolific sources of secondary

metabolites with a wide range of biological activities (Eamvijarn et al. 2013b;

Gomes et al. 2014; Prompanya et al. 2014, 2015).

8 ... Dethoup & al

Samson et al. (2007) revised the taxonomy of Aspergillus sect. Fumigati and

Neosartorya based on phenotypic and molecular characteristics, describing 33

species, including 23 with teleomorphic states. Since then, several new species

in A. sect. Fumigati have been reported: Eamvijarn et al. (2013b) described

A. siamensis from coastal forest soil in Samaesarn Island, Thailand; Hubka et

al. (2013) described A. waksmanii from New Jersey soil and A. marvanovae

from contaminated water in the Czech Republic; Matsuzawa et al. (2014a,b)

described A. caatingaensis and A. pernambucoensis from semi-desert soil in

Brazil and A. huiyaniae from desert soil in Xinjiang, China; and Novakova et

al. (2014) described A. brevistipitatus, A. conversis, and A. wyomingensis from

reclamation site soils in Wyoming, USA.

In our ongoing search for bioactive compounds from fungi against plant

pathogens, unusual isolates of Aspergillus sect. Fumigati have been isolated

from the marine sponge Rhabdermia sp. Here, we describe and illustrate the

sponge isolates as a new species, A. similanensis.

Materials & methods

Fungal Isolation’

The marine sponge Rhabdermia sp. was collected at a coral reef in the Similan Islands

National Park (8°39’09”'N 97°38’27”E), Phangnga province, Thailand. The sponge tissue

was washed three times with sterile sea water, cut into 0.5 x 0.5 cm pieces, and placed on

half potato dextrose agar (PDA) with 70% of sea water and 300 mg L"' of streptomycin

sulphate. The plates were incubated at 28°C for 7 days.

Hyphal tips from a pure fungal strain were transferred onto PDA slants and stored in

the Culture Collection, Department of Plant Pathology, Kasetsart University, Bangkok,

Thailand (KUFA) for subsequent identification and deposited in a metabolically inactive

state at BIOTEC Culture Collection, Pathumthani, Thailand (BCC).

Morphological Identification

Macroscopic characteristics were observed from the colonial growth pattern (i.e.,

color and texture) on malt extract agar (MEA), cornmeal agar (CM), Czapek agar (CzA),

oatmeal agar (OA), and Czapek yeast extract agar (CYA), after incubation at 25°C, 37°C,

and 45°C for 7 days (Samson et al. 2007). Colony colors were designated according

to Rayner (1970). After incubation, microscopic characteristics were thoroughly

examined under a light microscope in a slide preparation using sterile distilled water as

the mounting medium. Ascospore ornamentation was examined by scanning electron

microscope (SEM: JEOL JSM 6400).

DNA extraction and molecular Identification

DNA was extracted from young mycelia following a modified Murray & Thompson

(1980) method. Primer pairs Bt2a and Bt2b were used for B-tubulin gene amplification

(Glass & Donaldson 1995), cmd5 and cmd6 for calmodulin gene amplification (Hong

et al. 2005), and ACT-512F and ACT-783R for actin gene amplification (Carbone &

Kohn 1999). The PCR products were sequenced using dideoxyribonucleotide chain

Aspergillus similanensis sp. nov. (Thailand) ... 9

termination method (Sanger et al. 1977) (Macrogen Inc., South Korea). DNA sequences

were edited using Finch TV software. The gene sequences were aligned and phylogenetic

trees were generated using Clustal X software (Thompson et al. 1997) utilizing neighbor-

joining analysis (Saitou & Nei 1987). The distances between sequences were calculated

based on the two-parameter model by Kimura (1980). A bootstrap was conducted with

1000 replications (Felsenstein 1985).

Taxonomy

Aspergillus similanensis Dethoup, sp. nov. FIGs 1-4

MycoBank MB810782

“Aspergillus similanensis” Prompanya et al., Mar. Drugs 12: 5160. 2014, nom. nud.

“Aspergillus similanensis” Prompanya et al., Mar. Drugs 13: 1342. 2015, nom. nud.

Differs from other Aspergillus species by its finely reticulate ascospores with two low

equatorial crests, the production of a yellowish colony on MEA, and extremely restricted

growth on CzA.

Type: Thailand, Phangnga Province, Similan Island, a dried culture from marine sponge,

Rhabdermia sp., collected from a coral reef, April 2010, T. Dethoup (Holotype, BCC

75436; ex-type cultures KUFA 0012, KUFA 0013; GenBank KM095494, KC920701,

KM202074).

EryMo.ocy: Referring to Similan Island where the sponge was collected for fungal

isolation.

Colonies on MEA growing rapidly, attaining a diameter of 35 mm in 7 days at

25°C, consisting of a floccose mycelium, producing abundant ascomata on the

mycelial felt, white to creamish, margins broad, thin and plane; conidiogenesis

absent; exudate none; odor lacking; reverse luteous (R12).

Colonies on CzA growing extremely restrictedly, attaining a diameter of

8-10 mm in 14 days at 25°C, producing only white mycelium covering the

mycelial plug. Colonies on CYA attaining a diameter of 15-20 mm in 7 days at

25°C, velvety, floccose at the central, consisting of a compact basal felt, furrowed,

margins irregular, pale luteous (R11) to sulphur yellow (R15); ascomata and

conidiogenesis absent; exudates and soluble pigment absent; reverse wrinkle,

slightly zonate, rust (R39).

Colonies on CM spreading broadly, attaining a diameter of 35 mm in 7 days

at 25°C, plane, velvety, consisting of thin mycelial felt, producing abundant

developing ascomata over the entire surface, margins thin and submerged,

white in color; conidiogenesis lacking; reverse luteous (R12).

Colonies on OM showing the same colonial characteristic as on CM,

attaining a diameter of 30 mm in 7 days at 25°C, plane, velvety, consisting

of thin mycelial felt, producing abundant developing ascomata at the central

colony, margins thin and submerged, white in color; conidiogenesis lacking;

reverse luteous (R12).

10 ... Dethoup & al

fo 26 SET

Fic. 1. Aspergillus similanensis (KUFA 0012): A. colony on MEA 25°C, 7d; B, C. conidial heads;

D. conidia; E. asci; F. ascospores; G, H. ascospores (SEM). Scale bars: A = 3 cm; B, C, E = 10 um,

D, F=5 um.

Homothallic.

At 37°C, colonies on all media except CzA growing more rapidly than at

25°C, but no growth at 45°C.

Mycelium composed of branched, septate, smooth-walled, hyaline hyphae,

2-3 um wide. Conidial heads small, short radiate, conidiophores 120-170 x 3-4

um wide in the middle, thick and smooth-walled, frequently septate at basal

area, vesicles subglobose, pyriform or subclavate 7-8 um wide in diameter;

uniseriate, phialides hyaline, 6-7 x 2-2.5 um, covering the upper half of the

vesicle, conidia pale green, globose to subglobose, 3-4 Um, smooth walled.

Aspergillus similanensis sp. nov. (Thailand) ... 11

Ascomata non-ostiolate, superficial, globose, 120-150 um, ripening within

10-14 days. Covering consisting of loosely hyaline, 4-5 um wide, branched,

septate, smooth-walled hyphae. Asci 8-spored, globose to subglobose,

10-11 um, evanescent at maturity. Ascospores hyaline, lenticular, 5-5.5 um

including two low equatorial crests, 1-1.5 lum wide, spore body 4—4.5 um and

with the convex surfaces fine reticulate ornamentation.

CoMMENTS—The strain KUFA 0012 produced lenticular ascospores, convex

surfaces decorated with fine reticulation with two low equatorial crests

(Fics 1G-H). Ascospores of this species are similar to those of A. huiyaniae

and A. pernambucoensis, which were recently reported from soils. In both of

those species the convex surface ornamentation of the ascospores is irregularly

ribbed to slightly reticulate. However, strain KUFA 0012 can be distinguished

from the two other species by its smaller ascospore size as well as colony

morphology on CzA in term of sporulation rate, colony coloration, and growth

rate. Phylogenetic analysis of the B-tubulin gene comparing KUFA 0012 and 43

other strains for comparison (Matsuzawa et al. 2014b) and the calmodulin gene

comparing KUFA 0012 with 10 related species revealed a close relationship

with Aspergillus huiyaniae (Fics 2-3), while actin gene analysis placed KUFA

0012 in the same clade with N. fennelliae and N. sublevispora (Fic. 4). The

6-tubulin, calmodulin, and actin DNA sequences were additionally analyzed

using BLAST software (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The B-tubulin

gene analysis showed KUFA 0012 closely related (98%) to N. fennelliae, and

the calmodulin and actin gene analyses revealed a 97% of similarity with

N. fennelliae.

Our phylogenetic analysis placed KUFA 0012 in a clade closely related to

A. huiyaniae, N. fennelliae, and N. sublevispora. Despite its morphologically

similar ascospores with A. huiyaniae, the KUFA 0012 strain produces smaller

ascospores with two low equatorial crests. KUFA 0012 also differs from

N. sublevispora in its distinct ascospore ornamentation presenting finely

reticulate ascospores with two well separated crests instead of regularly

microtuberculate ascospores with two closely appressed crests. Although the

gene analysis revealed a close phylogenetic relationship with N. fennelliae, the

strains are clearly separated by mating type: KUFA 0012 strain is homothallic

and N. fennelliae was described as heterothallic.

Phylogenetic and morphological analyses showed that the marine-derived

taxon is an undescribed species, which we have named Aspergillus similanensis.

As recently reported by our research group (Prompanya et al. 2014, 2015), the

ethyl acetate extract from KUFA 0013 contains seven new natural products—

chevalone E; pyripyropene E and T; similanamide; and similanpyrone A, B,

12 ... Dethoup & al

0.02

60 A, unilateralis CBS 126.56 (AB248301)

N, warcupii NRRL 35723 (EU220283)

N. assulata KACC 41691 (DQ114123)

N. papuensis CBS 841.96 (AY870738)

100 NN. stramenia CBS 498.65 (AY870766)

N. aurata CBS 466.65 (AF057318)

A, turcosus KACC 42091 (DQ534143)

N. tatenoi CBMFA 22 (DQ114130)

A, marvanovae CCM 8003 (HE974387)

N, nishimurae CBS 116047 (DQ534075)

ee -A. waksmanii NRRL 179 (EF669794)

‘A, pernambucoensis IFM 61342 (AB743856)

A. caatingaensis TFM 61335 (AB743854)

NN. tsunodae 1FM 57609 (AB488755)

99 NN, multiplicata TEM 46955 (DQ114129)

NN, shendaweii T1FM57611 (AB488754)

N. galapagensis CBS 117522 (DQ534145)

N. udagawae TFM 46972 (AB248302)

N. indohii FM 53615 (AB488757)

400 N, aureola CBS 105.55 (AB057319)

‘A, viridinutans CBS 127.36 (AF134779)

2 57 N. takakii CBMFA 884 (AB787221)

LN. paulistensis IPM 46585 (AB488758)

N, spinosa CBS 483.65 (AF057329)

N. coreana KACC 41659 (AY870758)

56|_| 100 A, lentulus FAS (AY738513)

A, fumisynnematus TEM. 42277 (AB248076)

66 N. fischeri CBS 544.65 (AF057322)

98 A. fumigatus CBS 545,65 Y685151)

‘A. novofumigatus IBT 16806 (DQ094886)

98 A. fumigatiaffinis IBT 12703 (DQ094883)

NN. australensis CBS 112.55 (AY870739)

NN. fennelliae CBS 598.74 (DQ114127)

99 N. sublevispora YFM 53598 (AB488759)

N. denticulata CBS 652.73 (DQ114125)

56 A. huiyaniae TEM 57847 (AB787220)

"A. similanensis KUFA 0012 (KM095494)

N. hiratsukae NHL 3008 (AF0S7324)

N, spathulata CBS 40889 (AF057327)

100 N. tsurutae IFM 56811 (AB488760)

N. quadricincta CBS 133.52 (AF057326)

77 A. duricanlis CBS 481.65 (A¥057313)

75 A, brevipes CBS 118.53 (AF057311)

N. glabra CBS 111.55 (AY870734)

N. pseudofischeri CBS 208.92 (AY870743)

A, clavatus CBS 514.65 (AB489851)

Fig. 2. Neighbor-joining tree from sequences of the B-tubulin gene. Numbers at nodes indicate

the percentage of bootstrap sampling (>50%), derived from 1000 samples. A. = Aspergillus,

N. = Neosartorya.

Aspergillus similanensis sp. nov. (Thailand) ... 13

N. glabra CBS 111.55 (AY870693)

N. hiratsukae NHL 3008 (AY870699)

N. multiplicata IFM 46955 (DQ114137)

A. waksmanii NRRL 179 (EF669863)

N. nishimurae CBS 116047 (DQ534150)

A. novofumigatus IBT1 6806 (DQ094893)

N. fennelliae CBS 598.74 (DQ114135)

N. sublevispora IFM 53598 (AB488767)

A, huiyaniae IFM 57847 (AB787564)

‘A. similanensis KUFA 0012 (KC920701)

N. tatenoi CBMFA22 (DQ114139)

‘A. clavatus CBS 514.65 (AB489852)

Fic. 3. Neighbor-joining tree from sequences of the calmodulin gene. Numbers at nodes indicate

the percentage of bootstrap sampling (>50%), derived from 1000 samples. A. = Aspergillus,

N. = Neosartorya.

N. stramenia CBS 498.65 (DQ534188)

A. marvanovae CCM 8003 (HE974401)

N. nishimurae CBS 116047 (DQ534186)

A. unilateralis CBS 126.56 (DQ094847)

A. fumisynnematus IFM 42277 (AB488769)

A, duricaulis CBS 481.65 (DQ094854)

N. pseudofischeri CBS 208.92 (DQ534187)

‘A. huiyaniae IFM 57847 (AB787567)

N. fennelliae CBS 598.74 (DQ534121)

N. sublevispora IFM 53598 (AB488776)

A, similanensis KUFA 0012 (KM202074)

A. clavatus CBS 514.65 (AB489853)

Fic. 4. Neighbor-joining tree from sequences of the actin gene. Numbers at nodes indicate

the percentage of bootstrap sampling (>50%), derived from 1000 samples. A. = Aspergillus,

N. = Neosartorya.

14 ... Dethoup & al

and C—as well as seven previously described compounds. Interestingly,

this new species was isolated from a marine sponge, an unusual habitat for

teleomorphic states of Aspergillus sect. Fumigati, which have mainly been

reported from soils and food (Eamvijarn et al. 2013a, Matsuzawa et al. 2014a,b).

Despite the limited distribution in marine environments, we have previously

reported several species of Neosartorya such as N. paulistensis, N. laciniosa, and

N. tsunodae from marine sponges collected from Thai waters (Gomes et al.

2014). Chemical analysis of these species resulted in the isolation of several

bioactive compounds (Eamvijarn et al. 2013a, Gomes et al. 2014). Since we

have found only one strain of A. similanensis, we consider this species rare and

presumably with limited distribution.

Acknowledgments

The authors are grateful to Prof. Dr. Stephen W. Peterson and Prof. Narong

Singburaudom for acting as pre-submission reviewers and for their valuable comments

and suggestions. This project was supported by the Thailand Research Fund and

Kasetsart University Research Development Institute.

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MY COTAXON

January-March 2016—Volume 131, pp. 17-24

http://dx.doi.org/10.5248/131.17

First record of Mycena plumipes in Romania,

with notes on its presence on pine cones

VASILICA CLAUDIU CHINAN”* & LUCIAN FUSU

Faculty of Biology, Alexandru Ioan Cuza University,

Iasi, Bd. Carol I, No. 20A, 700505, Romania

*CORRESPONDENCE TO: vasilechinan@yahoo.com

ABSTRACT — Mycena plumipes was found in Romania on fallen and partially buried cones

of Pinus nigra and Picea abies. The analysis of ribosomal DNA internal transcribed spacer

sequences showed that the specimens collected in Romania on P. nigra belong to M. plumipes.

This is the first record of M. plumipes in Romania and confirms that it grows on pine cones

(as well as spruce cones), based on molecular and morphological data. A detailed description

and illustrations of macroscopic and microscopic aspects of the Romanian specimens are

given.

Key worps — Basidiomycota, Mycena sect. Fragilipedes, Mycena strobilicola, habitat

Introduction

Mycena plumipes is a vernal, cone-inhabiting species that belongs to M. sect.

Fragilipedes (Fr.) Queél. (Robich 2006). It was described by Kalchbrenner

(1873) under the name “Agaricus Collybia plumipes” |i.e., Agaricus (Collybia)

plumipes]. In his original description, Kalchbrenner did not mention its habitat

very precisely, stating the substrate as “ad conos pinuum’” (i.e., “on pine cones”),

but in the lectotype figure the basidiomata are illustrated on what appears to be

a Picea cone (rather than a Pinus cone). In addition, Kalchbrenner mentioned

that Agaricus plumipes is accompanied in its habitat by Agaricus esculentus

Wulfen [= Strobilurus esculentus (Wulfen) Singer], a species that grows in

spring on Picea cones (Gulden 1966). Moreau (2003) evaluated these unclear

aspects to conclude, based on the iconography and accompanying species, that

a cone very similar to one of Picea serves as the substrate of Agaricus plumipes.

Also, Moreau showed that Agaricus plumipes is an earlier synonym of Mycena

18 ... Chinan & Fusu

strobilicola J. Favre & Ktihner, which also was described from Picea cones

(Kithner 1938), and went on to propose the combination Mycena plumipes.

In Romania, this species was found on fallen cones in plantations of both

Pinus nigra J.F. Arnold and Picea abies (L.) H. Karst. Here we report the first

records of M. plumipes in Romania and demonstrate the conspecificity of

the specimens collected from cones of Norway spruce and black pine using

molecular markers.

Materials & methods

Specimens of Mycena plumipes were collected in April 2011 and 2012 from fallen

and partially buried cones of Pinus nigra and Picea abies. Fragments from fresh and

dried basidiomata were mounted in Congo Red and Melzer’s reagent for examination

under the light microscope (LM). The specimens were identified using Breitenbach

& Kranzlin (1991), Kihner (1938), Elborne et al. (1992), Emmett et al. (2008), Maas

Geesteranus (1988), Moreau (2003), Robich (2003, 2006), and Roux (2006). The

collected basidiomata were dried and deposited in the Herbarium of the Alexandru

Ioan Cuza University, Iasi, Romania (I). The description is based on the Romanian

specimens collected by the authors.

In order to verify that specimens growing on Pinus and Picea cones are conspecific

(and to confirm the species identity), we targeted the nuclear ribosomal internal

transcribed spacer (ITS) region, recently formally proposed as the primary fungal

barcode marker (Schoch et al. 2012). The ITS DNA sequences were generated from dried

basidiomata using primers ITSIF 5’-cTTGGTCATTTAGAGGAAGTAA-3’ (Gardes & Bruns

1993) and ITS4, 5’-TCCTCCGCTTATTGATATGC-3’ (White et al. 1990) and sequenced

at a commercial facility (Alvalab, Santander, Spain). The obtained ITS sequences are

deposited in GenBank (accession numbers JX297424-297427). Sequences of Mycena

sect. Fragilipedes retrieved from GenBank were aligned with the ClustalW algorithm

(Larkin et al. 2007) with a gap opening penalty of 10 and a gap extension penalty of 1.

The most appropriate substitution model was selected using a Maximum Likelihood

(ML) criterion. The ML analysis with 1000 bootstrap replicates was conducted using the

Tamura 3-parameter model with a discrete Gamma distribution (T92 + G) indicated

by BIC as the best-fit model. Nucleotide-sequence divergences were calculated using

the K2P model (Nei & Kumar 2000). All phylogenetic analyses were performed using

MEGA 6.0.5 (Tamura et al. 2013).

Taxonomy

Mycena plumipes (Kalchbr.) P.-A. Moreau, Bull. Mycol. Bot. Dauphiné-Savoie

43(171): 5 (2003) PLATE 1

Basidiomata 40-70 mm high. Pileus 15-30 mm diam., hemispherical,

campanulate or conical, with a broad or truncate umbo; finely fibrillose under

the lens, hygrophanous, sometimes finely translucently striate at the edge; the

colour variable: brown, light brown, ochraceous brown, or greyish brown,

margin concolorous or paler. Lamellae 26-35 reaching the stipe (with 3-5

Mycena plumipes on pine cones (Romania) ... 19

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PLATE 1. Mycena plumipes: a, b. basidiomata on Pinus nigra cones; c. basidiomata on Picea abies

cone; d. cheilocystidia (LM); e. caulocystidia (LM)

20 ... Chinan & Fusu

intervening lamellulae), ventricose, adnate-emarginate with decurrent tooth,

<5 mm broad, white, whitish to pale grey, turning to pink with age, edge entire.

Spore deposit white. Stipe 30-70 x 2-3 mm, central, cylindrical, straight to

slightly curved, equal, hollow, glabrous, grey brown, apex paler to whitish and

minutely pruinose, base at the contact zone with the cone covered with coarse,

long, whitish fibrils. Context whitish to pale grey. Odor nitrous.

Basidia 25-30 x 6.5-7.5 um, 4-spored. Basidiospores 7-10 x 4-5 um,

smooth, dacryoid, phaseoliform to ellipsoid, amyloid. Cheilocystidia 30-80 x

9-17 um, lageniform, fusiform, apically gradually or abruptly narrowed into a

longer or shorter neck. Some cheilocystidia have granular refringent secretions

on the neck. Pleurocystidia similar to cheilocystidia. Caulocystidia with long

cylindrical excrescences, 35-120 x 4-7 um. Pileipellis hyphae 2-4 um wide,

smooth, rarely with lateral cylindrical excrescences. Clamp connections very

rare, present at the hyphae of stipe trama.

Hasirat - On fallen and often buried cones of Pinus nigra and Picea abies,

solitary or in small groups (2-5 basidiomata), spring (April).

SPECIMENS EXAMINED: ROMANIA, Iasrt County: Breazu Village, 47°12’45”N

27°31'16”E, 111 m alt., on cones of PB nigra, 17.1V.2011, Chinan (I 137185); 2.IV.2012,

Chinan (I 137329; GenBank JX297426); near Hlincea Village, 47°07’04’”N 27°34’56’E,

104 m alt., on cones of P._ nigra, 1.1V.2012, Chinan (I 137328; GenBank JX297425);

Tomesti Village, 47°07’47”N 27°42’14”E, 55 m alt., on cones of P nigra, 16.1V.2011,

Chinan (I 137184); 1.1V.2012, Chinan (I 137327; GenBank JX297424); NEAMT COUNTY,

near Leghin Village, 47°14’12”N 26°13’58”E, 508 m alt., on cones of P. abies, 5.1V.2012,

Chinan & Fusu (I 137330; GenBank JX297427).

MOLECULAR ANALYsiIs — ITS sequences of three Romanian M. plumipes

specimens were identical (I 137327 and I 137328 from P. nigra, and I 137330

from P. abies), and the fourth specimen (I 137329 from P. nigra) differed

by only three transitions (= 0.0038 divergence). The only publicly available

sequence of M. plumipes (Gene Bank JF908440.1, as M. strobilicola) differs in

only one nucleotide indel from the sequences of Romanian specimens I 137327,

I 137328, and I 137330. In the ML tree, all these sequences are recovered as a

highly supported clade, with a bootstrap value of 99 (Fic. 1).

Discussion

Mycena plumipes is widely distributed in Europe (Breitenbach & Kranzlin

1991, Derbsch & Schmitt 1987, Moreau 2003, Robich 2003, Gulden 1966,

Gerhold 1989), but it is also mentioned in the Altai Mountains (Singer 1938)

and Asiatic Turkey (Solak et al. 1999). It is easy to identify in the field due to

its habitat on cones, its nitrous smell, and the whitish fibrils on the base of the

stipe.

The Romanian specimens collected from cones of P. nigra and P. abies

morphologically agree with the description provided by Moreau (2003).

Mycena plumipes on pine cones (Romania) ... 21

Mycena strobilicola 456b

Mycena plumipes | 137327

99 || Mycena plumipes | 137330

Mycena plumipes | 137328

Mycena plumipes | 137329

Mycena polygramma 439b

Mycena leptocephala KF225583.1

Mycena zephirus 54g

Mycena vitilis 136f

Mycena citrinomarginata 317h

Mycena viridimarginata 104h

Mycena algeriensis 390b

95 -— Mycena silvae-nigrae 515a

Mycena ainetorum 393

Mycena robusta 4436

Mycena aetites 412a

Mycena laevigata JQ358808.1

Mycena semivestipes TENN61770

Mycena subcana 531

Mycena maurella 314a

0.05

Fic. 1. ML tree based on ITS sequences. Bootstrap values >50, based on 1000 replications, are

shown near nodes. Sequences are labelled with voucher number or GenBank accession number.

Newly generated sequences are presented in bold font.

In comparing our observations with published data, an important aspect refers

to the striation of the pileus, as only some of the specimens from Romania

are finely translucently striate at the edge. Moreau (2003) and Roux (2006)

describe the edge of the pileus as slightly striate, whereas Kiihner (1938), Maas

Geesteranus (1988), Breitenbach & Kranzlin (1991), and Robich (2003) state

that the pileus is not striate.

The differences between the ITS sequences of M. plumipes from Romania

are well below the mean intraspecific sequence divergence of 0.025 reported

for fungi (Schoch et al. 2012). This confirms the conspecificity of Mycena

specimens from the cones of P. abies and P. nigra. The morphologically based

identification of the species is further confirmed by the high similarity of

our sequences with a sequence available in Gene Bank (accession number

JF908440.1) and derived from a specimen collected from Italy, identified as

M. strobilicola (= M. plumipes), in the herbarium of the Museum of Natural

History in Venice (Osmundson et al. 2013).

In terms of habitat, in Europe M. plumipes is described by most authors

as growing on Picea cones (Breitenbach & Kranzlin 1991; Derbsch & Schmitt

1987; Emmett et al. 2008; Gulden 1966; Kiithner 1938; Maas Geesteranus 1988;

22 ... Chinan & Fusu

Moreau 2003; Robich 2003, 2006; Roux 2006). However, in some regional

species lists it (as M. strobilicola) has been reported on pine cones. Gerhold

(1989, 1991, 1993, 1994) mentions its presence on pine and spruce cones in

Austria, without specifying the species of pine. The species is further reported

on pine cones in Asiatic Turkey (Anatolia), specifically on the cones of Pinus

brutia Ten. (Gezer et al. 2007), P. nigra (Gezer et al. 2007, Tirkoglu 2008,

Turkoglu et al. 2007), and P. pinea L. (Solak et al. 1999), but without mentioning

the diagnosis of this species or highlighting its presence on pine cones. Our

results based on molecular analysis and morphology confirm the presence of

M. plumipes on pine cones as previously reported from Austria and Turkey, an

aspect that in the past has been poorly documented and overlooked.

In Romania we found M. plumipes in conifer plantations on fallen and

buried cones of Picea abies and Pinus nigra, along with other cone-inhabiting

vernal species: Strobilurus tenacellus (Pers.) Singer, S. stephanocystis (Kihner &

Romagn. ex Hora) Singer (on black pine cones), and S. esculentus (on Norway

spruce cones). From the published data it appears that elsewhere, M. plumipes

has been found in native forests. Since Pinus species are widely distributed, it is

intriguing why to date this fungus has been reported on pine cones only from

Austria and Turkey.

We found M. plumipes in April, specifically in depressions in the soil where

the cones gather and the water from melting snow stagnates. The Romanian

collections confirm the vernal phenology of M. plumipes (Gulden 1966,

Kiihner 1938, Moreau 2003, Tirkoglu et al. 2007). However, Einhellinger

(1977) found this species in Germany in early July. In Asiatic Turkey it seems

to have a particular phenology; beside springtime, it has been reported in

October (Gezer et al. 2007), November (Gezer et al. 2007, Solak et al. 1999),

and December (Tiirkoglu 2008, Solak et al. 1999).

Acknowledgments

We thank Giovanni Robich (Italy) and Pierre-Arthur Moreau (France) for providing

access to important literature. The authors are grateful to Vladimir Antonin (Czech

Republic), Giuseppe Venturella (Italy), and Shaun R. Pennycook (New Zealand) for

reviewing the manuscript and for valuable comments on previous versions of this paper.

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Vorkommen und Beschreibungen. Saarbriicken.

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Moore, Teil 2. Ber. Bayer. Bot. Ges. 48: 61-146.

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Elborne SA, Laessge T, Ostmoe KH. 1992. Mycena (Pers. : Fr.) Roussel. 152-169, in: L Hansen,

H Knudsen (eds). Nordic Macromycetes, vol. 2. Polyporales, Boletales, Agaricales, Russulales.

Nordsvamp, Copenhagen.

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Knudsen, J Vesterholt (eds). Funga Nordica. Agaricoid, boletoid and cyphelloid genera.

Nordsvamp, Copenhagen.

Gardes M, Bruns TD. 1993. ITS primers with enhanced specificity for basidiomycetes — application

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http://dx.doi.org/10.1111/j.1365-294X.1993.tb00005.x

Gerhold N. 1989. Contribution to the cartography of macrofungi (Macromycetes) in Austria in

1987 and 1988. Ber. Naturwiss.-med. Ver. Innsbruck 76: 7-24.

Gerhold N. 1991. Contribution to the cartography of macrofungi (Macromycetes) in Austria in

1989 and 1990. Ber. Naturwiss.-med. Ver. Innsbruck 78: 19-34.

Gerhold N. 1993. Contribution to the cartography of macrofungi in Austria in 1992 (Macromycetes).

Ber. Naturwiss.-med. Ver. Innsbruck 80: 15-37.

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Ber. Naturwiss.-med. Ver. Innsbruck 81: 15-32.

Gezer K, Isloglu M, Tiirkoglu A, Alli H. 2007. Macrofungi of Honaz Mountain (Denizli). Turk. J.

Bot. 31: 253-261.

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Carolum Kalchbrenner observatorum et delineatorum, I. Pestini, Typis Athenaei.

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http://dx.doi.org/10.1016/B978-0-12-372180-8.50042-1

MY COTAXON

January-March 2016—Volume 131, pp. 25-30

http://dx.doi.org/10.5248/131.25

Passalora caesalpiniicola sp. nov.

from India on Caesalpinia bonduc

SHAMBHU KUMAR! & RAGHVENDRA SINGH?

"Herbarium Division, Birbal Sahni Institute of Palaeobotany,

53, University Road, Lucknow, U.P, India 226007

*Centre of Advanced Study in Botany, Institute of Science, Banaras Hindu University,

Varanasi, U.P, India 221005

*CORRESPONDENCE TO: skumartaxon@gmail.com

ABSTRACT — Passalora caesalpiniicola on Caesalpinia bonduc (Leguminosae, Caesalpinioideae)

is described and illustrated as a new species. It differs in several characters from the only

other Passalora sp. recorded on Caesalpinia and can be differentiated from morphologically

similar Passalora spp. on caesalpinioid hosts.

KEY worps — _ cercosporoid fungi, mycodiversity, morphotaxonomy, foliicolous

hyphomycetes

Introduction

Passalora Fr. belongs in the complex of cercosporoid fungi (Capnodiales,

Mycosphaerellaceae). In its current circumscription, this genus is characterized

by having pigmented conidiophores, thickened and darkened conidiogenous

loci, either consistently internal or internal and external mycelium in vivo,

and solitary to catenate, pigmented conidia with thickened and darkened hila

(Braun et al. 2013, 2014, 2015; Crous & Braun 2003; Kamal 2010). During the

last decade a large number of fungal species from all over the world have been

recombined in Passalora (Braun et al. 2013, 2014, 2015; Crous & Braun 2003),

particularly from India (Kamal 2010). Recently, additional Passalora species

have been described from India (Singh et al. 2012, 2013; Kumar & Singh

2015), suggesting that the diversity of such fungi is still insufficiently known

in this region. A leaf-spotting cercosporoid hyphomycete corresponding to the

26 ... Kumar & Singh

current concept of Passalora has recently been collected on Caesalpinia bonduc

in the Nichlaul forest of Uttar Pradesh, India. As it clearly differs from the only

Passalora species previously described on Caesalpinia (also from India) and

other morphologically similar Passalora species on hosts in Caesalpinioideae,

we therefore propose here a new species, Passalora caesalpiniicola.

Materials & methods

Living leaves with fungal symptoms were collected from a subtropical forest (Terai,

Nichlaul) in Uttar Pradesh, India. The collections were carried to the laboratory and

processed by standard techniques set forth in Hawksworth (1974) and Savile (1962).

After the sun-dried and pressed leaves were placed in air tight polyethylene bags, they

were kept in paper envelopes with collection notes. Specimens were sectioned by hand

or scraped for microscopic observation. Sections from infected leaf areas were mounted

in clear lacto-phenol cotton blue mount for observation under an Olympus BX-51

light microscope. Detailed morphological characters were observed (and camera-

lucida line drawings made) at 400x and 1000x magnifications, and 30 conidia, hila,

and conidiophores and 20 stromata (extremes shown in parentheses) were measured.

Identification was made with reference to the current literature and available resident

expertise. The holotype is deposited in Herbarium Cryptogamiae Indiae Orientalis,

Indian Agricultural Research Institute, New Delhi, India (HCIO) and an isotype was

retained in Mycological Herbarium of Birbal Sahni Institute of Palaeobotany, Lucknow,

India (BSIPMH). Description and nomenclatural details have been deposited in

MycoBank (www.MycoBankorg) and Faces of Fungi (www.facesoffungi.org).

Classification of the new taxon is based on current concepts (Braun et al. 2013, 2014,

2015; Cannon & Kirk 2007; Crous & Braun 2003; Farr & Rossman 2015; Kirk et al. 2008;

Seifert et al. 2011).

Taxonomy

Passalora caesalpiniicola Sh. Kumar & Raghv. Singh, sp. nov. FIGs 1, 2

MycoBaNnk MB 812867

Differs from Passalora caesalpiniae by its longer and wider conidiophores and its longer,

wider, smooth, and solitary conidia.

Type: India, Uttar Pradesh, Nichlaul forest, Mahrajganj, on living leaves of Caesalpinia

bonduc (L.) Roxb. (Leguminosae, Caesalpinioideae), 13 February 2007, coll. Shambhu

Kumar (Holotype, HCIO 48665; isotype, BSIPMH 30).

EryMoLoGcy: the species epithet is derived from the name of the host genus.

Infection spots hypogenous, circular to irregular, dark brown to black, 1-4

mm diam. Coronises hypophyllous, effuse, brown. Mycelium internal.

Sexual morph: not developed. Asexual morph: Stromata well-developed,

sub-epidermal, pseudoparenchymatous, olivaceous-brown, 28-44 um diam.

CONIDIOPHORES macronematous, fasciculate (up to 6), arising from stromata,

erumpent, simple, erect to procumbent, divergent, straight, smooth, thick-

Passalora caesalpiniicola sp. nov. (India) ... 27

Figure 1. Passalora caesalpiniicola on Caesalpinia bonduc: a. Host plant. b. Infection spots on leaf.

c. Infection spots on leaflet. Scale bars = 20 mm.

walled, <3x transversely euseptate, olivaceous to dark brown, 80-135 x 5-7

um. CONIDIOGENOUS CELLS integrated, terminal, polyblastic, conidiogenous

loci (scars) thickened and darkened, 2-3 um wide. Conip1a solitary, simple,

dry, acropleurogenous, smooth, thin-walled, 3-5 times transversely septate,

straight to curved, obclavate to obclavate-cylindrical, base rounded, apex

obtuse to acute, olivaceous to olivaceous-brown, 25-65 x 5-10 um, hilum

thickened and darkened, 2-3 um wide.

ComMMENTS— The only Passalora species previously described from Caesalpinia

is P. caesalpiniae (Bhalla et al.) U. Braun et al. (Hernandez-Gutiérrez & Dianese

2009; Bhalla et al. 2001, as Phaeoramularia caesalpiniae from C. bonducella

[= C. bonduc] in India). Passalora caesalpiniae differs from P. caesalpiniicola by

its amphigenous leaf spots, smaller stromata (8.5-13 x 4.5-20.5 um), shorter

narrower verruculose conidiophores (8-47 x 2.5-5 um) arranged in larger

fascicules, and its shorter narrower catenate conidia (4-30.5 x 2.5-6 um; Bhalla

et al. 2001).

28 ... Kumar & Singh

1 RARE

Pirkei sh

een

Cae

hey

FiGuRE 2. Passalora caesalpiniicola (holotype, HCIO 48665): a. Infection spots. b. Stromata.

c. Conidiophores. d. Conidia. Scale bars: a = 20 mm, b = 20 um.

Passalora caesalpiniicola sp. nov. (India) ... 29

Five other Passalora species with solitary conidia and caesalpinioideous

resemble P. caesalpiniicola: P. aenea (Cif.) U. Braun & Crous differs by its shorter

conidia (19-53 x 4-6 um, 2—5-septate; Braun & Crous 2003), P. bauhiniicola

U. Braun by its longer conidia (35-70 x 6-8 um, 3-8-septate; Braun 2001),

P. cercidicola (Ellis) U. Braun by its longer conidia (20-80 x 4-8 um, 3-5-septate;

Braun 1995), P. schizolobii M.J. Wingf. & Crous by its longer verruculose

conidia with more septa (40-90 x 3-4 um, 1-8-septate; Wingfield et al. 2006),

and P. chamaecristae (Ellis & Kellerm.) U. Braun by its narrower conidia (31-65

x 4-7 um, 1-5-septate; Braun & Mel'nik 1997).

Acknowledgements

The authors thank Prof Dr Kamal, DDU Gorakhpur University, Gorakhpur,

for helpful taxonomic advice. We are grateful to Prof. Dr Uwe Braun and Dr

Rafael F. Castafieda Ruiz for helpful comments and corrections. Dr. Shaun Pennycook’s

nomenclature review is greatly appreciated. We are much obliged to the Director of

Birbal Sahni Institute of Palaeobotany (Lucknow) for providing necessary facilities.

Author thanks are also due to the Curator, HCIO, IARI (New Delhi), for accepting and

accessioning the type specimen. We are deeply grateful to Science and Engineering

Research Board (SERB), Department of Science and Technology (DST), Government of

India, New Delhi for financial assistance to the first author as Fast Track Young Scientist

Start-Up Research Project (SB/YS/LS-288/2013).

Literature cited

Bhalla K, Sarbhoy AK, Kulshrestha M, Kushwaha KPS. 2001. New species of Phaeoramularia,

Pseudocercospora and Stenella from Western Ghats of India. Microbiol. Res. 156: 107-112.

http://dx.doi.org/10.1078/0944-5013-00056

Braun U. 1995. Miscellaneous notes on phytopathogenic hyphomycetes (II). Mycotaxon 55:

223-241.

Braun U. 2001. Taxonomic notes on some species of the Cercospora complex (VII) Fungal Diversity

8: 41-71.

Braun U, Melnik VA. 1997. Cercosporoid fungi from Russia and adjacent countries. Trudy Bot.

Inst. Komarova, Ross. Akad. Nauk. 20: 1-130.

Braun U, Nakashima C, Crous PW. 2013. Cercosporoid fungi (Mycosphaerellaceae) 1.

Species on other fungi, Pteridophyta and Gymnospermae. IMA Fungus 4(2): 265-345.

http://doi:10.5598/imafungus.2013.04.02.12

Braun U, Crous PW, Nakashima C. 2014. Cercosporoid fungi (Mycosphaerellaceae) 2. Species

on monocots (Acoraceae to Xyridaceae, excluding Poaceae). IMA Fungus 5(2): 203-390.

http://doi:10.5598/imafungus.2014.05.02.04

Braun U, Crous PW, Nakashima C. 2015. Cercosporoid fungi (Mycosphaerellaceae) 3. Species on

monocots (Poaceae, true grasses). IMA Fungus 6(1): 25-97.

http://doi:10.5598/imafungus.2015.06.01.03

Cannon PF, Kirk PF. 2007. Fungal families of the world. Wallingford, UK: CAB International.

456 p.

Crous PW, Braun U. 2003. Mycosphaerella and its anamorphs: 1. Names published in Cercospora

and Passalora. CBS Biodiversity Series 1. 571 p.

30 ... Kumar & Singh

Farr DE, Rossman AY. 2015. Fungal databases, Systematic Mycology and Microbiology Laboratory,

ARS, USDA. Retrieved April 30, 2015, from http://nt.ars-grin.gov/fungaldatabases/

Hawksworth DL. 1974. Mycologist’s handbook. CMI, Kew. 231 p.

Hernandez-Gutiérrez A, Dianese JC. 2009. New cercosporoid fungi from the Brazilian Cerrado 2.

Species on hosts of the subfamilies Caesalpinioideae, Faboideae and Mimosoideae (Leguminosae

s. lat.). Mycotaxon 107: 1-24. http://dx.doi.org/10.5248/107.1

Kamal. 2010. Cercosporoid fungi of India. Bishan Singh Mahendra Pal Singh Publication,

Dehradun (UK), India. 351 p.

Kirk PF, Cannon PF, Minter DW, Stalpers JA. 2008. Dictionary of the fungi. 10th ed. Wallingford,

UK: CAB International. 711 p.

Kumar S, Singh R. 2015. Passalora musicola sp. nov. - a new Indian hyphomycete. Sydowia 67:

21-23. http://dx.doi.org/10.12905/0380.sydowia67-2015-0021

Savile DBO. 1962. Collection and care of botanical specimens. Can. Dept. Agr., Res. Br. Publ. 1113.

http://dx.doi.org/10.5962/bhI.title.53755

Seifert K, Morgan-Jones G, Gams W, Kendrick B. 2011. The genera of hyphomycetes. CBS

Biodiversity Series 9. 997 p.

Singh R, Chaurasia B, Shukla K, Upadhyaya PP. 2012. Passalora aseptata, a new

cercosporoid fungus from northeastern Uttar Pradesh, India. Mycotaxon 120: 461-463.

http://dx.doi.org/10.5248/120.461

Singh R, Kumar S, Saini DC, Upadhyaya PP, Kamal, Braun U. 2013. Diversity of Passalora on Ficus.

Mycol. Progr. 12: 637-643. http://dx.doi.org/10.1007/s11557-012-0870-6

Wingfield MJ, Crous PW, Groenewald JZ. 2006. Passalora schizolobii. Fungal Planet no. 2.

MY COTAXON

January-March 2016—Volume 131, pp. 31-44

http://dx.doi.org/10.5248/131.31

First record of Russula anthracina and its ectomycorrhiza

associated with Himalayan cedar from South Asia

SANA JABEEN’?, ABDUL REHMAN NIAZI’ & ABDUL NASIR KHALID?

"Harvard University Herbaria, Department of Organismic and Evolutionary Biology,

Harvard University, Cambridge, MA 02138, USA

’Department of Botany, University of the Punjab,

Quaid-e-Azam Campus-54590, Lahore, Pakistan

* CORRESPONDENCE TO: ectomycorrhzae@gmail.com

AsBstTRACT — Russula anthracina and its ectomycorrhiza are identified on the basis of

morphological characters and molecular analysis. The species is characterized by brittle

and blackening basidiomata with pinkish lamellae. Ectomycorrhizal morphotypes are

characterized by monopodial pinnate ramification and the presence of typical russuloid

cystidia on outer mantle surface. Molecular phylogenetic analysis clustered the ITS sequences

generated in this study with other R. anthracina sequences. This is the first report of

R. anthracina from Pakistan and South Asia.

Key worps — Cedrus deodara, Russula sect. Compactae, Swat

Introduction

The genus Russula Pers. (Agaricomycetes, Russulales, Russulaceae) is

represented by more than 750 species distributed worldwide (Kirk et al. 2008,

Das et al. 2013, Arora & Nguyen 2014, Razaq et al. 2014, Dutta et al. 2015).

From Pakistan, 26 species of Russula have been reported to date (Ahmad et al.

1997, Niazi et al. 2006, Niazi 2008, Razaq et al. 2014, Jabeen et al 2015). Russula

species are important ecologically as ectomycorrhizal symbionts associated

with many kinds of forest trees (Richardson 1970; Bills et al. 1986; Villeneuve

et al. 1989, 1991; Gardes & Bruns 1996; Buyck et al. 1996). Many species are

harvested worldwide for consumption (Fogel 1975, Fogel & Trappe 1978, Hu &

Zeng 1992, Guo 1992, Rammeloo & Walleyn 1993, Buyck 1994).

Russula anthracina is characterized in R. sect. Compactae, which consists

of blackening species. Taxa in the section are quite easy to identify as a group.

32 ... Jabeen, Niazi & Khalid

Fic. 1. Map of Pakistan showing sampling sites.

Their squat, robust, and brittle basidiomata are initially white but rapidly

become brown to black in all parts (Kibby 2001). However within the section

species are less easily distinguished due to their intergrading colors and obscure

morphological and anatomical variations. Here, combining morphological and

molecular analyses greatly helps in the correct identification of taxa (Arora &

Nguyen 2014).

The goal of the current study was to collect ectomycorrhizal fungal fruit

bodies and their ectomycorrhizae associated with Himalayan cedar from

Pakistan. During the field survey (2012-14) in monsoon season, abundant

Russula anthracina fruiting bodies were found in coniferous forests of Swat

district, Khyber Pakhtunkhwa, Pakistan. The area is surrounded by mountains

and has a typical dry temperate climate (Stucki & Khan 1999). The mountains

are dominated by forests of Cedrus deodara (Roxb. ex D. Don) G. Don along

with Pinus spp. and Quercus oblongata D. Don [= Q. incana Roxb., nom. illeg.]

(Champion et al. 1965). Russula anthracina constituted the most abundant

macrofungal taxon observed during 2013 in Kalam (Fic. 1), a lush green area

along the river Swat. Russula anthracina fruiting bodies and ectomycorrhizae

associated with Himalayan cedar were also collected from Mushkun (Fic. 1).

Russula anthracina ectomycorrhiza with Himalayan cedar (Pakistan) ... 33

Nuclear ribosomal DNA sequence analyses of the ITS region from fruit

bodies and ectomycorrhizal tissues were combined with detailed morphological

examination to confirm our Pakistani collections as R. anthracina.

Materials & methods

Collection, morphological and anatomical study of basidiomata

Specimens were photographed in the field using a Nikon D708S digital camera. Fresh

morphological characters were recorded and colors were designated using Munsell

Color System (1975) after which the specimens were preserved by drying over hot air.

For detailed anatomical examination, tissues from lamellae, pileipellis and stipitipellis

were mounted on glass slides and observed in Phloxine (1%) for better contrast, Melzer’s

reagent for amyloid basidiospore ornamentation, and KOH (5%) for colored hyphae

using a Meiji Techno MX4300H microscope. Anatomical features were measured using

a Carl Zeiss Jena ocular micrometer and line drawing were made using a Leitz Wetzlar

camera lucida.

The abbreviation ‘n/b/p’ indicates ‘n’ basidiospores measured from ‘b’ basidiomata

from ‘p’ collections. Basidiospore dimensions are given as length x width (1 x w), with

extreme values given in parenthesis. Q = |/w ratio of individual spores; avQ = average Q

of all spores + standard deviation. Collector's numbers prefixed with K refer to Kalam

and SJ (basidiomata) and SA (ectomycorrhizae) refer to sequence codes. Voucher

specimens were deposited in the Herbarium, Department of Botany, University of the

Punjab, Lahore, Pakistan (LAH).

Collection, isolation, morphological and anatomical study of ectomycorrhiza

15-cm’ soil blocks were dug a few centimetres away from tree trunks using extreme

care to insure that the fine roots in the soil block belonged to the selected tree species.

Twenty soil cores were taken from each sampling site. In laboratory, each soil core was

soaked in water for few hours to loosen the adhering soil particles and then put on a 2

mm sieve under running water to separate the roots from the soil. The ectomycorrhizae

(EcM) were carefully sorted out into morphotypes under incandescent light and then

under a Meiji Techno EMZ-5TR stereomicroscope. The morphotypes were cleaned

under the stereomicroscope using a fine brush. Morphologically identical morphotypes

were kept in McCartney bottles in distilled water for later examination. Replicates

of these morphotypes were kept at 8 °C in eppendorf tubes containing 1 mL of 2%

CTAB buffer for molecular analysis. Morphological characters were noted under the

stereomicroscope. For anatomical details, mantle layers (inner and outer) and radiating

hyphae were mounted in trypan blue stain (Agerer 1991). Hyphae were measured using

an ocular micrometer and drawn using a camera lucida.

DNA extraction, amplification and sequencing

For genomic DNA extraction, 2 mg of fungal tissue was taken from the gills in

an eppendorf tube, 2-4 ectomycorrhizal root tips were taken in a separate eppendorf

tube. DNA was extracted following modified CTAB method (Bruns 1995). Using

forward primer ITSIF (5’-cTTGGTCATTTAGAGGAAGT-3’) and reverse primer ITS4

(5’-TCCTCCGCTTATTGATATGC-3’) (White et al. 1990), internal transcribed spacer (ITS)

34 ... Jabeen, Niazi & Khalid

regions along with the central 5.8S region of nuclear ribosomal DNA (nrDNA) were

amplified following Gardes & Bruns (1993). The PCR products were sent to Macrogen

Inc. (Korea) for sequencing.

Molecular phylogenetic analysis

Consensus sequences were generated from the sequences obtained by both primers

in BioEdit software. These sequences were then BLAST searched at NCBI (http://www.

ncbi.nlm.nih.gov/). Sequences with closest match were selected from GenBank to

reconstruct phylogeny (sequences with less query cover and negative E values were not

included). Published sequences of the most closely related species were also included in

the final data set. Boletus reticuloceps (M. Zang et al.) Q.B. Wang & Y.J. Yao was chosen as

outgroup (Shimono et al. 2004). Multiple sequences were aligned using online MUSCLE

by EMBL-EBI (http://www.ebi.ac.uk/Tools/msa/muscle/). The sequences were trimmed

with the conserved motifs 5’-(...GAT)CATTA... and ...GACCT(CAAA...)-3’ and the

alignment portions between them were used for phylogenetic analysis. Maximum

likelihood (ML) analysis was performed using General Time Reversible model (Nei &

Kumar 2000) in MEGA6 software (Tamura et al. 2013) to test the phylogeny at 1000

bootstraps. Percentage identity and divergence in nrDNA-ITS of the taxa were analysed

using MegAlign (DNAStar, Inc.). Sequences generated in this study were submitted to

GenBank.

Russula anthracina Romagn., Bull. Mens. Soc. Linn. Lyon 31: 173 (1962) —_ Fies 2, 3

BASIDIOMATA medium to large. PILEUS 4.5-8.6 cm broad, depressed from

the middle; black to greyish black (1OYR1/2) to grey (10YR2/2), dark brown

(10YR2/4) to light brown (10R3/6) and pinkish (SYR9/2) in the form of

random patches; surface dry, smooth, reflective; margins smooth, incurved

when young becoming straight at maturity. LAMELLAE <0.3 cm broad, pinkish

(SYR9/2), regular, adnexed, subdistant to close, edge entire. LAMELLULAE

frequent, single-tiered, variable in length. Stipe 3.3-5.1 x 1.1-2.5 cm, white,

central, cylindrical, slightly narrower towards the apex. Surface smooth to

finely wrinkled.

Basiprospores [n/b/p = 40/4/2] (5.4-)7.3-8.0(-8.3) x (4.7-)6.1-7.0(-7.8)

um, Q = 1.03-1.23, avQ = 1.12 + 0.04; globose to subglobose, echinulate, echines

<0.2 um; apiculus prominent; guttulate, pale yellow in 5% KOH, amyloid in

Melzer'’s reagent. BAsip1A 40.6-66 x 9.7 - 10.8 um, clavate, wall <3 um, pale

yellow in 5% KOH, guttulate. PLEUROCySTIDIA 68.4-110 x 5.2-5.9 um, fusoid,

guttulate, pale yellow in 5 % KOH. CHEILocystTIpIA 52.7-71 x 5.4-6.4 um,

fusiform, guttulate, pale yellow in 5% KOH. PILerPELuis hyphae 3.8-5.1 um

wide, filamentous, branched, pale yellow to transparent in 5% KOH, septa

infrequent, clamp connections and pileocystidia not observed. STIPITIPELLIS

hyphae 3.1-4.7 um diam., filamentous, branched, hyaline to pale yellow, rarely

septate, clamp connections not observed.

Russula anthracina ectomycorrhiza with Himalayan cedar (Pakistan) ... 35

Fic. 2. Russula anthracina basidiomata. A. LAH35009; B. LAH35008. Scale bars = 0.5 cm.

36 ... Jabeen, Niazi & Khalid

Fic. 3. Russula anthracina (LAH35008). A. basidiospores; B. basidia; C. pleurocystidia;

D. cheilocystidia; E. hyphae from stipitipellis; F hyphae from pileipellis. Scale bars: A = 4.5

um; B, F = 12 um; C = 20.5 um; D = 10 um; E= 14 um.

Russula anthracina ectomycorrhiza with Himalayan cedar (Pakistan) ... 37

Fic. 4. Russula anthracina ectomycorrhiza (LAH-EM2-2013). A. ectomycorrhizal system;

B. cystidia; C. inner mantle layer; D. outer mantle layer; E. emanating hyphae. Scale bars: A = 0.7

mm; B = 8.5 um; C = 12 um; D = 14.5 um; E= 25 um.

38 ... Jabeen, Niazi & Khalid

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA PROVINCE, SWAT

District, Kalam, 2070 m asl, on soil under Cedrus deodara, 3 Sep 2013, Sana Jabeen K3;

SJ5 (LAH35008; GenBank KR011879); Mushkun, 2500 m asl, under Cedrus deodara, 5

Sep 2013, Sana Jabeen K4-22; SJ19 (LAH35009; GenBank KRO11880).

Morphological characterization of EcM FIG. 4

ECTOMYCORRHIZAL SYSTEM monopodial pinnate, 0.7-1.2 cm long, axis <1

mm diam., unramified ends straight, cylindrical, greyish brown (10YR7/4) to

dark brown at maturity, tip greyish brown (10YR7/4). MANTLE greyish brown

to transparent, distinct, smooth surface, lustre matte, cortical cells visible under

the mantle surface at some tips. RHIZOMORPHS not observed. EMANATING

HYPHAE transparent, rare, sometimes frequent at few root tips, straight,

cylindrical rarely branched. Cystip1a frequent.

OUTER MANTLE LAYER Cells 14.6 x 8.1 um, INNER MANTLE LAYER Cells

12.9 x 6.1 um. Cells in both layers pseudoparenchymatous, yellowish to

transparent, angular to somewhat roundish or elongated (mantle type L; Agerer

1987-2002), contents clear.

EMANATING HYPHAE elongated, transparent, straight, cylindrical, <3.9 um

in diameter, wall <0.7 um thick, surface smooth, septate, septa frequent, 2.0-49.3

um apart, clamp connections rare, ornamentation absent, contents clear,

ramification rather frequent, Y-shaped, anastomoses H-shaped, frequent,

without septa and clamp connections. Cystip1a lageniform, frequent, 23.9 um

long, strongly inflated base, <6.8 um diam., attachment point <5 um wide, neck

straight, 4.9 um diam. with 3.7 um tip.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA PROVINCE, SWAT

District, Mushkun, 2500 m asl, associated with Cedrus deodara, 5 Sep 2013, Sana

Jabeen K4-5; SA99 (LAH-EM2-2013; GenBank KRO11881).

Molecular phylogenetic characterization FIG. 5

Sequencing of the Russula anthracina PCR products using ITS1F/ITS4

yielded 697-752 base pairs. A 598-base pair consensus sequence was obtained

by trimming the motifs. BLAST of R. anthracina (KRO11881) revealed it as

99% identical with KF679819, FN669239, and KM576561 (100% query

cover, 0.0 E value) and a 99% identity with JF908673 (93% query cover,

Fic. 5. Molecular phylogenetic analysis of Russula spp. inferred by the Maximum Likelihood

method. The percentage of trees in which the associated taxa clustered together is shown next to

the branches. Initial tree for the heuristic search was obtained by applying the Neighbor-Joining

method to a matrix of pairwise distances estimated by the Maximum Composite Likelihood (MCL)

approach. The tree is drawn to scale, with branch lengths measured in the number of substitutions

per site. The analysis involved 64 nucleotide sequences. New sequences generated from Pakistan

are marked with @. There were a total of 828 positions in the final dataset.

Russula anthracina ectomycorrhiza with Himalayan cedar (Pakistan) ... 39

77 p— GU371295 Russulg livescens

AY061700 Russula insignis

JO622344 Russila pulverulenta

KF245530 Russula foetens

AY239335 Gymnamyces parksil

so KG152107 Gymnormyces fallax

KFO02757 Russula subfoetens

HES 47707 Russula foetentoides

HO677769 Russula iota

3 KF245527 Russula lauroceras!

AJ438037 Gymnomyces ammophilus

JN681188 Russula cerslens

“AB2Z11275 Russula sororia

7 JQ622327 Russula arioenclens

AF418612 Russuia acruginea

AF418611 Russula parazurea

| 60 48 EUG18425 Russula maria

46 13 L EU819437 Russula virescens.

oF Soe 8610 Russula vesca

oa AF418809 Russula heterophylla

oS AY061716-Russula roselpes

pe JQ711999 Russula turci

HO604850 Russula miurrillil

86 AF418627 Russula solaris

- JO888199 Russula paludosa

59 AF4188371 Russula firmula

AF418630 Russula veternosa

AY245542 Russula californiensis

GU234063 Russula chamitese

FJ@45432 Russula decolorans

DO422022 Russula risigallina

KFO02762 Russula gilva

HQ604848 Russula lutea

AF230888 Russula postigna

og ¢wF 908682 Russula amethystina

‘| 100 > EU5981 97 Russula ecceftrica

= ~EUS98163. Russula eccentrica

go DQ422027 Russula polyphylla

KF 306038 Russula cantharellicola

KF308037 Russula cantharellicola

°81 KF306036 Russula cantharellicola

100, AB291746 Russula subnigricans

764 S57 AB291744 Russula subnigricans

; ACS581314 Russula nigricans

joo) EUS97075 Russula nigricans

100, JFS34355 Russula albonigra

7 KF 306041 Russula albonigra

100, AB291765 Russula densifolla

58 AB291767 Russula densifolla

if, 99; J0888194 Russula adusta

S271 JF908669 Russula adusta

EU303008 Russia dissimulans

bs £U526006 Russula cascadensis

| | JF834370 Russula aff acrifolia

og |r JF834363 Russula acrifolia

I KF850401 Russula acrifolia

42 ty DQ42 1998 Russula acrifolla

17> 7 @ KRO1 1880 Russula anthracina

aol H@ KRO1 1881 Russula anthracina

“1 1h@ KRO11879 Russula anthracina

gsqiyJF808673 Russula anthracina

KM576561 RusSula anthracina fas R. sp.)

491 FN669239 Russula anthracina (as R,. sp.)

EU2Z31968 Boletus reticuloceps

100

Ingratae

Hetérophyilae

Tenellae

Compactaé

Out group

AO ... Jabeen, Niazi & Khalid

0.0 Evalue). Published sequences from different Russula sections were included

to reconstruct a phylogeny with Boletus reticuloceps (EU231968) as outgroup.

The analysis revealed four major clades (/compactae, /heterophyllae, /ingratae,

/tenellae) corresponding with the taxonomic sections (Fic. 5). Species within

these clades were clustered with strong bootstrap, supported by morphological

characters. Within /compactae, R. acrifolia Romagn., R. adusta (Pers.) Fr.,

R. albonigra (Krombh.) Fr., R. anthracina, R. cantharellicola Arora & N.H.

Nguyen, R. cascadensis Shaffer, R. densifolia Secr. ex Gillet, R. dissimulans

Shaffer, R. eccentrica Peck, R. nigricans Fr., R. polyphylla Peck, and R. subnigricans

Hongo clustered with 76% bootstrap support.

Discussion

Russula anthracina can be distinguished from R. acrifolia by its pinkish

tinged lamellae (Romagnesi 1967, Galli 1996, Sarnari 1998), which are pale

cream in R. acrifolia. Anatomically, in R. anthracina the pileus cuticle lacks

pileocystidia, which are numerous with frequently diverticulate tips in

R. acrifolia. Russula albonigra is distinguished from R. anthracina and

R. acrifolia by its rapidly blackening flesh and rather large, unforked pilocystidia

(Rayner 1985, Kibby 2001).

Russula anthracina is an ectomycorrhizal symbiont of both deciduous and

coniferous trees (Gminder et al. 2000, Bahram et al. 2011, Suz et al. 2014). We

describe and illustrate this ectomycorrhiza here for the first time in detail.

Morphologically it differs from the ectomycorrhiza of the closely related

R. acrifolia (Agerer 1996) by its monopodial pinnate branching pattern and

greyish brown to transparent mantle surface with cellular hyphae and typical

russuloid cystidia without an apical knob.

In phylogenetic analysis, our Pakistani sequences showed a 0.2-1% genetic

divergence from R. acrifolia and clustered with European R. anthracina

sequences from Austria, Estonia, and Italy with 85% bootstrap support. Russula

anthracina is a European species (Romagnesi 1967, Petersen & Vesterholt 1993,

Einhellinger 1994, Hallingback & Aronsson 1998, Gminder et al. 2000, Piltaver

et al. 2002, Legon & Henrici 2005, Kranzlin 2005, Adam¢ik et al. 2006, Holec &

Beran 2006, Dimou et al. 2008, Ronikier & Adam¢ik 2009, Bahram et al. 2011,

Cabo 2012, Kasom & Karadelev 2012, Osmundson et al. 2013, Suz et al., 2014),

but it has also been recorded in America (Roberts 2007, Kuo 2009) and Africa

(El Kholfy et al. 2011, Kinge et al. 2013). From western Asia, it is reported from

Turkey (Yalz et al., 2006, Giing Ergénil et al. 2013, Sesli & Denchev 2014).

Our Russula anthracina collections represent an addition to the mycobiota of

Pakistan and a first report of the species from South Asia.

Russula anthracina ectomycorrhiza with Himalayan cedar (Pakistan) ... 41

Acknowledgements

We are highly indebted to Higher Education Commission (HEC), Pakistan for

financial assistance under Indigenous PhD Fellowships (Phase II). Thanks to all lab

fellows accompanied for the collection. Sincere thanks to Prof. Donald H. Pfister

(Harvard University Herbaria, Harvard University, MA, USA) for providing the

opportunity for the first author to work in his lab. Thanks to Dr. Lorenzo Pecoraro

(Tsinghua University, Shenzhen, China) for the review. The authors are grateful to

Dr. T.K. Arun Kumar (The Zamorin’s Guruvayurappan College, Kerala, India) for his

valuable comments and suggestions to improve the manuscript. Thanks are also due to

Dr. Shaun Pennycook for his precious time in editing the nomenclature.

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http://dx.doi.org/10.1371/journal.pone.0062419

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MY COTAXON

January-March 2016—Volume 131, pp. 45-48

http://dx.doi.org/10.5248/131.45

Sympodiosynnema, a new genus of dematiaceous hyphomycetes

from southern China

JI-WEN X14, YING-RuI Ma, JIAN-MEI Gao, ZHUANG LI & XIU-GUO ZHANG

Department of Plant Pathology, Shandong Agricultural University, Taian, 271018, China

*CORRESPONDENCE TO: sdau613@163.com, liz552@126.com

ABSTRACT — Sympodiosynnema gen. nov. is illustrated and described from dead stems of

an unidentified plant in Hainan, southern China. The genus is characterized by solitary

dry naviculiform conidia, sympodially proliferating conidiogenous cells, and synnematous

conidiophores.

Key worps — Sympodioplanus, conidial fungi, taxonomy

Introduction

During a survey of dematiaceous hyphomycetes colonizing diverse plant

habitats from the forests of Hainan, China, a fungus that does not match any

existing genera was collected growing on unidentified dead twigs. A review

of the literature and morphological studies revealed that it represents an

undescribed genus (Ellis 1971, 1976, Subramanian 1971, Matsushima 1975,

1983, 1985, 1989, 1993, 1995, Carmichael et al. 1980, Castafieda-Ruiz 1986,

Castaneda-Ruiz & Kendrick 1990a,b, 1991, Wu & Zhuang 2005, Seifert et al.

2011).

The new genus, Sympodiosynnema, is described and illustrated. It is

characterized by synnematous conidiophores and polyblastic and sympodially

proliferating conidiogenous cells that produce solitary naviculiform dry

conidia. Specimens are deposited in the Herbarium of Department of Plant

Pathology, Shandong Agricultural University, Taian, China (HSAUP) and

the Mycological Herbarium, Institute of Microbiology, Chinese Academy of

Sciences, Beijing, China (HMAS).

46 ... Xia & al.

Sympodiosynnema J.W. Xia & X.G. Zhang, gen. nov.

MycoBAnk MB 815649

Differs from Sympodioplanus by possessing synnematous conidiophores.

TYPE SPECIES: Sympodiosynnema elegans J.W. Xia & X.G. Zhang

EryMoLoGy: Sympodio- referring to the similar genus Sympodioplanus; synnema,

referring to the synnematous conidiophores.

ASEXUAL FUNGUS. COLONIES on the natural substrate effuse, brown. Mycelium

mostly immersed, composed of branched, septate, hyaline to brown, smooth

hyphae. CONIDIOMATA synnematal unbranched, erect, with brown stalks,

consisting of compact aggregation of parallel conidiophores. CONIDIOPHORES

macronematous, erect, unbranched, septate, pale brown to brown, divergent

towards the distal part of the synnema. CONIDIOGENOUS CELLS polyblastic,

integrated, terminal, sympodial, hyaline to pale brown. Conidial secession

schizolytic. Conip1a solitary, dry, naviculiform, euseptate, pale brown.

Sympodiosynnema elegans J.W. Xia & X.G. Zhang, sp. nov. FIG. 1

MycoBank MB 815650

Differs from Sympodioplanus capensis by its synnematous conidiophores with

sympodial proliferations and its larger conidia and from S. goaensis by its synnematous

conidiophores with sympodial proliferation and its more septate conidia.

Type: China, Hainan Province: Jianfengling, on dead stems of unidentified broadleaf

tree, 22 Apr. 2014, J.W. Xia (Holotype, HSAUP H6447; isotype, HMAS 245682).

EryMo_oey: elegans, referring to the elegant conidia.

CoLoniEs on the natural substrate effuse, brown. Mycelium mostly immersed,

composed of branched, septate, hyaline to brown, smooth, 1.5-3 um wide

hyphae. CONIDIOMATA synnematal, unbranched, erect, with brown stalks,

consisting of a compact aggregation of parallel conidiophores, terminating in

brown fertile heads, <300 um long, 25-35 um wide in the middle with a base

<45 um wide and an apex 110 um wide. CONIDIOPHORES macronematous,

erect, unbranched, septate, pale brown to brown, 3-5 um wide, divergent

towards the distal synnema. CONIDIOGENOUS CELLS polyblastic, integrated,

terminal, sympodial, thin-walled, smooth, hyaline to pale brown, bearing flat

scars, sometimes slightly obscured. Conidial secession schizolytic. CONIDIA

solitary, dry, naviculiform, 3-5-euseptate, smooth, pale brown, 22.5-28 x

6-8 um.

ComMENTS - Sympodiosynnema demonstrates unique morphological and

ontogenetic features. Its macronematous conidiophores form distinct synnemata

with a sympodial proliferation of the conidiogenous cells and produce

solitary dry naviculiform conidia. Conidial fungi characterized by sympodial

proliferation represent a large group, among which only Sympodioplanus

SS ae

‘

| =

< =

x =

ran

me,

—

Sympodiosynnema elegans gen. & sp. nov. (China) ... 47

Fic. 1. Sympodiosynnema elegans (holotype, HSAUP H6447).

A. synnema; B, C. conidia; D. conidiogenous cells and conidia;

E. conidiogenous cells and conidiogenous loci.

A8 ... Xia & al.

R.C. Sinclair & Boshof (Sinclair et al. 1997) has conidiogenous cells and conidia

that are morphologically similar to Sympodiosynnema. Sympodioplanus,

however, is distinguished by mononematous conidiophores.

In conidial shape, Sympodiosynnema elegans is most similar to

Sympodioplanus capensis R.C. Sinclair & Boshof, which differs by its smaller

conidia (13-16 x 2.5-4 um; Sinclair et al. 1997). The also similar S. goaensis

J. Pratibha can be distinguished by its less septate conidia (1-2-septate; Pratibha

2013).

Acknowledgments

The authors express gratitude to Dr. Rafael E Castafteda-Ruiz and Dr. Eric H.C.

McKenzie for serving as pre-submission reviewers and for their valuable comments and

suggestions. This project was supported by the National Natural Science Foundation of

China (Nos. 31093440, 31230001) and the Ministry of Science and Technology of the

People’s Republic of China (Nos. 2006FY120100).

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32. 53 p.

Castaneda-Ruiz RF, Kendrick WB. 1990b. Conidial fungi from Cuba: II. Univ. Waterloo Biol. Ser.

33.61 p.

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Canada. Univ. Waterloo Biol. Ser. 35. 132 p.

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England. 608 p.

Ellis MB. 1976. More dematiaceous hyphomycetes. Commonwealth Mycological Institute, Kew,

Surrey, England. 507 p.

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Kobe, Japan. 209 p, 415 pl.

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Matsushima T. 1989. Matsushima mycological memoirs 6. Published by the author, Kobe, Japan. 100 p.

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Matsushima T. 1995. Matsushima mycological memoirs 8. Published by the author, Kobe, Japan. 54 p.

Pratibha J. 2013. Sympodioplanus goaensis sp. nov. from Goa, India. Mycotaxon 125: 145-148.

http://dx.doi.org/10.5248/125.145

Seifert K, Morgan-Jones G, Gams W, Kendrick B. 2011. The genera of hyphomycetes. CBS

Biodiversity Series 9. 997 p.

Sinclair RC, Boshoff S, Eicher A. 1997. Sympodioplanus, a new anamorph genus from South Africa.

Mycotaxon 64: 365-374.

Subramanian CV. 1971. Hyphomycetes: an account of Indian species, except Cercosporae. Indian

Council Ag. Res., New Delhi. 930 p.

Wu WP, Zhuang WY. 2005. Sporidesmium, Endophragmiella and related genera from China. Fungal

Divers. Res. Ser. 15. 351 p.

MY COTAXON

January-March 2016—Volume 131, pp. 49-60

http://dx.doi.org/10.5248/131.49

New records from Lithuania of fungi alien to Europe

JuRGA MOTIEJUNAITE! , ERNESTAS KUTORGA’, JONAS KASPARAVICIUS’,

VAIDOTAS LyGIs' & GODA NORKUTE’

' Institute of Botany of Nature Research Centre,

Zaliyjy Ezery Str. 49, Vilnius, LT-08406 Lithuania

? Department of Botany and Genetics, Vilnius University,

Saulétekio Av. 7, Vilnius, LT-10222 Lithuania

“ CORRESPONDENCE TO: jurga.motiejunaite@botanika.It

ABSTRACT — First records from Lithuania of the ascomycete Ophiostoma novo-ulmi and

basidiomycetes Clathrus archeri, Leucocoprinus cepistipes, and Stropharia rugosoannulata are

presented. All four species are alien to Europe and two, C. archeri and S. rugosoannulata, have

never been recorded in the eastern part of the Baltic Sea region before. Also, a reassessment of

the status in Lithuania of the alien pathogen Hymenoscyphus fraxineus and its close relative,

the non-pathogenic H. albidus indigenous to Europe, indicates that only H. fraxineus occurs

in Lithuania. Descriptions of the examined fungi are presented, and remarks on their habitats

and distribution are provided.

Key worps — anthropogenic habitats, distribution, invasive species, non-native fungi,

saprobes

Introduction

Precise data on diversity and distribution of fungi, their geographical

ranges, and habitat requirements are often lacking even for comparatively well-

studied regions. Fragmentary baseline knowledge of fungal taxonomy, ecology,

and geographical distribution is probably one reason why research on alien

organisms and their effect on ecosystems has so far paid insufficient attention

to fungi (PySek et al. 2008), with the exception of some invasive pathogenic

species that cause conspicuous damage to vegetation or wildlife (Desprez-

Loustau et al. 2010). Until recently, the spread of alien mycorrhizal and saprobic

macromycetes in particular was treated as a mycological curiosity, although

50 ... Motiejiinaité & al.

lately the economical and ecological importance of alien non-pathogenic fungi

has been acknowledged (Richardson et al. 2000, Desprez-Loustau et al. 2007,

Vellinga et al. 2009, Nunez & Dickie 2014). Nonetheless, the general knowledge

of geographical distribution of alien fungi is far from complete (Kreisel 2006,

Desprez-Loustau et al. 2010, Santini et al. 2013) as information on new

localities of already established alien fungi and on new arrivals continuously

accumulates. Moreover, accurate identification is often an issue for reports on

the occurrence of alien macromycetes — e.g., Amanita phalloides (Fr.) Link

in North America; Pringle & Vellinga 2006 — and plant pathogens — e.g.,

Hymenoscyphus fraxineus (Baral & Bemmann 2014, Gross et al. 2014) and

subspecies and hybrids of Ophiostoma novo-ulmi (Brasier 1991, Brasier & Buck

2001, Brasier & Kirk 2001) — in Europe.

In Lithuania, the first records of fungi (including aliens) appeared almost 200

years ago; since then the number of non-native species records has continuously

increased, although so far there is still no comprehensive list of such fungi in

the country. Here we report and discuss the first cases of occurrence of four

fungal species that are alien in Europe, and revise the status of an invasive ash

dieback pathogen, Hymenoscyphus fraxineus, and its close relative indigenous

saprobe, H. albidus (Gillet) W. Phillips, in Lithuania.

Materials & methods

EXAMINATION OF FUNGAL FRUIT-BODIES. Fresh basidiomata of Leucocoprinus

cepistipes and Stropharia rugosoannulata and dried basidiomata of Clathrus archeri

were studied. Additionally, color photographs of C. archeri in situ were examined.

Microscopical features were examined under a Nikon Eclipse Ci-S light microscope.

Squashed preparations were mounted in water and in 5% KOH solution. All

measurements were made in water. Herbarium specimens previously identified as

Hymenoscyphus albidus were examined for presence of simple septa or croziers at the

bases of asci. Squashed sections or fragments of ascomata were mounted in 3% KOH

+ Lugol’s iodine solution or Congo Red and studied under an Olympus BX51 light

microscope. All descriptions are based on Lithuanian material.

MOLECULAR IDENTIFICATION OF OPHIOSTOMA NOVO-ULMI ISOLATES. A total of 49

O. novo-ulmi isolates were recovered from wood of wilting branches of 20 Ulmus glabra

and 29 U. minor trees growing in various parts of Lithuania (for details of the localities

see Norkuté 2013). In the laboratory, the fungi were isolated and cultured according

to Brasier (1981) and Bakys et al. (2009). All 49 isolates that were morphologically

similar to O. novo-ulmi based on cultural descriptions by Brasier (1991) and Kirisits &

Konrad (2004) were assigned for DNA extraction and grown for two weeks on liquid

Hagem media in static cultures at room temperature. Protocols for DNA extraction

and PCR amplifications followed Karen et al. (1997), and the ribosomal ITS region was

sequenced by Macrogen Inc. (Seoul, Korea) using two primers (ITS1F and ITS4; White

et al. 1990). All obtained sequences were identical and identified via a NCBI BLAST

Four new European agaric records (Lithuania) ... 51

database (www.ncbi.nlm.nih.gov) search as O. novo-ulmi. The representative sequence

of the Lithuanian O. novo-ulmi (LPM ON3; for details see below) has been deposited at

GenBank (accession no. KJ677112).

PCR-RFLP of the cerato-ulmin (cu) and colony type (COL1) genes was used to

identify O. novo-ulmi subspecies and hybrids following the protocol developed by

Konrad et al. (2002): DNA of all 49 isolates was amplified using DreamTaq™ Green DNA

polymerase (Fermentas, Vilnius, Lithuania) following the manufacturer's instructions

with a different annealing temperature for each primer (68°C for cu, 58°C for CoL1).

The restriction enzymes Bfal and HphI (Fermentas, Vilnius, Lithuania) were used in the

RFLP process according to manufacturer instructions and following recommendations

by Konrad et al. (2002). Restriction products were visualized in 2% agarose gel

(90 min at 120 V in sodium borate (SB) buffer; Fic. 1). Assignment of the screened

O. novo-ulmi isolates to subspecies or hybrids followed Konrad et al. (2002).

SPECIMEN ACCESSIBILITY. All specimens cited below are deposited in fungal

collections of either the Herbarium of the Institute of Botany of Nature Research Centre,

Vilnius, Lithuania (BILAS) or the Herbarium of Vilnius University, Vilnius, Lithuania

(WI). Isolates of O. novo-ulmi are stored in the culture collection of the Laboratory of

Phytopathogenic Microorganisms at the Institute of Botany of Nature Research Centre,

Vilnius, Lithuania (LPM).

Nomenclature follows MycoBank Fungal Databases (http://www.mycobank.org).

Clathrus archeri (Berk.) Dring, Kew Bull. 35: 29. 1980.

Immature basidiomata globose to ovoid, dirty whitish, growing solitary or

gregarious. Mature basidiomata with 4-6 (most commonly five) receptaculum

arms initially fused at the tips, later splitting and spreading outwards; arms

tapered to apices, bright red on the inner (upper) surface and pale red elsewhere,

finely reticulately pitted, with dark green gleba. Receptaculum of rounded cells,

40-50 um diam. Gleba emitting a strong carrion-like odor (persistent even in

dried material). Stem with the base encased in a whitish volva. Basidiospores

cylindric-ellipsoid, hyaline, greenish in mass, smooth-walled, 4.5-6 x

2-2.5 um. Basidia not observed.

SPECIMEN EXAMINED: LITHUANIA, KreTINGA DISTR., northern edge of Kartena

village, 55°56’N 21°28’E, 1 Sept 2011, E. Slusnyté (BILAS 49286).

HABITAT IN LITHUANIA: On soil on a semi-open slope, covered with shrubs and in

herbaceous vegetation between planted young Picea abies and Pinus sylvestris.

Notes: Originally described from Tasmania, and indigenous in Australia and

New Zealand, Clathrus archeri has spread to Europe, North America, and Asia

as an alien species. Since it was first recorded from France in 1914, the fungus

has spread widely across western and central Europe (Michael et al. 1986,

Pegler et al. 1995, Hansen & Knudsen 1997, Birsan et al. 2014). The records

of C. archeri closest to Lithuania are from Poland, mostly from the southern

part where it has a status of a spreading species, although the reports from

52 ... Motiejiinaité & al.

northern Poland are few and at the present time confined to the coastal areas

with a milder climate (Miadlikowska 1995, Halama et al. 2010, Szczepkowski

& Obidzinski 2012). In Lithuania, the fungus was found in the west, also

characterized by a milder climate influenced by the Baltic Sea. Kreisel (2006)

noted that elsewhere in Europe, C. archeri continues to spread northwards,

where it has already become established in southern Sweden (M. Jeppson,

personal communication). Our present record is the first for the eastern Baltic

Sea region.

Hymenoscyphus fraxineus (T. Kowalski) Baral, Queloz & Hosoya, IMA Fungus 5:

79. 2014.

Ascomata developing on blackened (stromatized) petioles of fallen previous

year's leaves of Fraxinus excelsior, gregarious, superficial, stipitate, cup-shaped,

1-4 mm diam. Hymenium shallowly concave or plane, whitish or cream to

ochraceous. Margin even or sinuate, hairless. Receptacle concolorous with the

hymenium, somewhat striate. Stipe obconical to cylindrical, concolorous with

the receptacle, blackened at the base, <1.2 mm long. Exterior covered by short

hyphal outgrowths, especially on stipe. Ectal excipulum a textura prismatica

(-porrecta), cells with slightly gelatinized walls. Medullary excipulum a hyaline

textura intricata. Asci arising from croziers, cylindric-clavate, 8-spored, 85-120

x 8-14 um, apical pore blue in Lugol's solution. Ascospores cylindrical to

cylindric-clavate, straight or slightly curved, often inequilateral, usually

scutuloid, with internal droplets, aseptate or occasionally 1-septate, 16-24 x

3-5 um. Paraphyses cylindrical, apically unthickened or slightly inflated <3 um.

SPECIMENS EXAMINED: LITHUANIA, KEDAINIAI DISTR., Stebuliai forest, 55°19’N

24°07’E, 21 Sept 1999, E. Kutorga (BILAS 34422), 29 Aug 2000, E. Kutorga (WI 7344);

Vilainiai forest, 55°18’N 24°02’E, 21 Sept 1999, E. Kutorga (BILAS 34426), 30 Aug 2000,

E. Kutorga (WI 7345); PaSiliai forest, 55°14’N 23°58’E, 1 Jun 2000, E. Kutorga (WI 7341);

Lan¢citinava forest, 55°20’N 24°12’E, 29 Aug 2000, E. Kutorga (WI 7342); Berankiskis

forest, 55°14’N 24°08’E, 29 Aug 2000, E. Kutorga (WI 7343), 3 Oct 2001, E. Kutorga

(BILAS 25151), 2 Oct 2002, E. Kutorga (BILAS 34425); Juodkiskiai forest, 55°16’N

24°02’E, 30 Aug 2000, E. Kutorga (WI 7346); Silainéliai forest, 55°14’N 24°02’E, 3 Oct

2002, E. Kutorga (BILAS 33994); SrrvinTOs pDIsTR., Sirvintos forest, 55°03’N 24°58’E, 10

Sept 1997, E. Kutorga (WI 7409); UKMERGE DISTR., Taujénai forest, 55°26’N 24°39’E, 11

Sept 1997, E. Kutorga (WI 7410); Vitntus ciTy, Verkiai forest, 54°45’N 25°17’E, 25 Jun

2001, A. Kazlauskiené (WI 4714).

HaBITAT IN LITHUANIA: Ash dieback symptoms occur in most habitats where E excelsior

grows. The ascomata of H. fraxineus were registered in leaf litter in different forests,

although most commonly in mixed deciduous stands. Ascomata were also recorded

from parks and nurseries under diseased trees, but these records were not confirmed

microscopically.

Notes: The causal agent of ash dieback, pathogenic H. fraxineus, is an alien

invasive ascomycete in Europe that is replacing the closely related saprobic

Four new European agaric records (Lithuania) ... 53

indigenous species, H. albidus (Queloz et al. 2011, 2012; McKinney et al. 2012;

Baral et al. 2014; Gross et al. 2014). Hymenoscyphus fraxineus is well known

from the characteristic disease symptoms (Gross et al. 2014) and molecular

data obtained from both isolated fungal cultures and symptomatic ash tissues.

In Lithuania, crown dieback of E excelsior was first observed in 1996 and by

2001 it had already become widespread (Juodvalkis & Vasiliauskas 2002).

The disease has devastated ash stands all over the country and currently is in

its chronic phase (Plitra et al. 2011, Bakys 2013, Lygis et al. 2014). Initially,

its primary causal agent was not properly diagnosed (Lygis et al. 2005,

Vasiliauskas et al. 2006). The fungus was correctly identified only in 2008 when

DNA sequence of a fungus (labelled as Hymenoscyphus sp. 970 by Lygis et al. in

2005) isolated in 2001 from a root collar of declining E excelsior was assigned to

H. fraxineus (GenBank AY787704, as Chalara fraxinea).

Since 1997, stromatized F. excelsior leaf petioles with numerous ascomata

of Hymenoscyphus sp. collected in Lithuania have been deposited in herbaria

BILAS and WI. The ascomata were first assigned to the saprobic H. albidus

(Treigiené et al. 2010). Recently, Zhao et al. (2012) and Baral & Bemmann

(2014) observed subtle microscopic differences in ascomatal structures between

closely related H. albidus and H. fraxineus and underlined the importance

of combining detailed morphological studies with molecular analysis in the

H. albidus / H. fraxineus species complex. They reported that the presence

or absence of croziers at the ascus base could distinguish H. albidus from

H. fraxineus, differences thatare strictly correlated with molecular characteristics.

Therefore, we re-examined all herbarium specimens microscopically in order

to clarify the status of the two Hymenoscyphus species in Lithuania. As the asci

of all studied specimens had croziers, they were assigned to H. fraxineus. ‘This

suggests that there is no official proof of previous or current occurrence of

H. albidus in Lithuania. It is worth mentioning that the Lithuanian collections

of H. fraxineus specimens with ascomata (collected between 1997-2002)

represent one of the earliest herbarium records of this fungus in Europe.

Leucocoprinus cepistipes (Sowerby) Pat., J. Bot. 3: 336. 1889.

Basidiomata growing in clusters. Pileus 2-6 cm diam, conic-campanulate

when young, later convex to plano-convex, with an obtuse umbo. Surface dry,

white to cream-colored, fibrillose, soon developing small white to cream-colored

scales. Context white, soft, very thin. Lamellae free, narrow, white, becoming

pallid to pale buff, edges white-floccose. Stipe 3-10 x 0.3-0.6 cm, cylindrical,

hollow; base swollen, stipe surface above the annulus white and smooth, below

— finely white-floccose, turning brownish when rubbed. Annulus fibrillose-

membranous, white, with a somewhat crenate margin. Odor and taste mild,

54 ... Motiejiinaité & al.

not distinctive. Basidiospores ellipsoid to ovoid, smooth, hyaline, with a small

germ pore, 6-11 x 5-7 um. Basidia clavate, with 4 sterigmata, without basal

clamp, 22-35 x 10-12 um. Cheilocystidia clavate, sometimes rostrate, 25-60 x

8-15 um. Pleurocystidia absent.

SPECIMENS EXAMINED: LITHUANIA, VILNtus city, Valakupiai, 54°44’N 25°19’E, 10

Jun 2005, V. Urbonas (BILAS 30747); Fabijoniskés, 54°44’N 25°15’E, 28 May 2009, V.

Kleizaité (WI 7278).

HABITAT IN LITHUANIA: on soil in a greenhouse and in a flowerpot in a private

apartment.

Notes: Leucocoprinus cepistipes is of tropical origin, native to Asia and Africa;

it has been introduced to Europe with a plant material and is apparently

spreading (Vellinga 2001). In central and northern Europe it grows almost

exclusively indoors (Pidlich-Aigner et al. 2002, Knudsen & Vesterholt 2008,

Gierczyk et al. 2011); however in western and southern Europe the fungus has

been found also outdoors on humus-rich soil, woodchips, compost or manure.

In northern Poland, L. cepistipes was once recorded outdoors, growing on a

horticultural waste dump (Gierczyk et al. 2011); however, there are no data of

its overwintering. In Latvia (Riga city), the fungus was found in a lawn strip

between a street and a sidewalk (Daniele & Meiere 2011; I. Daniele, personal

communication). In Lithuania, L. cepistipes has been found exclusively indoors.

Ophiostoma novo-ulmi Brasier, Mycopathologia 115: 155. 1991.

No sexual and asexual fruiting structures of O. novo-ulmi were observed

in Lithuania. Disease symptoms were in accordance with those provided by

Anonymous (2009). Characteristics of the cultures coincided with those

provided by Brasier (1991).

MATERIAL EXAMINED: (for geographical coordinates see Norkuté 2013).

subsp. novo-ulmi: LITHUANIA, Brrzal pDIsTR., Karajimiskis, from Ulmus glabra,

May 2011 (LPM ON13); Kaunas pistTR., Raudondvaris, from U. glabra, Nov 2011 (LPM

ON43); KEDAINIAI DISTR., Aristava, from U. minor, Jun 2011 (LPM ON14, ON15,

ON18, ON19, ON21-ON34, ON36-ON39, ON42); PASVALYS DISTR., Gulbinénai, from

U. glabra, May 2011 (LPM ON10); Paberliai, from U. glabra, May 2011 (LPM ON11,

ON12); PLUNGE pDisTR., Kuliai, from U. glabra, Jun 2011 (LPM ON48-ON51); RokIsKis

DISTR., Dusetos, from U. glabra, Jun 2011 (LPM ON44-ON47).

subsp. novo-ulmi x subsp. americana hybrid: LITHUANIA, KEDAINIAI DISTR.,

Aristava, from Ulmus minor, Jun 2011 (LPM ON16, ON17, ON20, ON35, ON40,

ON41); PLUNGE pisTR., Kuliai, from U. glabra, Jun 2011 (LPM ON52, ON53); VILNIUS

city, Verkiai park, from U. glabra, 12 Jul 2010 (LPM ON3; GenBank KJ677112);

VILNIUS DISTR., VerkSionys, from U. glabra, 19 Jul 2010 (LPM ONS, ON6); Dukstos,

from U. glabra, 19 Jul 2010 (LPM ON7, ON8).

HABITAT IN LITHUANIA: According to information provided by the Department of

Forest Sanitary Protection at Lithuanian State Forest Service (personal communication),

Dutch elm disease symptoms occur in most habitats where elms (Ulmus spp.) grow.

Four new European agaric records (Lithuania) ... 55

Notes: Ophiostoma ulmi (Buisman) Nannf. s. str. caused the original Dutch

elm disease epidemic in Europe and North America in the mid-1900’s (Gibbs

1978). Ophiostoma novo-ulmi, a more aggressive pathogen of elms, largely

replaced O. ulmi during the second half of the 20" century (Brasier 1991).

Seemingly, O. ulmis. str. is already absent in most of the European countries as

no recent data on occurrence of this species is available. Ophiostoma novo-ulmi

has two subspecies - O. novo-ulmi Brasier subsp. novo-ulmi (Eurasian, EAN)

and O. novo-ulmi subsp. americana Brasier & S.A. Kirk (North American,

NAN) (Brasier & Kirk 2001). The species is invasive in Europe: NAN was

introduced from North America in 1960's and EAN spread across Europe from

east to west at about the same time (Brasier & Gibbs 1973, Brasier 1990, 1996).

In Lithuania, the outbreak of Dutch elm disease and the causative agent

O. ulmis. str. were first recorded in mid-20" century (Zuklys 1958), butafterwards

this pathogenic ascomycete was not observed. Our molecular identification

of fungal isolates from wood of wilting elm branches collected in 2010-2011

from different regions of Lithuania has demonstrated that all isolates represent

O. novo-ulmi. Fic. 1 shows the RFLP banding patterns of cu and CoL1 genes

from seven Lithuanian O. novo-ulmi isolates digested with restriction enzymes

Hphl and Bfal; according to the cu and coL1 gene fragment sizes (see Konrad

et al. 2002), isolates LPM ON47-ONS51 (lanes 1-5) correspond with subspecies

EAN, while LPM ON52 and ONS3 (lanes 6 and 7) correspond with EAN x

Fic. 1. RFLP banding patterns of cerato-ulmin gene cu from Ophiostoma novo-ulmi digested

with restriction enzyme HphlI (lane numbers labelled “c”) and colony type gene coll digested with

restriction enzyme Bfal (lane numbers labelled “I”) after separation on a 2% agarose gel. Lane

numbers represent seven O. novo-ulmi isolates: 1 = ON47; 2 = ON48; 3 = ON49; 4 = ON50;

5 = ON51; 6 = ON52; 7 = ON53 (see also Norkuté 2013). M = DNA marker, Thermo Scientific

GeneRuler Low Range DNA Ladder‘ (Thermo Fisher Scientific Inc.).

56 ... Motiejiinaité & al.

NAN hybrid. On the basis of similar RFLP patterns from all 49 Lithuanian

O. novo-ulmi isolates, 36 were identified as EAN and 13 as EAN x NAN hybrid

(Norkuté 2013). Our results suggest that O. novo-ulmi has replaced O. ulmi

s. str. in Lithuania.

Stropharia rugosoannulata Farl. ex Murrill, Mycologia 14: 139. 1922.

Basidiomata numerous, growing solitary or gregarious. Pileus 5-15 cm

diam, convex to broadly convex, surface dry, smooth, wine red to reddish

brown, or pale brownish, sometimes cracked in old age (both color types were

present in the Lithuanian sample); the margin with partial veil remnants.

Context white, not changing color upon exposure. Lamellae broadly adnate,

gray when young, later gray blue, black violet, close. Stipe 7-15 x 3 cm, central,

cylindrical to slightly clavate, brittle; surface above the annulus white and

finely striate, surface below with longitudinal fibrils, which are whitish at first

and later pale yellowish on a whitish background; base with white mycelial

threads. Annulus compact, pendent, white; surface finely striate on its upper

side; margin splitting causing annulus to become stellate. Odor pleasant,

taste mild. Basidiospores brownish-violet, ellipsoid to ovoid, smooth, thick

walled, with a germ pore, 10-13 x 7.5-9 um. Basidia clavate, with 4 sterigmata,

without basal clamp, 26-33 x 8-9.5 um. Cheilocystidia ventricose, clavate or

lageniform, with apical protrusion, not changing color in KOH, 30-45 x 11-22

uum. Pleurocystidia fusiform-ventricose, some also with contents not changing

color in KOH, 28-50 x 10-13 um. Pileipellis periclinal, composed of brown-

pigmented, 5-13 um thick hyphae; some septa with clamps.

SPECIMEN EXAMINED: LITHUANIA, VILNIUs DISTR., Gineitiskés, 54°44’N 25°11’E, 9

Oct 2011, V. Buda (BILAS 50367).

HABITAT IN LITHUANIA: on wood chips in a garden among shrubs of Ribes spp.

Notes: The fungus has apparently been introduced to Lithuania with imported

gardening woodchips, a substrate known as a rich source of alien macromycete

species (Shaw & Kibby 2001). Stropharia rugosoannulata is a widely cultivated

mushroom (although not in Lithuania) that has also recently been observed

in natural and semi-natural environments elsewhere in Europe (Breitenbach

& Kranzlin 1995, Knudsen & Vesterholt 2008). At least in central Europe, the

species is likely to be ephemeral as it usually disappears with deterioration

of its substrate (Voglmayr & Krisai-Greilhuber 2002). This hypothesis is

supported by Shaw et al. (2004), who state that substrates like woodchips made

of ornamental trees can sustain diverse albeit short-lived fungal communities.

This is the first record of S. rugosoannulata in the eastern Baltic region.

Four new European agaric records (Lithuania) ... 57

Acknowledgments

Authors are grateful to E. Slusnyté (Kretinga district, Kartena) and V. Buda (Vilnius)

for providing the specimens of Clathrus archeri and Stropharia rugosoannulata. Our

special thanks are extended to a local photographer (who wishes to remain anonymous)

for providing ample and detailed photographic material of C. archeri basidiomata and

its habitat. The authors are indebted to M. Wrzosek (University of Warsaw, Poland),

I. Saar (University of Tartu, Estonia), and H.J. Knudsen (Natural History Museum of

Denmark) for reviewing the manuscript. We also acknowledge the accurate and helpful

improvement of the manuscript by S. Pennycook (Manaaki Whenua Landcare Research,

Auckland, New Zealand).

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MY COTAXON

January-March 2016—Volume 131, pp. 61-85

http://dx.doi.org/10.5248/131.61

Some stromatic pyrenomycetous fungi from northern Thailand —

2. Annulohypoxylon and Ustulina

LARISSA N. VASILYEVA! , STEVEN L. STEPHENSON? & KEVIN D. HypDE?4

‘Institute of Biology & Soil Science, Far East Branch of the Russian Academy of Sciences,

Vladivostok 690022, Russia

*Department of Biological Sciences, University of Arkansas, Fayetteville, Arkansas 72701, USA

*Institute of Excellence in Fungal Research e& *School of Science, Mae Fah Lung University,

Chiang Rai 57100, Thailand

“ CORRESPONDENCE TO: vasilyeva@biosoil.ru

ABSTRACT—Fourteen species of Annulohypoxylon and one species of Ustulina collected

in northern Thailand are discussed. Only three of the species (Annulohypoxylon

bahnphadengense, A. bogoriense comb. nov., A. urceolatum) have been reported previously

from southeastern Asia. The other twelve species (A. chiangmaiense, A. derelictum,

A. dipterocarpi, A. maesaeense, A. microbovei, A. morisuspectum, A. neglectum,

A. paratruncatum, A. planodiscum, A. pseudonitens, A. sordidum, Ustulina pseudozonata) are

proposed as spp. nov., described, and illustrated.

KEY worps—Ascomycota, taxonomy, Xylariaceae

Introduction

In our first paper in this series (Vasilyeva et al. 2012), we emphasized that

Thailand is the portion of the Indo-Malayan center of biodiversity characterized

by a unique assemblage of vascular plants and, consequently, many associated

species of fungi. As numerous xylariaceous fungi are host-specific (Rogers

1979, Whalley 1985, 1996, Stadler 2011) and those within the Indo-Malayan

center of biodiversity would be expected to be likewise, it can be difficult to

trace host-fungus associations in tropical forests, where dead branches are

mixed together on the forest floor and the tree (or trees) from which they have

fallen are not easy to identify. Nonetheless, we anticipate that future research

will reveal definite substrate preferences for southeastern Asian species for

which associations are now unknown.

62 ... Vasilyeva, Stephenson & Hyde

In attempting to follow a biogeographical approach to taxonomy, we made

a preliminary assessment of Annulohypoxylon species that might be found in

Thailand. Several species and varieties are known only from southeastern Asia

(Ju & Rogers 1996, Juet al. 2004, Fournier et al. 2010), including Annulohypoxylon

bahnphadengense (Thailand), A. bovei var. microsporum (J.H. Mill.) Y.M. Ju et al.

(China, Japan, Philippines, Taiwan), A. discophorum (Penz. & Sacc.) Y.M.

Ju et al. (Indonesia), A. elevatidiscum (Y.M. Ju et al.) Y.M. Ju et al. (Taiwan),

A. ilanense (Y.M. Ju & J.D. Rogers) Y.M. Ju et al. (Taiwan), A. maeteangense

J. Fourn. & M. Stadler (Thailand), A. microcarpum (Penz. & Sacc.) Y.M. Ju

et al. (Indonesia), A. moriforme var. microdiscum (Y.M. Ju & J.D. Rogers)

Y.M. Ju et al. (Taiwan), and A. urceolatum (Philippines, Sri Lanka, and Taiwan).

These were the first taxa checked when identifying our specimens collected in

Thailand. Although we recorded only two species (A. bahnphadengense and

A. urceolatum) again in 2011, we expect others will be represented by additional

collections in the future.

There are a few other species reported from both southeastern Asia and the

tropics of the western hemisphere, but their types and many other specimens

are mostly from the eastern hemisphere. For example, Annulohypoxylon nitens

(Ces.) Y.M. Ju et al., described originally from Malaysia, has been found also in

China, Philippines, Sri Lanka, and Taiwan. Its occurrence in Thailand has been

reported by Suwannasai et al. (2002, 2005), Thienhirun et al. (2003), Thienhirun

& Whalley (2004), Phosri et al. (2008), and Okane et al. (2008). Records of

A. nitens from Mexico and Puerto Rico (Ju & Rogers 1996), Argentina (Hladki

& Romero 2009), and Brazil (Pereira et al. 2010b) may represent a different

taxonomic entity (cf. Vasilyeva et al. 2012, Vasilyeva & Stephenson 2014). In

contrast to the southeastern Asian concentration of A. nitens, the distribution

of A. stygium (Lév.) Y.M. Ju et al. is centered primarily in the Caribbean Basin.

Specimens reported from Indonesia and Malaysia as Hypoxylon stygium

(e.g., Whalley & Whalley 2007) might be assigned to other species (such as

Hypoxylon bogoriense and H. stigmoideum Ces.) described previously from

these countries, and the unification of the three entities (H. bogoriense,

H. stigmoideum, H. stygium) into a single species (Ju & Rogers 1996) might

need further consideration. Collections from Thailand identified as H. stygium

(Whalley et al. 1995, Suwannasai et al. 2002, 2005, Thienhirun et al. 2003, Tang

et al. 2007, Phosri et al. 2008) might also represent different species.

Materials & methods

The specimens considered herein were collected in two provinces in northern

Thailand: Chiang Mai (Mae Taeng District near the villages of Pha Deng and Mae

Sae; and the Doi Suthep temple in the vicinity of the city of Chiang Mai) and Chiang

Annulohypoxylon & Ustulina spp. nov. (Thailand) ... 63

Rai (Doi Pui Mountain in the Muang District). Microscopic analyses were carried out

using standard techniques. Material was mounted in 10% KOH for examination of

stromatal pigments and dehiscence of the perispore, and Melzer’s iodine reagent was

used for examination of the ascal apical ring. Color designations follow Rayner (1970).

Photographs of stromata and associated structures were taken using a Nikon D40x

digital camera. Measurements were obtained from about 15 ascospores and 5 glomerate

stromata to represent the range of variation. The material from Thailand is deposited in

the herbarium of Mae Fah Luang University, Chiang Rai, Thailand (MFLU).

Taxonomy

Annulohypoxylon bahnphadengense J. Fourn. & M. Stadler,

Fungal Diversity 40: 30. 2010 Fics 1, 15a

SPECIMEN EXAMINED: THAILAND, CHIANG MAI PROVINCE, Mae Taeng District,

Pha Deng village, Mushroom Research Centre, on wood, 19 June 2011, L. Vasilyeva

(MFLU11-1165).

Note: Annhulohypoxylon bahnphadengense, characterized by _ effused-

applanate, dull black to shiny black stromata, yielding grayish sepia (106)

pigments in 10% KOH, ostiolar discs 0.25-0.3 mm diam., ascospores 6.5-8 x

3-3.5 um, with a faint straight germ slit spore-length, perispore dehiscent in

10% KOH, and described only recently by Fournier et al. (2010), was collected

again in same general locality.

Annulohypoxylon bogoriense (Hoéhn.) Lar.N. Vassiljeva, S.L. Stephenson &

K.D. Hyde, comb. nov. Figs 2, 15b

MycoBank MB 801406

= Hypoxylon bogoriense Hohn., Sitz. K. Akad. Wiss. Math.-Nat. K]., Abt. 1, 118: 341. 1909.

STROMATA effused, rather plane, with mostly inconspicuous (sometimes

conspicuous) perithecial mounds at the margins, surface brown or dark-

reddish-brown to the naked eye but pinkish between the black ostiolar discs

in direct light and under low magnification, shiny black around the individual

perithecia, with citrine (13) to olivaceous buff (89) KOH-extractable pigments.

PERITHECIA spherical, 0.3-0.4 mm diam., ostioles papillate, encircled with a

slightly concave or flat ostiolar discs 0.2-0.25 mm diam. Ascr not observed.

Ascosporss light brown, ellipsoid-inequilateral, 5-7 x 2.4-3 um, with a

straight germ slit shorter than the spore length; perispore dehiscent in 10 %

KOH.

SPECIMEN EXAMINED: THAILAND, CHIANG MAI PROVINCE, Mae Taeng District,

Pha Deng village, Mushroom Research Centre, on wood, 24 June 2011, L. Vasilyeva

(MFLU11-1166).

Notes: Hypoxylon bogoriense was listed as a synonym of Hypoxylon stygium

by Ju & Rogers (1996). Our illustration (Fic. 2) of the specimen from Thailand

64 ... Vasilyeva, Stephenson & Hyde

shows the pinkish stromatal surface between the black ostiolar discs, which is

similar to A. atroroseum (J.D. Rogers) Y.M. Ju et al. Annulohypoxylon stygium

and A. atroroseum share many similar features, but mature stromata are usually

shiny black in A. stygium, whereas those of A. atroroseum are rose-colored with

a black ostiolar region (Ju & Rogers 1996). However, the mature stromata in

our specimen from Thailand are not rose-colored; this tint is apparent only

in direct light and under low magnification when photographed, and the

actual color is dark brown or in accordance with Hohnel’s (1909) description

of Hypoxylon bogoriense as being violet-brown and darkening with the age.

Moreover, Rogers (1981) described H. atroroseum as having conspicuously

sunken ostiolar discs, whereas those in our specimen are slightly concave and

almost flat, thus slightly elevated above the surface. As Hohnel indicated, the

flat-convex tops of the perithecia, always at the margins but rarely in the middle

parts of the stromata, stick out (“Scheitel der randstandigen Perithecien stets,

seltener auch der mittelstandigen flachkonvex vorragend;” Hohnel 1909, p. 341),

and this condition corresponds to our specimen. Although H. atroroseum

has been reported from Thailand (Suwannasai et al. 2005, Phosri et al. 2008),

it was described originally from the west of central Africa (Gabon), and all

specimens from Thailand might represent H. bogoriense, originally described

from Indonesia.

Annulohypoxylon chiangmaiense Lar.N. Vassiljeva, S.L. Stephenson & K.D. Hyde,

sp. Nov. Fics 3, 15c

MycoBAank MB 801393

Differs from Annulohypoxylon bahnphadengense by its larger ascospores and its citrine

to olivaceous buff KOH-extractable pigments

Type: Thailand, Chiang Mai Province, Mae Taeng District, Pha Deng village, Mushroom

Research Centre, on wood, 22 June 2011, L. Vasilyeva (Holotype, MFLU11-1167).

EryMoLoGcy: refers to the province where the fungus was collected.

STROMATA effused-pulvinate, with perithecial mounds 1/2 to 1/4 exposed,

surface at first brownish-olivaceous, then dark brown, with black granules

immediately beneath surface and citrine (13) to olivaceous buff (89) KOH-

extractable pigments. PERITHECIA spherical, 0.4-0.5 mm diam., ostioles

papillate, encircled with flat ostiolar discs 0.3-0.35 mm diam. Asct in the spore-

bearing portions 70-80 x 4.5-5 um, the stipes <60 um, with a tiny apical ring

bluing in Melzer’s iodine reagent. Ascospores brown, ellipsoid-inequilateral,

8.5-9.5 x 4-5 um, with a straight germ slit slightly shorter than the length of the

spore; perispore dehiscent in 10 % KOH.

Notes: Annulohypoxylon chiangmaiense is superficially quite similar to

A. bahnphadengense, also collected from the same site. However, the pigments

Annulohypoxylon & Ustulina spp. nov. (Thailand) ... 65

Fics. 1-3. Stromata: 1. Annulohypoxylon bahnphadengense. 2. A. bogoriense. 3. A. chiangmaiense.

Scale bars: 1, 2, 3a = 0.4 mm; 3b = 1.7 mm.

66 ... Vasilyeva, Stephenson & Hyde

of A. bahnphadengense are grayish sepia and its light-brown ascospores

(Fic. 15a) are 6.5-8.4 um long (7.5 um in average). It is also noteworthy that

young portions of the stromata in A. chiangmaiense are brownish-olivaceous.

Annulohypoxylon derelictum Lar.N. Vassiljeva, S.L. Stephenson & K.D. Hyde,

sp. nov. Fic. 4

MycoBAank MB 801394

Differs from other Annulohypoxylon species in lacking KOH-extractable pigments.

Type: Thailand, Chiang Mai Province, Muang District, T. Thasai, on dead branch

of Dipterocarpus (?), in a dipterocarp forest, 30 June 2011, L. Vasilyeva (Holotype,

MFLU11-1168).

ErymMo oey: from the Latin derelictus, meaning ‘abandoned’ or ‘neglected’.

STROMATA effused-pulvinate, with perithecial mounds 1/4 exposed or

inconspicuous, surface dull black, blackish granules immediately beneath

surface, without apparent KOH-extractable pigments. PERITHECIA spherical,

0.3-0.5 mm diam., ostioles papillate and encircled with a flat or slightly concave

disc 0.2-0.3 mm diam. Asci not observed. Ascosporss brown to light brown,

ellipsoid-inequilateral, 8-10.5 x 4.5-5.2 um, with a straight germ slit almost the

length of the spore; perispore dehiscent in 10% KOH.

Note: The most prominent feature of A. derelictum is the lack of KOH-

extractable pigments, a condition not previously recorded for Annulohypoxylon.

Annulohypoxylon dipterocarpi Lar.N. Vassiljeva, S.L. Stephenson & K.D. Hyde,

sp. nov. Figs 5, 15g

MycoBAnk MB801395

Differs from Annulohypoxylon purpureonitens by its smaller ostiolar discs and larger

ascospores.

Type: Thailand, Chiang Mai Province, Muang District, T. Thasai, on dead branch

of Dipterocarpus (?), in a dipterocarp forest, 30 June 2011, L. Vasilyeva (Holotype,

MFLU11-1169).

EryMo_oey: refers to the genus of the probable host plant, with the specimen collected

in an almost pure dipterocarp forest.

STROMATA effused-pulvinate, with perithecial mounds 1/2 to 1/4 exposed or

inconspicuous, surface black to the naked eye but brownish under direct light,

blackish granules immediately beneath the surface, KOH-extractable pigments

vinaceous purple (101). PERITHECIA spherical, 0.3-0.5 mm diam., ostioles

papillate and encircled with a small concave disc about 0.1-0.2 mm diam. AscI

70-80 x 5-6 um long in the spore-bearing portions, the stipes 30-35 um long,

with an apical part not bluing in Melzer’s iodine reagent. AscosporEs brown to

Annulohypoxylon & Ustulina spp. nov. (Thailand) ... 67

light brown, ellipsoid-inequilateral, (8—)9-11(-12) x 4.5-5 um, with a straight

germ slit the length of the spore; perispore dehiscent in 10% KOH.

Notes: Annulohypoxylon dipterocarpi has vinaceous purple KOH-extractable

pigments and is characterized by effused pulvinate stromata similar to A. purpureo-

nitens (Y.M. Ju & J.D. Rogers) Y.M. Ju et al., known only from Brazil and Mexico

(Ju & Rogers 1996); A. purpureonitens differs in having larger ostiolar discs

(0.2-0.5 mm diam.) and smaller ascospores (6.5-10 ttm). The lack of a positive

Melzer’s iodine reaction in the asci is also diagnostic for A. dipterocarpi. When

the perithecia are crashed in a mount, a mass of tiny ellipsoid spores 1.5-3

um long appears together with asci and ascospores, suggesting a conidial state

within the stromata but probably instead a hyperparasitic anamorphic fungus.

Annulohypoxylon maesaeense Lar.N. Vassiljeva, S.L. Stephenson & K.D. Hyde,

sp. nov. Fics 6a, 15h

MycoBank MB801396

Differs from Annulohypoxylon moriforme by its larger ascospores.

Type: Thailand, Chiang Mai Province, Mae Taeng district, near Mae Sae village, on

the bark of an unidentified tree, 23 June 2011, S. Stephenson & T. Ko Ko (Holotype,

MFLU11-1170).

EryMo.oey: from the Mae Sae village where the specimen was collected.

STROMATA glomerate, 3-5 mm diam., often confluent, with perithecial mounds

1/2 to 1/4 exposed, surface dark brown, blackish granules immediately beneath

surface, with pale olivaceous but mostly slightly hazel (88) tinted KOH-

extractable pigments. PERITHECIA spherical, <1 mm diam., ostioles papillate

and encircled with a concave disc <0.3-0.35 mm diam. Ascr not observed.

Ascospores brown, ellipsoid-inequilateral, (8—)10-12(-12.5) x 4.5-5.5 um,

with a straight or oblique germ slit extending the length of the spore or slightly

shorter; perispore dehiscent in 10% KOH.

Notes: Based on the key to taxa of Hypoxylon sect. Annulata (Ju & Rogers 1996),

of the species characterized by having ascospores with a dehiscent perispore,

ostiolar discs 0.2-0.5 mm diam, and dull brownish mature stromata), only two

resemble A. maesaeense—A. moriforme (Henn.) Y.M. Juet al. and A. truncatum

(Schwein.) Y.M. Ju et al., both also reported from Thailand (Whalley et al. 1995,

Suwannasai et al. 2002, Thienhirun et al. 2003, Phosri et al. 2008, Okane et al.

2008).

Annulohypoxylon moriforme, said to occur in the tropics and subtropics

of both hemispheres (Ju & Rogers 1996), is distinguished from A. truncatum

and A. maesaeense by its smaller ascospores (6-9 x 2.5-4 um). One variety,

A. moriforme var. microdiscum from Taiwan, has larger ascospores comparable

68 ... Vasilyeva, Stephenson & Hyde

Fics. 4-6. Stromata: 4. Annulohypoxylon derelictum. 5. A. dipterocarpi. 6a. A. maesaeense.

6b. A. truncatum on Quercus (from Ouachita Mountains, Arkansas, VLA P-2156).

Scale bars: 4, 5 = 0.4 mm; 6a = 0.8 mm; 6b = 1 mm.

Annulohypoxylon & Ustulina spp. nov. (Thailand) ... 69

in size to those of A. maesaeense, but differs by having ostiolar discs smaller

than 0.2 mm diam.

Annulohypoxylon truncatum (Fic. 6b) differs from A. maesaeense in

having greenish olivaceous (90) to dull green (70) KOH-extractable pigments.

Moreover, A. truncatum has been reported to inhabit Quercus in temperate

regions, but species records actually have been indicated only from Mexico

and the southeastern United States (North Carolina, Georgia, Louisiana,

Massachusetts, Pennsylvania; Ju & Rogers 1996) as well as from Tennessee

(Vasilyeva et al. 2007) and Arkansas and Texas (our unpublished data).

Annulohypoxylon truncatum has also been recorded (as Hypoxylon truncatum)

from Japan (Abe 1990, Abe & Doi 2000) and Korea (Velmurugan et al. 2007),

not unexpected given that an appreciable number of pyrenomycetous fungi

occur in both eastern North America and temperate regions of eastern Asia

(Vasilyeva & Stephenson 2010). However, the eastern Asian material of

A. truncatum needs to be reconsidered, in light of the redetermination of a

specimen identified as “A. truncatum” from the Russian Far East (Vasilyeva

1998) to A. orientale Lar.N. Vassiljeva & S.L. Stephenson (Vasilyeva et al. 2013).

Annulohypoxylon microbovei Lar.N. Vassiljeva, S.L. Stephenson & K.D. Hyde,

sp. nov. Fics 7, 15d

MycoBank MB801397

Differs from other species of the Annulohypoxylon bovei complex by its very small

ostiolar discs.

Type: Thailand, Chiang Mai Province, vicinity of Chiang Mai city, near Doi Suthep

temple, Huai Kok Ma, on dead branches of unidentified tree, 21 June 2011, L. Vasilyeva

(Holotype, MFLU11-1171).

EryMo_ocy: refers to the similarity of this fungus to the Annulohypoxylon bovei

complex, albeit having smaller ostiolar discs.

STROMATA glomerate or effused-pulvinate, mostly confluent, with perithecial

mounds 1/4 to 3/4 exposed, surface dark brown, blackish granules immediately

beneath surface, with citrine (13) to hazel (88) tinted KOH-extractable

pigments. PERITHECIA spherical, 0.5-0.7 mm diam., ostioles papillate and

encircled with a slightly concave or flat disc 0.2-0.3 mm diam. Asci not

observed. Ascospores brown, ellipsoid-inequilateral, 8-11 x 4.5-5.5 um, with

a straight germ slit spore length; perispore dehiscent in 10% KOH.

Notes: The stromata and ostiolar discs in A. microbovei are similar to those

in A. bovei (Speg.) Y.M. Ju et al., especially as illustrated by Miller (1961,

Figs 158, 159). Annulohypoxylon bovei var. microsporum, widely distributed in

southeastern Asia, is also known from China, Japan, Philippines, and Taiwan

70 ... Vasilyeva, Stephenson & Hyde

(Ju & Rogers 1996) and shares a number of features (including ascospore size)

with A. microbovei but differs in its larger (0.3-0.7 mm diam) ostiolar discs.

Annulohypoxylon morisuspectum Lar.N. Vassiljeva, S.L. Stephenson & K.D. Hyde,

sp. Nov. Fics 8, 15e

MycoBank MB801398

Differs from Annulohypoxylon moriforme by its flat ostiolar discs and its brownish

stromatal surface.

Type: Thailand, Chiang Mai Province, vicinity of Chiang Mai city, near Doi Suthep

temple, Huai Kok Ma, on dead branches of unidentified tree, 21 June 2011, L. Vasilyeva

(Holotype, MFLU11-1172).

ETYMOLOGY: as compared with A. moriforme, with which it shares a number of similar

features.

STROMATA glomerate, 1.5-2.5 mm diam., often confluent, with perithecial

mounds 1/2 to 1/4 exposed, surface dark brown, blackish granules immediately

beneath surface, with greenish to citrine (13) KOH-extractable pigments.

PERITHECIA spherical, 0.4-0.5 mm diam., ostioles finely papillate and encircled

with a flat disc 0.2-0.3 mm diam. Asci 60-70 x 4-5 um long in the spore-

bearing portions, the stipes 35-50 um long, with an apical ring bluing in

Melzer's iodine reagent, discoid, 1.5-2 um broad. Ascospores brown to light

brown, ellipsoid-inequilateral, 7.5-10 x 3-4.5 um, with a straight germ slit

slightly shorter than the length of the spore; perispore of at least some spores

dehiscent in 10% KOH, but rarely.

Notes: Annulohypoxylon morisuspectum resembles A. moriforme and

A. truncatum, which are distinguished by their stromata with concave ostiolar

discs of the truncatum-type; A. moriforme is further distinguished by the

olivaceous shades of its stromatal surface (Ju & Rogers 1996).

Annulohypoxylon neglectum Lar.N. Vassiljeva, S.L. Stephenson & K.D. Hyde,

sp. Nov. Figs 9, 15i

MycoBank MB801400

Differs from Annulohypoxylon purpureopigmentum by its smaller ascospores.

Type: Thailand, Chiang Mai Province, vicinity of Chiang Mai city, near Doi Suthep

temple, Huai Kok Ma, on dead branches of an unidentified tree, 21 June 2011,

L. Vasilyeva (Holotype, MFLU11-1173).

EryMoLoey: refers to the small and inconspicuous, easily overlooked stromata.

STROMATA small, glomerate, 1.5-2 mm diam., often confluent, with perithecial

mounds 1/3 to 1/4 exposed, surface dark brown, almost black to the naked

eye, with a purplish tint under the dissecting microscope, blackish granules

immediately beneath surface, with dilute bay (6) to purplish KOH-extractable

Annulohypoxylon & Ustulina spp. nov. (Thailand) ... 71

Figs. 7-9. Stromata: 7. Annulohypoxylon microbovei. 8. A. morisuspectum. 9. A. neglectum.

Scale bars: 7 = 0.5 mm; 8 = 0.6 mm; 9 = 0.7 mm.

72... Vasilyeva, Stephenson & Hyde

pigments. PERITHECIA spherical, 0.2-0.3 mm diam., ostioles rather coarsely

papillate and encircled with a concave disc 0.15-0.2 mm diam. Asci1 40-50 x

3.5-4 um long in the spore-bearing portions, the stipes 15-20 um long, with an

apical ring bluing in Melzer’s iodine reagent, discoid, about 0.5-0.7 um broad.

Ascosporss light brown, ellipsoid-inequilateral, 4-6.3 x 2.5-3 um, with a

straight germ slit, perispore dehiscent in 10% KOH

Notes: Despite its inconspicuous habit, A. neglectum possesses features

uncommonly encountered in Annulohypoxylon, and their combination

in the same taxon is noteworthy. Very small ascospores were reported

for A. atroroseum, A. ilanense and A. stygium, but these species differ from

A. neglectum by their widely effused stromata; in addition, A. ilanense differs

by its brick colored KOH-extractable pigments (Ju & Rogers 1999), and

A. atroroseum and A. stygium by their greenish olivaceous (90) to dull green

(70) pigments (Ju & Rogers 1996).

Purplish KOH-extractable pigments are present in A. purpureonitens and

A. urceolatum; the pigments in A. neglectum are similar but lack a vinaceous

tinge (101 or 116). Annulohypoxylon minutellum (Syd. & P. Syd.) Y.M. Ju et al.

[= Hypoxylon cohaerens var. microsporum J.D. Rogers & Cand.] has vinaceous

to rusty KOH-extractable pigments (Hsieh et al. 2005), but this species does

not have annulate ostioles. Annulohypoxylon purpureopigmentum Jad. Pereira

et al. (Pereira et al. 2010a, Fig. 16) is characterized by purple vinaceous KOH-

extractable pigments, but the ascospores (6.5-8(-10) um long) are larger than

those of A. neglectum. The appearance of glomerate stromata with large coarse

papillae that almost fill up the concave discs is typical for A. neglectum and

for A. purpureopigmentum, which seems the most similar western hemisphere

species to A. neglectum.

Annulohypoxylon paratruncatum Lar.N. Vassiljeva, S.L. Stephenson & K.D. Hyde,

sp. nov. Figs 10a, 15f

MycoBank MB801401

Differs from Annulohypoxylon truncatum by its smaller ostiolar discs and its citrine to

olivaceous-buff KOH-extractable pigments.

Type: Thailand, Chiang Mai Province, Mae Taeng District, Pha Deng village, Mushroom

Research Centre, on the bark, 19 June 2011, L. Vasilyeva (Holotype, MFLU11-1174).

EryMo_oey: refers to the close similarity of the new fungus to A. truncatum.

STROMATA glomerate to effused-pulvinate, with perithecial mounds 1/2 to 1/4

exposed, surface dull black to the naked eye, dark brown under the dissecting

microscope, blackish granules immediately beneath surface, with citrine (13)

to olivaceous-buff (89) KOH-extractable pigments. PERITHECIA spherical,

0.3-0.4 mm diam., ostioles papillate and encircled with a concave disc about 0.2

Annulohypoxylon & Ustulina spp. nov. (Thailand) ... 73

Fics. 10-11. Stromata: 10a. Annulohypoxylon paratruncatum.

10b. A. truncatum (from Ouachita Mountains, Arkansas, VLA P-2156). 11. A. planodiscum.

Scale bars: 10a = 1 mm; 10b = 0.9 mm; 11 = 1.3 mm.

(-0.25) mm diam. Asc1 60-80 x 4.5-5 um long in the spore-bearing portions,

the stipes 15-20 um long, with an apical ring bluing in Melzer’s iodine reagent,

discoid, about 0.5 um broad. Ascosporgs brown to light brown, ellipsoid-

inequilateral, 8-10.5 x 4-4.6 um, with a straight germ slit shorter than length

of the spore; perispore dehiscent in 10% KOH.

Notes: Images of stromata show that A. paratruncatum (Fic. 10a) and

A. truncatum (Fic. 10b) are superficially very similar; however, A. truncatum,

which appears to have much larger perithecia and ostiolar discs, also differs by

its greenish olivaceous (90) to dull green (70) KOH-extractable pigments.

74 ... Vasilyeva, Stephenson & Hyde

Annulohypoxylon planodiscum Lar.N. Vassiljeva, S.L. Stephenson & K.D. Hyde,

sp. nov. Figs 11, 15)

MycoBank MB801402

Differs from Annulohypoxylon elevatidiscum by its glomerate stromata and flat ostiolar

discs.

Type: Thailand, Chiang Mai Province, Muang District, Doi Pui Mountain, on the trunk

of an unidentified tree, 30 June 2011, L. Vasilyeva (Holotype, MFLU11-1175).

EryMo_oey: refers to the mostly flat ostiolar discs.

STROMATA glomerate (seemingly effused-pulvinate when confluent), with

perithecial mounds 1/2 to 1/4 exposed, surface dull black (sometimes overlaid

with a white substance, blackish granules immediately beneath the surface),

with greenish-olivaceous (90) KOH-extractable pigments. PERITHECIA

spherical, 0.3-0.4 mm diam., ostioles papillate and encircled with a flat disc

0.2-0.3 mm diam. Asci 60-80 x 4.5-5 um in the spore-bearing portions, the

stipes 15-20 um long, with an apical ring bluing in Melzer’s iodine reagent,

discoid, about 0.5 um broad. Ascospores brown to light brown, ellipsoid-

inequilateral, 9-11.5 x 4-5 um, with a straight germ slit spore extending the

length of the convex side of the spore; perispore dehiscent in 10% KOH.

Notes: The combination of several key characters in the type of

A. planodiscum—greenish olivaceous KOH-extractable pigments, a 0.2-0.3 mm

diam ostiolar disc, a straight germ slit on the convex side of the ascospores, and

a perispore dehiscent in 10% KOH—are also found in A. elevatidiscum (Ju et al.

2004), which differs by its slightly larger ascospores and regularly effused-

pulvinate stromata. Although some A. planodiscum discs appear to be slightly

convex, the majority are flat, with its stromata initially glomerate and only later

confluent, like those of A. truncatum.

Annulohypoxylon pseudonitens Lar.N. Vassiljeva, S.L. Stephenson & K.D. Hyde,

sp. nov. Fics 12, 15l

MycoBAnk MB801403

Differs from Annulohypoxylon nitens by its smaller ascospores and its smaller ostiolar

discs.

Type: Thailand, Chiang Mai Province, vicinity of Chiang Mai city, near Doi Suthep

temple, Huai Kok Ma, on dead branches of an unidentified tree, 21 June 2011, L.

Vasilyeva (Holotype, MFLU11-1176).

EryMo_oey: refers to the similarity of this species with Annulohypoxylon nitens in a

number of features.

STROMATA effused-pulvinate, with perithecial mounds 1/2 to 1/4 exposed,

surface dull to shiny black in some parts, blackish granules immediately

beneath the surface, with citrine (13) KOH-extractable pigments. PERITHECIA

Annulohypoxylon & Ustulina spp. nov. (Thailand) ... 75

spherical, 0.3-0.5 mm diam., ostioles papillate and encircled with a concave

disc about 0.2 mm diam. Asci 60-65 x 4-4.5 um in the spore-bearing part,

stalks 20-25 um long, with an apical ring bluing in Melzer’s iodine reagent,

discoid, about 0.5 um broad. Ascosporgs brown to light brown, ellipsoid-

inequilateral, 5-7.5 x 2.5-3 um, with a straight or slightly oblique germ slit

shorter than the length of the spore; perispore dehiscent in 10% KOH.

Notes: Annulohypoxylon pseudonitens resembles A. nitens in having shiny

black mature stromata, somewhat exposed perithecial mounds, smaller

ascospores, and greenish tinged KOH-extractable pigments. However, the

ascospore length in A. pseudonitens does not exceed 7.5 um, overlapping

with the lower range cited for A. nitens (6.5-10(-11) um long; Ju & Rogers

1996). As A. nitens has been reported previously from Thailand (Suwannasai

et al. 2002, 2005, Thienhirun et al. 2003, Okane et al. 2008, Phosri et al. 2008),

we first assigned our specimen to A. nitens. However, the type specimen of

A. nitens [= Rosellinia nitens Ces.], treated as a synonym of Hypoxylon truncatum

(Schwein.) J. Mill. (Miller 1961), is cited as having 8-12 um long ascospores (cf.

Saccardo 1882, Miller 1961). In addition Hladki & Romero (2009) cite longer

(8-9 um) ascospores for a specimen of A. nitens from Argentina. The material

of A. nitens from China and Philippines, identified previously as Hypoxylon

bovei var. microsporum, also has 8-10 um long ascospores (Miller 1961, Ju &

Rogers 1996), exceeding the spore length of A. pseudonitens.

Annulohypoxylon bovei and its variety differ from A. pseudonitens by their

mainly glomerate stromata.

Annulohypoxylon sordidum Lar.N. Vassiljeva, S.L. Stephenson & K.D. Hyde,

sp. nov. Figs 13, 15k

MycoBank MB801404

Differs from Annulohypoxylon moriforme by its effuse stromata and pale fawn KOH-

extractable pigments.

Type: Thailand, Chiang Mai Province, Mae Taeng District, Pha Deng village, Mushroom

Research Centre, on wood, 20 June 2011, L. Vasilyeva (Holotype, MFLU11-1177).

EryMo_ocy: derived from the Latin sordidus, meaning ‘dirty-looking’

STROMATA effused-pulvinate, with perithecial mounds 1/2 to 1/4 exposed,

surface dull, dark brown, blackish granules immediately beneath surface, with

dilute fawn (87) KOH-extractable pigments. PERITHECIA spherical, 0.3-0.4

mm diam., ostioles papillate and encircled with a tiny concave disc up to 0.1

mm diam. Asci 80-85 x 4.5-5 um in the spore-bearing part, stalks 15-20 um

long, with an apical ring bluing in Melzer’s reagent, discoid, about 0.5-0.7 um

broad. Ascosporss brown to light brown, ellipsoid-inequilateral, 9-11(-12) x

76 ... Vasilyeva, Stephenson & Hyde

Fics. 12-14. Stromata: 12. Annulohypoxylon pseudonitens. 13. A. sordidum. 14. A. urceolatum.

Scale bars: 12, 13 = 0.6 mm; 14= 1.4mm.

4-5.5 um, with a straight germ slit slightly shorter than the length of the spore;

perispore infrequently dehiscent in 10% KOH.

Notes: Annulohypoxylon sordidum has very small ostiolar discs similar to

those of A. moriforme var. microdiscum, but both A. moriforme varieties are

characterized by basically glomerate and blackish stromata with olivaceous

shades, as well as greenish olivaceous (90) to dull green (70) KOH-extractable

pigments.

Annulohypoxylon & Ustulina spp. nov. (Thailand) ... 77

The stroma of A. sordidum illustrated in Fic. 13 is somewhat similar to that

of A. pyriforme (Y.M. Ju & J.D. Rogers) Y.M. Ju et al. Ju & Rogers 1996,

Fig. 14G) in having rather prominent perithecial mounds and small ostiolar

discs. Annulohypoxylon pyriforme differs in its slightly smaller (8-10 x 3.5-4

um) pyriform ascospores, a perispore that is always indehiscent in 10% KOH,

and dull green to dark green KOH-extractable pigments. Annulohypoxylon

sordidum may represent a western hemisphere counterpart of A. pyriforme,

known only from Venezuela (Cerro de la Neblina).

Annulohypoxylon urceolatum (Rehm) Y.M. Ju, J.D. Rogers & H.M. Hsieh,

Mycologia 97: 861. 2005. Fic. 14

SPECIMEN EXAMINED: Thailand, Chiang Mai Province, Muang District, Doi Pui

Mountain, on the branch of an unidentified tree, 30 June 2011, L. Vasilyeva (Holotype,

MFLU11-1178).

Notes: Annulohypoxylon urceolatum is described in Ju & Rogers (1996, as

Hypoxylon urceolatum). As indicated earlier, this species has been found only

in southeastern Asia, but our record is the first for Thailand. We observed all

the main features of this species such as the vinaceous purple KOH-extractable

pigments, ascospores that are 9-14 x 3.5-4.5 um, and a perispore that in our

specimen is indehiscent in 10% KOH.

Key to the species of Annulohypoxylon considered in this paper

(New species indicated in bold font)

1. Stromata without apparent KOH-extractable pigments, effused-pulvinate;

ASCOSPOKES BLO SMT OTS 6 he Fel: a oe Rete sate acto = Sette Wi oo Be A. derelictum

if Sthoniata with KO? excteactaDle PICIMeMtS oe PON el PEE LEN bela on ae aap ow et 2

2. KOH-extractable pigments grayish-sepia, stromata widely effused,

ascospores 6.5-8.4 um long ............. eee eee eee eee A. bahnphadengense

2: KOH extractable proments OF Otnek COLORS 4:9. 088 Go neath Ge in td Gosase WE in ane Een Eos 3

3; KOH extractable pisments purplish-tinged:! 6 roc ct eat iiees files foe oe oie ee C0 4

sekO-extractable pisinients: OF Other COlGtS OP sr su a gS yo SRE ce SL ATEES go a Stal on al naval 8

He SUL ORAALACLOMVEL ALC set oir Met att Met oe Reon con ana cate AN ge ee eg Re gh ‘)

A, SUPOMIALa-WAGELY CH USE Ps. faye 4 fags Fe fouge Be age re Touge ne Meages ee Pgeeeean ee eye lea 6

5s ASCOSPOLES’4=G-S-LIONG 5. Sx eosin tg Wert KA ge xe ne een eA ge A. neglectum

5: ASCOSPOLeS 6:58, MONS et Ane et Let Lat Le A. purpureopigmentum

Gz ASCOSPORES B12 (DON Ge at. 5 Saeed a ake cages aongsa ke ngs ch eg hab vec opNcob abe ogtet alte cAba tes 7

GAScospokes Up toa Lent lon. ane tater. Neale s bees hme 0 Bel A. urceolatum

7, OstidlamdiscsQsd 0-2 mint dain 93.2 ecko ead rpeeail hated: oeeetades io A. dipterocarpi

7. Ostiolar discs larger, 0.2-0.5 mm diam ....................004. A. purpureonitens

78 ... Vasilyeva, Stephenson & Hyde

8. KOH-extractable pigments greenish-olivaceous, dull green, citrine,

OLTACE OUST DUTT, AVA ZC Bante Meehan mtn an ge he ot rte Naoto tas Fee i]

8. KOH-extractable pigments brick colored ........... 0.0... cee e ee eee A. ilanense

9. Stromata glomerate, often confluent and then becoming effused-pulvinate ...... 10

OU Stromatatataneewidely etruseds Mott Mache ide hort tari pa heed preparer dees prs 18

LOL Osticlatedisceotthe Dovel type A An clon eaten tees hehe ti 11

10aOstiolamdisesof the trunentuncty pe tc tte tie otto fets Blane Beds Pie 13

PlvOstiolardises0:2-0.3- mire AVA oie nt ied nck ate asthe gts als nee eyes eye eee 12

11. Ostiolar discs 0.3-0.7 mm diam..............--.-006- A. bovei var. microsporum

12. Ascospores narrower (3-4.5 um), perispore almost indehiscent in 10% KOH

ail east as stalliys WS pea a We ocd a bh Frida bh Sri a ths Sradtya bh Sraet ath Srsehaa be bend A. morisuspectum

12. Ascospores wider (4.5-5.5 um), perispore dehiscent in 10% KOH ... A. microbovei

TS Ascospares G7 (9): party Loin sat Se ach nike po nto dh je nce eo sw BE Ee ne A. moriforme

Le Ascospores BD UMS 225 ob Sty ee Stk hae eee eee cinta clam ee ties bins 14

14. Ostiolar discs do not exceed 0.2 mm diam ........ 0... eee eee 15

I Ostiolardises:G:2-O-4 wir tatty ur oe ie nareen Moke Bele; ads «ky oe oly op Soke fh Bok 16

15. KOH-extractable pigments citrine to olivaceous-buff,

ASCOSPOres Sa LOS OG x dase a pak evn Eps ods Pas rte pease ep A. paratruncatum

15. KOH-extractable pigments greenish olivaceous to dull green,

ascospores 10-12 (nrlone Ph ale adie lie steels A. moriforme var. microdiscum

16. KOH-extractable pigments close to hazel tint ................... A. maesaeense

16. KOH-extractable pigments greenish-olivaceous to dull green ................ 17

PCISLIO ATA ISES CO MCAV C4 a8 Gite ner a Sasa tantt battnrtt harem ins ean ss ase A. truncatum

V-Ostiolar discs flat s+. f24.0203 es ex ck eee heey ncee eens ees A. planodiscum

18; Ostiolar dises:of: Dover Pe se < ccenis ena oe asin weet ane e Late oes Voom ee Heil A. nitens

T8yQistlolardises- Ob GU MCAHMIAY Pe? oxo. Bantex Hodes ea Boies enn Bade ex Brdes 7% Donkey rh Sande 76 aa 19

19: -Ostiolaidiscs up to:0Ml tim diame tan San onde ee A. sordidum

POF OstiGlar discelaroer Jae hoy ae ot oe A aN SAY hOB SO eT Gh EN 20

DO. Ostiolam discs Derr. hiatal e 5a! Payer a Pucca Se PS cee Pm Pca Re ES A. pyriforme

207 @OSHOlab aise SOE TAAL ADT Se oy ox bs Psi a th esa bh Freya hy Prelate beagle a bh bred ab droetea eS 21

il. ASecospores o=7 [lid LONG, fas -acex hae eee kee ete lage emg ete Relay eens 22

21. Ascospores 8.5-9.5 um long ...... eee eee ee eee eee A. chiangmaiense

22. Stromatal surface pinkish between black ostioles ...................2 200 ee 23

22s Stromatal surfacesuniformly: Dae «cise glee namo nee ena see EY ne els Moet Y Hil 24

23. KOH-extractable pigments citrine to olivaceous-buff, ostiolar discs slightly concave

OF alMOst Hat xf x5 eh ma ease a ely elo 8 Teh efat tod eeu oY Pog Pe aay 8 A. bogoriense

23. KOH-extractable pigments greenish olivaceous to dull green, ostiolar discs

COMSPICUOUSIY SUNKEN: 9 A oer aa! hie ah AAT oe UR US A. atroroseum

Annulohypoxylon & Ustulina spp. nov. (Thailand) ... 79

Fic. 15. Ascospores: a. Annulohypoxylon bahnphadengense. b. A. bogoriense. c. A. chiangmaiense.

d. A. microbovei. e. A. morisuspectum. f. A. paratruncatum. g. A. dipterocarpi. h. A. maesaeense.

i. A. neglectum. j. A. planodiscum. k. A. sordidum. |. A. pseudonitens. Scale bars: a = 7.5 um;

b-d, f, i= 4 um; e = 3 um; g, h, j-1= 5 pm.

24, KOFI-extractable pigments citrine. 1s iva Zkeweibb-sshbest boars A. pseudonitens

24. KOH-extractable pigments greenish olivaceous to dull green ......... A. stygium

80 ... Vasilyeva, Stephenson & Hyde

Ustulina pseudozonata Lar.N. Vassiljeva, S.L. Stephenson & K.D. Hyde,

Sp. Nov. Fic. 16

MycoBAnk MB801405

Differs from Ustulina zonata by its larger ascospores and light-brownish stromata; and

from U. deusta by its wider ascospores, longer germ slit, and light-brownish stromata.

Type: Thailand, Chiang Mai Province, Mae Taeng district, near Mae Sae village, on the

bark of an unidentified tree, 23 June 2011, L. Vasilyeva (Holotype, MFLU11-1179).

EryMoLocy: refers to some similarity with Ustulina zonata.

STROMATA effused-pulvinate with the appearance of a flat elongated cake,

2.5-5 x 1.5-2.5 cm, light-brownish, with wavy and greyish margins, surface

undulate, irregularly cracked, and very finely papillate, ostioles almost

umbilicate. PERITHECIA tubular, 1-1.2 mm long, 0.4-0.6 mm wide. Ascr in the

spore-bearing portions 180-200 x 12-14 um, stipes up to 200 um long, with

an apical ring bluing in Melzer’s iodine reagent, urn-shaped, 7-7.5 x 3-4 um.

Ascospores brown, unicellular, fusoid-inequilateral, smooth, (27.5-)30-35

(-37.5) x 8-10(-12) um, with a straight germ slit extending almost the length

of the spore.

Notes: The name Ustulina is currently treated as a synonym of Kretzschmaria

(Martin 1970, Leess@e 1994, Rogers & Ju 1998), but within Kretzschmaria sensu

lato two taxonomic complexes can be clearly distinguished, the kretzschmarioid

complex and the ustulinoid complex (Rogers & Ju 1998). Both complexes were

accepted within a single genus based primarily on similar cultural features

(Rogers & Ju 1998), but many other groups of xylariaceous genera (e.g.,

Annulohypoxylon, Daldinia, and Hypoxylon in one instance and Entoleuca,

Nemania, and Rosellinia in another) known to share cultural features are

nevertheless accepted as independent taxonomic entities (Stadler 2011).

We believe that Ustulina deserves recognition as a separate genus; among

the ustulinoid taxonomic complex, the most closely related species to

U. pseudozonata are U. deusta (Hoffm.) Lind and U. zonata (Lév.) Sacc. Rogers

& Ju (1998) distinguish these two species in their key provided based on

U. deusta having ascospores 27-35(-38) x 7-9 um, a germ slit much shorter

than the spore length, and a northern temperate distribution vs. U. zonata

having ascospores 21-34 x 8.5-12 um, a germ slit that is either the length of the

spore or nearly so, and a subtropical and tropical distribution. The specimen

from northern Thailand has the range in ascospores length characteristic of

U. deusta but a germ slit that fits U. zonata, and this combination is enough to

characterize it as a separate species.

Ustulina zonata, described from Java, has been mentioned repeatedly as a

pathogen that causes root decay of trees in southeastern Asia (Brooks 1915,

Annulohypoxylon & Ustulina spp. nov. (Thailand) ... 81

Fic. 16. Ustulina pseudozonata. a. Stromata (photo by Randy Darrah). b. Ascus top with an apical

apparatus and an ascospore. c. Ascospores. Scale bars: a = 7 mm; b = 7 um; c = 8 um.

Petch 1928, Nandris et al. 1987, Muraleedharan & Baby 2007, Barthakur 2011).

The only color illustration found on the Internet (http://www.ava.gov.sg) shows

U. zonata as having carbonaceous fruit bodies hidden among grass and roots.

The common name of the disease (charcoal stump root) seems to emphasize the

black color of the stromata, although Petch (1928, p. 241) wrote that stromata

usually referred to as representing U. zonata are “fairly frequently reddish

on the surface, wholly or in part, but the majority of the specimens are, until

82 ... Vasilyeva, Stephenson & Hyde

weathered, grey or purple-grey; when weathered, they are, of course, black?

He was unable to locate Léveillé’s type of U. zonata but found Léveillé’s specimen

of “U. deusta’, which did not have zones on the fragment of a stroma, but had a

reddish tinge. According to Petch, Léveillé distinguished Ustulina zonata from

U. vulgaris (= U. deusta) by its reddish color and zoned surface. None of these

features is mentioned by Rogers & Ju (1998), who indicate only that U. zonata

has stromata, perithecia, ostioles, and asci similar to those found in U. deusta.

In our specimen of U. pseudozonata from Thailand, the fully mature stromata

are light brownish or sand-colored. ‘This is the color reported for the maturing

stromata in Kretzschmaria milleri J.D. Rogers & Y.M. Ju from the Caribbean

Basin (French Guiana, Guyana, Mexico, Venezuela).

“Ustulina zonata” from southeastern Asia might represent a species

complex waiting for separation into several separate taxa. Interestingly, almost

all specimens listed under the description of U. zonata by Rogers & Ju (1998)

are from the Caribbean Basin (Cuba, French Guiana, Guadeloupe, Mexico,

Puerto Rico). Only one specimen from Taiwan was cited (Rogers & Ju 1998) for

U. zonata, which differs from U. pseudozonata by its ascospores with pinched

ends. Among the synonyms Rogers & Ju (1998) give for Kretzschmaria zonata is

Hypoxylon deustum f. madagascariensis (Henn.) Hendr., which Dennis (1963)

reported as having ascospores 20-28 x 7-10 um and which Rogers & Ju noted

very likely represented Kretzschmaria zonata. The specimens from Thailand

and Madagascar are not conspecific, primarily due to the spore size differences

that ostensibly demonstrate the heterogeneity of the Ustulina zonata ‘complex:

Given the economic importance of U. zonata as a pathogen of many trees and

shrubs, further taxonomic investigations are warranted.

Discussion

In this paper, we have increased the number of examples of closely

related species known from the Caribbean and Indo-Malayan centers of

biodiversity (cf. Vasilyeva et al. 2012). As noted in earlier treatments of species

from temperate latitudes of northeastern Asia and eastern North America

(Vasilyeva & Stephenson 2010, 2011), the morphological differences between

species in the western and eastern hemispheres are rather insignificant and

sometimes pertinent to only a single character (e.g., a difference in ascospore

size, of anamorphs, or in stromatal pigments). This enigma currently has no

biogeographic explanation but does demand an accumulation of relevant

examples to solve.

Specimens of six different Annulohypoxylon taxa (as Hypoxylon bovei

var. microsporum, H. moriforme, H. nitens, H. purpureonitens, H. stygium,

H. stygium var. annulatum) reported from Thailand by Suwannasai et al. (2002)

Annulohypoxylon & Ustulina spp. nov. (Thailand) ... 83

were said to differ from taxa described previously by Ju & Rogers (1996) due

to a high level of morphological variation in the species involved. However,

the differences might as easily be explained by the completely different species

composition of the fungal assemblage found in Thailand. In fact, morphological

variation within Hypoxylon and Annulohypoxylon is not very high, as both the

same characters (stromatal pigments and shapes, dehiscent or indehiscent

perispore, etc.) and the same ranges of variation (e.g., size of ascospores) repeat

themselves infinitely (cf. Vasilyeva & Stephenson 2010) while combining

differently with one another other for a particular taxon. Diversity lies not

in morphological variation per se, but rather in the combinations of features

characterizing different species.

Acknowledgments

The research reported herein was funded in part by a grant (OISE-1042602) from the

National Science Foundation to the University of Arkansas. The support provided by the

Global Research Network for Fungal Biology and King Saud University is also gratefully

acknowledged. Thanks are extended to Randy Darrah for providing the image used

for Figure 16a. We are also thankful to Liliane Petrini (Breganzona, Switzerland) and

Katherine Winsett (North Carolina State University, Raleigh, USA) for the thorough

reading of our manuscript and their valuable comments.

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Vasilyeva LN, Stephenson SL, Hyde KD, Bahkali AH. 2012. Some stromatic pyrenomycetous fungi

from northern Thailand - 1. Biscogniauxia, Camillea and Hypoxylon (Xylariaceae). Fungal

Diversity 55: 65-76. http://dx.doi.org/10.1007/s13225-011-0150-9

Vasilyeva LN, Ma HX, Stephenson SL. 2014 [“2013”]. Biogeographical patterns in pyrenomycetous

fungi and their taxonomy. 3. The area around the Sea of Japan. Mycotaxon 126: 1-14.

http://dx.doi.org/10.5248/126.1

Velmurugan N, Han SS, Sa DM, Lee YS. 2007. Consideration of Hypoxylon truncatum, based on

morphological characteristics of Korean collection. Korean Journal of Electron Microscopy

37: 281-287;

Whalley AJS. 1985. The Xylariaceae: some ecological considerations. Sydowia 38: 369-382.

Whalley AJS. 1996. The xylariaceous way of life. Mycological Research 100: 897-922.

http://dx.doi.org/10.1016/S0953-7562(96)80042-6

Whalley MA, Whalley AJS. 2007. Ascomycota: Xylariales. 95-115, in: EBG Jones et al. (eds).

Malaysian Fungal Diversity. Mushroom Research Center, Malaysia.

Whalley AJS, Hywel-Jones NL, Jones EBG, Whalley MA. 1995. A preliminary account of the

genera Biscogniauxia and Hypoxylon in the Chanthaburi and Chon Buri Provinces of South

East Thailand. Sydowia 47: 70-81.

MY COTAXON

January-March 2016—Volume 131, pp. 87-94

http://dx.doi.org/10.5248/131.87

Taxonomic re-evaluation and phylogenetic position of

Hemibeltrania cinnamomi within Xylariales

KUNHIRAMAN C. RAJESHKUMAR’, SAYALI D. MARATHE!,

KARADKA MADHUSUDHANAN? & RAFAEL F. CASTANEDA-RUvIZ?

‘National Fungal Culture Collection of India, Biodiversity and Palaeobiology Group,

Agharkar Research Institute, G.G. Agarkar Road, Pune, India

*Department of Botany, St. Alberts College, Cochin 18, Ernakulum, Kerala, India

?Instituto de Investigaciones Fundamentales en Agricultura Tropical ‘Alejandro de Humboldt’

(INIFAT), Académico Titular de la Academia de Ciencias de Cuba,

Calle 1 Esq. 2, Santiago de Las Vegas, C. Habana, Cuba, C.P. 17200

*CORRESPONDENCE TO: rajeshfungi@gmail.com

ABSTRACT — Hemibeltrania cinnamomi associated with leaf blotch disease of Cinnamomum

malabatrum (Lauraceae) is described and illustrated. Phylogenetic LSU and ITS sequence

analyses support the fungus as a species of Hemibeltrania associated with other Beltrania-like

genera. A synoptic table of Hemibeltrania species is provided.

KEY worps — asexual Ascomycetes, plant pathogen

Introduction

When revisiting Beltrania and related genera, Pirozynski (1963) introduced

the genus Hemibeltrania (typified by H. cinnamomi) for a causal fungus of

cinnamon leaf spot in Sierra Leone characterized by conidia described as

ellipsoid to obovate and 12-16 x 10-12 um. The phylogenetic placement of

Hemibeltrania is unknown.

In December 2014, a survey was conducted to investigate microfungal

diversity in the natural forests of Kaladi and the surrounding area within

the southern Western Ghats, India. An unusual species of Hemibeltrania

was discovered, which causes foliar blotches in Cinnamomum malabatrum.

The present study aims to authenticate the species of Hemibeltrania using

morphological characters and resolve the phylogenetic position of the genus

using ITS and LSU sequence data.

88 ... Rajeshkumar & al.

Materials & methods

ISOLATES AND MORPHOLOGY—Conidia were directly isolated from the surface of

a leaf spot and observed under a Nikon stereomicroscope (Model SMZ-1500 with

Digi-CAM). Single conidial cultures were established on 2% Potato Dextrose Agar (PDA)

and 2% Malt Extract Agar (MEA) plates (Crous et al. 2009). For morphotaxonomic

studies and photomicrographs a Carl Zeiss Image Analyzer 2 microscope was used.

Conidia and conidiophores were mounted in lactic acid cotton blue and measured using

AxioVision REL.4.8 software, with 30 observations per structure. Colony characteristics

in culture were studied on MEA and PDA (Crous et al. 2009). Herbarium specimens

were deposited in Ajrekar Mycological Herbarium (AMH 9667), and the culture

was accessioned and preserved in the National Fungal Culture Collection of India

(NFCCI-3695), Agharkar Research Institute, Pune, India.

DNA EXTRACTION, AMPLIFICATION, AND PHYLOGENETIC ANALYSES—Colonies of

H. cinnamomi were grown on MEA plates, and genomic DNA was extracted following

the rapid salt-extraction method of Aljanabi & Martinez (1997). Fragments containing

the region encoding the 28S nrDNA (LSU) and ITS 1-5.88 nrDNA-ITS 2 (ITS) were

amplified using primer pairs LROR (Rehner & Samuels 1994) and LR7 (Vilgalys &

Hester 1990) for LSU and primer pairs ITS4 and ITS5 (White et al. 1990) for ITS. PCR

conditions, sequence alignments, and phylogenetic analyses followed the protocols

of Crous et al. (2006). Thirty LSU sequences of the order Xylariales and an outgroup

of two sequences (Trichoderma and Lulworthia) were downloaded from GenBank.

Twenty-seven ITS sequences of the order Xylariales and an outgroup of two sequences

(Trichoderma and Lulworthia) were downloaded also for phylogenetic analyses. The ITS

and LSU regions were analysed by the Maximum Parsimony method with bootstrap

(BS) analysis using 1000 replications in MEGA6 (Molecular Evolutionary Genetics

Analysis version 6.0). Sequence data were deposited in GenBank. Family placement of

the sequences follows Shirouzu et al. (2010) and Hidayat et al. (2014).

Taxonomy

Hemibeltrania cinnamomi (Deighton) Piroz., Mycol. Pap. 90: 32 (1963) Fis 1, 2

SYMPTOMS: necrotic, reddish brown to brick brown, amphigenous, irregular.

ON THE NATURAL SUBSTRATE: CONIDIOPHORES distinct, single or slightly

branched toward the apex, erect, straight or flexuous, cylindrical, slightly

sinuate, 4—8-septate, smooth, dark brown at the base, pale brown toward the

apex, thin-walled, 130-250 x 3.5-6 um, arising from a radially lobed basal cell,

10-11.5 um diam. CONIDIOGENOUS CELLS mono- and polyblastic, integrated

or discrete, terminal, determinate or sympodial extended, denticulate;

denticles cylindrical. Conidial secession schizolytic. Conip1a solitary,

limoniform, broadly ellipsoidal or obovate to irregular, rounded or obtuse at

the apex, sometimes attenuate, subulate or rounded at the base with a small frill

at the base, acrogenous becoming acropleurogenous after several sympodial

extensions, unicellular, smooth, 12-25 x 8-13 um, hyaline, subhyaline to pale

olivaceous.

Hemibeltrania cinnamomi reconsidered ... 89

Fic. 1. Hemibeltrania cinnamomi (AMH 9667).

a-f: Conidiophore branching. g-l: Denticulate conidiogenous cells. m-o: Conidiophore bases.

Bars: a-f = 50 um; g-o = 10 um.

90 ... Rajeshkumar & al.

Fic. 2. Hemibeltrania cinnamomi (AMH 9667).

a-j: Conidia. k-n: Conidial attachments. Bars: 10 um.

Colonies on MEA slow to moderate growing, 35-45 mm diam after 7 days,

grayish or mouse gray, later turning dark gray, velutinous, margin irregular,

reverse blackish to blackish blue. Conidia 12-17 x 9.5-12 um.

SPECIMEN EXAMINED: INDIA. KALApDI: Western Ghats, Kerala, 10°16’N 76°43’E, on

leaves of Cinnamomum malabatrum (Burm. f.) J. Pres! (Lauraceae) December 2014,

K.C. Rajeshkumar & K. Madhusudhanan (AMH 9667; culture NFCCI 3695; Genbank

KT119564, KT119565).

Phylogenetic analyses

LSU (KT119565): based on MegaBLAST search of GenBank LSU sequences,

the closest hits are: Xylaria sp. (JQ862647.1, identities = 96%, gaps 0.2%);

Hemibeltrania cinnamomi reconsidered ... 91

97 EU552155.1| Sarcostroma bisetulatum CBS:122695

83 — DQ278924.1| Sarcostroma restionis strain CBS 118154

96 JN871212.1| Seimatosporium eucalypti strain CPC 159

AB593733.1| Seimatosporium elegans

73 AB593740.1| Seimatosporium mariae

KC005809.1| Seiridium phylicae CPC:19965

36 AF382370.1| Monochaetia monochaeta strain CBS 191.82

57 33 AF382371.1| Monochaetia karstenii

AB593723.1| Immersidiscosia eucalypti

KF251708.1| Phlogicylindrium eucalyptorum strain CBS 111689

— AY346259.1| Apiospora setosa

55 DQ471018.1| Apiospora montagnei

= KF144961.4| Arthrinium rasikravindrii Apiosporaceae

86 AB220350.1| Arthrinium phaeospermum

95 AB220348.1| Arthrinium sacchari

EU040241.1| Subramaniomyces fusisaprophyticus strain CBS 418.95

DQ810233.1| Pseudomassaria carolinensis voucher 9502

KJ869183.1| Beltraniopsis neolitseae strain CPC 22168

2 KJ869185.1| Beltraniella endiandrae strain CPC 22193

AB496426.1|Beltraniella botryospora strain: TMQa1A18

KT119565.1| Hemibeltrania cinnamomi NFCCI 3695

99 AB496423.1| Beltrania rhombica strain: 10353

7 KJ869215.1| Beltrania pseudorhombica strain CPC 23656

EU040235.1| Parapleurotheciopsis inaequiseptata strain MUCL 41089 “]Incetae sedis

Amphisphaeriaceae

Beltrania clade

AY083834.1| Hyponectria buxi “]Hyponectriaceae

99 JQ746546.1| Diatrype disciformis ‘

65 1DQ836903.1| Eutypa lata ] mi acs

DQ836306.1| Graphostroma platystoma _] Graphostromataceae

55 EU552100.1| Anthostomella leucospermi ] Xylariaceae

60 AY327480.1| Xylaria hypoxylon strain ATCC 42768

AF452043.1| Clypeosphaeria phillyreae “] Clypeosphaeriaceae

JN941453.1| Trichoderma gelatinosum strain NBRC 104900 }Hypocreales orcrou

58 DQ522856.1| Lulworthia grandispora isolate AFTOL-ID 424 ‘Jluworthiales

Fic. 3. LSU phylogenetic tree generated from Maximum Parsimony (MP) analyses of the aligned

sequences of Hemibeltrania cinnamomi and allied taxa of the order Xylariales, with Trichoderma

and Lulworthia as outgroup. Percentage bootstrap support (250%) is shown on the branches.

The MP tree was obtained using the Subtree-Pruning-Regrafting algorithm with search level 1

in which the initial trees were obtained with the random addition of sequences (10 replicates).

The bootstrap test was conducted with 1000 replicates.

Xylaria sp. (JQ862650.1, identities = 96%, gaps = 0.2%); and Xylaria badia

(JQ862643.1, identities = 96%, gaps = 0.2%).

ITS (KT119564): A MegaBLAST of the ITS sequence showed that the closest

hits are: Hemibeltrania sp. CL12WA (JQ621881.1, identities = 99%, gaps 0.4%);

Beltraniella endiandrae CPC 22193 (KJ869128.1, identities = 93%, gaps = 3%);

and Beltrania pseudorhombica CPC 23656 (KJ869158.1, identities = 92%,

gaps = 4%).

The ITS and LSU phylogenetic analyses supported the genus Hemibeltrania

as closely related to Beltrania, Beltraniopsis, Beltraniella, and Subramaniomyces

and forming a unique monophyletic lineage in the Xylariales and closely allied

to Amphisphaeriaceae and/or Apiosporaceae (Fics 3, 4).

92 ... Rajeshkumar & al.

GU905994.1| Beltrania querna BCRC 34620

KM35731 7.1| Beltrania rhombica

KJ869158.1| Beltrania pseudorhombica CPC 23656

GU797390.1| Betrania rhombica

KJ869128.1| Beltraniella endiandrae strain CPC 22193

GU9059 93. 1| Beltraniella portoricensis BCRC 34690

KJ512150.1| Bekraniella portoricensis Beltrania clade

EF029240.1| Beltrania querna ICMP:15825

KJ869126.1| Beltraniopsis neolitseae CPC 22168

JQ621881.1| Hemibeltrania sp. CL12WA

10 1KT119564.1| Hemibeltrania cinnamomi NFCCI 3695

EU040241.1| Subramaniomyces fusisaprophyticus CBS 418.95

JF 440983.1| Pseudomassana fallax

EU040235.1| Parapleurotheciopsis inaequiseptata MUCL 41089 ‘JIncetae sedis

AB594793.1| lmmersidiscosia eucalypt

DQ278923.1| Sarcostroma restionis strain CBS 118153

AB594801.1| Seimatosporium elegans Amphispheriaceae

NR_120230.1| Seiridium phylicae CPC 19962

JX 262802 .1| Monochaetia monochaeta isolate 18

EU040223.1| Phlogicylindrium eucalyptorum strain CBS 120221

JN688916.1| Apiospora montagnei strain H3_83

NR_121561.1| Arthrinium pterospermum

(_ 15.1| Graphostroma platystoma CBS 270.87 “]Graphostromataceae

AY662394.1| Eutypa lata “]Diatryp aceae

A993 138.1| Xylaria hypoxylon CBS1 21680

AlM993148.1| Xylaria hypoxylon voucher M:M-0125974 piv

AM993143.1| Xylana hypoxylon CBS1 22619

AF009808.1| Clypeosphaeria mamillana _]Clypeosphaeriaceae

AF222865.1| Trichoderma virens “JHypoceales

Kl27 2368. 1| Lulworthia sp. “]Lulworthiales | SSRN

EE}

} piosporaceae

Fic. 4. ITS phylogenetic tree generated from Maximum Parsimony (MP) analyses of the aligned

sequences of Hemibeltrania cinnamomi and allied taxa, with Trichoderma and Lulworthia as

outgroup. Percentage bootstrap support (250%) is shown on the branches. The MP tree was

obtained using the Subtree-Pruning-Regrafting algorithm with search level 1 in which the initial

trees were obtained with the random addition of sequences (10 replicates). The bootstrap test has

1000 replicates.

Discussion

Hemibeltrania comprises 12 accepted species (MycoBank 2015; TABLE 1).

Taxonomic study of the present collection revealed that in H. cinnamomi,

conidial shapes and sizes are more variable than the original description:

conidia are longer than 10 um, while conidial shapes range from limoniform,

obovate, and broadly ellipsoid to irregular. The symptomatology and other

taxonomic characters, however, are identical to the type description given by

Pirozynski (1963). Considering the variable conidial shape and size observed in

AMH 9667, we propose a broad species concept for H. cinnamomi that might

be expanded in the future based on re-examination and phylogenetic analysis

of H. nectandrae, which shows some affinity with H. cinnamomi.

Earlier studies have shown that Beltrania and related genera

(Beltraniella, Beltraniopsis, Subramaniomyces) are phylogenetically allied

with Amphisphaeriacaeae, Xylariales (Shirouzu et al. 2010, Hidayat et al.

2014). Shirouzu et al. (2010) also noted that Beltraniella anamorphs and

Hemibeltrania cinnamomi reconsidered ... 93

TABLE 1. Comparative morphology of accepted Hemibeltrania species

naa CONIDIA CONIDIOPHORE SIZE

SHAPE SIZE (um) (um)

H. cinnamomi Obovoid/broadly ellipsoid/limoniform — 12-25 x 8-13 130-250 x 3.5-6

H. convallariae Ovoid 14-24 x 10-15 <2000 x 13-23

H. cymbiformis Cymbiform 21-24 x 3-4.5 $140 x 2.2-4.5

H. decorosa Fusiform 17-22 x 3.5 <250 x 4-6.5

H. echinulata Ovoid 12-14 x 5.5-7 140-300 x 3.5-5

H. laxa Obclavate/navicular 13-17 x 2-3 50-350 x 3-7

H. malaysiana Ellipsoid/navicular 13-20 x 3-5 <400 x 4-7

H. mitrata Mitriform 14-17 x 4.5-5.5 35-85 x 3-4.5

H. nectandrae Limoniform 15-23 x 10-13 <250 x 4-6

H. ovalispora Obovoid 12-147 2750 x 4-6

H. saikawae Clavate/cylindrical 14-19 x 2.5-3 10-155 x 2-3

H. urbanodendri Ovoid/obovoid/ biconic/limoniform 12-25 x 6-10 55-106 x 4-6

Pseudomassaria teleomorphs are phylogenetically linked. Our phylogenetic

analysis of H. cinnamomi does support Hemibeltrania within the Xylariales.

However, the family in which it should be placed remains ambiguous.

Acknowledgements

We are indebted to Dr. Elaine Malosso (Federal University of Pernambuco, Brazil)

and Dr. De-Wei Li (The Connecticut Agricultural Experiment Station Valley Laboratory,

USA) for reviewing this manuscript. Thanks are also due to Department of Science and

Technology (DST), Government of India, New Delhi, for providing financial support

and the Director, ARI, for providing facilities. RFCR is grateful to the Cuban Ministry

of Agriculture and “Programa de Salud Animal y Vegetal” (project P131LH003033) for

facilities. We acknowledge the assistance 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 and Dr. Shaun Pennycook’s nomenclatural reviews are greatly

appreciated.

Literature cited

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for PCR-based techniques. Nucleic Acids Research 25: 4692-4693.

http://dx.doi.org/10.1093/nar/25.22.4692

Crous PW, Verkley GJM, Groenewald JZ, Samson RA (eds). 2009. Fungal biodiversity.

CBS Laboratory Manual Series. Centraalbureau voor Schimmelcultures, Utrecht, Netherlands.

Crous PW, Slippers B, Wingfield MJ, Rheeder J, Marasas WFO, Phillips AJL, Alves A, Burgess T,

Barber P, Groenewald JZ. 2006. Phylogenetic lineages in the Botryosphaeriaceae. Studies in

Mycology 55: 235-253. http://dx.doi.org/10.3114/sim.55.1.235

Hidayat I, Harahap I, Rahayu G. 2014. The first phylogenetic study of Kiliophora (Fungi, anamorphic

Xylariales). Mycosphere 5: 78-84. http://dx.doi.org/10.5943/mycosphere/5/1/3

MycoBank. 2015. MycoBank: an online mycological database. http://www.mycobank.org

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Pirozynski KA. 1963. Beltrania and related genera. Mycological Papers 90: 1-37.

Rehner SA, Samuels GJ. 1994. Taxonomy and phylogeny of Gliocladium analysed from

nuclear large subunit ribosomal DNA sequences. Mycological Research 98: 625-634.

http://dx.doi.org/10.1016/S0953-7562(09)80409-7

Shirouzu T, Hirose D, Tokumasu S, To-Anun C, Maekawa N. 2010. Host affinity and phylogenetic

position of a new anamorphic fungus Beltraniella botryospora from living and fallen leaves of

evergreen oaks. Fungal Diversity 43: 85-92. http://dx.doi.org/10.1007/s13225-010-0037-1

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

ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: 4238-4246.

White TJ, Bruns T, Lee J, 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, San Diego.

http://dx.doi.org/10.1016/B978-0-12-372180-8.50042-1

MY COTAXON

January-March 2016— Volume 131, pp. 95-102

http://dx.doi.org/10.5248/131.95

Ramularia hydrangeicola sp. nov. with distinctive traits on

Hydrangea serrata f. acuminata in Korea

JI-HyuUN PARK & HYEON-DONG SHIN*

Division of Environmental Science and Ecological Engineering, Korea University,

Seoul 02841, Korea

*CORRESPONDENCE TO: hdshin@korea.ac.kr

ABSTRACT — A new species, Ramularia hydrangeicola, causing leaf spots was observed on

Hydrangea serrata f. acuminata. Morphological characteristics and phylogenetic analyses

support this fungus as a distinct new species. The new fungus is described, illustrated,

and compared with R. hydrangeae, R. hydrangeae-macrophyllae, and R. philadelphi, three

additional species on hosts of the Hydrangeaceae.

Key worps — anamorphic Mycosphaerella, plant pathogen

Introduction

Ramularia is an anamorph-typified genus comprising hyphomycetous

asexual morphs partly associated with sexual morphs of the genus

Mycosphaerella (Mycosphaerellaceae). Based on the new rules of the ICN

(Art. 59) and the new “one fungus one species” principles, Mycosphaerella

has been reduced to synonymy with Ramularia since the type species of

Mycosphaerella, M. punctiformis, forms a Ramularia anamorph, R. endophylla,

and clusters with other Ramularia species (Verkley et al. 2004, Braun et

al. 2013). The older name Ramularia has priority and is now applied as

holomorph name for this monophyletic unit composed of asexual and sexual

morphs (Braun et al. 2013, Wijayawardene et al. 2014). Most Ramularia species

are phytopathogenic fungi that cause leaf spots, necrosis, or chlorosis; several

species are economically important pathogens. Some Ramularia species are

saprophytic or hyperparasitic (Braun 1998). Ramularia species are considered

host-specific, like other fungi of the Mycosphaerellaceae. Species of Ramularia

96 ... Park & Shin

and other allied genera have conventionally been morphologically delimited

according to their host plant families and genera (Braun 1995).

Since 2002, a previously undescribed leaf spot has been observed on

Hydrangea serrata f. acuminata during field surveys of cercosporoid fungi in

Korea. The morphological characteristics of this fungus were quite different from

those of other similar hyphomycetes observed on Hydrangea and were similar

to those of Ramularia as described by Braun (1998). In this study, this novel

species from Korea is described and illustrated on the basis of morphological

characteristics and phylogenetic analyses. This species is compared with the

three Ramularia species previously described on hydrangeaceous hosts:

R. hydrangeae, R. hydrangeae-macrophyllae, and R. philadelphi.

Materials & methods

Fresh material and air-dried specimens were used for morphological examinations.

The fungal structures were detached from the infected leaves and mounted in a few

drops of distilled water or 3% KOH on a glass slide. These slides were examined by

bright field- and differential interference contrast (DIC)-light microscopy, by using an

Olympus BX51 microscope (Olympus, Tokyo, Japan) for measurements and a Zeiss

AX10 microscope equipped with AxioCam MRc5 (Carl Zeiss, Gottingen, Germany) for

imaging. Voucher specimens were deposited in the Korea University Herbarium, Seoul,

Korea (KUS).

To obtain a pure isolate, infected tissue with abundant sporulation was excised

and placed in a drop of distilled water on a glass slide. A loop of conidial suspension

was streaked onto 2% water agar plates supplemented with 100 mg/L of streptomycin

sulfate. After 2 days of incubation at 25°C, single conidial colonies were transferred onto

potato dextrose agar (PDA). Two monoconidial isolates were deposited in the Korean

Agricultural Culture Collection, National Institute of Agricultural Science, Jeonju,

Korea (KACC).

The fungal mycelium was harvested from the culture on PDA and genomic DNA

was extracted by means of a DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA,

USA). The internal transcribed spacer (ITS) region and the large subunit (LSU) of

rDNA were amplified by using primers ITS1 and ITS4 (White et al. 1990) for ITS, and

LROR (Rehner & Samuels 1994) and LR7 (Vilgalys & Hester 1990) for LSU. The PCR

amplicons were purified with the help of a QIAquick PCR purification Kit (Qiagen

Inc.) and were sequenced by applying the Macrogen Sequencing Service (Macrogen,

Seoul, Korea). The raw sequences were edited by means of DNASTAR version 5.05

(Lasergene, Madison, WI, USA). For phylogenetic analyses, all available ITS and LSU

sequences that closely matched the extracted sequences were retrieved from the NCBI

GenBank database. Sequence datasets for the two genomic loci were aligned on the

base of an online version of MAFFT 7 (Katoh & Standley 2013). A phylogenetic tree

was constructed by the neighbor-joining method using MEGA 6 (Tamura et al. 2013).

Branches of the inferred tree were tested by a bootstrap analysis of 1000 replicates.

Furthermore, a Bayesian inference analysis was performed according to MrBayes version

Ramularia hydrangeicola sp. nov. (Korea) ... 97

3.1.2 (Ronquist & Huelsenbeck 2003). Best-fit substitution models, which were obtained

by applying MrModeltest version 2.2 (Nylander 2004), were used to ascertain the best

nucleotide substitution model setting for each data partition. K80+I+G and GTR+I+G

as nucleotide substitution models were used for ITS and LSU datasets. Branch support

was calculated as posterior probability (PP) and neighbour-joining bootstrap (NJ-BS).

The phylogenetic tree generated from the ITS sequence alignment was deposited in

TreeBASE (http://www.treebase.org/) under accession number $17946.

Taxonomy

Ramularia hydrangeicola J.H. Park & H.D. Shin, sp. nov. FIG. 1

MycoBank MB813449

Differs from Ramularia hydrangeae, R. hydrangeae-macrophyllae, and R. philadelphi

in having conspicuous, hypophyllous, pinkish or reddish tinged caespituli and longer,

wider conidia that are pinkish tinged, smooth, guttulate and mostly constricted at the

septa.

TyPeE: On living leaves of Hydrangea serrata f. acuminata (Siebold & Zucc.) E.H. Wilson

(Hydrangeaceae): Korea, Yangpyeong, Okcheon-myeon, 37°34’45”N 127°27’28”E,

18 October 2007, H.D. Shin (Holotype: KUS-F23039; ex-type culture, KACC43597;

GenBank KT265707, KT265708)

ErymMo ocy: The epithet is derived from the host plant genus.

LEAF SPOTS amphigenous, scattered to confluent, angular to irregular, vein-

limited, distinct, small, 1-5 mm diam., or up to 10 mm when confluent, at

first visible as pinkish patches on the lower surface, pale greenish on the upper

surface, indistinct discolorations, later becoming brown to dark brown on

both sides. CAEspiTuLI hypophyllous, conspicuous, pinkish or with reddish

tinge due to abundant fungal fructification. MycELIum internal; hyphae

branched, hyaline or with faint pinkish tinge, 3-5 um wide, occasionally with

superficial secondary mycelium emerging through the substomatal cavity.

STROMATA substomatal or sometimes intraepidermal. CONIDIOPHORES 3-8

in loose fascicles, emerging through stomata, solitary conidiophores arising

from superficial hyphae, hyaline throughout or partly with pinkish tinge,

0-3-septate, simple, straight to curved, usually geniculate-sinuous, or 0-3

times geniculate near the apex, 12.5-40 x 2.5-5.0 um. CONIDIOGENOUS CELLS

integrated, terminal, intercalary, proliferating sympodially, 3.5-15 um long;

conidiogenous loci conspicuous, thickened and darkened, 1.2-2.1 um diam.

Conip1a solitary or catenate, sometimes in short branched chains, cylindrical

to obclavate-ellipsoid, hyaline or with pinkish tinge, smooth, guttulate, mostly

uniseptate, occasionally 2-5-septate, constricted to non-constricted at the

septa, obtuse to subobtuse at both ends, sometime gently attenuated towards

each end, 18-45 x 3-7 um; hilum minute, 1.2-1.8 um diam., conspicuously

thickened, darkened, and slightly protuberant. CoLontes on PDA attaining

98 ... Park & Shin

Fic. 1. Ramularia hydrangeicola. A: symptoms on lower leaves of Hydrangea serrata f. acuminata

with distinctive traits; B, C: close-up of the leaf lesions showing pinkish caespituli; D: conidiophores

emerging through the substomatal cavity; E-G: conidiophores arising from superficial hyphae;

H-J: catenate conidia; K—N: conidia ranging from hyaline to reddish tinged; O: 6-week-old

colonies formed on potato dextrose agar (D-N = holotype, KUS-F23039; O = KACC43597).

Scale bars: D—N = 20 um.

Ramularia hydrangeicola sp. nov. (Korea) ... 99

ca. 10 mm diam. after 6 weeks at 25°C under 12-h fluorescent light, velvety,

pinkish to whitish, margins undulate, consisting of dense aerial mycelium and

producing several droplets of slimy exudate. TELEOMORPH unknown.

ADDITIONAL SPECIMENS EXAMINED: on Hydrangea serrata f. acuminata: KOREA,

Jesu, 14 Sep. 2002, H.D. Shin (KUS-F19005); 2 Nov. 2007, H.D. Shin (KUS-F23141);

PYEONGCHANG, 27 Sep. 2002, H.D. Shin (KUS-F19080); 20 Sep. 2003, H.D. Shin

(KUS-F19700); 9 Oct. 2008, H.D. Shin (KUS-F23778; culture, KACC44254; GenBank

KT265709, KT265710); YANGPYEONG, 28 Aug. 2008, H.D. Shin (KUS-F23614);

CHUNCHEON, 7 Oct. 2008, H.D. Shin (KUS-F23759); GAPYEONG, 13 Oct. 2011,

H.D. Shin & J.H. Park (KUS-F26314); Wonyju, 18 Sep. 2014, H.D. Shin & J.H. Park

(KUS-F28177).

Results & discussion

Several mycosphaerellaceous fungi have been described on Hydrangea

species, including Cercospora, Pseudocercospora, Passalora, Phacellium, and

Ramularia spp. (Braun 1998, Crous & Braun 2003, Braun & Hill 2008, Farr &

Rossman 2015). Our new species, R. hydrangeicola, is characterized by having

mostly uniseptate conidia with thickened, darkened hila, either formed solitary

or catenate, ranging from cylindrical to obclavate-ellipsoidal in shape and from

hyaline to reddish tinged in color. These characteristics are consistent with the

main characteristics of Ramularia (Braun 1998).

Three Ramularia species have been recorded on hosts of the family

Hydrangeaceae: R. hydrangeae Y.L. Guo & U. Braun was described on

H. bretschneideri from China (Braun 1998), based on material originally

misdetermined as R. saxifragae (Guo 1993); R. hydrangeae-macrophyllae

U. Braun & C.F. Hill was described on H. macrophylla from New Zealand

(Braun & Hill 2008); and H. philadelphi Sacc., originally described from Italy,

has been recorded on Philadelphus spp. from Europe, North America, and Asia

(Braun 1998, Farr & Rossman 2015).

Ramularia hydrangeae differs from R. hydrangeicola in having inconspicuous,

greyish white caespituli and hyaline, verruculose, shorter (8-35 um), and

narrower (2-5 um) conidia (Braun 1998); R. hydrangeae-macrophyllae differs

in having inconspicuous caespituli and shorter (4-18 um), narrower (1.5-2.5

um), and 0-1-septate conidia (Braun & Hill 2008); and R. philadelphi differs in

having amphigenous, greyish white caespituli and hyaline, verruculose, shorter

(8-20 um), and narrower (2.5-5.0 um) conidia (Braun 1998).

Phacellium hydrangeacearum U. Braun on Jamesia americana from the

USA is another ramularioid fungus on a hydrangeaceous host, but easily

distinguished by its synnematous conidiomata and verruculose conidia

(Braun 1998).

100 ... Park & Shin

1.00100] KT 265708 Ramularia hydrangeicola

KT265710 Ramularia hydrangeicola

ee? GU214693 Ramularia sp.

EU019284 Ramularia pratensis var. pratensis

GU214690 Ramularia sp.

GU214692 Ramularia coleosporii

GU214694 Ramularia uredinicola

—_— GU 214486 Ramularia grevilleana

EU019285 Ramularia sp.

1.00/100] FJ493208 Phaeophleospora eugeniicola

FJ493209 Phaeophleospora eugeniicola

1.00/100 | GQ852624 Passalora vaginae

GU214461 Passalora vaginae

1.00/93] GU214481 Pseudocercospora eucalyptorum

GU253788 Pseudocercospora eucalyptorum

GU214483 Pseudocercospora vitis

GU253844 Pseudocercospora vitis

EU019262 Cladosporium cladosporioides

0.57/63

0.005

Fic. 2. LSU rDNA phylogenetic tree generated by a neighbor-joining method by using

MEGA6 with 1000 bootstrap replications based on sequences of Ramularia and allied genera.

Branch support is indicated above the branches (PP/NJ-BS). Sequences of R. hydrangeicola are

shown in bold and their well-supported clade is highlighted in gray.

The holotype and paratype specimens of R. hydrangeicola yielded 1172-bp

LSU and 495-bp ITS sequences. The LSU sequences were congruent between

the two isolates, whereas their ITS sequences differed by three base-pairs.

A GenBank BLAST search revealed that the LSU sequences had 99% similarity

to GU214693 (Ramularia sp.), GU214690 (Ramularia sp.), and GU214692

(R. coleosporii); and the ITS sequences had 94% similarity to GU214693

(Ramularia sp.), EU019284 (R. pratensis), and KJ504787 (R. decipiens). Based

on the molecular phylogenetic trees inferred from the neighbor-joining

method and Bayesian analyses using ITS and LSU sequences, R. hydrangeicola

formed an independent clade with high bootstrap values: PP = 1, NJ-BS = 100%

(Fics 2, 3). Moreover, the LSU phylogeny indicated that this species belongs in

the genus Ramularia, and the ITS phylogeny revealed that it forms a separate

lineage among Ramularia species. Although there are no comparable sequence

data for R. hydrangeae, R. hydrangeae-macrophyllae, and R. philadelphi,

R. hydrangeicola clearly differs morphologically from these species. Therefore,

R. hydrangeicola is recognized as a new species based on the present

morphological and molecular data.

Ramularia hydrangeicola sp. nov. (Korea) ... 101

4.00400 7——KT265707 Ramularia hydrangeicola

KT265709 Ramularia hydrangeicola

GU214693 Ramularia sp.

DQ466083 Ramularia carthami

0.93/94 HQ728118 Ramularia cynarae

EF535676 Ramularia lamii var. lamii

KF924738 Ramularia coleosporii

EF535671 Ramularia sp.

GU214692 Ramularia coleosporii

o3/4 GU214694 Ramularia uredinicola

KF251223 Ramularia pratensis

871 EU019284 Ramularia pratensis

KJ504787 Ramularia decipiens

GU214690 Ramularia sp.

EU040238 Ramularia aplospora

KJ504806 Ramularia pratensis

p.ssiag JN7 12499 Ramularia stellenboschensis

EU707899 Ramularia proteae

p.79/89 KJ504775 Ramularia glennii

KJ504777 Ramularia glennii

KJ504792 Ramularia eucalypti

KJ504795 Ramularia eucalypti

DQ885902 Ramularia miae

0.90/98

0.82/58

0.85/84

0.86/88

KC149564 Mycosphaerella pini

0.01

Fic. 3. ITS rDNA phylogenetic tree generated by a neighbor-joining method by using MEGA6 with

1000 bootstrap replications based on sequences among Ramularia species in GenBank database.

Branch support is indicated above the branches (PP/NJ-BS). Sequences of R. hydrangeicola are

shown in bold and their well-supported clade is highlighted in gray.

Acknowledgements

The authors are grateful to Dr. Uwe Braun and Dr. Chiharu Nakashima for their

critical review and comments on the manuscript. This work was supported by a grant

from Regional Subgenebank Support Program of Rural Development Administration,

Republic of Korea and also by the BK21 Plus Program in 2013-2015 funded by National

Research Foundation of Korea (NRF).

Literature cited

Braun U. 1995. A monograph of Cercosporella, Ramularia and allied genera (phytopathogenic

hyphomycetes). Vol. 1. IHW-Verlag, Miinchen.

Braun U. 1998. A monograph of Cercosporella, Ramularia and allied genera (phytopathogenic

hyphomycetes). Vol. 2. IHW-Verlag, Miinchen.

Braun U, Hill CF. 2008. New species and new records of foliicolous hyphomycetes from

New Zealand. Australasian Mycologist 27: 45-56.

Braun U, Nakashima C, Crous PW. 2013. Cercosporoid fungi (Mycosphaerellaceae) 1.

Species on other fungi, Pteridophyta and Gymnospermae. IMA Fungus 4: 265-345.

http://dx.doi.org/10.5598/imafungus.2013.04.02.12

Crous PW, Braun U. 2003. Mycosphaerella and its anamorphs: 1. Names published in Cercospora

and Passalora. CBS Biodiversity Series 1: 571 p.

102 ... Park & Shin

Farr DE, Rossman AY. 2015. Fungal databases. Syst. Mycol. Microbiol. Lab., Online publication,

ARS, USDA. Retrieved July 1, 2015.

Guo YL. 1993. Foliicolous hyphomycetes of Xiaowutai in Hebei Province. Mycosystema 6: 91-102.

Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment software version

7: improvements in performance and usability. Mol. Biol. Evol. 30: 772-780.

http://dx.doi.org/10.1093/molbev/mst010

Nylander JAA. 2004. MrModeltest version 2.0. Program distributed by the author. Uppsala

University, Uppsala, Sweden.

Rehner SA, Samuels GJ. 1994. Taxonomy and phylogeny of Gliocladium analysed from nuclear

large subunit ribosomal DNA sequences. Mycol. Res. 98: 625-634.

http://dx.doi.org/10.1016/s0953-7562(09)80409-7

Ronquist F, Huelsenbeck JP. 2003, MrBayes 3: Bayesian phylogenetic inference under mixed

models. Bioinformatics 19: 1572-1574. http://dx.doi.org/10.1093/bioinformatics/btg180

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: Molecular Evolutionary

Genetics Analysis version 6.0. Mol. Biol. Evol. 30: 2725-2729.

http://dx.doi.org/10.1093/molbev/mst197

Verkley GJM, Crous PW, Groenewald JZ, Braun U. 2004. Mycosphaerella punctiformis

revisited: morphology, phylogeny, and epitypification of the type species of the

genus Mycosphaerella (Dothideales, Ascomycota). Mycol. Res. 108: 1271-1282.

http://dx.doi.org/10.1017/s0953756204001054

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 SB, 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. San Diego, Academic Press.

http://dx.doi.org/10.1016/B978-0-12-372180-8.50042-1

Wijayawardene NN, Crous PW, Kirk PM et al. 2014. Naming and outline of Dothideomycetes -

2014 including proposals for the protection or suppression of generic names. Fungal Divers.

69: 1-55. http://dx.doi.org/10.1007/s13225-014-0309-2

MY COTAXON

January-March 2016—Volume 131, pp. 103-110

http://dx.doi.org/10.5248/131.103

Phylogenetic placement of the secotioid fungus

Araneosa columellata within Agaricus

ScoTT T. BATES?, ROBERT M. CHAPMAN’, MELISSA B. ISLAM‘,

ANNA SCHWABE*’, ERIK C.P. WARDENAAR? & VERA S. EVENSON*

"Department of Biology, Purdue University Northwest, Westville, IN 46391, USA

*Las Cruces, NM 88012, USA

The Bell Museum of Natural History, 10 Church St. SE, Minneapolis, MN 55455, USA

“Denver Botanic Gardens, 909 York St., Denver, CO 80206, USA

* CORRESPONDENCE TO: stbates@purdue.edu

ABSTRACT —Recently collected material was used to evaluate the phylogenetic position of the

secotioid fungus Araneosa columellata. We amplified the nuclear ribosomal RNA gene (ITS1,

ITS2, and LSU) and found Ar. columellata to be a member of the genus Agaricus (Agaricaceae,

Agaricales), necessitating its transfer to that genus as Agaricus columellatus comb. nov.

A detailed description is provided along with illustrations, and a lectotype is designated.

Key worps — Basidiomycota, molecular phylogeny, Sonoran Desert, taxonomy

Introduction

DNA-based studies demonstrate multiple origins of gastroid (angiocarpic

forms) and secotioid (stipitate angiocarpic forms often with reduced lamellae)

species from within agaricoid (mushroom-forming) groups in the fungal

phylogeny (Hibbett et al. 1997, Vellinga et al. 2003, Lebel & Syme 2012). Modern

phylogenetic taxonomy (Queiroz & Gauthier 1992) stipulates that taxa be

circumscribed as monophyletic clades, and this has necessitated the taxonomic

reevaluation of these fungi. As a result, many familiar gastroid or secotioid taxa

have been transferred into genera once reserved solely for agaricoid forms.

For example, Endoptychum agaricoides was transferred to Chlorophyllum as

C. agaricoides (Vellinga 2002), while Endoptychum depressum was transferred

to Agaricus as Ag. inapertus (Vellinga et al. 2003).

104 ... Bates & al.

Long (1941) described the monotypic secotioid genus Araneosa (type

species: Araneosa columellata) within Arachniaceae (Coker & Couch 1928)

from material collected at two localities in southern Arizona. Species within

Arachnion, such as A. album, are superficially similar to Ar. columellata;

however, Araneosa is distinguished primarily by its stipitate habit and the

presence of a distinct columella within the gleba (Long 1941). Molecular

phylogenetic studies have been needed to determine the taxonomic position of

Ar. columellata (Moreno et al. 2007).

Several specimens of Ar. columellata collected by Long in the 1930-40s are

deposited in institutional collections throughout the United States and Canada

(BPI, FH, ILL, NY, NCU, UBC), many of which represent splits of the original

type material (isotypes) in various stages of disintegration. An exceptionally

well-preserved isotype (Fic. 1E) was located at the University of Illinois

Herbarium, and this specimen is selected as the lectotype here. Specimens

of this age are typically not desirable for DNA sequencing (Osmundson et al.

2013), and despite the need for new material, collections of this fungus have

rarely been made (Esqueda 1998). On a recent excursion to the southwestern

United States several fruiting bodies of Ar. columellata were collected. These

specimens were morphologically identical to those described in the protologue

(Long 1941) and yielded sufficient genomic DNA for Sanger sequencing of

the nuclear ribosomal RNA gene (ITS1, ITS2, and LSU). This allowed us to

examine the phylogenetic placement of Ar. columellata within the Agaricales.

Materials & methods

Original descriptions were made from material collected in the field, now deposited

in the Fungal Collection of the Bell Museum of Natural History Herbarium at the

University of Minnesota (MIN). An Olympus BH2 conventional bright field microscope

was used in gathering the micro-morphological data on material first infiltrated

with 95% aqueous ethanol and then mounted in water or 3% aqueous KOH. All

measurements were made at 1000x using an ocular reticle with units calibrated by an

optical micrometer. Spore statistics include: x, the arithmetic mean of the spore length

by spore width (+ SD) for n spores measured; Q, the quotient of spore length and spore

width in any one spore, indicated as the range of variation in n spores measured; Q the

mean of Q values. Classical descriptive methodologies follow those published previously

for angiocarpic fungi (Miller & Miller 1988), while color descriptions follow the system

outlined in the Methuen Handbook of Colour (Kornerup & Wanscher 1967). Standard

abbreviations for herbarium collections follow Index Herbariorum (Thiers 2016).

Genomic DNA was extracted following standard protocol of the Qiagen’s DNeasy

Kit, after first grinding glebal tissue and spores with a micro-pestle and then incubating

this material in the kit buffer for 10 min. at 65°C. To amplify nuclear ribosomal RNA

genes (ITS1, ITS2, and LSU), we used the primer pairs ITS1F/ITS4 (White et al. 1990,

Gardes & Bruns 1993) and LROR/LRS (Vilgalys & Hester 1990, Bunyard et al. 1994).

Agaricus columellatus comb. nov. (U.S.A.) ... 105

PCR reactions were carried out under the following thermal cycler conditions for LSU

(with modifications for ITS in parentheses): 94°C for 3 min. for initial denaturation;

followed by 35 cycles at 94°C for 30 sec. (1 min.), 54°C for 1 min., and 72°C for 1.5

min. (1 min.); then a final extension at 72°C for 4 min. (3 min.). PCR amplicons were

then imaged in gel electrophoresis to visualize the presence and relative concentration

of fungal DNA. Automated sequencing was carried out at the University of Chicago

Comprehensive Cancer Center DNA Sequencing & Genotyping Facility.

Complementary DNA sequences were compiled and manually edited using MEGA

6.0 software suite (Tamura et al. 2013). Additional sequences of closely related taxa

from within the Agaricales were culled from GenBank (https://www.ncbi.nlm.nih.gov/),

with accession numbers cited in the phylogenetic tree (Fic. 2). MEGA 6.0 was used to

align sequences and carry out the phylogenetic analysis. A total of 15 LSU nrRNA gene

sequences was aligned using the MUSCLE algorithm (gaps were treated as complete

deletions), which was used to carry out the maximum likelihood (ML) phylogenetic

analysis (NNI heuristic method, with the K2+G nucleotide substitution model chosen

under model selection analysis) and bootstrap testing (1000 replicates). The phylogenetic

tree was rooted using Psathyrella candolleana (Psathyrellaceae, Agaricales) as outgroup

for Agaricus (Agaricaceae, Agaricales; Matheny 2006).

Taxonomy

Agaricus columellatus (Long) R.M. Chapm., V.S. Evenson & S.T. Bates,

comb. nov. FIGURE 1

MycoBank MB 809082

= Araneosa columellata Long, Mycologia 33(4): 353 (1941).

Type: USA, Arizona, Santa Cruz Co., 7 miles from Nogales on State Highway 89; open

areas in mesquite—catclaw (Prosopis—Acacia) flats, elev. 3857 ft., 21.1X.1934, William H.

Long (7937) and Victor O. Sandberg (the original type collection no. 7937 from Long’s

Herbarium now exists only as widely distributed isotypes: BPI 736388, BPI 736396, FH

488386, NY 449250!, NY 780610!, NY 780611!, NCU-F-0000798, UBC 371. Lectotype

designated here: ILL 33529! (Fic. 1E)).

BASIDIOME 20-35 mm in diameter x 20-30 mm in height, secotioid,

subglobose, depressed-globose to broadly ovoid or pyriform, furnished with

a rudimentary stipe at the base that extends into the gleba as one unit to form

a distinct columella, the entire unit often detaching completely and leaving

a circular opening at the base of the peridial body; strpE-COLUMELLA UNIT

concolorous with the exoperidium, up to 30 mm long, with roughly half

(~15 mm) of the unit protruding from the base of the basidiome and the upper

half (~15 mm) inserted into the gleba, slightly bulbous at the base, up to 5 mm

in diameter and even along the stipe portion, somewhat swollen at the point

of insertion, and tapering toward the apex within the gleba as the columella,

developing longitudinal striations and becoming more firm when dry,

surrounded at the point of insertion by a thin layer of peridial tissue, this with

striations radiating out from the central point of the stipe and resembling velar

106 ... Bates & al.

tissue; PERIDIA slowly breaking apart to expose the gleba, often starting from

the basal opening formed as the stipe-columella unit detaches. ExoPERIDIUM

whitish to pale yellow (3A1-3), dull, coarsely to irregularly rugose overall,

though glabrous at the surface, thin, firm and persistent, cracking apart or

flaking off with age to expose portions of the endoperidium. ENDOPERIDIUM

yellowish gray (3B2-4B2), dull glabrous, thin, firm and persistent, slowly

breaking apart in conjunction with the exoperidium. GLEBA brown to grayish

brown (6E3-5), composed of labyrinthiform, sinuous tramal tissue, forming

numerous locules, the interior of which is a hymenial surface densely covered

with brownish spore mass, and an exterior composed of grayish, loosely woven

hyphal tissue, breaking apart into irregular shaped glebal fragments with age.

BASIDIOSPORES 4.0-5.6 xX 4.8-7.2 um [x = 4.8 + 0.5 x 6.1 + 0.7 um,

Q_ = 1.3, n = 20], subglobose to ovoid or occasionally irregular ovoid, smooth,

brownish in water mounts; pedicel rudimentary or up to 4 um in length;

STERIGMATAL REMNANTS mostly absent from mounts. CAPILLITIUM absent.

Perip1A composed of thin-walled hyphal elements.

Hasirat: Terrestrial and found in soil amongst Acacia, palo verde

(Parkinsonia), creosote (Larrea), mesquite (Prosopis) and jojoba (Simmondsia).

Agaricus columellatus occurs at lower elevations within the Arizona Upland

Subdivision of the Sonoran Desertscrub and Semidesert Grassland biotic

communities (Brown 1994).

DISTRIBUTION: In the United States, known only from southern Arizona.

Also reported from the state of Sonora in Mexico.

GENETIC DATA: Nuclear ribosomal RNA gene sequences for Agaricus

columellatus (MIN 938394) have been deposited in GenBank (ITS: KJ912899;

LSU: KJ912900).

ADDITIONAL SPECIMENS EXAMINED: USA, ARIZONA, GILA Co., AZ 288, north of

Globe, 33.67°N 110.96°W, alt. 707 m, in sand, amongst Acacia, palo verde, creosote

bush, and jojoba, 30.VIII.2012, R.M. Chapman 1385 (MIN 938394); AZ 288, north of

Globe, 33.67°N 110.97°W, alt. 721 m, in sand, amongst acacia, palo verde, creosote bush,

and jojoba, 1.1X.2012, R.M. Chapman 1388 (hb. Chapman). Santa Cruz Co., 7 miles

north of Nogales, Arizona on Nogales-Tucson road, 30.1X.1939, William H. Long 8388

(paratype NY 2169336).

Results & discussion

Long (1941) described Araneosa columellata within Arachniaceae, stating

“peridioles subglobose to irregularly oblong to angular, walls rather thick

and firm, 90-268 microns by 134-402 microns, held loosely together by

delicate interwoven hyphae, easily separable from the endoperidium, leaving

its inner surface reticulate, the lamelliform arrangement of the peridioles

often permitting the gleba to split into flakes which easily crumble into the

Agaricus columellatus comb. nov. (U.S.A.) ... 107

7 ailos frua Nogales Ariz. om

Highway #29 21,1984

; MYGOLOGICAL COLLECTIONS

' Araneosa columelista W.H. Long

: tor substrate:

pie site: AZ Hwy. 89, 6-8miles from Nogales. Alt. 3857 (t

Santa Gru7 U.S.A.

County.

Perm LI NOS

yright reserved

Collector: W.H. Long & VO Sandberg No. 7937

Date: 21 Sep 1934 Accession Number 33529

UNIVERSITY OF ILLINOIS (ILL)

FiGureE 1. Agaricus columellatus. A. Field image of the gasterocarp (MIN 938394). B. Dried

specimens (MIN 938394, bar = 20 mm). C. Exposed gleba of dried specimen (MIN 938394).

D. Cross section through basidiome of dried specimen showing insertion of stipe-columella unit

(MIN 938394; bar = 20 mm). E. Lectotype (ILL 33529).

individual peridioles.” Although our material was morphologically identical

to Long’s (1941) concept of Ar. columellata, distinct peridioles did not form

in the gleba. Inspection of authentic material from Long (NY 449250, NY

780610, NY 780611, NY 2169336, and the lectotype ILL 33529), revealed in

these specimens the formation of irregularly shaped glebal fragments rather

than discrete peridioles, which is consistent with our material (Fic. 1C). While

the age of these specimens prevented us from confirming an exact match to

the Long specimens by molecular means, on the basis of morphology, our

specimens clearly represent a recent collection of Ar. columellata.

108 ... Bates & al.

Agaricus columellatus (KJ912900)**

Agaricus aridicola (AF261478)**

Agaricus diminutivus (AF482877)

Agaricus semotus (AY207136)

Agaricus arvensis (U11910)

Agaricus deserticola (AF261479)**

Agaricus sylvaticus (AY207137)

Agaricus campestris (AY207134)

Chlorophylium rachodes (U85277)

Lycoperdon perlatum (AF518630)*

100 Lycoperdon marginatum (AF261485)*

Macrolepiota clelandii (AF482882)

87 + Macrolepiota procera (AlM946456)

Coprinus comatus (NG027626)

Psathyrella candolleana (DQ110874)

fpeemenn nnn]

0.07

Figure 2. Molecular phylogeny (ML consensus tree) for the large subunit nuclear ribosomal

RNA gene dataset showing the inclusion of Agaricus columellatus (MIN 938394), within Agaricus

(Agaricaceae). Basidiome forms are agaricoid, unless otherwise indicated as gastroid (*) or

secotioid (**). Branch lengths are shown at scale, with the bar representing the number of base pair

changes along the branches, and nodes with 250% bootstrap support are shown. ‘The tree is rooted

with Psathyrella candolleana (Psathyrellaceae), and the GenBank accession numbers are given for

all taxa included in the tree.

Phylogenetic analysis of the LSU nrRNA gene alignment places our

collection of Ar. columellata within Agaricus (Agaricaceae; Fic. 2); therefore,

we transfer Araneosa columellata to this genus here as Agaricus columellatus.

While our LSU tree (Fic. 2) shows Ag. columellatus as sister to Ag. aridicola

(= Gyrophragmium dunalii), ITS sequence data (tree not shown) do not

support a sister relationship for these two taxa, which share only 95% sequence

similarity in the ITS region. Of the several Australian secotioid Agaricus species

reported in the study of Lebel & Syme (2012), Ag. columellatus was most closely

related to Ag. wariatodes; however, ITS sequence similarity between these two

taxa was likewise very low (95%). Thus our study suggests yet another example

of the independent origin of a unique secotioid fruiting body within the genus

Agaricus and of the angiocarpic form more broadly across the Agaricaceae.

The distribution of Ag. columellatus is restricted to the desert regions of the

southwestern U.S.A. and northern Mexico, with reports from the environs of

Tucson and Nogales, Arizona, as well as Hermosillo in Sonora, Mexico (Long

1941, Esqueda et al. 1998, Moreno et al. 2007). The recorded range extends

Agaricus columellatus comb. nov. (U.S.A.) ... 109

a linear distance of approximately 400 km (north to south), while this study

expands the distribution roughly another 170 km northward to the area of

Globe, Arizona. This distribution essentially spans the western edge of the

Sonoran Desert, though the area surrounding Nogales is technically considered

outside of its boundary (Brown 1994). More extensive collecting in the area

will almost certainly extend the range westward within the Sonoran Desert,

and potentially eastward into the Chihuahuan Desert. The occurrence of

Ag. columellatus within the arid-lands of North America reinforces the concept

that selective pressure of xeric climates may have driven the evolution of

angiocarpic forms from their agaricoid ancestors (Thiers 1984, Lebel & Syme

2012).

Acknowledgments

Specimen data were provided by institutions participating in the Macrofungi

Collections Consortium (NSF ADBC 1206197) project, which have been made

available through the Mycology Collections data Portal (http://mycoportal.org). The

authors thank Drs Bradley R. Kropp (Utah State University, Logan) and Else C. Vellinga

(University of California, Berkeley) for presubmission review.

Literature cited

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MY COTAXON

January-March 2016—Volume 131, pp. 111-121

http://dx.doi.org/10.5248/131.111

Morphological traits, DELTA system, and molecular analysis for

Gibellula clavispora sp. nov. from China

WAN-HAO CHEN’, YAN-FENG HAN’, ZONG-QI LIANG” & XIAO ZOU!

‘Institute of Fungal Resource, College of Life Sciences &

?Institute of Entomology, College of Agriculture

Guizhou University, Guiyang, Guizhou 550025, China

* CORRESPONDENCE TO: Zqliang2@hotmail.com

AsstrAct — Gibellula clavispora, a new fungus collected in Changbai Mountain of Jilin

Province, China, is illustrated and described based on morphological traits, the DELTA

(DEscription Language for TAxonomy) system, and molecular analysis. Gibellula clavispora

is characterized by cylindrical synnemata, aspergillate conidiophores with an apical vesicle,

obovoid metulae (8.6-10.8 x 2.2 um), clavate phialides (5.4-6.5 x 1.1-2.2 um), distinctly

clavate conidia, and the absence of Granulomanus-synanamorphic conidiogenous cells.

KEY WORDS — araneogenous fungus, numerical classification, phenotypic traits, phenetic

tree, Splitstree

Introduction

Cavara (1894: ) introduced the genus Gibellula with G. pulchra Cavara

as the type species. Gibellula species are highly specialized and apparently

obligate parasites of spiders with a worldwide distribution (Samson & Evans

1992). Since the genus was established, several more species have been

described on spiders (Petch 1932; Mains 1950; Samson & Evans 1973, 1977;

Humber & Rombach 1987). When Samson & Evans (1992) described four

new Gibellula species from infected spiders collected in South America, they

thoroughly discussed the taxonomy, host specificity, habitats, and teleomorph

connections and presented a key to the nine accepted Gibellula species.

Tzean et al. (1997, 1998), who conducted an extensive study of the insect

and spider pathogens from Taiwan since 1989, described three new Gibellula

taxa, including G. clavulifera var. major, G. unica, and G. dimorpha. Although

112 ... Chen & al.

more than 30 names have been published, most are treated as synonyms

(http://www.indexfungorum.org/Names/Names.asp).

After a survey of the synnematous entomogenous fungi in China, we

reported 15 species of Gibellula (Chen et al. 2014), using morphological

characters as the main identification criteria in the absence of any significant

body of gene sequences in GenBank or other genomic databases. In order

to make the taxonomy of Gibellula more effective and rational, additional

taxonomic approaches were required for identifying a new species in Gibellula.

DELTA (DEscription Language for TAxonomy), a computer program

developed for botanists (Dallwitz 1980) to generate taxonomic output

and interactive identification, is not yet widely used by mycologists. The

morphology-based DELTA expert system offers many advantages over the more

traditional taxonomic approaches for animals, plants, and microbes because it

uses digital standardization of morphological characters to determine which

are suitable for taxonomic identification (Li et al. 1993; Li 1996; Chang et al.

2000; Carney 2003; Chen & Chen 2008; Han et al. 2009, 2010) and international

communication (Chen et al. 2000a,b). We have been successful in classifying

Gibellula species using the DELTA system (Chen et al. 2014).

In this paper, we describe and illustrate a new Gibellula species, Gibellula

clavispora, which we identified by combining a classical morphological

approach with the DELTA system and ITS sequence analysis.

Materials & methods

Collection and morphological examination

A Gibellula specimen (GZUIF07815) was obtained from a naturally infected spider

collected on Changbai Mountain, Jilin Province, China, in July 2007. The specimen was

oven dried at 40°Cto prevent growth of fungal contaminants. Snippets of outer layer tissue

from the delaminated synnemata in the dried specimen were mounted in lactophenol

cotton blue solution and examined morphologically under a light microscope following

procedures set forth by Liang et al. (2005). The fungus is deposited in the Institute of

Fungal Resource, Guizhou University, Guiyang, China (GZUIF).

DNA extraction, amplification, and sequencing

DNA was extracted from the specimen according to Tigano-Milani et al. (1995) and

stored at -20°C. Tag enzyme and dNTP were obtained from Shanghai Sangon. The

internal transcribed spacer region (ITS1+5.8S rDNA+ITS2) was amplified by polymerase

chain reaction (PCR) using the primers ITS5 (5’-GGTGAGAGATTTCTGTGC-3’) and

ITS4 (5’-TCCTCCGCTTATTGATATGC-3’)(Han et al. 2013). After initial denaturation at

94°C for 5 min, the DNA was amplified for 35 cycles with denaturation at 94°C for 40 s,

annealing at 49°C for 40 s, and extension at 72°C for 1 min, followed by a final extension

at 72°C for 10 min. The PCR products were purified using the UNIQ-10 column PCR

product purification kit (no. SK1141; Sangon Biotech Co., Shanghai, China) according

Gibellula clavispora sp. nov. (China) ... 113

TABLE 1. Specimens and ITS sequences used in the phylogenetic analysis

(New sequences are in bold font.)

TAXON SPECIMEN/STRAIN COUNTRY GENBANK

Isaria farinosa (Holmsk.) Fr. CBS 262.58 Thailand AY624179

Gibellula clavispora GZUIF07815 China KJ857270

G. clavulifera var. clavulifera HN0801 China KP685596

G. leiopus GZUIFR-XS0805 China KP685597

G. pulchra XCH0827 China KP685595

Gibellula sp. BCC14505 Thailand GQ250021

Gibellula sp. NHJ13158 USA JN049864

Gibellula sp. BCC02757 Thailand GQ250020

Gibellula sp. BCMU GP01 Japan AB237661

Gibellula sp. MY2566 Thailand HM161738

Torrubiella arachnophila NHJ14150 Thailand HM161739

T. arachnophila EPF083 Thailand JX192719

T. flava NBRC 30612 Japan AB100609

T. ratticaudata ARSEF 1915 USA JN049837

to the manufacturer's protocol and sequenced with ITS5 and ITS4 at Sangon Biotech Co.

The full ITS sequence (GZUIF07815) was deposited as GenBank KJ857270.

Sequence alignment and phylogenetic analyses

Sequence data from the holotype specimen of the new species were compared with

ITS sequences obtained from GenBank. Representative sequences with similarities of

>90% are included in our analysis. Full ITS sequences from selected known allies were

also downloaded from GenBank (Benson et al. 2000). These sequences (TABLE 1) were

aligned by the CLUSTAL W program (Thompson et al. 1997) using the default settings

followed by manual refinements.

The sequences were analyzed by neighbor-joining (NJ) methods (Saitou & Nei 1987),

maximum parsimony (MP), and maximum likelihood (ML) methods using MEGA ver.

5 (Tamura et al. 2011). NJ analysis used the default parameters, with distance values of

samples calculated by the Kimura two-parameter model (Kimura 1980). ML analysis

followed the GITR+G+I model (six general time reversible substitution rates, assuming

gamma distributed with invariant sites). The MP tree was obtained using the Subtree-

Pruning-Regrafting (SPR) algorithm (Nei & Kumar 2000) with search level 1, in which

the initial trees were obtained with the random addition of sequences (ten replicates).

Bootstrap values of the three methods were obtained from 1000 replications per

analysis. Other parameters followed the default conditions. Isaria farinosa (Holmsk.) Fr.

was designated as outgroup.

A network was created by Splitstree version 4.10 (Huson & Bryant 2006) using the

sequences from the phylogenetic tree except for the outgroup. The running conditions

were P-distance, Unrooted Neighbor Net.

114... Chen & al.

DELTA system

Fifteen Gibellula species were used in the study (TABLE 2). A data matrix

(TABLE 3) was generated using published character data from species descriptions

(Chen et al. 2014). These characters were then used to generate a phenetic tree (Fic. 3;

Chen et al. 2014) in DELTA using the PCLASS program in accordance with the program

CONFOR using the Editor “todis” directives.

TABLE 2. Gibellula spp. used for constructing the DELTA database

TAXON

G. alata Petch

G. brunnea Samson & H.C. Evans

G. clavata Samson & H.C. Evans

G. clavispora

G. clavulifera var. alba Humber & Rombach

G. clavulifera (Petch) Samson & H.C. Evans var. clavulifera

G. clavulifera var. major Tzean et al.

G. curvispora Z.Q. Liang et al.

G. dabieshanensis B. Huang et al.

G. dimorpha Tzean et al.

G. leiopus (Vuill. ex Maubl.) Mains

G. mainsii Samson & H.C. Evans

G. mirabilis Samson & H.C. Evans

G. pulchra Cavara

G. unica L.S. Hsieh et al.

TABLE 3. Phenetic characters and their states for species of Gibellula.

1. Host a. spider; b. insect

2. Synnemata a. present; b. absent

3. Synnema state a. solitary; b. in pairs; c. scattered; d. clustered

4. Synnema shape a. clavate; b. cylindrical

5. Synnema tip a. bulbous; b. acuminate

6. Conidiophores a. penicillate; b. aspergillate

7. Conidiophore surface a. smooth; b. rough

8. Conidiophores bases a. pigmented; b. hyaline

9. Conidiophore length (um)

10. Vesicle a. present; b. absent or hardly developed

11. Metulae shape a. ovoid; b. ellipsoidal; c. obovoid; d. clavate; e. cylindrical

12. Phialide shape a. clavate; b. cylindrical; c. ellipsoidal

13. Phialide neck a. present; b. absent

14. Phialide length (um)

15. Phialide width (um)

16. Conidium shape a. clavate; b. ovoid; c. allantoidal; d. fusiform; e. ellipsoid

17. Conidium surface a. smooth; b. apiculate

18. Conidium length (yum)

19, Conidium width (um)

20. Granulomanus synanamorph a. present; b. absent

21. Granulomanus conidiophores a. rough; b. smooth

Gibellula clavispora sp. nov. (China) ... 115

Results

Phylogenetic analysis

For MP analysis of the 14-taxon ITS dataset (150 positions), the consistency

index was 0.892857, the retention index was 0.911756, and the composite

index was 0.814076 (0.785714) for all sites (parsimony-informative sites).

ML analysis of the same dataset produced a tree with a log likelihood of -349.60.

The ITS-based phylogram (Fic. 1) clearly supports the new species, Gibellula

clavispora, in an independent clade.

_Gibellula sp. GQ250020

| Gibellula sp. GQ250021

| T. arachnophila JX192719

96196195 Gibellula sp. AB237661

| Gibellula sp. JN049864

79/95/95 Gibellula sp. HM161738

r |G. pulchra KP685595

THTS/8A | | Torrubiella arachnophila HM161739

| G. clavulifera var. clavulifera KP685596

721-156

Torrubiella ratticaudata JN049837

>-— G. leiopus KP685597

Torrubiella flava AB100609

73/65/85

Gibellula clavispora KJ857270

'— Isaria farinosa AY624179

Fic. 1. Phylogenetic tree generated based on the ITS sequences. Statistical support values (250%)

are shown at nodes, for neighbor-joining/maximum parsimony/maximum likelihood.

Taxonomy

Gibellula clavispora Z.Q. Liang, Wan H. Chen & Y.E. Han, sp. nov. Fig, 2

MycoBank MB 810567

Differs from other Gibellula species by its cylindrical synnemata, aspergillate

conidiophores, obovoid metulae, clavate phialides, and distinctly clavate conidia.

Type: China, Jilin Province, Changbai Mountain, spider, 15 August 2007, A.Y. Liu

(Holotype, GZUIF07815).

Erymo oey: referring to the shape of conidia.

Host: a species of spider completely covered by white mycelium. SYNNEMATA

solitary, arising from the host, cylindric, slender, scattered, attenuated, and

composed of parallel, multiseptate longitudinal hyphae. HypHar branched,

hyaline, smooth. ConipiopHoreEs hyaline, septate, arising from aerial

mycelium or synnemata, scattered, 96-113 um long, septa conspicuous,

smooth or occasionally roughened along the length, narrowing abruptly to a

slender apex, and terminating in a swollen vesicle. VESICLE obovate, hyaline,

smooth-walled. PHIALIDEs clavate, smooth-walled, with a short neck, hyaline,

116 ... Chen & al.

un: Pa

Pathe

rit oy. «

Fic. 2. Gibellula clavispora (holotype, GZUIF07815) A: infected spider; B: conidiophores arising

from the synnema; C-E: conidiophores with smooth or occasionally rough surface; F: conidia.

Scale bars: C, E, F = 10um.

8.6-10.8 x 2.2 um. METULAE obovate, hyaline, smooth-walled, 7.6-8.6 x 3.2

um, borne on vesicle. Conrp1A clavate, hyaline, smooth-walled, single, 5.4-6.5

x 1.1-2.2 um, occasionally ellipsoidal, 2.2-4.3 x 1.1 um. Neither teleomorph

nor Granulomanus synanamorph observed.

CoMMENTs: Three other Gibellula species produce clavate conidia—G. alata,

G. brunnea, and G. mainsii (TABLE 4). Gibellula alata is distinguished by its short

slender synnemata terminating in a bulbous outgrowth from which a number

of conidiophores and diagnostic wing-like structure arise. Gibellula brunnea

Gibellula clavispora sp. nov. (China) ... 117

TABLE 4. Morphological comparison between Gibellula clavispora and allied species.

Seaeees SYNNEMA GRANULOMANUS PHIALIDE SHAPE CONIDIAL SHAPE

APEX SYNANAMORPH & SIZE (um) & SIZE (um)

G. alata bulbous absent clavate, oblong-oval or clavate,

9-13 x 3-4 4-9 x 2-4

G. brunnea acuminate present cylindrical, fusiform or sl. clavate,

10-13 x 3-4 8-10 x 2-2.5

G. clavispora acuminate absent clavate, clavate,

8.6-10.8 x 2.2 5.4-6.5 x 1.1-2.2

G. mainsii acuminate absent cylindrical, fusiform or sl. clavate,

10-13 x 3-4 8-10 x 2-2.5

differs from G. clavispora in its Granulomanus synanamorph state, while

G. mainsii differs in its cylindrical phialides.

G.dimorpha

G.brunnea

G.mirabilis

G.clavata

G.dabieshanensis

Le etre : G.unica I

G.pulchra

G.leiopus

- - G. clavispora B

G.mainsti

G.curvispora

Galata Cc

G.clavulifera var. clavulifera

G.clavulifera var. alba

G.clavulifera vat. major

Fic. 3. Phenetic tree generated for Gibellula spp. by DELTA.

DELTA system analysis

DELTA system analysis is a numerical character classification. It was not

granted special priority, so that all characters selected were weighted equally in

this study. The phenetic tree (Fic. 3) based on the morphological characteristics

presented in TABLE 3, separates Gibellula into several distinct groups. The

new species G. clavispora was grouped together with G. pulchra, G. leiopus,

118 ... Chen &al.

G. mainsii, and G. curvispora on a branch nested within the other species of

this genus.

Discussion

The solitary septate conidiophores of this new species are scattered on

the host body or synnemata, usually with small but distinct terminal vesicle

bearing one or two orders of the tightly clustered metulae characteristic of

Gibellula species (Humber & Rombach 1987). Gibellula is further distinguished

from allied genera by its spider-specific species characterized by septate

conidiophores that narrow abruptly to a slender apical neck (Samson et al.

1992). It is obvious that G. clavispora belongs in Gibellula and represents a new

species morphologically distinguished by cylindrical synnemata, aspergillate

conidiophores, obovoid metulae, clavate phialides, distinctly clavate conidia,

and the absence of conidiogenous cells of Granulomanus synanamorph.

The DELTA system analysis produced a clustering diagram dividing Gibellula

into two distinct groups (I-II) and three subgroups (A-C). The two groups are

separated by aspergillate (Group I) and penicillate (Group II) conidiophores,

which is consistent with the findings of Tzean et al. (1997).

Group II taxa—G. clavulifera var. clavulifera, G. clavulifera var. alba,

G. clavulifera var. major—are characterized by solitary and attenuated

synnemata, penicillate conidiophores, and the absence of vesicles.

Group I is divided into three subgroups. Subgroup A, comprising

G. dimorpha, G. mirabilis, G. clavata, G. dabieshanensis, and G. unica, is

characterized by solitary or paired, attenuated synnemata, aspergillate

conidiophores, cylindrical phialides, and fusiform conidia. Subgroup B

(G. pulchra, G. leiopus, G. clavispora, G. mainsii, G. curvispora, and G. brunnea)

is characterized by cylindrical and scattered synnemata and the absence of

a Granulomanus synanamorph. Subgroup C, containing only G. alata, is

distinguished by the flattened-globose head and wing-like extension at the

terminal apex of its synnemata.

DELTA system analysis revealed that some characters, such as conidiophore

shape and the presence/absence of vesicle and Granulomanus synanamorph,

could help us conveniently identify species. The phenetic tree places the

new species G. clavispora on an independent branch, as separated by its

clavate conidia, aspergillate conidiophores, and absence of Granulomanus

synanamorph.

The ITS-derived phylogram (Fic.1) also placed G. clavispora in an

independent clade. The phylogenetic and phenetic trees were not congruent,

possibly due to inherent differences between morphological and molecular

characters.

Gibellula clavispora sp. nov. (China) ... 119

Gibellula clavis ora KJ857270

G. leiopus KP685597

T. flava AB100609 Torrubiella ratticaudata JN049837

G. clavulifera var. clavulifera KP685596

G. sp. GQ250021.. 7. arachnophila HM161739 T. arachnophila 3X192719

G. sp. HM161738 G. sp. JN49864 G. pulchra KP685595_" G. sp. AB237661 —_G. sp. GQ250020

Fic. 4. Reconstruction of the Neighbor-Net network from taxa in Fie. 1.

We investigated the incongruence in the horizontal processes using

Splitstree4 (Huson & Bryant 2006), which produced a recomputed neighbor-

net (Fic.4) with a 99.999 fit and 99.999 LS fit. The result revealed an ambiguous

signal present between G. clavispora and other species, with G. clavispora on a

separate branch in the neighbor-net. Combining morphology and phylogenetic

tree, Neighbor-Net analyses support Gibellula clavispora as a new taxon in

Gibellula.

Acknowledgements

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

Prof. Richard A. Humber & Prof. Bo Huang. We thank Prof. A.Y. Liu for collecting the

specimen and providing the ecological record about this fungus. We also warmly thank

Dr. S.R. Pennycook and Dr. L.L. Norvell for editorial review and revisions. The research

is supported by Major Program of National Natural Science Foundation of China

(31093440, 31493010 & 31493011), the National Basic Research Priorities Program of

China (2013FY110400) and Youth Foundation of Guizhou Province (No. 2013-05).

120 ... Chen &al.

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MY COTAXON

January-March 2016—Volume 131, pp. 123-133

http://dx.doi.org/10.5248/131.123

Olpitrichum sphaerosporum: a new USA record

and phylogenetic placement

DeE-WEI L1”?, NEIL P. SCHULTES? & CHARLES VOSSBRINCK*

"The Connecticut Agricultural Experiment Station, Valley Laboratory,

153 Cook Hill Road, Windsor, CT 06095

?Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University,

Nanjing, Jiangsu 210037, China

*The Connecticut Agricultural Experiment Station, Department of Plant Pathology and Ecology,

123 Huntington Street, New Haven, CT 06511-2016

‘The Connecticut Agricultural Experiment Station, Department of Environmental Sciences,

123 Huntington Street, New Haven, CT 06511-2016

* CORRESPONDENCE TO: neil.schultes@ct.gov

ABSTRACT — Olpitrichum sphaerosporum, a dimorphic hyphomycete isolated from the

foliage of Juniperus chinensis, constitutes the first report of this species in the United States.

Phylogenetic analyses using large subunit rRNA (LSU) and internal transcribed spacer (ITS)

sequence data support O. sphaerosporum within the Ceratostomataceae, Melanosporales.

KEY worps — asexual fungi, Chlamydomyces, Harzia, Melanospora

Introduction

Olpitrichum G.F. Atk. was erected by Atkinson (1894) and is typified by

Olpitrichum carpophilum G.F. Atk. Five additional species have been described:

O. africanum (Saccas) D.C. Li & TY. Zhang, O. macrosporum (Farl. Ex Sacc.)

Sumst., O. patulum (Sacc. & Berl.) Hol.-Jech., O. sphaerosporum, and O. tenellum

(Berk. & M.A. Curtis) Hol.-Jech. This genus is dimorphic, with a Proteophiala

(Aspergillus-like) synanamorph.

Chlamydomyces Bainier and Harzia Costantin are dimorphic fungi also

with a Proteophiala synanamorph (Gams et al. 2009). Melanospora anamorphs

comprise a wide range of genera including Acremonium, Chlamydomyces,

124 ... Li, Schultes & Vossbrinck

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Phylogeny of Olpitrichum sphaerosporum, new to U.S.A. ... 125

Harzia, Papulaspora, Paecilomyces, Phialophora, and Proteophiala (Ciferri

1958, Kendrick & DiCosmo 1979, Cannon & Hawksworth 1982, Rehner &

Samuels 1995, Hawksworth et al. 1999, Davey et al. 2008, Kirk et al. 2008, Gams

et al. 2009). Some Papulaspora species reportedly also have a Proteophiala

synanamorph (Gams & Domsch 1969, Domsch et al. 1980, Seifert et al.

2011). The phylogenetic relationship among genera that have a Proteophiala

synanamorphic state remains uncertain.

No teleomorph for Olpitrichum has yet been reported, and a search of

GenBank showed no DNA sequence data for Olpitrichum. Here we present

the LSU and ITS DNA sequences for Olpitrichum sphaerosporum. Among the

genera sharing the Proteophiala synanamorph, we found no DNA sequence data

for Chlamydomyces, two ITS sequences for Harzia acremonioides, one ITS and

one LSU for H. cameroonensis, and one LSU for Papulaspora sepedonioides. The

systematic assignment of Olpitrichum is ambiguous, except that the members

of this genus belong to the asexual fungi of Pezizomycotina, Ascomycota (Index

Fungorum 2015).

Several studies have been conducted to elucidate the phylogenetic

relationships of Melanospora and its allies (Rehner & Samuels 1995, Zhang &

Blackwell 2002, Zhang et al. 2006, Hibbett et al. 2007). These studies having

indicated Melanospora as a sister taxon to the Ceratostomataceae (Zhang

& Blackwell 2002, Hibbett et al. 2007), the new order Melanosporales was

proposed in Hibbett et al. (2007) to accommodate Melanospora and its allies.

Phylogenetic analysis using LSU sequence data indicated that Papulaspora

sepedonioides Preuss also belongs to Melanosporales (Davey et al. 2008).

Olpitrichum sphaerosporum, isolated from the foliage of Juniperus chinensis

in Windsor, Connecticut, constitutes the first record of this fungus in the

United States. Here we shed new light on the placement of O. sphaerosporum

based upon ITS and LSU DNA sequence analyses.

Materials & methods

A sample of Juniperus chinensis with a purported foliar disease was submitted to the

Inquiry Office of The Connecticut Agricultural Experiment Station Valley Laboratory

for diagnosis in February 2014. The sample was placed in a plastic bag with a piece of wet

paper towel for three days at room temperature. Following incubation, a fungus grew on

the necrotic part of the foliage. A sterile needle was used to transfer the fungal spores

to a 2% malt extract agar (MEA) plate. Fungal isolates were grown on MEA at 25°C for

7 days. Conidiophores and conidia were mounted in 85% lactic acid. All microscopic

observations were made under an Olympus BX40 microscope with Nomarski differential

interference contrast optics. Photomicrographs were taken with an Olympus Microfire

digital camera (Goleta, CA). Measurements of the fungal structures were statistically

analyzed for means and standard deviations with 95% confidence interval of means.

126 ... Li, Schultes & Vossbrinck

DNA was extracted from a petri plate-grown colony according to the

procedure in ZR Fungal/Bacterial DNA MicroPrep Kit (Zymo Research, Irvine,

CA, USA). Oligonucleotides ITS1 (5’-rccetacGTGAacctGcGGc-3’) and ITS4

(5’-TCCTCCGCTTATTGATATGC-3’) were used with isolated genomic DNA to amplify a

fragment corresponding to the small subunit rDNA internal transcribed spacer (ITS)

region by polymerase chain reaction (PCR) (Gardes & Bruns 1993). Oligonucleotides

5.8SR (5° TCGATGAAGAACGCAGCG 3’) and LR7 (5’-TACTACCACCAAGATCT-3’) were

used to amplify the ITS region and a portion of the large subunit rDNA region by PCR

(Gargas & DePriest 1996). The resulting PCR products were purified using QIA quick

PCR Purification columns (Qiagen, Valencia, CA, USA). The ITS PCR product was

sequenced using oligonucleotides ITS1, ITS4, ITS2 (5’-GCTGCGTTCTTCATCGATGC-3’),

and ITS3 (5’-GCATCGATGAAGAACGCAGC-3’). The LSU PCR product was sequenced using

primers LROR (5’-ACCCGCTGAACTTAAGC-3’), LR7 (5’-TACTACCACCAAGATCT-3’),

LR5 (5’-TCCTGAGGGAAACTTCG-3’), LR3R (5’-GTCTTGAAACACGGACC-3’), and LR16

(5’-TTCCACCCAAACACTCG-3’). DNA sequence information was deposited to GenBank

as accession KT221061 for the ITS region and KT221062 for the LSU region.

Sequence similarity searches and comparisons were conducted using MegaBLAST

(Wheeler et al. 2003) against the NCBI nucleotide database. Harzia, Olpitrichum,

and several additional allied genera were chosen for phylogenetic analysis (TABLE 1).

Xylaria hypoxylon (ATCC 42768) was designated as outgroup.

Sequences were aligned using ClustalW (Thompson et al. 1994). Phylogenetic

analyses were conducted using the neighbor joining and maximum likelihood

procedures in MEGA6 (Tamura et al. 2013). A bootstrap test was carried out with 1000

replicates.

Results

Taxonomy

Olpitrichum sphaerosporum Matsush., Icon. Microfung. Matsush. Lect.: 103

(1975) Bre. it

Cotonigs on MEA effuse, cottony, pale ochraceous to yellow-buff, attaining

100 mm in diam. at 25°C in 7 days; aerial hyphae abundant; some mycelia

reaching the lid of the plate in 7 days. CONIDIOPHORES erect, simple or sparsely

branched at right angles, (100-—)150-—400(-500) um long, 4-6(-7) um wide at the

base and the middle, 4-6 septate, thick-walled, smooth. Terminal fertile part of

conidiophores 5-15 um long, often extending, with 3-6 denticle conidiogenous

cells. Conidiogenous loci denticles arranged in irregular verticils, tapering to

the apex, 4-15 um long, 7-9 um wide at the base, rarely constricted at the base,

often branched and developing new denticles or extending and forming new

conidiophores. Conip1A unicellular, solitary, subhyaline to pale ochraceous,

spherical or subspherical, smooth or verruculose, sometimes with a small

papilla at the base, (18—)19-23(-25) x (15-)18-22(-25) um (n = 30), with wall

0.5-1 um thick. Conidial secession rhexolytic.

Phylogeny of Olpitrichum sphaerosporum, new to U.S.A. ... 127

Fic. 1. Olpitrichum sphaerosporum (UAMH 11865) a. Conidiophore and conidia; b. Conidiogenous

cells and an immature conidium; c. Conidia; d. Proteophiala synanamorph. Scale bars: a = 20 um;

b=5um;c,d=10 um.

Synanamorph Proteophiala (Aspergillus-like), scattered in basal hyphae;

conidiophores colorless, smooth, phialides 6-7.5 x 3-4 um, conidia catenate,

obovoid with a truncate base, colorless, smooth, 2-4 x 1.5-3 um in diam.

128 ... Li, Schultes & Vossbrinck

MATERIAL EXAMINED: USA, CONNECTICUT, Windsor, 41°51’04”N 72°39’39’W, dead

foliage of Juniperus chinensis L. (Cupressaceae), Feb 24, 2014, coll. De-Wei Li, UAMH

11865 (DWL2014-6).

HostTs/SUBSTRATES: saprobe, on dead foliage of Juniperus chinensis, dead

pericarp of Camellia japonica L., decaying leaf of palm.

DISTRIBUTION: Japan (Matsushima 1975), Mexico (Heredia Abarca & Arias

Mota 2008), USA.

Note: Olpitrichum sphaerosporum represents a new record for the USA.

Phylogenetic analyses

Maximum likelihood and neighbor joining analyses produced the same

results regarding the relationships within the Melanosporales. LSU sequence

analyses indicated that Olpitrichum sphaerosporum and members of the

Ceratostomataceae, Melanosporales—Harzia, Melanospora, Papulaspora,

Sphaerodes, Vittatispora—form a monophyletic lineage (Fic. 2), clearly

Melanospora tiffanyae AY 15630

Melanospora tiffanyae FJ748915

72 Melanospora zamiae AY046579

Harzia cameroonensis KF777216

@ Olpitrichum sphaerosporum KT221062

Papulaspora sepedonioides EU518666

Melanosporales

Sphaerodes compressa AY015633

Sphaerodes fimicola AY015628

Melanospora brevirostris AY015627

Sphaerodes quadrangularis GQ354530

_ Melanospora singaporensis AY015629

Vittatispora coorgii DQ017375

Scortechinia conferta AY695272 _]Coronophorales

Chaetosphaerella phaeostroma AY695264

Coronophorales

Bertia tropicalis AY695263

Hirsutella stilbelliformis var. myrmicarum GQ866967 _]Hypocreales

Tolypocladium capitatum AY489721 “]Hypocreales

Corollospora maritima AF491260 |] Hypocreales

Petriella setifera DQ470969

Microascales

Microascus trigonosporus U47835

Xylaria hypoxylon U47841 |] Xylariales

0.02

Fic. 2. LSU sequence maximum likelihood analysis of Olpitrichum sphaerosporum and related

species (1000 bootstrap replicates; bs values indicated). Species names precede sequence accession

numbers. Xylaria hypoxylon U47841 represents the outgroup. The scale bar indicates the number

of expected changes per site.

Phylogeny of Olpitrichum sphaerosporum, new to U.S.A.... 129

Harzia acremonioides HQ288051

73 | | Harzia acremoniocides HQ698593

@ Olpitrichum sphaerosporum KT221061

Harzia cameroonensis KF777163

Sphaerodes fimicola JQ034510

Melanospora pascuensis AJ011312

Sphaerodes pseudofimicola JN709487

Melanospora tiffanyae FJ748921

Xylaria hypoxylon AY327477

0.05

Fic. 3. ITS sequence maximum likelihood analysis of Olpitrichum sphaerosporum and allied

fungi (1000 bootstrap replicates; bs values indicated). Species names precede sequence accession

numbers. Xylaria hypoxylon AY327477 represents the outgroup. The scale bar indicates the number

of expected changes per site.

supporting Olpitrichum sphaerosporum within the Ceratostomataceae.

ITS sequence analyses resolved the phylogenetic relationship between Olpitrichum

sphaerosporum and Harzia species more finely and grouped them in the same

clade, indicating that O. sphaerosporum and Harzia (Fic. 3) are congeneric.

Discussion

This is the first report of Olpitrichum sphaerosporum from the United States.

Among the six Olpitrichum species, only O. sphaerosporum develops spherical

conidia. The Connecticut isolate differs slightly from the Japanese type isolate

(Matsushima 1975) in having longer conidiophores.

The Connecticut isolate grows very slowly on DG18 media (less than 2 mm

at 25°C in 7 days). It completely lost viability when stored in water at 4°C for

6 months, and only a few conidia survived when cryopreserved at -70°C for a

year. This poses a challenge for culture maintenance.

Olpitrichum is morphologically similar to Chlamydomyces and Harzia

(Holubova-Jechova 1974, Seifert et al. 2011) and all display a Proteophiala

synanamorph (Seifert et al. 2011). Chlamydomyces and Harzia are both

connected to the same sexual genus, Melanospora (Rehner & Samuels 1995).

As noted above, no rDNA sequence information for Olpitrichum and

Chlamydomyces was available prior to this report. Both our phylogenetic

130 ... Li, Schultes & Vossbrinck

analyses (Fics 2, 3) and the morphologies of the asexual and sexual states

suggest that phylogenetically Olpitrichum, Harzia, Melanospora, and Sphaerodes

are closely related.

Harzia Costantin was erected by Costantin (1888) and typified by

H. acremonioides (Harz) Costantin. Harzia acremonioides, H. cameroonensis,

and Olpitrichum sphaerosporum were included in the phylogenetic ITS

sequence analysis. As our results reveal that Olpitrichum and Harzia are closely

related (Fic. 3), we conclude that either Harzia should remain a paraphyletic

genus or that Olpitrichum sphaerosporum should be transferred to Harzia. With

no culture of the type species, Olpitrichum carpophilum, currently available,

it is premature to merge the two genera until the type specimen or verifiable

material from the type species is available for DNA sequencing.

Previous studies have placed Harzia, Melanospora, Sphaerodes, Vittatispora,

and Papulaspora sepedonioides within Ceratostomataceae, Melanosporales

(Zhang & Blackwell 2002, Chaudhary et al. 2006, Davey et al. 2008, Lackner

et al. 2014, Index Fungorum 2015), and Seifert et al. (2011) speculated that

Olpitrichum might belong to Ceratostomataceae, Melanosporales according to

its morphology. Our LSU sequence analysis groups Olpitrichum sphaerosporum

in the same clade as the aforementioned genera (Fic. 2) and clear molecular

evidence supports Olpitrichum sphaerosporum within the Ceratostomataceae,

Melanosporales.

The studies of Zhang & Blackwell (2002) and Chaudhary et al. (2006)

grouped Sphaerodes and Melanospora in the same clade, and Chaudhary et al.

(2006) suggested that Sphaerodes and Melanospora are congeneric based on

their phylogenetic analysis. Neither study included anamorphic genera, such as

Olpitrichum and Harzia. Since the anamorph of Sphaerodes remains unknown

and no DNA sequence data are available for the type species, Sphaerodes

episphaerium (W. Phillips & Plowr.) Clem., the true phylogenetic relationships

of Sphaerodes with Melanospora and other closely related asexual genera

(Olpitrichum and Harzia) remain to be determined.

Olpitrichum, Chlamydomyces, and Harzia share the same Proteophiala

synanamorph. However, the phylogenetic relationship of Chlamydomyces with

Olpitrichum and Harzia remains unknown. From our results we could not

determine the relationships among Melanospora, Sphaerodes, and Papulaspora.

Papulaspora is a phylogenetically heterogeneous genus, and its members have

been linked to numerous teleomorphs including Ascobolus, Chaetomium,

Cubonia, Hypomyces, Microthecium, Lophium, Melanospora, Neurospora, and

Sordaria (Seifert et al. 2011). Papulaspora sepedonioides is the type species of

the genus. Thus, Papulaspora taxa not phylogenetically related to Melanospora

should be excluded from Papulaspora and moved to other taxonomic groups.

Phylogeny of Olpitrichum sphaerosporum, new to U.S.A.... 131

However, as Melanospora is apparently also polyphyletic, further studies on

these genera are necessary.

Acknowledgments

The authors express their sincere gratitude to Dr. Bryce Kendrick and Dr. Rafael

E Castaneda Ruiz for their critical reviews of the manuscript. The authors thank

Dr. Elisabeth Cowles, Eastern Connecticut State University, and Yale University Library

for their assistance in obtaining otherwise unavailable literature. The authors are very

grateful to Dr. James A. LaMondia of The Connecticut Agricultural Experiment Station

(CAES) for his editorial help and the Inquiry Office of CAES Valley Laboratory for

providing the sample. Dr. Lorelei Norvell’s editorial review and Dr. Shaun Pennycook’s

nomenclature review are appreciated.

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MY COTAXON

January-March 2016—Volume 131, pp. 135-139

http://dx.doi.org/10.5248/131.135

Sarcogyne saphyniana sp. nov.,

a saxicolous lichen from northwestern China

LAzzAT NurTAr’, KERRY KNUDSEN? & ABDULLA ABBAS"

‘College of Life Science, Arid Land Lichen Research Center of Western China,

Xinjiang University, Urumdi, 830046 , P. R. China

*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: zxg_lichen@163.com

ABSTRACT — A new species of Sarcogyne in the badiofusca group is described from northwest

China. It differs from Acarospora badiofusca in having an algal layer interrupted by wide

hyphal bands.

KEY worps — Acarosporaceae, diversity, phylogeny, taxonomy

Introduction

The crustose lichen genus Sarcogyne Flot. (Acarosporaceae) is characterized

by black or red lecideine apothecia with or without a carbonized exciple,

a non-carbonized epihymenium, a hymenium with or without branching

paraphyses, and asci containing 50-100 or more simple hyaline ascospores

(Knudsen & Standley 2007, Fletcher & Hawksworth 2009). The thallus may

be endolithic or epilithic. Species occur on non-calcareous or calcareous

rock, on soil in biological soil crusts, and one species (S. lapponica (Ach.)

K. Knudsen & Kocourk.) is known only from a historic collection on wood

(Knudsen & Kocourkova 2008). The genus contains approximately 40 species,

by our estimate. However, it contains many more names than species, because

the monographer Magnusson included in Sarcogyne many species with a

carbonized epihymenium later transferred to Polysporina Vézda, but which

actually belong in Acarospora A. Massal. where they have yet to be transferred

(Magnusson 1935 a, & b; Westberg et al. 2015). Recent phylogenetic studies have

shown that many current Acarospora species should be referred to Sarcogyne,

136 ... Nurtai, Knudsen & Abbas

including such well-known species as Acarospora glaucocarpa (Ach.) Kérb.,

A. cervina (Ach.) A. Massal., and A. badiofusca (Nyl.) Th. Fr. (Reeb et al. 2004,

Miadlikowska et al. 2014, Westberg et al. 2015). However, the backbone of the

most recently published phylogenetic tree is weak, suggesting that Sarcogyne is

not monophyletic; it is expected that the genus will eventually be divided into

several genera (Westberg et al. 2015). For the sake of nomenclatural stability,

some current Acarospora species (such as A. badiofusca) have not yet been

transferred to Sarcogyne, although they clearly do not belong to Acarospora

Ss. Str.

In this paper we describe a new Sarcogyne species from the mountains of

Xinjiang in northwestern China that belongs to the A. badiofusca group, a

group characterized as having algal layers that may or may not be interrupted

by hyphal bundles (Knudsen et al. 2014). The only member of this group that

has thus far been sequenced is A. badiofusca, which phylogenetic analyses

place at the end of a long branch with A. cervina (which has an interrupted

algal layer) but embedded within Sarcogyne (Westberg et al. 2015). Our new

species, which has lecideine apothecia and a non-carbonized epihymenium,

anatomically also fits the current concept of Sarcogyne.

Materials & methods

The examined specimens (including the holotype) are preserved in Arid Land

Lichen Research Center of Western China, Xinjiang University, Urumqi, China (XJU-

NALH). They were morphologically examined with Olympus STM7 microscope and

photographed taken with a Nikon DS-Fi2 and Canon PC1200. Measurements were

made in water and amyloid reaction was tested with Lugol’s (I). The lichen was checked

for secondary metabolites in Solvent C using thin layer chromatography techniques

(TLC) (Culberson, 1972).

Taxonomy

Sarcogyne saphyniana A. Abbas, Nurtai & K. Knudsen, sp. nov. FIGS 1, 2

MycoBank MB 815828

Differs from Acarospora badiofusca by its algal layer divided by wide hyphal bands into

palisades and clumps of algal cells.

Type — China. Xinjiang, Tianshan Grand Canyon, 43°18.89’N 87°19.51’E, alt. 2257

m, sandstone, 10 Jul 2014, A. Abbas, B. Memet, L. Nurtai 20140414 (Holotype, XJU-

NALH).

EtymMoLocy — Sarcogyne saphyniana is named in honor of Saphyn, a wise man from

Tartar Republic who in 1910 came to Xinjiang, where he helped the poor people and

established a school.

Thallus squamulose to areolate, the areoles or squamules round to angular,

sometimes imbricate, 0.3-2.1 mm wide, ca 400-800 um thick. Upper surface

Sarcogyne saphyniana sp. nov. (China) ... 137

F 1G. 1. Sarcogyne saphyniana (holotype): Thallus with apothecia. Scale bar = 1 mm.

usually pale brown, sometimes dark brown, smooth, epruinose, sometimes

slightly shiny. Lower surface corticated and dark, with a stipe. Upper cortex

35-55 um thick, its upper layer reddish brown, lower layer hyaline. Algal

layer, <280 um thick, interrupted by wide hyphal bands, algal cells 7.5-18 um

in diam. Medulla white, prosoplectenchymatous, <385 um thick, continuous

with stipe. Apothecia 1-4 per thallus per unit, round to irregular, 0.3-1.4 mm

wide, emerging from the thallus as they mature and projecting above the

thallus. Disc reddish-brown, smooth to fissured, margin formed of the exciple,

color blackish-brown or same color as disc. Exciple <45 um wide, expanding

around the disc <100 um wide. Hymenium hyaline, 66-80 um high, I+

blue. Epihymenium reddish-brown, <18 um high. Paraphyses 2.0-2.5 um

wide, apices reddish-brown, barely expanded. Asci clavate, 40-7 x 2-17 um.

Ascospores hyaline, simple, broadly ellipsoid, 3.5-5.5 x 2-2.5 um (N = 50).

Subhymenium <50 um high. Hypothecium, hyaline, <45 um high. Pycnidia

not observed. Lacking secondary metabolites.

ADDITIONAL SPECIMENS EXAMINED — CHINA. XINJIANG. Altay Mountains, Two-River

Source Nature Reserve, 47°35’N 88°39’E, alt. 1075 m, shale, 18 May 2014, A. Abbas,

G. Sahedat 20140412 (XJU-NALH); Altay Mountain Grape Scenic Spot, 47°48’N 88°04’E,

alt. 917 m, shale, 16 May 2014, A. Abbas, G. Sahedat 20140418 (XJU-NALH); Tianshan

Mountain, Xiao Q Zi Village, 44°08’N 81°42’E, alt. 1490 m, sandstone, 30 Jul 2007,

A. Abbas 20077730 (XJU-NALH); Shi Ren Gou, 43°45’N 87°50’E, alt. 1345 m, 24 Jul 2013,

sandstone, A. Abbas, G. Nazarbek, L. Nurtai 20137224 (XJU-NALH).

Sarcogyne saphyniana belongs to the badiofusca group (Knudsen et al.

2014) characterized by emergent lecideine apothecia with a non-carbonized

margin. Acarospora badiofusca has a similarly low hymenium but differs in its

uninterrupted algal layer. Sarcogyne saphyniana has an algal layer interrupted

138 ... Nurtai, Knudsen & Abbas

layer in thin section of the thallus; D. Thallus with apothecia. Scale bars: A= 50 um; B = 20 um;

C= 100 um; D= 1mm.

by wide hyphal bands as does another member of the badiofusca group, the

European species A. irregularis H. Magn., which differs in its higher (80-140 um)

hymenium (Knudsen et al. 2014). The third member of the badiofusca group,

A. boulderensis H. Magn. from North America, is distinguished by its higher

hymenium and an algal layer interrupted by narrow hyphal bands that do

not radically break up the algal layer into palisades and clumps of algal cells

(Knudsen et al. 2014). Acarospora cervina is distinguished from S. saphyniana

by its higher (85-100 um) hymenium, narrowly ellipsoid ascospores (4-5 x

2 um), and pruina on the margins or entire thallus (Magnusson 1929; for an

example of the typical A. cervina phenotype, see Wirth et al. 2013). All species

discussed above have an I+ blue hymenium. Of the five Sarcogyne species

previously reported in China, three have been collected in Xinjiang (Wei 1991,

Abbas 1998, 2000).

Acknowledgments

We thank our two reviewers, M. Gdkhan Halici (Erciyes Universitesi, Turkey) and

J.C. Lendemer (New York Botanical Garden, USA). The work of Kerry Knudsen was

Sarcogyne saphyniana sp. nov. (China) ... 139

financially supported by the grant “Environmental aspects of sustainable development

of society” 42900/1312/3166 from the Faculty of Environmental Sciences, Czech

University of Life Sciences Prague. The work of Abdulla Abbas was financially

supported by National Science Fund of China (2013FY110400, 30960003, 30460001).

We cordially thank Rachel M. Destree and Joshua D. Destree for editorial help.

Literature cited

Abbas A, Wu JN. 1998. Lichens of Xinjiang, Sci-Tech & Hygiene Publishing House of Xinjiang.

Urumadi. 178 p.

Abbas A, Mijit H, Tumur A, Wu JN. 2001. A checklist of the lichens of Xinjiang, China. Harvard

Papers in Botany 5(2): 359-370.

Culberson CE. 1972. Improved conditions and new data for the identification of lichen products by

a standardized thin-layer chromatographic method. Journal of. Chromatography 72: 113-125.

Fletcher A, Hawksworth D. 2009. Sarcogyne A. Massal. (1851). 829-830, in: CW Smith et al. (eds).

The Lichens of Great Britain and Ireland. British Lichen Society, Natural History Museum, U.K.

Knudsen K, Kocourkova J. 2008. A study of lichenicolous species of Polysporina (Acarosporaceae).

Mycotaxon 105: 149-164.

Knudsen K, Standley SM. 2007. Sarcogyne. 289-296, in: TH Nash III et al. (eds). Lichen Flora of

the Greater Sonoran Desert Region, Volume 3. Lichens Unlimited, Arizona State University,

Tempe.

Knudsen K, Kocourkova J, Nordin A. 2014. Conspicuous similarity hides diversity in the Acarospora

badiofusca group. Bryologist 117(4): 319-328. http://dx.doi.org/10.1639/0007-2745-117.4.319

Magnusson AH. 1929. A monograph of the genus Acarospora. Kungliga Svenska Vetenskaps-

Akademiens Handlingar, ser. 3, 7. 400 p.

Magnusson AH. 1935a. Acarosporaceae, Thelocarpaceae. 1-318, in: AE Zahlbruckner (ed.)

Rabenhorst’s Kryptogamen-Flora von Deutschland, Osterreich, und der Schweiz. 2nd edition.

Band 9. Abt. 5. Borntraeger, Leipzig.

Magnusson AH. 1935b [“1934”]. On the species of Biatorella and Sarcogyne in America. Annales

de Cryptogamie Exotique 7: 115-145.

Miadlikowska J, Kauff FE, Hégnabba F, Oliver, JC, Molnar K, Fraker E, Gaya E, Hafellner J,

Hofstetter V, Gueidan C, Otalora MAG, Hodkinson B, Kukwa M, Liicking R, Bjork C,

Sipman HJM, Burgaz AR, Thell A, Passo A, Mylly L, Goward T, Ferndndez-Brimem S,

Hestmark G, Lendemer JC, Lumbsch HT, Schmull M, Schoch CL, Sérusiaux E, Maddison

DR, Arnold AE, Lutzoni FE, Stenroos S. 2014. A multigene phylogenetic synthesis for

the class Lecanoromycetes (Ascomycota): 1307 fungi representing 1139 infrageneric

taxa, 317 genera and 66 families. Molecular Phylogenetics and Evolution 79: 132-168.

http://dx.doi.org/10.1016/j.ympev.2014.04.003

Reeb V, Lutzoni FE, Roux C. 2004. Contribution of RPB2 to multilocus phylogenetic studies of

the euascomycetes (Pezizomycotina, Fungi) with special emphasis on the lichen-forming

Acarosporaceae and evolution of polyspory. Molecular Phylogenetics and Evolution 32:

1036-1060. http://dx.doi.org/10.1016/j-ympev.2004.04.012

Wei JC. 1991 An enumeration of lichens in China. International Academic Publishers.

Westberg M, Millanes AM, Knudsen K, Wedin M. 2015. Phylogeny of the Acarosporaceae

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Diversity 73:145-158. http://dx.doi.org/10.1007/s13225-015-0325-x

Wirth V, Hauck M, Schultz M. 2013. Die Flechten Deutschlands. 2 Bd. Eugen Ulmer, Stuttgart.

1244 p.

MY COTAXON

January-March 2016—Volume 131, pp. 141-144

http://dx.doi.org/10.5248/131.141

Mirandina uncinata sp. nov.

from submerged leaves from Brazil

PATRICIA OLIVEIRA FIUZA’, JOSIANE SANTANA MONTEIRO’,

Luis FERNANDO PASCHOLATI GUSMAO! & RAFAEL E. CASTANEDA-RUIZ?

"Universidade Estadual de Feira de Santana, Depto. de Ciéncias Biolégicas, Lab. de Micologia,

Avenida Transnordestina s/n, Bairro Novo Horizonte, 44036-900, Feira de Santana, Brazil

?Inst. de Investigaciones Fundamentales en Agricultura Tropical Alejandro de Humboldt’,

Calle 1 Esq. 2, Santiago de Las Vegas, C. Habana, Cuba, C.P. 17200

* CORRESPONDENCE TO: lgusmao@uefs.br

ABSTRACT — Anewspecies, Mirandina uncinata, collected on submerged leaves of Calophyllum

brasiliense in the Brazilian semiarid region, is described and illustrated. Mirandina uncinata

is characterized by polyblastic, integrated, terminal, sympodially extended conidiogenous

cells and cylindrical-fusiform, uncinate toward the apex, 5-7-septate, pale brown conidia.

Key worps — freshwater fungi, conidial fungi, taxonomy

Introduction

An undescribed Mirandina species was collected during an investigation

of conidial fungi associated with submerged decaying leaves of Calophyllum

brasiliense in a riparian forest of Chapada Diamantina in Brazil's semiarid

region. It is described here as new.

Materials & methods

Samples of submerged leaves of C. brasiliense were collected in Piata, Bahia

State, from November 2013 to January 2015. The material was placed in plastic bags

and taken to the laboratory and processed according to Castafieda-Ruiz (2005). The

samples were washed, placed in Petri dish moist chambers, and stored in a polystyrene

box with sterile water plus glycerol for 30 days. Slide mounts were prepared in PVL

(polyvinyl alcohol and lactic acid) and measurements were made at a magnification

of x1000. Microphotographs were obtained with an Olympus microscope (model

BX51) equipped with bright field and Nomarski interference optics. The specimens and

142 ... Fiuza & al.

Fic. 1. Mirandina uncinata (ex HUEFS 216003). A. Conidia; B-D. Conidiophores and

conidiogenous cells; E. Conidiophore and conidium. Bars: A, B, D, E = 20 um; C = 10 um.

Mirandina uncinata sp. nov. (Brazil) ... 143

holotype are deposited in the Herbarium of Universidade Estadual de Feira de Santana,

Brazil (HUEFS).

Taxonomy

Mirandina uncinata Fiuza, J.S. Monteiro, R.F. Castafieda & Gusmao, sp. nov. Fic. 1

MycoBank MB813056

Differs from Mirandina flagelliformis by cylindrical-fusiform, pale brown conidia that

are mostly 5-7-septate and uncinate toward the apex.

Type: Brazil, Bahia, Piata, Serra da Tromba, 13°07’S 41°50’W, on submerged decaying

leaves of Calophyllum brasiliense Cambess. (Clusiaceae), 21. XI. 2014, coll. P.O. Fiuza

(Holotype: HUEFS 216003).

ErymMo_oey: Latin, uncinata, referring to the hooked conidia.

CoLonizs on the natural substratum effuse, hairy, hypophyllous, brown.

Mycelium mostly immersed in the substrate. CONIDIOPHORES distinct, single,

unbranched, cylindrical, straight to geniculate toward the apex, 2-7-septate,

brown at the base becoming paler to the apex, smooth, 20-100 x 3-5 um.

Conidial secession schizolytic. CONIDIOGENOUS CELLS polyblastic, integrated,

mostly terminal, sometimes intercalary, indeterminate, sympodially elongated,

slightly denticulate, brown to pale brown, 20-25 x 3-4 um, slightly cicatrized at

the loci. Conrv1a solitary, cylindrical-fusiform, attenuate, truncate at the base,

rostrate, uncinate toward the apex, (1-)5-7(-11)-septate, pale brown, smooth,

40-95 x 2-4.5 um.

ADDITIONAL SPECIMENS EXAMINED: BRAZIL, ParA, Belém, Ilha do Combu, 1°29 S

48°25 W, on submerged decaying twig 29 X. 2011, coll. J.S. Monteiro (HUEFS 216005);

Parque Estadual de Utinga, 1°25 S 48°27 W, on submerged decaying twig, 30 VII. 2012,

coll. J.S. Monteiro (HUEFS 216004).

Note: The genus Mirandina G. Arnaud ex Matsush., typified by M. corticola,

is characterized by distinct, unbranched, brown conidiophores and polyblastic,

denticulate, integrated, sympodial elongated conidiogenous cells. The

conidia are cylindrical, fusiform to obclavate, unicellular or septate, hyaline,

and subhyaline (Kirk 1986). Ten species have been accepted in Mirandina

(Castafieda-Ruiz & Kendrick 1991, Castafieda-Ruiz et al. 1997, de Hoog

1985, Kirk 1986, Matsushima 1975, 1980, 1987; Matsushima & Matsushima

1996, Paulus et al. 2003, Ma et al. 2015). Mirandina uncinata resembles

M. flagelliformis Matsush., which differs by its narrowly obclavate hyaline

conidia that are flagelliform toward the apex and slightly constricted at the

septa (Matsushima 1987, Matsushima & Matsushima 1996).

Acknowledgments

The authors express their sincere gratitude to Dr. De- Wei Li and Xiu-Guo Zhang for

their critical review of the manuscript. The authors are grateful to Programa de Pesquisa

144 ... Fiuza & al.

em Biodiversidade do Semiarido (PPBio Semiarido - CNPq/MCTI - Proc. 558317/2009-0)

for financial support. POF thanks the ‘Coordenagao de Aperfeigoamento de Pessoal de

Nivel Superior’ (CAPES) and the ‘Programa de Pds-graduacao em Botanica PPGBot/

UEFS’ JSM thank the Postdoctoral fellowship (CAPES - 071/2012). RFCR is grateful

to ‘Ciéncia sem Fronteiras’ and to Cuban Ministry of Agriculture and ‘Programa de

Salud Animal y Vegetal, project P131LH003033 for facilities. We acknowledge the

assistance provided by Dr. P.M. Kirk and Drs. V. Robert and A. Decock through the

Index Fungorum and MycoBank websites. Dr. Lorelei Norvell’s editorial and Dr. Shaun

Pennycook’s nomenclatural reviews are greatly appreciated.

Literature cited

Castaneda-Ruiz, RF. 2005. Metodologia en el estudio de los hongos anamorfos. 182-183, in: Anais

do V Congresso Latino Americano de Micologia. Brasilia.

Castafieda-Ruiz RE, Kendrick, WB. 1991. Ninety-nine conidial fungi from Cuba and three from

Canada. University of Waterloo Biology Series 35: 1-132.

Castafeda-Ruiz RF, Guarro J, Figueras MJ, Gene J, Cano, J. 1997. More conidial fungi from La

Gomera, Canary Islands, Spain. Mycotaxon 65: 121-131.

Hoog, GS de. 1985. Taxonomy of the Dactylaria complex. IV. Dactylaria, Neta, Subulispora and

Scolecobasidium. Studies in Mycology 26: 1-60.

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from the British Isles. Transactions of the British Mycological Society 86: 409-428.

http://dx.doi.org/10.1016/S0007-1536(86)80185-1

Ma YR, Xia JW, Zhang XG, Castafieda-Ruiz, RF. 2015. New species of Phaeomonilia and Mirandina

from dead branches in China. Mycotaxon 130(3): 775-781. http://dx.doi.org/10.5248/130.775

Matsushima T. 1975. Icones microfungorum a Matsushima lectorum. Nippon Printing Co, Osaka.

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mycological memoirs no. 1. Matsushima Fungus Collection, Kobe, Japan.

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Kobe, Japan.

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Paulus B, Gadek P, Hyde, KD. 2003. Two new species of Dactylaria (anamorphic fungi) from

Australian rainforests and an update of species in Dactylaria sensu lato. Fungal Diversity

14: 143-156.

MYCOTAXON

January-March 2016—Volume 131, pp. 145-152

http://dx.doi.org/10.5248/131.145

Pleurothecium bicoloratum &

Sporidesmiopsis pluriseptata spp. nov. from Brazil

JOSIANE SANTANA MONTEIRO’, LUIS FERNANDO PASCHOLATI GUSMAO*

& RAFAEL E CASTANEDA-RUIZ?

' Universidade Estadual de Feira de Santana,

Avenida Transnordestina, s/n, Novo Horizonte, 44036-900, Feira de Santana, Brazil

? Instituto de Investigaciones Fundamentales en Agricultura Alejandro de Humboldt’ (INIFAT),

Calle 1 Esq. 2, C.P. 17200, Santiago de Las Vegas, C. Habana, Cuba

“ CORRESPONDENCE TO: gusmao@uefs. br

ABSTRACT — Two new species of microfungi, Pleurothecium bicoloratum and Sporidesmiopsis

pluriseptata, found on decaying plant materials are described and illustrated. Pleurothecium

bicoloratum, also collected in Venezuela, is distinguished by broad allantoid, bicolored,

3-septate, smooth conidia with dark olivaceous-brown central cells and hyaline ends;

S. pluriseptata, thus far known only from Brazil, is characterized by obclavate, smooth, dirty

brown conidia with up to 23 septa.

KEY worps — asexual fungi, taxonomy, leaf litter

Introduction

During a survey of hyphomycetes associated with plant litter from the

Amazon forest (Amapa state) and semi-arid region (Bahia state), two

undescribed Pleurothecium and Sporidesmiopsis species were collected. The

specimens, which could not be assigned to any species currently accepted in

those genera, are described here as new.

Materials & methods

Leaf and wood litter samples were transported to the laboratory where they were

placed in Petri dish moist chambers and stored in a 170 L polystyrene box with 200 mL

sterile water plus 2 mL glycerol, at 25°C for 30 days (Castafieda-Ruiz 2005). Mounts

were prepared in polyvinyl alcohol, lactic acid, and phenol (PVL) and examined and

146 ... Monteiro, Gusmao & Castafieda-Ruiz

photographed using an Olympus BX51 microscope equipped with bright field and

Nomarski interference optics. All measurements were made at a magnification of

x1000. The type specimens are deposited in the Herbarium of Universidade Estadual de

Feira de Santana, Brazil (HUEFS).

Taxonomy

Pleurothecium bicoloratum R.F. Castafieda, J.S. Monteiro & Gusmao sp. nov.

FIGS 1-3A

MycoBank MB 813040

Differs from all other Pleurothecium species by its bicolored conidia with dark olivaceous

brown to black central cells and hyaline end cells.

Type: Brazil, Bahia State, Abaira, Distrito de Catolés, Mata do Cigano, 13°15’S 41°54’W,

on decaying wood of an unidentified plant, 09.1.2015; coll. J.S. Monteiro (Holotype:

HUEFS 215982).

ErymMo oey: Latin, bicoloratum, referring to the bicolored conidia.

~~ 6,0)

Fic. 1. Pleurothecium bicoloratum (ex holotype HUEFS 215982).

Conidiophores, conidiogenous cells, and conidia. Bar = 20 um.

Pleurothecium and Sporidesmiopsis spp. nov. (Brazil) ... 147

F G H 1 |

Fic. 2. Pleurothecium bicoloratum (ex holotype HUEFS 215982). A, H. Conidia;

B-G, I. Conidiogenous cells and conidia. Bars = 10 um.

148 ... Monteiro, Gusmao & Castafieda-Ruiz

Cotontgs effuse, hairy dark brown or black. Mycelium mostly immersed in

substrate. CONIDIOPHORES macronematous, mononematous, erect, straight to

slightly flexuous toward the apex, single, 5—10-septate, smooth, dark brown

below, pale brown toward the apex, 300-470 x 5—7.5 um. CONIDIOGENOUS

CELLS polyblastic, integrated, terminal, sometimes becoming intercalary, 20-90

x 4-5 um sympodially elongated, denticulate, denticles narrow cylindrical,

hyaline, 3-4 x 1-1.5 um. Conidial secession schizolytic. Conrp1a solitary,

broad allantoid, smooth, 3-septate, bicolor, with dark olivaceous-brown to

black central cells, hyaline end cells, smooth, 19-24 x 7-12 um.

ADDITIONAL SPECIMEN EXAMINED: VENEZUELA, ARAGuUA STATE: Forest near Colonia

“Tovar”, 10°25’N 67°17’W, on decaying petiole of Bactris setulosa H. Karst. (Arecaceae),

16.XII.2003; coll. J. Mostacero (HUEFS 216011).

Note: Hohnel (1919) established Pleurothecium for P. recurvatum (Morgan)

Hohn. The genus is characterized by distinct brown conidiophores and

polyblastic sympodially extended denticulate conidiogenous cells. The conidia

are solitary, unicellular or septate, hyaline or pigmented (Arzanlou et al. 2007,

Cooper 2005, Goos 1969, Matsushima 1975, 1980, Matsushima & Matsushima

1996, Réblova et al. 2012, Subramanian & Bhat 1989, Wu & Zhang 2009).

Pleurothecium leptospermi J.A. Cooper and P. recurvatum are superficially

similar to P. bicoloratum in having paler end cells and darker central cells,

but P. leptospermi has brown denticles and fusiform 3-septate brown smaller

(15-18 x 4-5 um) conidia, while the allantoid conidia of P. recurvatum are

hyaline initially and narrower (18—23(—30) x 5-7 um). The conidia of other

described Pleurothecium species clearly differ from P. bicoloratum (see Fie. 3).

Pleurothecium magnum Subram. & Bhat (Subramanian & Bhat 1989) should

be excluded from Pleurothecium: it more closely resembles Kylindria species

and is distinguished by sympodial extensions of the inner conidiogenous cell

layers, some enteroblastic percurrent regenerations, and conidia eccentrically

attached to flattened denticulate conidiogenous loci—none of which are

characters assigned to Pleurothecium.

Key to Pleurothecium species

PEGG midia tm i cellu Lats wg hia ve 9 sSta lw sta wy oes io ee ea g e ction oe esa ne be cSa eh boats Web watia es bs 2.

Wy HORI TASSPUAle se acl ieee to A abo cS ie RG wg Eo UR og Td osha GRR ARLE 3

2. Conidia ellipsoidal to oblong or obovoid, hyaline, 5-8 x 2.5-3 um .. PB semifecundum

2. Conidia narrowly obovoid, pale brown, 8.5-13.5 x 4-5 um ......... P. obovoideum

CLOG) GNP Topp LE ed wr oA Teen Tee ea toes oN EON cee cea cent seen Ul tee Et GS 4

Se Conidia with MOre Tall, ONE SEP ELINA Scho ocitclis ania ante bone desea lh drantua bh oes ab fe 5

4. Conidia clavate, pale brown, 15-32 x 3-4um ....... 2. eee eee eee eee P. clavatum

4. Conidia cylindrical, hyaline to pale brown, 14-21 x 1.5-2 um........ P. malayense

Pleurothecium and Sporidesmiopsis spp. nov. (Brazil) ... 149

Fic. 3. Representative conidia of Pleurothecium spp. (re-drawn from the literature).

A. P. bicoloratum. B. P. clavatum. C. P. leptospermi. D. P. malayense. E. P. obovoideum.

E P. pulneyense. G. P. recurvatum. H. P. semifecundum. Bars = 20 um.

150 ... Monteiro, Gusmao & Castafieda-Ruiz

5. Conidia 3-septate, always hyaline, cylindrical to fusiform, slightly curved, guttulate,

Pi ato] be Cir boat og LON any net (ao a FLD aee! fgeent! Ty Rear eoe” ry eee! Fy RD ert FD P. pulneyense

5. Conidia 3-septate, sometimes or always pigmented ....................000 000% 6

6. Conidia fusiform, curved, brown with median cells darker than end cells,

NSS PA GAM EN hye ay geben! big gibt sade Made Nh! Rade oh? Mains P. leptospermi

GC ONT AU a OT Up corde Rye. ae dege yea ee Bipot dee Rake cay Sale eee ies ee Renter Renda ort Bade; woe Bows 7

7. Conidia always with dark olivaceous-brown to black median cells and

hyalinevenc cells M6227 3¢ FEL Ate oc oa, s og ok wont gin © = phace &- Bt ne P. bicoloratum

7. Conidia at first hyaline, later pale brown with median cells darker than

the“enidicellsss1S 231230) ae 7 Wn) . Minis Mow « Mawteo « Maun « hie P. recurvatum

Sporidesmiopsis pluriseptata J.S. Monteiro, Gusmao & R.F. Castafieda sp. nov.

FIG 4

MycoBAnk MB 813041

Differs from all other Sporidesmiopsis spp. by its larger and multiseptate conidia.

Type: Brazil. Amapa State: Serra do Navio, 00°54’N 51°59’W, on decaying leaves of

Elaeis sp. (Arecaceae), 14.X.2014; coll. H.M.P. Sotao (Holotype: HUEFS 216009).

ErymMo_oey: Latin, pluri-, meaning many several, + -septata, refers to the septa.

Cotonigs effuse, hairy, black. Mycelium mostly immersed in substrate.

CONIDIOPHORES macronematous, mononematous,_ erect, straight,

branched above, cylindrical, <22-septate, sometimes with 1-2 enteroblastic

regenerations, smooth, black, 160-320 x 6-10 um, 0-7-septate, branches more

or less cylindrical, sometimes slightly sinuate, dark brown, 7-30 x 4-5 um.

CONIDIOGENOUS CELLS monoblastic, mostly discrete, lateral, determinate,

sometimes integrated, terminal, doliiform or shortly cylindrical, dark brown,

smooth, 3-7 x 4-5 um. Conidial secession schizolytic. Conrp1A solitary,

obclavate, straight or curved, acropleurogenous, 11-23-euseptate, brown, pale

brown toward the apex, smooth, 80-130 x 6-8.5um, smooth, sometimes with

a rudimentary, hyaline, mucous tunica at the apical cell.

Note: Subramanian & Bhat (1989) established Sporidesmiopsis for S. malabarica

Subram. & Bhat. The genus is characterized by conidiophores that are distinct,

erect, branched toward the apex, and conidiogenous cells that are monoblastic,

discrete, and short cylindrical or doliiform (Xia et al. 2014). Sporidesmiopsis

dennisii (J.L. Crane & Dumont) Bhat et al., which is superficially similar to

S. pluriseptata, is distinguished by its 10-15-septate, smooth or verruculose,

dark brown conidia, paler toward the apex, 69-92 x 11-13 um (Bhat &

Kendrick 1993, Crane & Dumont 1978).

Pleurothecium and Sporidesmiopsis spp. nov. (Brazil) ... 151

D ; )

H I : |

Fic. 4. Sporidesmiopsis pluriseptata (ex holotype HUEFS 216009). A-G. Conidia;

H-K Conidiophores, conidiogenous cells and conidium. Bars: A~J = 20 um; K = 10 um.

152 ... Monteiro, Gusmao & Castafieda-Ruiz

Acknowledgments

The authors express their sincere gratitude to Dr. De-Wei Li and Dr. Xiu-Guo

Zhang for their critical review of the manuscript. The authors thank the National

Council for Scientific and Technological Development (CNPq) (Proc. 142014/2011-7

and 303062/2014-2) and “Programa de Pesquisa em Biodiversidade do Semiarido

(PPBio Semiarido -CNPq/MCTI) for supporting this study. JSM thank the Postdoctoral

fellowship (CAPES - 071/2012). RFCR is grateful to Cuban Ministry of Agriculture and

“Programa de Salud Animal y Vegetal’, project P131LH003033 Cuban for facilities.

We acknowledge the assistance provided by Dr. P.M. Kirk and Drs. V. Robert and

G. Stegehuis through the Index Fungorum and MycoBank websites. Dr. Lorelei Norvell’s

editorial and Dr. Shaun Pennycook’s nomenclatural reviews are greatly appreciated.

Literature cited

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morphotaxonomic revision of Ramichloridium and allied genera. Studies in Mycology

58: 57-93. http://dx.doi.org/10.3114/sim.2007.58.03

Bhat DJ, Kendrick WB. 1993. Twenty-five new conidial fungi from the Western Ghats and the

Andaman Islands (India). Mycotaxon 49: 19-90.

Castaneda-Ruiz RE 2005. Metodologia en el estudio de los hongos anamorfos: 182-183, in: Anais

do V Congresso Latino Americano de Micologia. Brasilia.

Cooper JA. 2005. New Zealand hyphomycetes fungi: additional records, new species

and notes on interesting collections. New Zealand Journal of Botany 43: 323-349.

http://dx.doi.org/10.1080/0028825X.2005.9512957

Crane JL, Dumont KP. 1978. Two new hyphomycetes from Venezuela. Canadian Journal of Botany

56: 2613-2616. http://dx.doi.org/10.1139/b78-313

Goos RD. 1969. The genus Pleurothecium. Mycologia 61: 1048-1053. http://dx.doi.org/10.2307/3757488

Hohnel F. 1919. Fiinfte vorlaufige Mitteilungen mykologischer Ergebnisse (Nr. 399-500). Berichte

der Deutschen Botanischen Gesellschaft 37: 153-161.

Matsushima T. 1975. Icones microfungorum a Matsushima lectorum. Nippon Printing Co, Osaka.

Matsushima T. 1980. Saprophytic microfungi from Taiwan, part 1, hyphomycetes. Matsushima

Mycological Memoirs no. 1. Matsushima Fungus Collection, Kobe, Japan.

Matsushima K, Matsushima T. 1996. Fragmenta mycologica — I. Matsushima mycological memoirs

no. 9: 31-40. Matsushima Fungus Collection, Kobe, Japan.

Réblova M, Seifert KA, Fournier J, Stépanek V. 2012. Phylogenetic classification of Pleurothecium

and Pleurotheciella gen. nov. and its dactylaria-like anamorph (Sordariomycetes)

based on nuclear ribosomal and protein-coding genes. Mycologia 104: 1299-1314.

http://dx.doi.org/10.3852/12-035

Subramanian CV, Bhat DJ. 1989 [“1987”]. Hyphomycetes from South India I. Some new taxa.

Kavaka 15: 41-74.

Wu YM, Zhang TY. 2009. New species of Phialosporostilbe and Pleurothecium from soil. Mycotaxon

110: 1-4. http://dx.doi.org/10.5248/110.1

Xia JW, Ma LG, Castafeda-Ruiz RF, Zhang XG. 2014. A new Sporidesmiopsis and three new records

of other dematiaceous hyphomycetes from southern China. Nova Hedwigia 98: 103-111.

http://dx.doi.org/10.1007/S10267-011-0152-1

MY COTAXON

January-March 2016—Volume 131, pp. 153-176

http://dx.doi.org/10.5248/131.153

New leptonioid species from New South Wales

and northeastern Queensland, Australia

Davip L. LARGENT"*, KERRI L. KLUTING’,

NOELLE M. ANDERSON? & SARAH E. BERGEMANN?

‘Biological Sciences, Humboldt State University, 1 Harpst St, Arcata CA 95521, USA

*Department of Evolutionary Biology, Evolutionary Biology Centre, Uppsala University,

Uppsala, SE-752 36, Sweden

Evolution & Ecology Group, Biology Department, Middle Tennessee State University,

PO Box 60, Murfreesboro TN 37132, USA

* CORRESPONDENCE TO: mrp@humboldt1l.com

ABSTRACT —Leptonia subpanniculus, L. newlingii, L. sabulosa, and L. substricta are described

as new species based on morphological and molecular characters. Phylogenetic relationships

and morphological similarities are discussed for these and other closely related species.

Key worps — Basidiomycota, Entolomaceae, taxonomy

Introduction

The morphologically defined genus Leptonia (Fr.) P. Kumm. is polyphyletic

(Co-David et al. 2009). In the most recent taxonomic study of Leptonia,

phylogenetic analyses strongly support Entoloma euchroum (Pers.) Donk [=

Leptonia euchroa (Pers.) P. Kumm.], the type species of Leptonia and Entoloma

subg. Leptonia (Fr.) Noordel. emend. Morozova et al. (2014), in a clade separated

from the Inocephalus-Cyanula and Nolanea-Claudopus clades (Morozova

et al. 2014). These authors have emended Entoloma subg. Leptonia to make

it monophyletic and consistent with Entoloma sect. Leptonia (Noordeloos

2004) and Leptonia subg. Leptonia (Largent 1994). Leptonia subg. Leptonia

sensu Largent is a diverse subgenus that includes species that are mycenoid,

collybioid, or tricholomatoid; have a conico-convex or plano-convex pileus, a

fibrillose-striate or flocculose-scaly (rarely almost smooth) stipe, lamellae with

154 ... Largent & al.

sterile, fertile or heterogeneous edges, cheilocystidia present or absent, weakly

to sharply angled (almost nodulose) basidiospores, a more or less a trichoderm

pileipellis of inflated elements with intracellular (and often with additional

encrusting) pigments, and clamp connections; and lack brilliant granules.

Here we describe and illustrate four new species from Australia: L. sabulosa,

L. subpanniculus, L. substricta, and L. newlingii. Our molecular phylogeny

supports these species and L. ambigua from Australia (Largent et al. 2013b)

within Leptonia subg. Leptonia sensu Largent (1994). We also discuss the

morphological and phylogenetic relationships among species in this clade.

Materials & methods

Macromorphological and micromorphological features

Techniques and equipment for collecting and describing basidiomata in the field and

GPS coordinates follow Largent et al. (2011a,b). Kornerup & Wanscher (1978) color

terms and factors determined from mathematical analyses used in the descriptions are

in Largent et al. (2013a,b). Digitized microphotographs were made using a Lumenera

Infinity 2-5C ccd digital camera attached to the trinocular head of a compound

microscope; all microscopic measurements were obtained using Infinity Analyze

software 6.5.2. All collections from New South Wales cited in ‘Additional collections

examined’ were deposited in The Plant Pathology Herbarium, Orange Agricultural

Institute, Orange, New South Wales, Australia (DAR) while those from Queensland

were deposited in the Australian Tropical Herbarium, Cairns, Queensland, Australia

(CNS). All holotype and isotype collections are deposited in the herbaria designated

using acronyms from Thiers (2012).

DNA sequences and phylogenetic analyses

DNAs were extracted from silica-dried tissues according to Largent et al. (2011b).

The nuclear large subunit ribosomal RNA (nrLSU) and the second largest subunit of

the ribosomal RNA polymerase (RPB2) genes were PCR amplified following protocols

described in prior publications (Largent et al. 2011a,b, 2013a,b). Partial mitochondrial

small subunit ribosomal RNA (mtSSU) sequences were obtained using two different

primer combinations: either 1) with MS1 and MS2 following White et al. (1990) or 2)

by modifying the MS1 and MS2 primers to increase the success of PCR amplification

(MS1-RhoF2, 5’ to 3’ — GTC AAT GCT CGM AAG AGT GAA CTA and MS2-RhoRI1,

5’ to 3’ — ACA TGC TTC ACT YCG TTT GCT). The PCR protocol using the newly-developed

primers is as follows: initial incubation at 94°C for 1 min, 39 cycles of 94°C for 1 min,

56°C for 1 min, 72°C for 1 min 30 sec, and a final temperature of 72°C for 7 min.

Sequences were generated on an ABI3130xl following the sequencing and precipitation

protocols in Largent et al. (2011b). After sequencing, forward and reverse sequences

were edited and all ambiguities in chromatograms were visually confirmed using

Sequencher 4.2.2 (Gene Codes Corp., Ann Arbor, MI).

Leptonia spp. nov. (Australia) ... 155

TABLE 1. Collections used in the phylogenetic analyses.

New sequences generated for this study are shown in bold. Square bracketed annotations

indicate species names applied in GenBank that differ from those in the phylogram (Fie. 1).

COLLECTION GENBANK ACCESSION No.

SPECIES

IDENTIFIER mtSSU LSU RPB2

Ae pase aa ae MCA1978 GU384583 GU384609 _GU384632

A. sericella DLL9524 KR869912 KR869940 KR869961

A. stylophora DLL9805 KR869911 KR869939 KR869960

Catathelasma imperiale 11CA001A KR869913 KR869941 KC816994

Claudopus minutoincanus DLL9871 HQ731511 HQ731514 HQ731517

C. parasiticus 20 GQ289317 GQ289177 GQ289248

[= Entoloma parasiticum]

10617 TJB KR233902 KR233872 KR233924

Claudopus sp. DLL9735 KR869910 KR869938 KR869959

C. viscosus DLL9788 HQ731513 HQ731516 HQ731518

Clitocella fallax 256680KM KR869909 AF261283 KC816937

[= Rhodocybe fallax]

Clitopilopsis hirneola REH8490 GU384587 GU384611 KC816904

[= Rhodocybe hirneola]

Clitopilus aff. hobsonii DLL9586 KJ021688 KJ021698 KC816912

Cl. apalus 26394WAT KR869907 KR869936 KC816906

Entocybe haastii DLL10087 JQ793645 JQ793652 JQ793659

[= Entocybe sp.]

Entoloma. abortivum 6693 TJB GU384595 GU384616 GU384642

5772 TJB KR233897 KR233869 KR233926

E. allochroum JVG 1060902-1 KC898488 KC898522 —_—

E. caespitosum GDGM 27564 JQ993070 JQ320130 JQ993078

E. callichroum var. venustum L Wo E17/10 KC898490 KC898523 —

E. chytrophilum L Dahncke 855 KC898479 KC898519 —

LE262994 KC898480 KC898520 —

E. dichroum JVG 1070821-4 KC898486 KC898527 —

LE234260 KC898487 KC898528 —

E. discoloratum DLL10217 JQ793646 JQ793653 JQ793660

E. euchroum LE262995 KC898483 KC898516 —

E. eugenei LE253771 7 KC898529 =;

LE254347 — KC898530 —

E. ferruginans 11CA032 KJ021689 KJ021699 KJ021693

E. fragilum MCA2415 KJ021690 KJ021700 KJ021694

E. indoviolaceum PM700 GQ289312 GQ289172 GQ289243

E. lampropus LE262991 KC898470 KC898505 —_

UPS:F-176490 KC898471 KC898506 —

E. lepidissimum LE254871 KC898493 KC898531 —

E. myrmecophilum 231 GQ289314 GQ289174 GQ289245

E. nidorosum 9971 TJB GU384596 GU384617 GU384643

E. pallideradicatum 255 GQ289316 GQ289176 GQ289247

E. pallidocarpum GDGM 28828 JQ993074 JQ410331 JQ993080

E. percoelestinum LE254327 KC898496 KC898526 =

Continued on p. 156

156 ... Largent & al.

TABLE 1, continued

SPECIES

E. placidum

E. prunuloides

E. pygmaeopapillatum

E. readiae

E. rhodopolium

E. sublaevisporum

E. tectonicola

E. tjallingiorum var. alnetorum

[=E. alnetorum|

E. tjallingiorum var. laricinum

E. tjallingiorum var.

tjallingiorum

E. transmutans

E. undatum

E. valdeumbonatum

E. vezzenaense

E. violaceotinctum

Inocephalus hypipamee

“I. lactifluus”

I. luteus

I. plicatus

I. virescens

Lepista nuda

Leptonia ambigua

L. boardinghousensis

L. coelestina

[= Entoloma coelestinum|

L. decolorans

L. exilis

L. newlingii

L. odorifera

L. omphalinoides

L. poliopus

L. sabulosa

L. subpanniculus

L. substricta

COLLECTION

IDENTIFIER

JVG1060830-6

LIP JVG 1070823T

il5

G00111402

LE254343

UPS:F-016378

LE254317

155

189

241

DLL10088

DLL9981

DLL10071

7962 TJB

6562 TJB

DLL9691

DLL9972

11CA041

DLL9872

DLL10086

LE258103

DLL9533

DLL9530

DLL10004

DLL10026

6534 TJB

DLL9800

DLL10209a

DLL10058

DLL10427

DLL9647

DLL9668

DLL9697

DLL10070

DLL10227

DLL10260

DLL9852

GENBANK ACCESSION No.

mtSSU

KC898482

GQ289324

GQ289325

GQ289326

GQ289327

KC898478

GQ289336

KC898473

KC898477

KC898474

KC898475

GQ289340

GQ289342

GQ289343

GQ289344

JQ793643

JQ624603

JQ624604

JQ624605

KR869908

KJ021692

JQ756399

JQ756398

KC898494

KR233883

KR869906

KR869897

KR869898

KR233899

JQ756401

JQ756402

KR869895

KR869896

KR869891

KR869892

KR869893

KR869889

KR869890

KR869894

LSU

KC898515

GQ289184

GQ289185

GQ289186

GQ289187

KC898518

GQ289196

KC898508

KC898513

KC898509

KC898511

GQ289200

GQ289202

GQ289203

GQ289204

JQ793650

JQ624608

JQ624609

AF261304

KR869944

JQ624610

KR869937

KJ021705

JQ756414

JQ756413

KC898524

KR233852

KR869935

KR869926

KR869927

KR233870

JQ756417

JQ756418

KR869924

KR869925

KR869919

KR869921

KR869922

KR869917

KR869920

KR869918

KR869923

RPB2

GQ289255

GQ289256

GQ289257

GQ289258

GQ289266

GQ289268

GQ289270

GQ289271

GQ289272

JQ793657

KR869958

JQ624616

JQ624617

KR869957

KJ136110

JQ756429

JQ756428

KR233914

KR869956

KR869951

KR233927

JQ756432

JQ756433

KR869950

KR869946

KR869947

KR869948

KR869949

Leptonia spp. nov. (Australia) ... 157

TABLE 1, cooncluded

COLLECTION GENBANK ACCESSION No.

SPECIES

IDENTIFIER mtSSU LSU RPB2

L. trichomata 11CA006 KR233873 KR233840 KR233903

L. umbraphila DLL9640 JQ756407 JQ756422 JQ756438

DLL9766 JQ756408 JQ756423 JQ756439

Mycena aff. pura 11CA007 KR869914 KR869942 KC816995

Nolanea bicoloripes DLL9532 KR869904 KR869933 KR869954

N. cetrata DLL9531 KF738927 KF738942 KF771346

N. cf. conferenda 11CA014 KF738935 KF738946 KF771351

N. conferenda 6 GQ289300 GQ289160 GQ289231

[= Entoloma conferendum|

N. hebes 46 GQ289310 GQ289170 GQ289241

[= Entoloma hebes]

N. hirtipes DLL9525 KR869905 KR869934 KR869955

N. rigidipes DLL9528 KR869902 KR869932 KR869953

N. sericea DLL9527 KR233882 KR233851 KR233913

11CA055 KF738933 KF738944 KF771349

Nolanea sp. DLL9976 KR869903 KR869931 KR869952

N. strictior var. isabellina 7710 TJB GU384594 GU384618 GU384641

[= Entoloma. strictius

var. isabellinum]

Panellus stipticus 11CA052 KR869915 KR869943, KC816996

Pouzarella farinosa DLL9934 HQ876538 HQ876516 HQ876495

P lasia DLL9662 HQ876551 HQ876529 HQ876507

P. pamiae DLL9794 HQ876539 HQ876517 HQ876496

P. pilocystidiata DLL9848 HQ876542 HQ876520 HQ876499

Rhodocybe pruinosostipitata MCA1492 GU384608 GU384627 GU384653

R. roseiavellanea 8130 TJB KR869901 KR869930 KC816982

R. spongiosa MCA2129 GU384604 GU384628 GU384657

Rhodophana nitellina 7861 TJB KR869900 KR869929 KC816959

Tricholoma flavovirens 11CA038 KJ021691 KJ021704 KC816997

Trichopilus porphyrophaeus 6957 TJB — AF261290 —

A total of 281 sequences (fifty-nine newly generated for this study and 222 obtained

from GenBank) were used to infer phylogenetic relationships (TABLE 1). Sequences

were aligned with MAFFT v. 7 (default settings) and manually adjusted using Mesquite

2.75 (Mesquite Software Inc., Austin, TX). Intronic regions in the RPB2 and hyper-

variable regions and large introns in the mtSSU were excised before analyses. Aligned

sequences were 1,078 bp (nrLSU), 585 bp (mtSSU), and 1501 bp (RPB2), and six

mtSSU and 28 RPB2 sequences were coded as missing data (TABLE 1). A Maximum

Likelihood (ML) analysis with 1000 replicates and specifying a GTRGAMMA model in

RAxML-HPC2 XSEDE 8.0.24 (Stamatakis 2006) using the CIPRES Science Gateway 3.1

(Miller et al. 2010) was conducted on a concatenated alignment of the three gene regions.

Each gene region was partitioned separately and the RPB2 region was partitioned across

all codon positions for a total of five partitions in the dataset. Supports for branches

were generated using 1000 bootstrap (BS) iterations, and BS support values =70 were

considered significant.

158 ... Largent & al.

Results & discussion

Our ML phylogenetic analysis (Fic. 1) statistically supports (BS = 80)

monophyly of Leptonia subg. Leptonia (= Entoloma subg. Leptonia) and

separates the Leptonia clade from other entolomatoid clades: Inocephalus-

Cyanula, Claudopus, Nolanea, Pouzarella, Prunuloides, Rhodopoloid, and

Rhodocybe-Clitopilus as previously demonstrated by Morozova et al. (2014).

Within the Leptonia clade, Morozova et al. (2014) identified two subclades

that correspond taxonomically to Entoloma sect. Dichroi O.V. Morozova et al.

and E. sect. Leptonia as well as a number of species with unknown affinity to

either section. Our analysis does not support the monophyly of these sections

(Fic. 1: boxes A, B). Our results support the four new species described here,

L. ambigua from Australia (Largent et al. 2013b), E. indoviolaceum Manim. &

Noordel. from India, and the Eurasian species described by Morozova et al.

(2014) all within L. subg. Leptonia.

Leptonia newlingii, L. sabulosa, and L. subpanniculus from Australia,

Entoloma indoviolaceum from India, E. dichroum (Pers.) P. Kumm. from

eastern and western Europe, E. eugenei Noordel. & O.V. Morozova from Russia,

and L. substricta from Australia (Fic. 1: box A) all have violet or blue-black

basidiomata and basidiospores with 5-7 more or less acute angles (PLATES. 2F,

4E, 6D, 8C). Although these seven species collectively resemble E. sect. Dichroi

sensu Morozova et al. (2014), a phylogenetic relationship is not statistically

supported (Fic. 1). Our analysis supports only one phylogenetic relationship

for these species, with L. newlingii and E. indoviolaceum the most closely

related (BS = 71, Fic. 1).

These seven species cluster into two groups—one with Leptonia newlingii,

L. sabulosa, L. subpanniculus, and Entoloma indoviolaceum and the other with

E. dichroum, E. eugenei, and L. substricta. However this clustering pattern was

not statistically supported (Fic. 1). Within the two groups, the species have

morphologically similar basidiospores. The group containing L. sabulosa has

basidiospores that are distinctly angular but slightly rounded or with very

slightly protruding angles, contrasting with the acutely angled basidiospores of

E. dichroum, E. eugenei, and L. substricta that lack protruding angles.

Our analysis also places L. ambigua in a sub-clade with E. euchroum

(= L. euchroa), E. lampropus (Fr.) Hesler, E. placidum (Fr.) Noordel.,

E. tjallingiorum var. alnetorum (Monthoux & Rollin) O.V. Morozova et al.,

FiGurRE 1. Topology of the maximum-likelihood phylogram showing branches with statistical

supports 270% based on 1000 bootstrap replicates. Shaded species highlight the species from

Australia described in this report. The labeled boxes show (A) Entoloma sect. Dichroi and

(B) E. sect. Leptonia as defined by Morozova et al. (2014)—see also Results & discussion.

Leptonia spp. nov. (Australia) ... 159

0.04 substitutions/site

100 Leptonia trichomata 11CA006

eptonia decolorans DLL9533

Entoloma transmutans 155

15, Atboleptonia stylophora DLL9805

99 Alboleptonia angustospora MCA1978

Entoloma caespitosum GDGM 27564

400 Leptonia poliopus DLL10209A

Leptonia exilis DLL9530.

100 Inocephalus hypipamee DLL10071

; inocephalus Tp pames, DLL9981

100 Leptonia umbraphila DLL964!

Leptonia umbraphila DLL9766 i

100 "Inocephalus lactifluus" 7962 TJB

a Inocephalus virescens DLL9972

Inocephalus plicatus DLL9691

Inocephalus iuteus 6562 TJB

Leptonia omphalinoides DLL9800

Trichopilus porphyrophaeus 6957 TJB

Leptonia odorifera 6534 TJB

—— Entoioma tectonicola 115

Alboleptonia sericella DLL9524

Leptonia boardinghousensis DLL10086

Claudopus minutoincanus DLL9871

100 Entoloma undatum 18

= 100 Setoe ab Riv tin 6993 TUB

ntoloma abortivum

99 Entoloma abortivum 5772 TJB Claudopus clade

100;- Claudopus parasiticus 20

100 Claudopus parasiticus 10617 TJB

Claudopus viscosus DLL9788

Entoloma,vezzenaense 241. .

povstnerener Leptonia subpanniculus DLL100 700 rs

: Leptonia subpanniculus DLL9668 A

: Leptonia subpanniculus DLL10260

; 100 Leptonia subpanniculus: DLL9647

eptonia suppanniculus A

H beeen Spence us BEE38357

i (77,92, Leptonia newlingii DLL 10004

: Leptonia newlingli DLL10026

Entoloma indoviolaceum PM700

95, Leptonia sabulosa DLL10427:

H Leptonia sabulosa DLL.10058 . H

: 100 Leptonia eugenei LE253771:

H A Leptonia eugenei LE254347!

i Leptonia substricta DLL9852 i

Inocephalus-Cyanula clade

lolyoiq “yas

Leptonia subg. Leptonia

Entoloma Galingior aur var. tlallingiorum LE254317 qi

Entoloma tjallingiorum var. laricinum LE254343 :

: 100, Entoloma lampropus LE26299

i Entoloma lampropus UPS:F-176490

i 77 Entoloma placidum JVG10608306

Leptonia ambigua DLL9872

i Entoloma euchroum LE262995

80 i 100, Entoloma chytrophilum L Pannete 855

78 i Entoloma sublaevisporum LIP JVG 1070823T

Entoloma percoelestinum LE254327

Entoloma lepidissimum LE254871

71. 100 Nolanea cf. conferenda 11CA014

Sar Nolanea conferenda 6

Nolanea rigidipes DLL9528

80 Nolanea strictus var, isabellina 7710 TJB

89, Nolanea cetrata DLL9531

Entoloma pallideradicatum 255

96... Nolanea sericea DLL9527

ocp2e} Nolanea sericea 11CA055 Nolanea clade

98 Entoloma valdeumbonatum 189

Entoloma pygmaeopapillatum 32

82 100 Nolanea hirtipes DLL9525

Entoloma hebes 46

Nolanea bicoloripes DLL9532

98 Nolanea sp. DLL9976

Entoloma readiae 102 :

Pouzarella lasia DLL9662

4100 99 Pouzarella farinosa DLL9934

eluojda’] ‘pa

Pouzarella pamiae DLL9794 Pouzarella clade

—— Pouzarella pilocystidiata DLL9848

Entoloma myrmecophilum 231

100 Entoloma ferruginans 11CA032

Entoloma pallidocaroum GDGM28828 Rhodopoloid clade

90 Entoloma rhodopolium 8

Entoloma nidorosum 9971 TJB

Entoloma prunuloides 40

92 Entoloma discoloratum DLL10217

87 Entoloma violaceotinctum DLL10088

Entoloma fragilum MCA2415

Entocybe haastii DLL10087 ae

: euigales apalus 26394WAT

Clitocella fallax 25668OKM _

a SOPs aff. Nea UE ig

itopilopsis hirneola -Clitopi

Rhodbcybe pruinosotipitata MCA1492 REPOS eo Ier ge

Rhodocybe roseiavellanea 8130 TJB

Rhodocybe Spongiess MCA2129

Rhodophana nitellina 7861 TJB

100 Mycena aff. pura 11C A007

a: a Panellus stipticus 11CA052

92 Catathelasma imperiale 11CA001A outgroup

$5 _~>———~ Tricholoma flavovirens 11CA038

Lepista nuda 11CA041

Prunuloides clade

100

160 ... Largent & al.

E. tjallingiorum var. laricinum O.V. Morozova et al., and E tjallingiorum

Noordel. var. tjallingiorum (BS = 77, Fic. 1). All these species have obscurely

angled basidiospores and dark violet to blue-black squamulose pilei that

become brown.

Taxonomy

Leptonia subpanniculus Largent & Bergemann, sp. nov. PiaTEs 1, 2

MycoBank MB 813877

Differs from Entoloma panniculus by its dark purplish violet colors, mild and not

distinctive taste and odor, and larger basidiospores.

Type — Australia, Queensland, Cook Region, Mt. Hypipamee National Park 17°25’35”S

145°29’16”E, in soil protected by roots, 9 March 2012, DL Largent 10260 (holotype BRI;

isotype CNS; GenBank KR869890 (mtSSU), KR869918 (LSU)).

EryMoLoGy — named for the similarity to E. panniculus.

BASIDIOMATA mycenoid to collybioid. PiLeus 10-38 mm broad, 5-13 mm

high, at first conic, conic-campanulate, or campanulate (but not umbonate),

expanding to broadly campanulate and umbonate to broadly convex with

an obscure umbo, eventually convex without an umbo; when young covered

entirely with minute erect dark purplish violet (17 or 18F6-7) squamules that

with pileal expansion remain erect at the center and becoming semi-erect to

appressed from margin to the edge; the squamules remaining dark purplish

violet at the tips but becoming dark grayish violet (18E6) elsewhere and

greyish violet (17C-D4-5) at the edge; the surface always dull and opaque,

neither hyrophanous nor translucent-striate; the edge fibrillose, typically

extending 1-1.5 mm beyond the lamellae, decurved then plane, even then

eroded to crenulate. CONTEXT 1.0-1.5 mm thick above stipe, 0.5 mm thick at

the edge; violaceous (17F4). OpDor not distinctive, mild. TasTE not distinctive

at first, later slowly becoming pungent. LAMELLAE 6-18 mm long, 2-9 mm

broad; yellowish white to pale yellow when young, becoming grayish orange

(5B3) from basidiospores, edges concolorous; sinuate, subdistant, sigmoid

and narrow at first, becoming moderately broad, eventually broad in larger

basidiomata; edges smooth. Stipe 10-61 mm long and width 2-3 mm at apex,

2-4 mm in middle, and 2-9 mm at base; rarely equal, typically clavate; + solid

and stuffed but not fragile; apex either white and glabrous or (when young)

entirely covered with dark purplish violet (17 or 18F6-7) dense fibrils and

punctate-squamules; when mature rimulose with dense fibrils and scattered

punctae forming darker reticulate ridges around grayish violet fibrillose gaps;

basal tomentum scarce to absent; basal rhizoids absent. VEIL possibly present

as indicated by the fibrils on the otherwise white stipe apex and the purplish

violet fibrils on the crenulate pileal edge.

Leptonia spp. nov. (Australia) ... 161

PiaTE 1. Leptonia subpanniculus (DLL10260 holotype): A. basidiomata;

B. pileus surface (left), stipe surface, and lamellae (right): Bars = 14 mm.

BASIDIOSPORES 5-7 distinct angles, at times the angles slightly more

pronounced and protuberate, in profile view isodiametric to heterodiametric,

on average heterodiametric, apex typically rounded-triangular, rarely truncate,

frequently with a suprahilar depression just above the adaxial side of the hilar

appendage; 9.2-13.4 x 6.0-9.5 um (x = 11.1 + 0.9 x 7.7 + 0.6 um; E = 1.1-1.8;

Q =1.44 + 0.1; n/11 = 242). Basip1a 2-4-sterigmate, subclavate and somewhat

tapered to the base, 31-54 x 10-17 um (x = 43.3 + 3.9 x 12.4 + 2.9 um;

162 ... Largent & al.

E = 2.4-4.8; Q =3.44 + 0.6; n/3 = 25). LAMELLAR EDGE fertile. CHEILOCYSTIDIA

absent. PLEUROCYSTIDIA absent. LAMELLAR HYPHAE subparallel, 99-159 x

6-13 um (n/1 = 6). PILEIPELLIS from 100-300 um thick, in young basidiomata

the hyphae typically entangled along the entire surface with the terminal 2-5

cells forming a distinct trichodermium, in older basidiomata a trichodermium

on the disc, forming semi-erect clusters towards the margin and forming a

periclinal layer of entangled hyphae at the edge. Prteocystip1A cylindric

to cylindro-clavate; typically long, 62-233 x 9-16 um (x =123.6 + 49.78 x

11.1 + 2.1 um; E = 5.5-17.3; Q =11 + 3.3; n/2 = 15). PILEUS TRAMA hyphae

subparallel in radial section, 179-298 x 8-15 um (n/1 = 3). STIPITIPELLIS

similar to the pileipellis along the entire length, but forming a cutis at the apex.

CAULOCYSTIDIA Similar to the pileocystidia, 48-204 x 6-11 um (n/2 = 15). STIPE

TRAMAL HYPHAE Subparallel, 200-367 x 6.0-12.9 um (n/1 = 4). OLEIFEROUS

HYPHAE rare to absent in the trama. BRILLIANT GRANULES typically absent but

at times very scattered in basidia. Lipoip GLOBULES absent. PIGMENTs in the

suprapellis hyphae cytoplasmic and brownish to purplish, in the hyphae of

the subpellis and outer pileal trama both cytoplasmic and purplish as well as

encrusted and dark brownish; pigments eventually producing a purplish grey

exudate in water, 10% NH,OH, and 3% KOH. CLAMP CONNECTIONS abundant

in all tissues.

ECOLOGY & DISTRIBUTION — Solitary or scattered in soft crumbly soil at the

bottom of a moss-covered depression or along a path in soil protected by roots

in a closed canopy forest just above the falls in Mt. Hypipamee National Park or

within moss-covered trenches in open canopy in Longlands State Forest, mid

to late March.

ADDITIONAL COLLECTIONS EXAMINED — AUSTRALIA. QUEENSLAND, Cook Region,

Mt. Hypipamee National Park, 17°25’35”S 145°29’12”E, 14 March 2009, DL Largent

9647; 18 March 2009, DL Largent 9668; 17°25’35”S 145°29’16”E, 17 March 2011, DL

Largent 10034; 17°25’35”S 145°29'15”E, 25 March 2011, DL Largent 10070; 17°25’35”S

145°29'17”E, 14 March 2012, DL Largent 10282; 17°25’37”S 145°29’15”E, 20 March 2012,

DL Largent 10306; Herberton National Park, Longlands Gap State Forest, 17°27'07”S

145°28’33”E, 24 March 2009, DL Largent 9697; 17°27'17”S 145°28’33”E, 1 March 2012,

DL Largent 10227; 10 March 2012, DL Largent 10266; 17°27'16”S 145°28’34’E, 14

March 2012, DL Largent 10285.

DISTINCTIVE CHARACTERS—Mycenoid to collybioid, purplish violet

basidiomata with a possible veil, stipe typically developing reticulate or punctate

ridges in age; basidiospores heterodiametric, averaging 11 x 8 um, distinctly

angular (occasionally with slightly protuberant angles), apex in profile view

often rounded or truncate and with an adaxial depression, suprapellis with

cytoplasmic brownish (occasionally purplish) pigments, and the subpellis with

both cytoplasmic purplish and encrusting dark brownish pigments.

Leptonia spp. nov. (Australia) ... 163

y we

—

ral

PLATE 2. Leptonia subpanniculus (DLL10260 holotype): A. stipitipellis longitudinal section at apex;

B. pileipellis radial section near disc; C. cylindro-clavate and narrowly obclavate pileocystidia

(terminal cells); D. pileipellis subpellis and outer pileal trama; encrusted and cytoplasmic pigment;

and clamp; E. basidia and basidioles; FE. basidiospores. Bars: A = 60 um; B = 50 um; C, E = 12 um;

D= 15 um; F= 8 um.

ComMMENTS—L. subpanniculus is morphologically very similar to Entoloma

panniculus (Berk.) Sacc. from Tasmania, which differs in its deep blue

coloration, farinaceous odor, oily taste, and basidiospore size (9-12 x 6-8 um)

(Noordeloos & Gates 2012).

Leptonia substricta Largent & Bergemann, sp. nov. PLATES 3, 4

MycoBank MB 813880

Differs from Entoloma strictum by its mycenoid basidiomata with yellowish white to

pale yellow adnexed lamellae, mild taste and odor, and obclavate caulocystidia.

164 ... Largent & al.

Type — Australia, New South Wales, central Hunter District, Myall Lakes National

Park, 32°32’30”S 152°18’39”E, in sandy soil, 10 April 2010, DL Largent 9852 (holotype

DAR; GenBank KR869894 (mtSSU), KR869923 (LSU), KR869949 (RPB2)).

EryMOLoGy — named for its similarity to E. strictum.

BASIDIOMATA mycenoid. PrLEus 10-20 mm broad, 8-15 mm high, at first

evenly conic to campanulate, in age convex and obscurely umbonate, edge

decurved, even when young, becoming crenulate in age; surface matted

squamulose becoming matted fibrillose towards the edge; indigo-blue (18F3)

to dark violet (18F6) with the squamule tips more blackish purple; opaque,

neither translucent nor hygrophanous. CoNTEXT 0.5-1.0 mm. above the stipe.

Opor & Taste mild. LAMELLAE 8-9 mm long, 3-4 mm broad, adnexed,

narrow to moderately wide, close to subdistant, edges eroded in age; bluish

white at first, then yellowish white (442-3) to pale yellow (4A3), becoming

orangish white (5A2-3) as spores mature; where injured becoming brownish;

edges concolorous. LAMELLULAE typically in 3 (rarely 4) series, 1-2 short,

1 medium, and 1 medium long. St1pz 40-90 x 2-3 mm, equal; solid but fragile

and snaps easily; surface at times longitudinally furrowed, densely appressed

fibrillose-squamulose; blackish blue (19F6), squamules with purplish black

punctate tips abundant at the apex, moderately abundant to scarce in the

middle, and very scattered to nearly absent at the base; base with abundant

to copious whitish blue tomentum, occasionally also with rhizoids projecting

10-20 mm into the soil.

BASIDIOSPORES 5-7 distinct angles, in profile subisodiametric to

heterodiametric (on average heterodiametric), apex typically rounded, rarely

truncate; 8-12 x 6-10 um (x = 10.5 + 0.9 x 7.7 + 0.8 um; E = 1.1-1.7; Q =1.38

+ 0.14; n/2 = 59). Basip1a 4-sterigmate, clavate, broad, 28-50 x 7-14 um

(x =41.1+48.5 x 11 + 2.3 um; E = 3.4-4.1; Q =3.71 + 0.28; n/2 = 9). LAMELLAR

EDGE fertile. CHEILOCYSsTIDIA absent. PLEUROCysTIDIA absent. LAMELLAR

TRAMAL HYPHAE subparallel, short to moderately long, 34-192 x 6.5-14 um

(x = 113 + 47 x 10 + 2.6 um; E = 4-19; Q =11.7 + 5.0; n/2 = 9). PILEIPELLIS at

first a trichodermium over the entire surface, 150-300 um deep, composed of

3-5 cells with the pileocystidia erect and the subterminal cells longitudinally

entangled, remaining so with age on central 2/3 but becoming semi-erect

towards the margin and nearly prostrate at the edge. PILEOCyYSTIDIA versiform

(clavate to cylindro-clavate, subulate to obclavate), 46-100 x 7-19.5 um

(x = 74.6 + 15.9 x 12.6 + 4.1 um; E = 3-11; Q =6.6 + 2.6; n/2 = 21). PILEAL

TRAMAL HYPHAE Subparallel 58-226 x 4-22 um (x = 119 + 63 x 12.1 + 6.3 um;

E = 4-52; Q =14.2 + 16.8; n/2 = 7). STIPITIPELLIS composed of clusters of

erect to semi-erect 2-5 celled-hyphae separated by a 2-3-celled prostrate cutis;

terminal cells differentiated. CauLocystip1A similar in shape but often much

Leptonia spp. nov. (Australia) ... 165

—pes 2 >

_— “SS a e

= I s

PLATE 3. Leptonia substricta (DLL 9852 holotype): basidiomata. Bar = 10 mm.

longer than the pileocystidia, 48-156 x 6-13 um (x = 81.8 + 35.8 x 9.2 + 1.7 um;

E = 4-19; Q =9.2 + 4.4; n/2 = 13). STIPE TRAMAL HYPHAE Subparallel, 200-367 x

6.0-13 um; n/2 = 9). OLEIFEROUS HYPHAE rare in the lamellar trama, absent

elsewhere. BRILLIANT GRANULES absent. LIPOID GLOBULES absent. PIGMENTS

in pilei-/stipitipellis brownish blue, cytoplasmic; in pileus subpellis dark brown,

encrusting. CLAMP CONNECTIONS in all tissues.

ECOLOGY & DISTRIBUTION—Barrington Tops National Park: scattered,

humicolous, subtropical gallery rainforest, mid-March; Mungo Brush, Myall

166 ... Largent & al.

PLATE 4. Leptonia substricta (DLL 9852 holotype): A. stipitipellis longitudinal section at apex;

B. pileipellis radial section near disc; C. basidia and basidioles; D. broadly obclavate pileocystidia

and cytoplasmic pigment; E. basidiospores. Bars: A = 125 um; B = 50 um; C = 9 um; D, E= 7 um.

Lakes National Park: exposed in sandy soil along the trail in a littoral rainforest,

early April.

ADDITIONAL COLLECTION EXAMINED —AUSTRALIA. New SouTtH WALES, central

Hunter District. Barrington Tops National Park, Williams Day Use Area, 16 March

2009, S Moore 1.

DISTINCTIVE CHARACTERS—Basidiomata mycenoid; pileus conic to

campanulate, appressed squamulose then matted squamulose to matted

fibrillose towards edge, indigo-blue to dark violet (18F6) with the squamule-

tips more blackish purple; stipe long and narrow, very fragile, blackish blue and

densely appressed fibrillose but with abundant to scarce purplish black-tipped

squamules; lamellar edge fertile; pileocystidia subulate to obclavate; Pilei-/

stipitipellis with brownish blue cytoplasmic pigments, pileus subpellis with

encrusting pigments; taste and odor mild.

ComMMENTS—Leptonia substricta morphologically resembles Entoloma strictum

G. Stev. (Horak 2008), which differs by its collybioid basidiomata, (arcuate)

Leptonia spp. nov. (Australia) ... 167

decurrent pink lamellae, farinaceous taste and odor, and clavate or vesiculose

caulocystidia. Entoloma cedretorum (Romagn. & Riousset) Noordel., from

southern France and Morocco, also close to L. substricta, differs in its white

lamellae, entirely wooly-tomentose stipe, and intracellular pigment.

Leptonia sabulosa Largent & Bergemann, sp. nov. PLATES 5, 6

MycoBank MB 813883

Differs from Entoloma endotum by its yellowish young lamellae, punctuate squamulose

stipe, mild taste, sandy habitat, and smaller basidiospores.

Type — Australia, New South Wales, central Hunter District, Tomaree National Park,

32°45'26”S 152°08’03’E, in sandy soil, 23 April 2012, DL Largent 10427 (holotype DAR,

GenBank KR869896 (mtSSU), KR869925 (LSU)).

Etymo.ocy — derived from the Latin sabulosus referring to the fruiting in sandy soil.

BASIDIOMATA tricholomatoid. PiLEus 27-64 mm broad, 7-12 mm high;

when young convex to broadly convex (not umbonate), expanding to plane

and broadly umbonate; at first uniformly appressed squamulose with flat

overlapping squamules that later collapse to form a uniformly velvet-like

surface; dull (neither translucent nor hygrophanous), center dark blackish blue

with a dark violet tint (18-19F5-7), somewhat lighter (18-19E5-7) towards the

margin, still lighter (18E5-8) on the very edge; outer margin incurved at first,

PiateE 5. Leptonia sabulosa (DLL10427 holotype): basidiomata. Bar = 25 mm.

168 ... Largent & al.

becoming decurved, then plane and eventually uplifted, the edge extending

beyond the lamellae by 1 mm. CoNTExT 2-5 mm thick above the stipe, solid,

watery grey. ODOR somewhat pungent. Taste mild. LAMELLAE 13-25 mm

long, 5-10 mm broad; sinuate, close to subdistant, moderately wide to wide,

edges smooth; initially yellowish white to orange-white (4-5A2), becoming

orange-white to grayish orange (5A-B2-3) from spores with concolorous edges.

LAMELLULAE in 3 tiers between lamellae, 2 short and 1 longer. Stipe 37-75 mm

long, 5-9 mm thick at apex and middle, 7-12 mm thick at base; stuffed at first

becoming hollow at the apex; overall densely fibrillose with the fibrils splitting

laterally and with abundant black-edged punctae from apex to base (at the

very base rare in some), at the base tomentum abundant, forming a broad pad;

blackish-blue (19F7-8) when young (becoming more greyish violet (18-19E5)

as the fibrils split apart), dark blue (19E6) at the base; basal tomentum staining

orangish (4A2-3).

BASIDIOSPORES 5-7 distinct angles (at times slightly more pronounced

and protuberate), in profile subisodiametric to heterodiametric (on average

decidedly heterodiametric, only rarely isodiametric), the apex typically rounded,

rarely truncate; 8-13 x 6-9 um (x = 10.4 + 0.8 x 7.0 + 0.6 um; E = 1.1-1.8;

Q =1.5 + 0.1; n/3 = 100). Basrp1a 4-sterigmate, clavate, 37-54 x 6.5-13.5 um

(x=45.1+45.1 x 11.0 + 1.4 um; E = 3-8; Q =4.2 + 0.9; n/3 = 27). LAMELLAR EDGE

fertile. CHEILOCYSTIDIA absent. PLEUROCYSTIDIA absent. LAMELLAR TRAMAL

HYPHAE subparallel, 46-135 x 5-23 um (x = 83.0 + 29.1 x 12.8 + 6.8 um;

E = 2-17; Q =8.3 + 4.8; n/1 = 12). PILEIPELLIS 122-321 um deep, a + erect

entangled layer of 3-6-celled hyphae; prteocystip1a cylindro-clavate to

broadly cylindro-clavate and often cystidiiform, 43-128 x 5-17 um (x = 80.5 +

21.9x 11.4+ 3.0 um; E = 2-16; Q =7.8 + 3.6; n/2 = 30). PILEAL TRAMAL HYPHAE

entangled, 13-28 um wide (n/1 = 7). STrpITIPELLis a layer of hyphae 4-6-cells

deep, composed of abundant clusters of semi-erect to erect anticlinal hyphae

interspersed among periclinal hyphae. CauLocystip1a cylindro-clavate,

51-86 x 7-13 um (x = 74.5 + 13.5 x 10.0 + 2.6 um; E = 5-12.5; Q =7.9 + 2.6;

n/2 = 10). STIPE TRAMAL HYPHAE 8-28 um diam (n/1 = 7). OLEIFEROUS HYPHAE

absent. BRILLIANT GRANULES scattered in the basidia. Liporp BoptrEs absent.

PIGMENTATION purplish blue, cytoplasmic in the pileipellis and stipitipellis.

CLAMP CONNECTIONS abundant in all tissues.

ECOLOGY & DISTRIBUTION—Scattered in sandy soil and leaves in the forest

between the beach and mangrove stands in northeastern Queensland, late

March; also beneath grass trees (species of Xanthorrhoea Sm.) and cycads

(species of Macrozamia Mig.) in central New South Wales, April.

Leptonia spp. nov. (Australia) ... 169

PiaTE 6. Leptonia sabulosa (DLL10427 holotype): A. pileipellis radial section near disc;

B. stipitipellis longitudinal section at apex; C. cylindro-clavate pileocystidia (terminal cells);

D. basidiospores; E. basidia and basidioles. Bars: A, B = 50 um; C = 80 um; D = 7 um; E= 11 um.

ADDITIONAL COLLECTIONS EXAMINED —AUSTRALIA. QUEENSLAND, Cook Region,

Yorkies Knob 16°49’01”S 145°43’50”E, 21 March 2011, DL Largent 10058. NEw SouTH

WALES, central Hunter District, Wyrrbalong National Park, 33°17’40”S 151°32’38.20’E,

P O'Sullivan NW 010; Tomaree National Park, 32°45’26”S 152°08’02”E; 24 April 2012,

DL Largent 10445.

DISTINCTIVE CHARACTERS—Basidiomata tricholomatoid; pileus dark blackish

blue with a dark violet tint; appressed squamulose pileus becoming velvety in

age; young yellowish tinged lamellae developing a decidedly grayish orange cast

when mature; cheilocystidia absent; basidiospores with slightly protuberant

angles; odor pungent, taste mild.

ComMENTS—Entoloma endotum Noordel. & G.M. Gates from Tasmania is

distinguished from L. sabulosa by its creamy pink young lamellae, fibrillose-

floccose or girdled stipe, a salty or bitter to acrid taste, and somewhat larger

basidiospores (10.5-12.7 x 7.9-8.5 um). Entoloma eugenei differs by its sterile

lamellar edges, pure white lamellae, blackish blue colors (violet tinted in the

170 ... Largent & al.

pileus), cylindrical, lageniform, or irregularly shaped cheilocystidia, and

strangulated pilei- and caulocystidia.

Leptonia newlingii Largent & Bergemann, sp. nov. PLATES 7, 8

MycoBank MB 813886

Differs from Entoloma indoviolaceum by its medium to large basidiomata, fertile

lamellar edges, slightly shorter basidiospores, and the cytoplasmic pigments in both

pilei- and stipitipellis.

Type — Australia, Queensland, Cook Region, Yorkies Knob Beach Forest, 16°48’59”S

145°43’49”E, in sandy soil, 11 March 2011, DL Largent 10004 (holotype BRI; isotype

cns; GenBank KR869897 (mtSSU), KR869926 (LSU)).

ETryMOLoGy — named for Peter Newling, who discovered it.

Piteus 11-51 x 5-18 mm, initially convex to campanulate (not umbonate),

then plane to uplifted and broadly umbonate; entirely punctate tomentose from

blunt erect pointed scales that flatten to form a uniformly appressed velvety

surface; dull, opaque but not translucent; blackish blue (19F5) when young,

becoming slightly lighter (19F4) on the disc and somewhat lighter (19E4)

elsewhere; the outer margin incurved then decurved to plane, the edge in age

crenulate to eroded, extending past lamellae by 0.5-1 mm. ConTExT 0.5-2 mm

thick above the stipe, violaceous to bluish white. ODOR mild. Taste mild

(occasionally somewhat pungent to farinaceous). LAMELLAE 7-25 x 2.5-8 mm;

adnexed to nearly adnate; at first close and narrow, then subdistant and

moderately broad to broad with smooth then eroded edges; initially pale yellow

(2A3) to yellowish white (4A2), becoming pale orange (5A3) as spores mature,

with concolorous edges. LAMELLULAE in 1 or 3 series, 1 medium or 2 short

(<3 mm) and 1 medium (<11 mm). STIPE 42-73 mm long, the 2-7 mm diam.

apex expanding to 7-15 mm base; at first apex punctate from erect squamules,

elsewhere densely fibrillose-squamulose, later remaining punctate at the apex

but becoming rimose and longitudinally recticulate-ridged elsewhere with

the ridges composed of fibrils interspersed with appressed squamules; at first

blackish blue (20F6), with the ridges lightening slightly (20D4), and then finally

dark blue (19E4); context stuffed then hollow, more or less yellowish orange

with a hint of orange (4-53); stipe base with scarce to moderately abundant

white (at times yellowish white, 2A2) tomentum and abundant yellowish white

rhizoids spreading into the soil.

BASIDIOSPORES 5-7 distinct to rounded angles, isodiametric to

heterodiametric (on average heterodiametric) in profile view, apex at first

somewhat rounded and + triangular (in some becoming truncate and in a few

with the angles slightly protuberant); 8.5-12.5 x 6.5-9 um (x = 10.4 + 0.7 x 7.7

+ 0.6 um; E = 1-1.6; Q =1.4 + 0.1; n/3 = 132). Basrp1a 4-sterigmate; clavate,

Leptonia spp. nov. (Australia) ... 171

PLATE 7 Leptonia newlingii (DLL10004 holotype): A. basidiomata (field); B. basidiomata (lab).

Bars = 30 mm.

tapered towards the base, 33-59 x 10-14.5 um (x = 46.0 + 6.6 x 11.9 + 1.1 um,

E = 3-4.6, Q =3.8 + 0.5, n/2 = 17). LAMELLAR EDGE fertile. CHEILOCYSTIDIA

absent. PLEUROCYSTIDIA absent. LAMELLAR TRAMAL HYPHAE subparallel,

moderately long and broad: 53-180 x 5-15 um (x = 94.5 + 31.3 x 8.7 + 2.9 um;

172 ... Largent & al.

E = 5-20; Q =11.5 + 4.0; n/2 = 16). PILEIPELLIS 90-250 um (n/1 = 3) deep,

composed of trichodermial tufts comprising 3-4 celled hyphae, loosely

entangled, and erect terminal cells. PlLEocystTip1< cylindro-clavate to clavate,

45-157 x 6.5-12.5 um (x = 95.8 + 27.9 x 9.6 + 1.7 um; E = 5-17; Q =10.2 + 3.2;

n/2 = 21). PILEAL TRAMAL HYPHAE Subparallel, entangled, short and cylindric

to cylindro-clavate; 80-240 x 9-22 um (x = 152.6 + 64.9 x 15.5 + 4.0 um;

E = 5-15; Q =9.9 + 3.6; n/2 = 8). STIPITIPELLIS composed of loosely entangled

hyphae with clavate terminal cells. CAuLocystiv1a similar to pileocystidia but

shorter and more slender, 50-123 x 5-8 um (x = 82.3 + 20.7 x 6.9 + 0.9 um;

E = 7-19; Q =12.2 + 3.2; n/3 = 21). STIPE TRAMAL HYPHAE subparallel and

entangled. OLEIFEROUS HYPHAE absent. BRILLIANT GRANULES scattered in

the basidia. LiporD GLOBULES absent. PIGMENTs in pileipellis and stipitipellis

bluish and cytoplasmic, soluble in 3% KOH, brownish pigments and encrusted

pigments absent. CLAMP CONNECTIONS abundant in all tissues.

ECOLOGY & DISTRIBUTION—Scattered in sandy soil and leaves, in forest

between the beach and mangrove stands or in soil under logs in vine forests,

in northeastern Queensland, mid-January to late March.

ADDITIONAL COLLECTIONS EXAMINED —AUSTRALIA. QUEENSLAND, Yorkies Knob

Beach Forest, 16°48’51”S 145°43’47”E, 1lm, 9 January 2011, P. Newling PN14b;

16°48’49”S 145°43’44”E, 14 March 2011, DL Largent 10026, 16°46’00’S 145°43’49”E, 22

March 2012, DL Largent 10313.

DISTINCTIVE CHARACTERS—Basidiomata at first collybioid but soon naucorioid

to nearly trichomatoid; pileus at first blackish blue and entirely tomentose

or erect squamulose, then dark blue and tomentose on disc and appressed

squamulose elsewhere; lamellae yellowish white; stipe fleshy, similar in color

to pileus, with ridges and bands of squamules; basal tomentum moderate to

abundant; stipe base with rhizoids penetrating the substrate.

ComMMENTS—Leptonia newlingii and E. indoviolaceum (from Kerala state in

India; Manimohan et al. 2006) are closely related phylogenetically (BS = 71,

Fic. 1). Morphologically, E. indoviolaceum is distinguished from L. newlingii

by its small to medium basidiomata, sterile lamellar edges, longer (11-14 um)

basidiospores, and encrusting pigments in the stipitipellis.

Entoloma panniculus from Tasmania can be distinguished from L. newlingii

by its smaller basidiomata, farinaceous odor and taste, and encrusting pigments

in the stipitipellis.

Since 2009, numerous L. newlingii populations have been observed in the

fall season, occurring abundantly in sandy soils in coastal vine forests just

north of Cairns in northeastern Queensland (P. Newling, pers. obs.).

Leptonia spp. nov. (Australia) ... 173

PLATE 8 Leptonia newlingii (DLL10004 holotype): A. pileipellis; B. pileocystidia; C. basidiospores;

D. caulocystidia; E. basidium; EF. pileocystidia. Bars: A = 170 um; B, D, F = 10 um; C = 8 um;

E=12um.

174 ... Largent & al.

PLaTE 9 Leptonia ambigua (DLL10130 holotype): A. basidiomata;

B. basidiospores; C. submoniliform pileipellis. Bars: A = 16 mm; B = 7.5 um; C = 10 um.

Leptonia ambigua Largent, Mycotaxon 125: 18 (2013). PLATE 9

CoMMENTS—Otur analysis supports the placement of L. ambigua within a

sub-clade that includes Entoloma euchroum (Fie. 1). Leptonia ambigua can be

distinguished from the other species within this sub-clade by its isodiametric

to subisodiametric basidiospores with 6-7(-8) angles and a pileipellis of

submoniliform clampless hyphae (PLATE 9C). Fora discussion of morphological

similarities and differences with other entolomatoid species see the description

of L. ambigua in Largent et al. (2013b).

Leptonia spp. nov. (Australia) ... 175

Acknowledgments

Materials required to complete this manuscript were provided by the Largent family

trust. The Australian Tropical Herbarium and the School of Marine and Tropical

Biology, James Cook University provided fieldwork and logistical support. The DNA

sequences generated in this study are based upon work supported by the National

Science Foundation under Grant No. DRI 0922922 awarded to Dr. Sarah Bergemann.

Comments by the two reviewers, Dr. Joseph FE Ammirati and Dr. Timothy J. Baroni,

and by the Nomenclature Editor, Dr. Shaun Pennycook, were also helpful. We wish to

thank Peter Newling for his assistance in collecting in northeastern Queensland and

Pam O'Sullivan and Skye Moore for their assistance in collecting in New South Wales.

Finally, David and Pamela Largent wish to especially thank Dr. Sandra Abell for being

an extraordinary research advisor and contact at James Cook University during the five

years of research in northeastern Queensland.

Literature cited

Co-David D, Langeveld D, Noordeloos ME. 2009. Molecular phylogeny and spore evolution of

Entolomataceae. Persoonia 23: 147-176. http://dx.doi.org/10.3767/003158509X480944

Horak E. 1980. Entoloma (Agaricales) in Indomalaya and Australasia. Beihefte zur Nova Hedwigia

65. 352 p.

Horak E. 2008. Agaricales of New Zealand. 1: Pluteaceae (Pluteus, Volvariella) Entolomataceae

(Claudopus, Clitopilus, Entoloma, Pouzarella, Rhodocybe, Richoniella). Fungi of New Zealand

Volume 5. Fungal Diversity Research Series 19. 305 p.

Kornerup A, Wanscher JH. 1978. Methuen handbook of colour 3rd ed. Richard Clay Ltd:

Chichester, Sussex.

Largent DL. 1994. Entolomatoid fungi of the western United States and Alaska. Mad River Press

Inc: Eureka, California.

Largent DL, Abell-Davis SE, Cummings GA, Ryan KL, Bergemann SE. 201la. Saxicolous

species of Claudopus (Agaricales, Entolomataceae) from Australia. Mycotaxon 116: 253-264.

http://dx.doi.org/10.5248/116.253

Largent DL, Bergemann SE, Cummings GA, Ryan KL, Abell-Davis SE, Moore S. 2011b. Pouzarella

(Agaricales, Entolomataceae) from New South Wales (Barrington Tops National Park) and

northeastern Queensland. Mycotaxon 117: 435-483. http://dx.doi.org/10.5248/117.435

Largent DL, Bergemann SE, Abell-Davis SE, Kluting KL, Cummings GA. 2013a. Three new

Inocephalus species with cuboid basidiospores from New South Wales and Queensland,

Australia. Mycotaxon 123: 301-309. http://dx.doi.org/10.5248/123.301

Largent DL, Bergemann SE, Abell-Davis SE, Kluting KL, Cummings GA. 2013b. Five Leptonia

species from central New South Wales and Queensland, Australia. Mycotaxon 125: 11- 35.

http://dx.doi.org/10.5248/125.11

Manimohan P, Noordeloos ME, Dhanya AM. 2006. Studies in the genus Entoloma (Basidiomycetes,

Agaricales) in Kerala State, India. Persoonia 19(1): 45-93.

Miller MA, Pfeiffer W, and Schwartz T. 2010. Creating the CIPRES Science Gateway for inference

of large phylogenetic trees. 1-8, in: Proceedings of the Gateway Computing Environments

Workshop (GCE).

Morozova OV, Noordeloos ME, Vila J. 2014. Entoloma subgenus Leptonia in boreal-

temperate Eurasia: towards a phylogenetic species concept. Persoonia 32: 141-169.

http://dx.doi.org/10.3767/003158514X681774

176 ... Largent & al.

Noordeloos ME. 2004. Entoloma s.l. Fungi Europaei, vol. 5a. Edizione Candusso, Italy.

Noordeloos ME, Gates GM. 2012. The Entolomataceae of Tasmania. Fungal Diversity Research

Series 22. 399 p.. http://dx.doi.org/10.1007/978-94-007-4679-4

Stamatakis A. 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with

thousands of taxa and mixed models. Bioinformatics 22: 2688-2690.

Thiers B. 2015. Index Herbariorum: a global directory of public herbaria and associated staff. New

York Botanical Garden's Virtual Herbarium. http://sweetgum.nybg.org/ih/ [accessed August

2015].

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

fungal ribosomal RNA genes for phylogenetics. 315-322, in: MA Innis et al. (eds).

PCR protocols: a guide to methods and applications. Academic Press, Inc. New York.

http://dx.doi.org/10.1016/B978-0-12-372180-8.50042-1

MYCOTAXON

January-March 2016—Volume 131, pp. 177-183

http://dx.doi.org/10.5248/131.177

Dictyoaquaphila appendiculata gen. & sp. nov.

from submerged wood from Brazil

JOSIANE SANTANA MONTEIRO’, LUCAS BARBOSA CONCEICAO’,

Marcos FABIO OLIVEIRA MARQUES’, Luis FERNANDO PASCHOLATI GUSMAO*

& RAFAEL F. CASTANEDA-RUIZ?

' Universidade Estadual de Feira de Santana,

Avenida Transnordestina, s/n, Novo Horizonte, 44036-900, Feira de Santana, Brazil

? Universidade do Estado da Bahia,

Rodovia Lomanto Jr., BR 407 Km 127, 48970-000, Senhor do Bonfim, Brazil

* Instituto de Investigaciones Fundamentales en Agricultura Alejandro de Humboldt’ (INIFAT),

Calle 1 Esq. 2, C.P. 17200, Santiago de Las Vegas, C. Habana, Cuba

* CORRESPONDENCE TO: lgusmao@uefs. br

ABSTRACT — Dictyoaquaphila appendiculata, collected from submerged wood in Brazil, is

described and illustrated as a new genus and species. It is distinguished by distinct, erect,

septate, subhyaline to very pale brown conidiophores, mono- and polyblastic conidiogenous

cells, and fusiform, elliptical to shortly clavate, brown separating cells. The conidia are

dictyoseptate, broad ellipsoidal-fusiform to prolate subglobose, dark brown, usually with

1-3 divergent, narrow cylindrical, hyaline appendages at the apical cell, and sometimes with

1-2(-3), narrow cylindrical, hyaline appendages arising from the basal cell.

Key worps — freshwater fungi, neotropic, taxonomy

Introduction

The diversity of conidial fungi in the Brazilian Amazon Forest has

received little attention, especially in freshwater habitats. During a survey

of hyphomycetes associated with submerged plant material in streams from

Para state, a conspicuous fungus was collected. This fungus shows remarkable

differences from all previously described genera (Seifert et al. 2011) and is

described here as new.

178 ... Monteiro & al.

Materials & method

Samples of submerged litter were placed in plastic bags for transport to the

laboratory, where they were placed in Petri dish moist chambers and stored at 25°C

for 30 days in a 170 L polystyrene box with 200 mL sterile water plus 2 mL glycerol

(Castafieda-Ruiz 2005). Mounts were prepared in PVL (polyvinyl alcohol, lactic acid,

and phenol) and measurements were made at a x 1000 magnification. Micrographs were

obtained with an Olympus BX51 microscope equipped with bright field and Nomarski

interference optics. The specimens including holotype are deposited in the Herbarium

of Universidade Estadual de Feira de Santana, Bahia, Brazil (HUEFS).

Taxonomy

Dictyoaquaphila J.S. Monteiro, L.B. Conc., M.E.O. Marques, Gusmao &

R.E. Castaneda gen. nov.

MycoBAnk MB 813243

Differs from Dictyodesmium by its effuse colonies, polyblastic and swollen conidiogenous

cells, fusiform separating cells, and dictyoseptate conidia with several cylindrical

filamentous hyaline appendages at one or both end cells.

TYPE SPECIES: Dictyoaquaphila appendiculata J.S. Monteiro et al.

EryMoLoGcy: Greek, dictyo-, referring to the transverse, longitudinal, and oblique

conidial septa; Latin, -aqua- meaning water; and Greek, —phila meaning loving.

Conidial fungi. CoLonizs on the natural substratum effuse, brown or dark

brown. CONIDIOPHORES distinct, single, unbranched, septate, subhyaline to pale

brown. CONIDIOGENOUS CELLS mono- and polyblastic, integrated or discrete,

determinate or indeterminate with sympodial extensions. CONIDIOGENOUS

Loc! flatted or slightly broad denticulate, subhyaline or pale pigmented.

SEPARATING CELLS swollen, fusiform, ellipsoidal or shortly clavate, brown or

dark brown. Conidial secession schizolytic. Conip1A solitary, dictyoseptate,

broad ellipsoid-fusiform to prolate subglobose, brown to dark brown, cuneate

to subulate or subconical at the end cells, with 0-3 divergent filiform or narrow

cylindrical, hyaline appendages at the ends.

Dictyoaquaphila appendiculata J.S. Monteiro, L.B. Conc., M.F.O. Marques,

Gusmao & R.F. Castafieda sp. nov. FIG 1-3

MycoBAank MB 813244

Differs from all Dictyodesmium spp. by its effuse and mono- or polyblastic colonies,

sometimes with sympodial extensions, and its indeterminate conidiogenous cells from

which arise swollen, fusiform, or shortly clavate separating cells and dictyoseptate

conidia, appendiculate at the end cells.

Type: Brazil, Para State: Santa Barbara, Parque Ecoldgico de Gumma, 1°13’S 48°17’W,

on decaying submerged wood in a river, 29.VII.2012, coll. J.S. Monteiro (Holotype:

HUEFS 216013).

Erymo ocy: Latin, appendiculata, referring to the small conidial appendages.

Dictyoaquaphila appendiculata gen. & sp. nov. (Brazil) ... 179

A B C

¢¢e@

D EK F G

e666@

9 6e6@

Fic. 1. Dictyoaquaphila appendiculata (ex holotype HUEFS 216013).

Conidia. Bar = 10 um.

180 ... Monteiro & al.

K L

Fic. 2. Dictyoaquaphila appendiculata (ex holotype HUEFS 216013).

A-I. conidiogenous cells, separating cells, and conidia; J-M. conidia. Bar = 10 um.

COLONIES on the natural substratum effuse, dark brown to black. Mycelium

mostly immersed. CONIDIOPHORES distinct, single or moderately branched,

erect, bent or flexuous, subhyaline or very pale brown, 0-3-septate, 20-40 x

3-5 um, smooth. CONIDIOGENOUS CELLS mono- and polyblastic, integrated,

sometimes discrete, determinate or indeterminate with sympodial extensions,

terminal and intercalary, subhyaline to very pale brown, 3-5 x 1.5-3 um.

Dictyoaquaphila appendiculata gen. & sp. nov. (Brazil) ... 181

Fic. 3. Dictyoaquaphila appendiculata (ex holotype HUEFS 216013).

A-H. conidia; I-J. conidiophores, conidiogenous cells and separating cells. Bar = 10 um.

182 ... Monteiro & al.

SEPARATING CELLS swollen, fusiform, ellipsoidal or shortly clavate, brown or

dark brown at the margin, pale brown at the middle, suhyaline at the ends,

sometimes in a short chains with a secondary cell, 7-10 x 3.5-5 um. Conidial

secession schizolytic. Conrp1A solitary, acrogenous, dictyoseptate, broad

ellipsoid-fusiform to prolate subglobose, 37-45 x 17-27 um, brown to dark

brown, black at the transverse septa, and subhyaline or hyaline at the cuneate

to subulate or subconical at the end cells, mostly with 0-3 divergent, filiform or

narrow cylindrical, hyaline, smooth apical appendages, 20-42 x 1-1.5 um and

sometimes 0-2(-3) divergent, filiform or narrow cylindrical, hyaline, smooth

basal appendages, 20-35 x 1-1.5 um.

ADDITIONAL SPECIMEN EXAMINED: BRAZIL, BAHIA STATE: Pindobacu, Serra da

Fumaga, on decaying submerged wood in stream, 7.XII.2012, L.B. Conceigao (HUEFS

216020).

Comments: Dictyoaquaphila is superficially similar to Dictyodesmium

S. Hughes, Dictyorostrella U. Braun, Muiaria Thaxt., and Xenostigmina Crous,

all of which have short distinct conidiophores and a blastic conidial ontogeny

but which lack separating cells and filiform appendages (Seifert et al 2011).

Filiform appendages, which are widely distributed in the aero-aquatic and

freshwater hyphomycete ecological group, probably function in anchoring

the conidia to substrates (Cai et al. 2006, Ingold 1975, Seifert et al. 2011).

The function of separating cells in freshwater hyphomycetes has not been

documented, although Kendrick (2003) did cover some hyphomycete genera

with biconic conidia attached at the separating cells.

Acknowledgments

The authors express their sincere gratitude to Dr. De-Wei Li and Prof. Bryce

Kendrick for their critical review of the manuscript. JSM is grateful to the “Coordenac¢ao

de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)” for Pos-doctoral fellowship

(proc. 071/2012). LFPG is grateful to the CNPq (Proc. 303062/2014-2). RFCR is grateful

to Cuban Ministry of Agriculture and “Programa de Salud Animal y Vegetal’, project

P131LH003033 Cuban for facilities. We acknowledge the assistance provided by

Dr. P.M. Kirk and Drs. V. Robert and G. Stegehuis through the Index Fungorum

and MycoBank websites. Dr. Lorelei Norvell’s editorial and Dr. Shaun Pennycook’s

nomenclatural reviews are greatly appreciated.

Literature cited

Cai L, Hyde KD, Tsui CKM. 2006. Genera of freshwater fungi. Fungal Diversity Research Series

18. 255 p.

Castaneda-Ruiz RE 2005. Metodologia en el estudio de los hongos anamorfos: 182-183, in: Anais

do V Congresso Latino Americano de Micologia. Brasilia.

Ingold CT. 1975. An illustrated guide of aquatic and water-borne hyphomycetes (fungi imperfecti)

with notes on their biology. Freshwater Biological Association, Scientific Publication 30. 96 p.

Dictyoaquaphila appendiculata gen. & sp. nov. (Brazil) ... 183

Kendrick B. 2003. Analysis of morphogenesis in hyphomycetes: new characters derived from

considering some conidiophores and conidia as condensed hyphal systems. Canadian Journal

of Botany 81: 75-100. http://dx.doi.org/10.1139/B03-008

Seifert K, Morgan-Jones G, Gams W, Kendrick B. 2011. The genera of hyphomycetes.

CBS Biodiversity Series 9. 997 p.

MY COTAXON

January-March 2016—Volume 131, pp. 185-192

http://dx.doi.org/10.5248/131.185

Anacoronospora diversiseptata gen. & sp. nov. from Brazil

JOSIANE SANTANA MONTEIRO’, Luis FERNANDO PASCHOLATI GUSMAO’ &

RAFAEL F, CASTANEDA-RUIZ?’

' Universidade Estadual de Feira de Santana,

Avenida Transnordestina, s/n, Novo Horizonte, 44036-900, Feira de Santana, Brazil

? Instituto de Investigaciones Fundamentales en Agricultura ‘Alejandro de Humboldt’ (INIFAT),

Calle 1 Esq. 2, C.P. 17200, Santiago de Las Vegas, C. Habana, Cuba

* CORRESPONDENCE TO: Igusmao@uefs. br

Asstract — The hyphomycete Anacoronospora diversiseptata, collected from decaying

leaves from Caatinga biome, Brazil, is described and illustrated as a new genus and species.

It is distinguished by mono- and polyblastic conidiogenous cells with successive sympodial

rectilinear or geniculate extension (sometimes delimited by one septum and followed by

enteroblastic percurrent extension), which process is repeated several times. The conidia are

oval to broad ellipsoidal, golden brown, and with a distoseptate main body, a subcylindrical

or doliiform basal cell with 0-4 mammillate protuberances, and a crown-shaped euseptate

apex with 3-4 divergent cylindrical branches.

KEY worps — asexual fungi, neotropic, taxonomy

Introduction

During a survey of hyphomycetes associated with plant litter from the semi-

arid region of Bahia State, a fungus was collected that differs remarkably from

all previously described genera (Seifert et al. 2011) and which we describe here

as new. Caatinga ecosystems are unique to Brazil and, with only about 1% of

the area under protection, are undoubtedly threatened. The type locality is a

small remaining fragment of natural or semi-natural vegetation surrounded by

farming and other developments. The discovery of this new genus here (and of

others already collected by the authors, but as yet undescribed) suggests that

the Caatinga may be as rich in fungi as in the many endemic animals and plants.

186 ... Monteiro, Gusmao, & Castafeda-Ruiz

Materials & methods

Samples of litter (leaves and wood) were collected in paper bags for transport to the

laboratory, where they were placed in Petri dish moist chambers and stored at 25°C

for 30 days in a 170 L polystyrene box with 200 mL sterile water plus 2 mL glycerol

(Castafieda-Ruiz 2005). Mounts were prepared in PVL (polyvinyl alcohol, lactic acid,

and phenol) and measurements were made at a magnification of x1000. Micrographs

were obtained with an Olympus BX51 microscope equipped with bright field and

Nomarski interference optics. The type specimens are deposited in the Herbarium of

Universidade Estadual de Feira de Santana, Bahia, Brazil (HUEFS).

Taxonomy

Anacoronospora J.S. Monteiro, Gusmao & R.E. Castafieda, gen. nov.

MycoBank MB 813219

Differs from Coronospora by its successive enteroblastic percurrent regeneration after

each holoblastic sympodial extension of the cicatrized conidiogenous cells and its

distoseptate and euseptate conidia with small basal protuberances.

TYPE SPECIES: Anacoronospora diversiseptata J.S. Monteiro et al.

EryMoLoGy: Greek, ana-, meaning upwards; Latin coronospora, referring to the

hyphomycete genus Coronospora.

Conidial fungi. CoLonizs on the natural substratum effuse, brown or dark

brown. Mycelium partly superficial and immersed. Conip1oPHoREs distinct,

single, unbranched, septate, brown or dark brown. CONIDIOGENOUS CELLS

mono- and polyblastic, integrated, with enteroblastic percurrent regenerations

and sympodial extensions. CONIDIOGENOUS LOCI evident, flattened, thick and

black. Conidial secession schizolytic. Conip14 solitary, at first acrogenous, later

acropleurogenous, ovoid, ellipsoidal, with several short protuberances on the

basal cell, distoseptate in the main body, crown-shaped at the apex, with several

divergent, aseptate or euseptate branches, brown to dark brown.

Anacoronospora diversiseptata J.S. Monteiro, Gusmao & R.F. Castafieda

sp. nov. FIGs 1-3

MycoBank MB 813220

Differs from all Coronospora spp. by its successive enteroblastic percurrent regenerations

after each holoblastic sympodial extension of the conidiogenous cells and solitary disto-

and euseptate, conidia with several short protuberances at the basal cell.

Type: Brazil, Bahia State: Abaira, Distrito de Catolés, Mata do Coqueiro, 13°14’S

41°43’W, on decaying leaves of unidentified plant, 11.1.2015, coll. J.S. Monteiro

(Holotype: HUEFS 215981).

EryMo_oey: Latin, diversi-, meaning turned different ways, diverse, contrary, different;

septata, referring to the septa.

Cotontgs on the natural substratum effuse, hairy, amphigenous, brown to

dark brown. Mycelium mostly immersed. ConipIopHoREs distinct, single,

Anacoronospora diversiseptata gen. & sp. nov. (Brazil) ... 187

Fic. 1. Anacoronospora diversiseptata (ex holotype HUEFS 215981): A-C, conidiophores,

conidiogenous cells, and conidium; D, E. conidiogenous cells and conidium; F. conidiophore and

conidiogenous cells. Bars = 10 um.

188 ... Monteiro, Gusmao, & Castafieda-Ruiz

Fic. 2. Anacoronospora diversiseptata (ex holotype HUEFS 215981): Conidia. Bar = 10 um.

Anacoronospora diversiseptata gen. & sp. nov. (Brazil) ... 189

Fic. 3. Anacoronospora diversiseptata (ex holotype HUEFS 215981):

Conidiogenous cells and conidia. Bar = 10 um.

erect, straight or slightly geniculate toward the apex, with up to 7 percurrent

regenerations, 5-10-septate, 70-150 x 5-7 um, dark brown below, brown to pale

brown toward the apex, somewhat fasciate after each enteroblastic regeneration,

190 ... Monteiro, Gusmao, & Castafieda-Ruiz

smooth. CONIDIOGENOUS CELLS mono- and polyblastic, integrated, with

enteroblastic percurrent regenerations and sympodial extensions, terminal

at first with a single terminal conidiogenous locus, then indeterminate, with

successive sympodial rectilinear or geniculate extension, sometimes delimited

by one septum and followed by enteroblastic percurrent extension, being

the same process repeated several times, producing terminal and intercalary

conidiogenous loci. Conidiogenous loci cicatrized, flat, dark brown or black.

Conidial secession schizolytic. Conip1A solitary, acropleurogenous, 30-45 x

14-19 um, ovoid to broad ellipsoidal, 4-5(-—7)-distoseptate at the main body,

golden brown, with subcylindrical, truncate, 5-6 um wide, cicatrized basal cell,

with 0-4 nipple-shaped protuberances radially arranged and crown-shaped,

euseptate at the apex, with a cuneiform or doliiform apical cell from which

arise 3-4 divergent, cylindrical, obtuse at the tip, 0-2-euseptate, pale golden

brown appendages, 10-20 x 3-4 um.

ComMENTS — The ontogenetic pattern in Anacoronospora can be classified using

the scheme for conidial development in Kirk et al. (2008). Conidial initiation

is holoblastic, delimitation by one septum, secession schizolytic, maturation

by diffuse wall-building, percurrent enteroblastic conidiogenous cell extension,

followed by further conidial initiation by replacement of apical wall-building;

melanized, with each successive conidium seceding before the next percurrent

elongation of the conidiogenous cell, but sometimes holoblastic sympodial

extension also occurs, producing two or more conidia. The combination of

sympodial and enteroblastic percurrent extension and regeneration with

flattened, thick, darkened conidiogenous loci in Anacoronospora separates

it from Coronospora M.B. Ellis, in which cicatrized loci are produced after

sympodial extensions of the polyblastic cicatrized conidiogenous cells disposed

in geniculate conidiophores (Ellis 1971, 1976, Matsushima 1975, 1985, Zhang

& Zhang 2004). Two described Coronospora species (Fic. 4), C. novae-zelandiae

Matsush. and C. uniseptata Matsush., have denticulate, non-cicatrized

conidiogenous loci, characters that deviate somewhat from those found in

Coronospora dendrocalami M.B. Ellis (the type species) and C. pallescens Meng

Zhang & TY. Zhang. Phylogenetic analysis is needed to elucidate the taxonomic

position of C. novae-zelandiae and C. uniseptata.

Acknowledgments

The authors express their sincere gratitude to Dr. De-Wei Li and Prof Bryce Kendrick

for their critical review of the manuscript. The authors thank the “Programa de Pesquisa

em Biodiversidade do Semiarido (PPBio Semiarido - CNPq/MCTI) for supporting

this study. LFPG is grateful to the CNPq (Proc. 303062/2014-2). JSM is grateful to

the “Coordenacao de Aperfeigoamento de Pessoal de Nivel Superior (CAPES)” for

Anacoronospora diversiseptata gen. & sp. nov. (Brazil) ... 191

Fic. 4. Representative conidia of Coronospora spp. (redrawn from literature):

A. C. dendrocalami; B. C. novae-zelandiae; C. C. pallescens; D. C. uniseptata. Bars = 20 um.

192 ... Monteiro, Gusmao, & Castafieda-Ruiz

Post-doctoral fellowship (proc. 071/2012). RFCR is grateful to Cuban Ministry of

Agriculture and “Programa de Salud Animal y Vegetal’, project P131LH003033 Cuban for

facilities. We acknowledge the assistance provided by Dr. P.M. Kirkand Drs. V. Robert and

G. Stegehuis through the Index Fungorum and MycoBank websites. Dr. Lorelei Norvell’s

editorial and Dr. Shaun Pennycook’s nomenclatural reviews are greatly appreciated.

Literature cited

Castafieda-Ruiz RF. 2005. Metodologia en el estudio de los hongos anamorfos: 182-183, in: Anais

do V Congresso Latino Americano de Micologia. Brasilia.

Ellis MB. 1971. Dematiaceous hyphomycetes. Commonwealth Mycological Institute, Kew, Surrey.

Ellis MB. 1976. More Dematiaceous hyphomycetes. Commonwealth Mycological Institute,

Kew, Surrey.

Kirk PM, Cannon PF, Minter DW, Stalpers JA. 2008. Ainsworth & Bisby’s dictionary of the fungi.

10" edition. CAB International Wallingford, U.K.

Matsushima T. 1975. Icones microfungorum a Matsushima lectorum. Nippon Printing Co, Osaka.

Matsushima T. 1985. Matsushima mycological memoirs no. 4. Matsushima Fungus Collection,

Kobe, Japan.

Seifert K, Morgan-Jones G, Gams W, Kendrick B. 2011. The genera of hyphomycetes.

CBS Biodiversity Series 9. 997 p.

Zhang M, Zhang TY. 2004. A new species of Coronospora from China. Mycosystema 23: 331-332.

MY COTAXON

January-March 2016—Volume 131, pp. 193-203

http://dx.doi.org/10.5248/131.193

Two new polypore species from the southwestern USA:

Fomitiporia fissurata and F. deserticola

JOSEF VLASAK** & JOSEF VLASAK JR.

‘Biological Centre of the Academy of Sciences of the Czech Republic,

Branisovskd 31, CZ-370 05 Ceské Budéjovice, The Czech Republic

* CORRESPONDENCE TO: vlasak@umbr.cas.cz

ABSTRACT — Based on molecular evidence and morphological studies, two new Fomitiporia

species are described from southwestern USA. Fomitiporia fissurata sp. nov., on living

Quercus, was previously misdetermined as FE robusta and is the only oak-inhabiting

Fomitiporia species in the region. Fomitiporia deserticola sp. nov. grows on cacti and desert

shrubs in Arizona and was previously misdetermined as FE. texana, which is a species found

only on juniper.

Key worps — Basidiomycota, Hymenochaetales, Hymenochaetaceae, molecular taxonomy

Introduction

Fomitiporia Murrill (Hymenochaetales, Basidiomycota) forms a monophyletic

clade within Hymenochaetaceae and is characterized by a perennial habit,

dextrinoid and cyanophilous basidiospores, and absence of setae in most species

(Dai et al. 2008; Dai 2010). Over the years, the interpretation of Fomitiporia

in the USA has changed and evolved. Murrill (1907, 1908) described many

American species that were not accepted by subsequent mycologists. During

the most of the 20th century, the European species F. robusta (P. Karst.) Fiasson

& Niemela, FE punctata (P. Karst.) Murrill, and F. hartigii (Allesch. & Schnabl)

Fiasson & Niemela were reported throughout the United States. Only E texana

(Murrill) Nuss and F. sonorae (Gilb.) Y.C. Dai from southwestern USA were

recognized as endemic American species (Gilbertson & Ryvarden 1987; Dai et

al. 2001).

Recently, the presence of F. robusta and FE. punctata in southern, western,

and eastern USA has been questioned (Fischer & Binder 2004; Vlasak & Kout

194 ... Vlasdk & Vlasak

201la) whereas other species, described by Murrill, have been confirmed.

For example, recognition of resupinate species Fomitiporia dryophila Murrill,

F. langloisii Murrill, and FE maxonii Murrill has been supported by molecular

phylogenetics (Decock et al. 2007). Pileate species E bakeri (Murrill) Vlasak

& Kout growing on birch and E calkinsii (Murrill) Vlasak & Kout growing on

oak from the eastern USA have also been confirmed (Vlasak & Kout 201 1a). In

addition, two new species—E cupressicola Amalfi et al. growing on Cupressus

arizonica in northern Mexico and F. polymorpha M. Fisch. on Salix from the

USA Pacific Coast—have been described (Fischer & Binder 2004; Amalfi

et al. 2012). In these studies, researchers noted that DNA sequence data,

ecological preferences, and biogeographical data were important in species

characterization because distinguishing microscopic features were absent.

Overholts (1953) realized that a peculiar form of Fomes robustus

[= Fomitiporia robusta], which was “less developed and nodulose-sessile,”

occurred on oaks in the western USA. Vlasak & Kout (2011a), who obtained a

sample of this taxon from California (voucher JV 0407/16-J), discovered that its

ITS rDNA sequence differed from all previously described Fomitiporia species.

Amalfi et al. (2012) obtained the same ITS sequence from a collection from

New Mexico designated Fomitiporia PS1 (phylogenetic species 1). We have

since collected many specimens of this oak-restricted and relatively common

fungus from southern California and Arizona, and describe it below as a new

species, EF fissurata.

Davidson & Mielke (1947) identified a different taxon found on cacti and

other desert plants in Arizona as a variety of Fomes robustus. Based on studies

of pure cultures, they concluded it was not conspecific with E texana, which

is known from the same region but grows on juniper. Pyropolyporus texanus

[= Fomitiporia texana] was considered conspecific with Fomes robustus by

Lowe (1957) and Lowe & Gilbertson (1961) but were later accepted as distinct

species, with the taxon growing on desert plants treated as conspecific with

F. texana (Gilbertson & Canfield 1972; Gilbertson & Ryvarden 1987). We

collected Fomitiporia specimens from both juniper and desert plants in Texas

and Arizona and conclude that they represent separate species. We describe

the taxon growing on desert plants as the new species Fomitiporia deserticola.

Materials & methods

Samples were collected in July 2004, spring and fall 2012, May 2013, and March

2014 at various localities in California and Arizona. The specimens were dried and

microscopically examined in Melzer’s reagent (IKI) and 5% KOH. All collected

specimens are deposited in herbarium of Josef Vlasak (JV); types deposited in Prague

Museum herbarium, Czech Republic (PRM). DNA isolation, nuclear ribosomal DNA

Fomitiporia fissurata and F. deserticola spp. nov. (U.S.A.) ... 195

i 13095 CA USA[KT381630

a 31205145 CA USA|KT381629]

a JV1305/2J hapl2 CAUSA(KT381626] type

4 5V1204/22.2) CAUSA|KT331621 Fomitoporia

i JV1206/2.4) CA USA[KT381623] fissurata

& JV1305/2J hapl1 CA USA[KT381627] type new sp.

A Fomitiporia sp. CA USA GU 126227

4JV1305/15 hapli CA USA|KT381625]

JVIZ0S 1 hapl2 CA USAIKTI81626]

a 3V1209/84 AZ USA[KT381624]

A SV1307/5) CA USAIKT381630|

A Fomitiporia sp. NM USA JQ087883

400; F. bakeri PA USA GU136219

F, bakeri NC USA GU 136221

F. robusta CZECH HO162313

@l) F. robusta CZECH GU136218

F. robusta ESTONIA AY340018

JV1208/6.1J F. polymomha CA USA[KT381639]

F. polymorpha USA GU 461956

F. punicata CHINA GU461975

F. punicata CHINA GU461974

F. harigii CZECH HQ162317

F. hartigi GERMANY AY340012

F, tsugina PA USA GU 594156

F. tsugina NH USA HQ162314

F. cupressicola MEXICO JQ087880

i—zl F. cupressicota MEXICO JQ087879

F. calkinsii TN USA GU136223

100) F. calkinsii TN USAGU136224

F. dryophila FL USA GU 126213

100L F. dryophila FL USA GU 136212

JV1403/9-J F. texana Juniper TX USA[KT381637]

JV1409134) F. texana Juniper TX USA[KT381636)

Miettinen16684 F. texana Juniper TX USA[KT381636]

@ 1V1209/46 O puntia AZ USA[KT381632] type

@ F. texana Morus AZUSA JQ087895 Fomitiporia

@ JV1209/41) Lamea AZ USAIKT381633] deserticola

@ JV1209/40-1 Covania AZ USAIKT381634] new sp.

@ JV1209/40-2 Covania AZ USAIKT381635

Phellinus igniarius GQ383711

t+———

O66

Fic. 1: Phylogenetic relationships of 36 Fomitiporia specimens inferred from ITS rRNA sequences.

Phellinus igniarius was used as outgroup. Topology from maximum likelihood (ML) analysis.

Support values along branches from ML bootstrap (500 replicates). Branch lengths are drawn

proportional to the number of substitutions per site. Triangles and black circles indicate specimens

of new species. GenBank numbers are presented at the end of taxon labels with new sequences

boxed.

196 ... Vlasak & Vlasak

ITS region sequencing, and comparative phylogenetic analyses methods follow Vlasak &

Kout (2011b). The evolutionary history was inferred by using the Maximum Likelihood

method; the analyses were conducted using MEGA 6 (Tamura et al. 2013).

Results

Phylogenetic analysis

Twenty ITS sequences of Fomitiporia specimens from California and Arizona

were generated for this study. A complete Fomitiporia ITS dataset, which

included 23 Fomitiporia sequences retrieved from GenBank and represented

all pileate Fomitiporia species occurring in the USA, produced an alignment

with 833 characters, of which 180 were parsimony informative. Phylogenetic

analyses strongly support all pileate Fomitiporia specimens collected on oak

in California and Arizona within the clade of the new species F. fissurata

(Fic. 1). Species in this clade are sister to the European species E robusta and the

American species F. bakeri, as expected from their morphological characters.

Fomitiporia specimens from various desert plants in Arizona (including cacti)

comprise another strongly supported clade, representing the new species

EF. deserticola, which is sister to the juniper-dwelling F. texana (Fie. 1).

Taxonomy

Fomitiporia fissurata Vlasak, sp. nov. FIGS 2, 3

MycoBank MB 813966

Differs from Fomitiporia robusta by its broader, rounded pileal margin, pileal surface

with deep arboriform fissures even in juvenile basidiomata, smaller basidiospores, and

a different ITS rDNA sequence.

Type: USA. California: Cleveland National Forest, Mt. Laguna, Quercus sp. (Fagaceae),

18 May 2013, J. Vlasdk Jr. (Holotype, PRM 922626; isotype, JV1305/2-J; GenBank

KT381627, KT381628).

EryMo_oey: fissurata (Lat.): referring to striking fissures on pileal surface of the fungus.

BASIDIOMES perennial, pileate, arising from a black, cracked, crust-like

outgrowth on the substrate, ungulate, up to 18 x 10 x 12 cm; margin 3-6 cm

broad, rounded; upper surface gray to blackish, often with white spore deposits,

at first glabrous and with a few wide sulcate zones, soon indurated to 1 cm

deep and cracking up irregularly, creating deep, often arboriform fissures; pore

surface yellowish brown; pores circular, 4-6 per mm, with thin to thick, entire

dissepiments; context yellowish-brown, shining at first, then woody hard,

up to 5 cm thick, with distinct white radial veins in older parts resulting in a

marmorate appearance of the context close to the substrate attachment; tube

layers distinct, light brown, tubes becoming white within from hyphae growing

in the tubes, woody, each layer up to 1 cm thick.

Fomitiporia fissurata and F. deserticola spp. nov. (U.S.A.) ... 197

Fic. 3: Fomitiporia fissurata specimen: JV 1209/54 (split collection: PRM 922627)

198 ... Vlasak & Vlasak

HYPHAL SysTEM dimitic, skeletal hyphae brownish, thick-walled, 2-4 um

diam., generative hyphae hyaline, thin-walled, simple septate, 2-4 um diam.

SETAE absent. BasipiA broadly ellipsoid, 4-sterigmate, 12-15 x 7-9 um, simple

septate at the base. Basip1ospores subglobose, hyaline, smooth, thick-walled,

strongly dextrinoid, 6-7 x 5-6(-6.5) um. White rot on living Quercus.

DISTRIBUTION & ECOLOGY. Common on living Quercus spp. in the US

southwest (California, Arizona, New Mexico); causing a white rot. No other

pileate Quercus-dwelling Fomitiporia species is known in the region.

ADDITIONAL SPECIMENS EXAMINED: USA. CALIFORNIA: Santa Cruz, Henry Covell

Redwoods State Park, Quercus, Jul 2004, J. Vlasak Jr. (JV0407/16J; GenBank GU136227,

GU136228); Thousand Oaks, Malibu Creek State Park, Quercus, 22 Apr 2012, J.

Vlasak Jr. (JV1204/22.2J; GenBank KT381621, KT381622); Saratoga, Castle Rock

State Park, Quercus, 2 Apr 2006, J. Vlasak Jr. (JV1206/2.4J; GenBank KT381623);

Cleveland National Forest, Pine Valley County Park, Quercus, 18 May 2013, J. Vlasak

Jr. JV1305/1J; GenBank KT381625, KT381626); Escondido, Mt. Palomar, Quercus, 23

May 2013, J. Vlasak Jr. (JV1305/4J; GenBank KT381629); Palm Springs, Mt. San Jacinto,

Quercus, 25 May 2013, J. Vlasak Jr. (JV1305/5J; GenBank KT381630); ARIZONA: Santa

Rita Mts., Madera Canyon, Quercus, 2 Sep 2012, J. Vlasak (JV1209/19); Chiricahua Mts.,

Chiricahua Nat. Monument Visitor Center, Quercus, 5 Sep 2012, J. Vlasak (JV 1209/54,

PRM 922627; GenBank KT381624); Cochise Stronghold National Monument, Quercus,

17 Jul 2013, J. Vlasak Jr. JV1307/5J; GenBank KT381631).

ComMMENTS. Fomitiporia robusta, which occurs in Europe, differs from

E fissurata by its pileal surface mostly covered with green algae and becoming

cracked only in old basidiomes. The cracks do not form arboriform fissures.

In addition, a black crust does not develop around the F. robusta basidiome,

which has a narrower rounded margin compared to EF fissurata. Finally,

F. robusta produces larger basidiospores (6-8.5 x 5.5-7 um). Fomitiporia

bakeri also resembles EF fissurata but is found only on birch in the eastern and

central USA. Fomitiporia calkinsii, which grows on oaks in the southeastern

USA, has a relatively sharp margin, a more thinly hardened crust that is rarely

or inconspicuously cracked, and a darker brown context and tube layers.

Fomitiporia polymorpha, growing primarily on willows in the western USA,

develops small, bulbous basidiomes, and EF hartigii and E tsugina are known

from gymnosperms.

Fomitiporia deserticola Vlasak, sp. nov. Fics 4-6(top)

MycoBank MB 813967

Differs from Fomitiporia texana by its angular, thin-walled pores, a different ITS rDNA

sequence, and smaller basidiospores.

Type: USA. Arizona: Tanque Verde, Opuntia versicolor Engelm. ex Toumey (Cactaceae),

4 Sep 2012, J. Vlasak 1209/46 (Holotype, PRM 934073; isotype, JV1209/46; GenBank

KT381632).

EtryMoLoey: deserticola (Lat.): referring to growth on desert plant substrates.

Fomitiporia fissurata and F. deserticola spp. nov. (U.S.A.) ... 199

~ Fie. 4: Fomitiporia deserticola specimens:

JV 1209/46, isotype (left); PRM 934073, holotype (right).

Fic. 5: Fomitiporia deserticola specimen: JV 1209/40.

200 ... Vlasak & Vlasak

BASIDIOMES perennial, pileate, distinctly ungulate, small to medium-sized,

usually 2-4 cm in diam., but sometimes up to 8 x 8 x 8 cm; upper surface

at first dark brown, tomentose, soon becoming blackened and deeply rimose,

usually with white spore deposits and cracks often partially filled by current

year context tissue; margin rounded, yellowish brown, tomentose; pore surface

pale brown; pores angular, somewhat irregular, 4-6 per mm, with thin to thick,

entire dissepiments; context woody hard, brown, up to 1 cm thick; tube layers

distinct, pale brown, becoming stuffed with light-colored mycelium, each layer

up to 1 cm thick.

HYPHAL SYSTEM dimitic, skeletal hyphae brownish, thick-walled, 2-4 um

diam, generative hyphae hyaline, thin-walled, simple septate, 2-4 um diam.

CysTIDIOLEs abundant, with an inflated base up to 8 um diam and a slender

neck, 10-30 um long and about 2 um thick. SETAE rare, similar to cystidioles

but thick-walled. Basrp1a subglobose, 4-sterigmate, 9-12 x 8-10 um, simple

septate at the base. Basip1ospores subglobose, hyaline, smooth, thick-walled,

strongly dextrinoid, 6-7.5 x 5.5-7 um.

DISTRIBUTION & ECOLOGY. On living cacti and desert shrubs in Arizona,

causing a white rot.

ADDITIONAL SPECIMENS EXAMINED:

Fomitiporia deserticola: USA, ARIZONA: Tanque Verde, Cowania stansburiana

Torr. (Rosaceae), 4 Sep 2012, J. Vlasak Jr. JV 1209/40, GenBank KT381634, KT381635);

Larrea tridentata (Sessé & Moc. ex DC.) Coville (Zygophyllaceae), 4 Sep 2012, J.Vlasak

Jr. JV1209/41J; GenBank KT381633); J. Vlasak Jr. (JV1209/42J).

Fomitiporia texana: USA, Texas: Austin, Juniperus ashei J. Buchholz

(Cupressaceae), 15 Aug 2013, O. Miettinen 16684 (JV1308/15; GenBank KT381636);

Balcones Canyonlands National Wildlife Ref., Juniperus sp., 21 Mar 2014, J. Vlasak Jr.

(JV 1403/9-J; GenBank KT381637); Pedernales Falls State Park, Juniperus sp., 22 Mar

2014, J. Vlasak Jr. (JV 1403/13-J; GenBank KT381638).

CoMMENTS. Fomitiporia texana, which occurs in the same region as

F. deserticola, can be distinguished by round thick-walled pores (Fic. 6,

bottom), cracks on pileal surface not filled with context tissue from current

year, larger basidiospores (mostly 8 um diam), and host specific on juniper.

Davidson & Mielke (1947) have also shown that culture characteristics of the

cactus and juniper taxa are different.

Gilbertson & Lindsey (1975) cited setae as characteristic of E texana,

although setae were mentioned neither in the original description (Murrill

1908) nor in the description of Fomes robustus on cacti (Davidson & Mielke

1947). Gilbertson & Ryvarden (1987) suggested that these structures represent

just cystidioles with thickened walls, and we agree with their interpretation.

Fomitiporia fissurata and F. deserticola spp. nov. (U.S.A.) ... 201

F. texana

Comparison of pores of Fomitiporia deserticola and

FIG

Imens

in about half of the spec

as they have been observed

Setae are uncommon

ion is

ifferenti

of both E texana and F deserticola, their value for species d

]

ited

202 ... Vlasak & Vlasak

Macroscopic features key to pileate Fomitiporia species occurring in the USA

1 Species with detached upper margin on otherwise resupinate basidiocarp .... 2

1 Distinctly pileate species, with mostly large, sessile or ungulate pilei ......... 4

2 CT SHG, ed ee Mic cen aetna dina Bias te liad oon inde one Bands ons Bandy or, odes apo Ss F. tsugina

2 On:dicotyledOnous WOSts: 25 2 obi g 2 Bay einai y end eee mey Lin Mun ernst kA aig sae ne 3

3 On Dodonea viscosa in a small area in Arizona.................006. EF sonorae

3 On Quercus in southern USA from Florida to Texas .............. EF. dryophila

4 On Betula in northeastern and midwestern USA .................00. E bakeri

4 Cin -Otherst bethates (Scie lanes e cetera ea hod lene Raden Sabine Baden Rade peated 5

5 CMCOMPCPOUS NOSIS: A Mets tees haat, eee one Mette Mee ae Meas Aeon sora 6

5 On dicotyledenGusdiostst! ss. nt xd sate ed see 88 “efi Def ed eng defy Od lenge me 7

6 On Juniperus in southern Texas and Arizona ..................006. F, texana

6 Onliving Cupressus in Mexico and southwestern USA .......... E cupressicola

7 On desert shrubs and cacti in southern Arizona ................ E deserticola

7 CEPITEC I CHE YS as ka Pa care RA i tged RA asda AA Ga ged AAR usere AB Bea GAB ea ge ees hatibene yh igine Ss 8

8 In southeastern USA, margin of typical pilei 0.5-1 cm thick,

pileus crust not at all or indistinctly cracked .................0.04. E calkinsii

8 In southwestern USA, pileal margin rounded and 2-5 cm thick,

crust deeply, irregularly cracked in old and juvenile pilei ........... E fissurata

Discussion

With the addition of two new Fomitiporia species from the western USA,

there are now nine North American pileate species. We do not include

F. robusta nor F. hartigii in our key, because neither Eurasian species occurs

in North America (Vlasak & Kout 2011a; Brazee 2013). Our DNA sequence

data results support the conclusions by Amalfi et al. (2012) and others that

geography, habitat, and host relationships are important factors in Fomitiporia

speciation. Accordingly, a key based on morphology, host and distribution only

(see above) can be used successfully to identify pileate species of Fomitiporia

in North America. Nevertheless, molecular methods are necessary to delineate

cryptic species.

Acknowledgments

The authors thank Otto Miettinen for sending a specimen of Fomitiporia texana.

Many thanks also to Y.-C. Dai (Beijing Forestry University, China) and K. Nakasone

for their contributions as reviewers. The research was funded by institutional support

RVO: 60077344.

Literature cited

Amalfi M, Raymundo T, Valenzuela R, Decock C. 2012. Fomitiporia cupressicola sp. nov., a

parasite on Cupressus arizonica, and additional unnamed clades in the southern USA and

Fomitiporia fissurata and F. deserticola spp. nov. (U.S.A.) ... 203

northern Mexico, determined by multilocus phylogenetic analyses. Mycologia 104: 880-893.

http://dx.doi.org/10.3852/11-196

Brazee NJ. 2013. Phylogenetic and differentiation analysis of the trunk rot pathogen Fomitiporia

tsugina in North America. Forest Pathology 43: 407-414. http://dx.doi.org/10.1111/efp.12047

Dai YC. 2010. Hymenochaetaceae (Basidiomycota) in China. Fungal Diversity 45: 131-343.

http://dx.doi.org/10.1007/s13225-010-0066-9

Dai YC, Zhang XQ, Zhou TS. 2001. New and noteworthy species of Hymenochaetaceae from China.

Mycosystema 20: 16-21.

Dai YC, Cui BK, Decock, C. 2008. A new species of Fomitiporia (Hymenochaetaceae, Basidiomycota)

from China based on morphological and molecular characters. Mycol. Res. 112: 375-380.

http://dx.doi.org/10.1016/j.mycres.2007.11.020

Davidson RW, Mielke JL. 1947. Fomes robustus, a heart-rot fungus on cacti and other desert plants.

Mycologia 39: 210-217. http://dx.doi.org/10.2307/3755009

Decock C, Herrera Figueroa S, Robledo G, Castillo G. 2007. Fomitiporia punctata (Basidiomycota,

Hymenochaetales) and its presumed taxonomic synonyms in America: taxonomy and

phylogeny of some species from tropical/subtropical areas. Mycologia 99: 733-752.

http://dx.doi.org/10.3852/mycologia.99.5.733

Fischer M, Binder M. 2004. Species recognition, geographic distribution and host-pathogen

relationships: a case study in a group of lignicolous basidiomycetes, Phellinus s.l. Mycologia 96:

799-811. http://dx.doi.org/10.2307/3762113

Gilbertson RL, Canfield ER. 1972. Poria carnegiea and decay of saguaro cactus in Arizona.

Mycologia 64: 1300-1311. http://dx.doi.org/10.2307/3757967

Gilbertson RL, Lindsey JP. 1975. Basidiomycetes that decay junipers in Arizona. Great Basin

Naturalist 35: 288-304.

Gilbertson RL, Ryvarden L. 1987. North American polypores. Fungiflora, Oslo.

Lowe JL. 1957. Polyporaceae of North America. The genus Fomes. State Univ. N.Y. Coll. For. Tech.

Publ. 80. 97 p.

Lowe JL, Gilbertson RL. 1961. Synopsis of the Polyporaceae of the southeastern United States.

J. Elisha Mitchell Sci. Soc. 77: 43-66.

Murrill WA. 1907-1908. Polyporaceae. North Am. Flora 9(1-2): 1-131.

Overholts LO. 1953. Polyporaceae of the United States, Alaska and Canada. University of Michigan

Press, Ann Arbor.

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA 6: Molecular

Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 30: 2725-2729.

http://dx.doi.org/10.1093/molbev/mst197

Vlasak J, Kout J. 2011a. Pileate Fomitiporia species in the USA. New combinations Fomitiporia

calkinsii and F. bakeri. Mycol. Progress 10: 445-452.

http://dx.doi.org/10.1007/s11557-010-0715-0

Vlasak J, Kout J. 2011b. Tropical Trametes lactinea is widely distributed in the eastern USA.

Mycotaxon 115: 271-279. http://dx.doi.org/10.5248/115.271

MY COTAXON

January-March 2016—Volume 131, pp. 205-209

http://dx.doi.org/10.5248/131.205

First report of Singerocybe in Thailand

JATURONG KUMLA’, NAKARIN SUWANNARACH’, SANTHITI VADTHANARAT’,

OLIVIER RASPE”? & SAISAMORN LUMYONG’™*

"Department of Biology, Faculty of Science, Chiang Mai University,

Chiang Mai, 50200, Thailand

*Botanic Garden Meise, Nieuwelaan 38, 1860 Meise, Belgium

Fédération Wallonie-Bruxelles, Service Général de Enseignement universitaire

et de la Recherche scientifique, Rue A. Lavallée 1, 1080 Brussels, Belgium

* CORRESPONDENCE TO: saisamorn.l@cmu.ac.th

ABSTRACT — Specimens collected in Thailand have been identified as Singerocybe

alboinfundibuliformis, based on morphology and ITS molecular analysis. A description and

illustration are provided. This is the first record of the genus from Thailand.

Key worps — Agaricales, Tricholomataceae, basidiomycete, gill fungus, taxonomy

Introduction

Harmaja (1988) erected Singerocybe (Agaricales, Tricholomataceae) with

S. viscida Harmaja as the type species. This genus is distributed worldwide

in temperate and tropical regions. Singerocybe species are saprotrophic and

usually grow on soils rich in humus, dead wood, or rotting leaves (Peck 1873,

Harmaja 1988, Takahashi 2000, Seok et al. 2009, Qin et al. 2014). Although

no Singerocybe species has been reported from Thailand (Chandrasrikul et al.

2011), during a taxonomic survey of macrofungi collected in northern Thailand,

we found specimens that corresponded to the description of Singerocybe

alboinfundibuliformis, a species previously reported from China, Korea, and

Japan (Takahashi 2000, Seok et al. 2009, Qin et al. 2014). Here, we describe and

illustrate the morphological characters of the Thai material and provide results

from ITS sequence analysis.

206 ... Kumla & al.

Materials & methods

Morphology studies

Basidiocarps were collected from Doi Suthep-Pui National Park, Chiang Mai

Province, Thailand in 2015 and wrapped in aluminum foil or kept in plastic specimen

boxes for transport to the laboratory where notes on macromorphological features

and photographs were taken within 24 h. Color names and codes follow Kornerup &

Wanscher (1978). The specimens were dried at 40—45 °C; sections of dried material

were mounted in 95% ethanol and rehydrated in distilled water, 3% KOH, or Melzer’s

reagent for microscopical examination; at least 50 measurements were made of each

structure. The collections are deposited at the Research Laboratory for Excellence in

Sustainable Development of Biological Resources, Faculty of Science, Chiang Mai

University, Thailand (SDBR-CMU).

Molecular studies

Genomic DNA of three specimens was extracted from fresh tissue using CTAB

according to Kumla et al. (2012). The internal transcribed spacer (ITS) region of

ribosomal RNA gene was amplified by polymerase chain reaction (PCR) with ITS4 and

ITS5 primers under the following thermal conditions: 94 °C for 2 min; 35 cycles of 95 °C

for 30 s, 50 °C for 30 s, 72 °C for 1 min; and 72 °C for 10 min. Negative controls lacking

fungal DNA were run to check for reagent contamination. PCR products were checked

on 1% agarose gels stained with ethidium bromide under UV light and purified using

NucleoSpin’ Gel and PCR Clean-up Kit (Macherey-Nagel, Germany) following the

manufacturer's protocol. The purified PCR products were directly sequenced. Sanger

sequencing was performed by 1°’ Base Company (Selangor, Malaysia) using the ITS4

and ITS5 PCR primers. Sequences were used to query GenBank database via BLAST

(http://blast.ddbj.nig.ac.jp/top-e.html).

Results

Taxonomic description

Singerocybe alboinfundibuliformis (Seok, Yang S. Kim, K.M. Park,

WG. Kim, K.H. Yoo & I.C. Park) Zhu L. Yang, J. Qin & Har. Takah.,

Mycologia 106: 1022 (2014). PLATE 1

Pileus 20-50 mm in diameter, infundibuliform, hollow down to the base

of stipe, smooth, white (4A1) to yellowish-white (4A2), margin involute when

young and incurved in age. Lamellae decurrent, <1.5 mm wide, white (4A1)

when young, becoming yellowish-white (4A2) to orange-white (5A2) in age.

Stipe central, 20-45 x 3-10 mm, cylindrical or tapering towards the base,

smooth, similar in color to the pileus. Odor strong. Pileipellis composed of

irregularly parallel hyphae that are 2-5 um wide, septate, and interspersed

with vesicles (20—45 x 3-10 um); vesicles also found at the stipe apex. Clamp

connections present. Basidia 22.0-33.0 x 4.5-6.0 um, clavate, 4-spored,

with basal clamp connections and long sterigmata (<5 um). Basidiospores

Singerocybe new to Thailand ... 207

PLATE 1. Singerocybe alboinfundibuliformis (SDBR-CMU-SGC72). A: basidiocarps; B: pileipellis;

C: basidia; D: basidiospores. Scale bars: A = 10 mm, B = 25 um, C = 10 um, D = 5 um.

208 ... Kumla & al.

3.0-6.5 x 2.0-4.0 um (n = 50), Q = 1.2-2.2, elliptical, smooth, thin-walled,

inamyloid. Spore print white.

SPECIMENS EXAMINED — THAILAND, CHIANG MAI PROVINCE, Muang District, Doi

Suthep-Pui National Park, Yod Doi Pui, 18°49’16”N 98°53’35’E, elevation 1495 m,

on rotting litter in mixed evergreen hill forest, 4 July 2015, Suwannarach N. (SDBR-

CMU-SGC61; GenBank KT373850); 4 August 2015, Kumla J. & Suwannarach N.

(SDBR-CMU-SGC85; GenBank KT373852); Doi Pui, 18°49’30”N 98°53’36’E, elevation

1515 m, on rich humus soil in mixed evergreen hill forest, 26 July 2015, Suwannarach N.

(SDBR-CMU-SGC72; GenBank KT373851).

Molecular analysis

The ITS sequences of specimens SDBR-CMUSGC61 (702 bp), SDBR-

CMUSGC72 (694 bp), and SDBR-CMUSGC85 (696 bp) were deposited in

GenBank. All ITS sequences obtained in this study showed 99% similarity with

Singerocybe alboinfundibuliformis JX514126 and JX514132.

Discussion

The presence of vesicles in the cuticle of the pileus and stipe support our

collections in Singerocybe. All three specimens collected in northern Thailand

were initially identified as S. alboinfundibuliformis based on descriptions by

Takahashi (2000) and Seok et al. (2009) and the key provided by Qin et al.

(2014). This species is easily distinguished from other Singerocybe species

by a pileus that is deeply infundibuliform down to the base of the tubular

stipe, the narrow and intervenose gills, and elliptical smooth basidiospores.

Singerocybe alboinfundibuliformis resembles S. humilis (Berk. & Broome) Zhu

L. Yang & J. Qin, and both are clearly distinguished from other species by their

deeply infundibuliform pilei, open tubular stipes, and obviously intervenose

gills (Takahashi 2000, Seok et al. 2009, Qin et al. 2014). However, S. humilis

is separated from S. alboinfundibuliformis by its very narrow gills with

interstices becoming transversely reticulate to subporoid and DNA sequence

analyses (Qin et al. 2014). Singerocybe humilis occurs in South Asia, while

S. alboinfundibuliformis appears restricted to East Asia (Takahashi 2000,

Seok et al. 2009, Qin et al. 2014). Singerocybe hydrogramma (Bull.) Harmaja,

S. phaeophthalma (Pers.) Harmaja, and S. viscida are known only from Europe

(Kuyper 1981, Harmaja 1988), S. adirondackensis (Peck) Zhu L. Yang &

J. Qin from North America (Peck 1873; Qin 2014); S. clitocyboides (Cooke &

Massee) Zhu L. Yang et al. from Australia and New Zealand (Pegler 1965), and

S. umbilicata Zhu L. Yang & J. Qin from East Asia (Qin et al. 2014).

The molecular analysis also confirmed the three Thai specimens as

S. alboinfundibuliformis. Therefore, the combination of morphological and

molecular characters help establish S. alboinfundibuliformis as a new record in

Thailand.

Singerocybe new to Thailand ... 209

Acknowledgements

This work was supported by grants from Chiang Mai University and Thailand

Research Fund, Research-Team Association Grant (RTA5880006). We are grateful to

Dr. Erik Hobbie for English proofreading. We also thank Dr. Eric H.C. McKenzie and

Dr. Steven L. Stephenson for their helpful comments and careful review of this article.

Literature cited

Chandrasrikul A, Suwanarit P, Sangwanit U, Lumyong S, Payapanon A, Sanoamuang N, Pukahuta

C, Petcharat V, Sardsud U, Duengkae K, Klinhom U, Thongkantha S, Thongklam S. 2011.

Checklist of mushroom (basidiomycetes) in Thailand. Office of Natural Resources and

Environmental Policy and Planning, Bangkok.

Harmaja H. 1988. Studies on the agaric genera Singerocybe n. gen. and Squamanita. Karstenia

27-71-7955

Kornerup A, Wanscher JH. 1978. Methuen handbook of colour. Eyre Methuen, London.

Kumla J, Bussaban B, Suwannarach N, Lumyong S, Danell E. 2012. Basidiome formation of an

edible wild, putatively ectomycorrhizal fungus, Phlebopus portentosus without host plant.

Mycologia 104: 597-603. http://dx.doi.org/ 10.3852/11-074

Kuyper TW. 1981. Notes on Clitocybe I. Persoonia 11: 385-386.

Peck CH. 1873. Report of the botanist. Annual Report on the New York State Museum of Natural

History 232: 27-135.

Pegler DN. 1965. Studies on Australasian Agaricales. Australian Journal of Botany 13: 323-356.

Qin J, Feng B, Yang ZL, Ratkowsky D, Gates G, Takahashi H, Rexer K, Kost GW, Karunarathna

SC. 2014. The taxonomic foundation, species circumscription and continental endemisms of

Singerocybe: evidence from morphological and molecular data. Mycologia 106: 1015-1026.

http://dx.doi.org/10.3852/13-338

Seok SJ, Kim YS, Park KM, Kim WG, Yoo KH, Park IC. 2009. New species of Agaricales.

Mycobiology 37: 295-299. http://dx.doi.org/10.4489/MYCO.2009.37.4.295

Takahashi H. 2000. Two new species and one new variety of Agaricales from central Honshu, Japan.

Mycoscience 41: 15-23. http://dx.doi.org/10.1007/BF02464381

MY COTAXON

January-March 2016—Volume 131, pp. 211-225

http://dx.doi.org/10.5248/131.211

Acremonium camptosporum isolated as an endophyte of

Bursera simaruba from Yucatan Peninsula, Mexico

Maria C. GONZALEZ’, ANTHONY E. GLENN?, RICHARD T. HANLIN?,

MARTHA L. Macias RUBALCAVA4, BLANCA E. HERNANDEZ BAUTISTA® &

ANA LuIsA ANAYA>

'Depto. Botanica, Instituto de Biologia, Universidad Nacional Autonoma de México,

Ciudad de México DF 04510 México

2 USDA, ARS, Toxicology & Mycotoxin Research Unit,

Russell Research Center, Athens GA 30604 USA

* Museum of Natural History Annex, University of Georgia,

4435 Atlanta Highway, Athens GA 30606 USA

*Depto. Productos Naturales, Instituto de Quimica, Universidad Nacional Auténoma de México,

Ciudad de México DF 04510 México

° Depto. Ecologia Funcional, Instituto de Ecologia, Universidad Nacional Autonoma de México,

Ciudad de México 04510, México

“ CORRESPONDENCE TO: mcgv@ib.unam.mx

ABSTRACT —An interesting endophytic fungus was isolated from the leaves of Bursera

simaruba, a tree in the semi-deciduous dry tropical forest at El Eden Ecological Reserve. The

cultured strain developed the morphology characteristic of the genus Acremonium—tapered

phialides and small aseptate conidia clustered in slimy heads. A detailed morphological

examination and 28S rDNA sequence analysis identified the Yucatan strain as Acremonium

camptosporum, an uncommon fungus found previously as a saprobe. The material is

described and illustrated as the first record of this species from Mexico.

KEY worpbs — anamorphic fungi, Ascomycota, clavicipitaceous fungi, Hypocreales, taxonomy

Introduction

Acremonium is a complex and diverse genus of Ascomycota with widespread

global distribution despite its simple morphology (Gams 1971, Glenn et al.

1996, Summerbell et al. 2011, Giraldo et al. 2014). Although Acremonium

212 ... Gonzalez & al.

species are usually isolated as saprobes from plant remains and soil, some taxa

are important opportunistic human and plant pathogens (Domsch et al. 2007,

Perdomo et al. 2011, Li et al. 2014, Oh et al. 2014). The systematic search for

and development of new sources of chemical compounds from Acremonium

species is also important (Rodriguez Saiz et al. 2004, Bloemendal et al. 2014).

Bursera simaruba, a frequent co-dominant tree of tropical semi-deciduous

forests in the Yucatan Peninsula, is important ecologically because it can

grow in poor, clay or sandy, saline soils. Mayan communities use this plant

medicinally for its analgesic, antimycotic, and anti-inflammatory properties

(Gomez Pompa et al. 2003). Bursera simaruba exhibits a notable phenotypic

variation associated with an ecotypic differentiation of some ecophysiological

characters (Becerra 1999). A search for novel allelochemical compounds from

endophytic fungi associated with plant communities at El Eden Ecological

Reserve has revealed a distinctive endophytic ascomycete diversity that can

serve as a source of new bioactive chemical compounds (Gonzalez et al. 2007,

2009; Macias Ruvalcaba et al. 2008, 2010). In a survey on the diversity of fungi

from El Eden Ecological Reserve, a strain (B101) with morphological characters

corresponding to Acremonium was isolated from the leaves of B. simaruba.

Materials & methods

Fungus isolation and phenotypic study

El Eden Ecological Reserve is located in the State of Quintana Roo in northeastern

Yucatan Peninsula (21°13’N 87°11’W). Three Bursera simaruba trees from a medium

semi-deciduous dry tropical forest ecosystem were randomly sampled; four branches

were collected from each tree for transport to the laboratory in labelled Zip-lock plastic

bags and processed within one hour of collection. One asymptomatic entire mature leaf

was removed from each branch, submerged in running sterile distilled water for 60 s,

and sectioned into 5 mm’ pieces with sterile scissors. The leaf segments were surface

cleaned by sequential washing with sodium hypochlorite solution 0.5% (v/v) (2 min) and

ethanol 70% (2 min), rinsed with sterile distilled water, and surface-dried under sterile

conditions (Arnold et al. 2001). Four 5 mm? leaf segments per Petri dish were plated on

malt extract agar culture medium (MEA, Difco™, Becton, Dickinson & Company, Sparks,

Maryland, USA) with 4g/L streptomycin sulfate and 5mg/L cyclosporine A added after

autoclaving (Dreyfuss 1986). Five Petri dishes were prepared for each leaf and incubated

at 25 °C under laboratory conditions and examined daily for a 28 d period. Each fungal

colony developed during that period was transferred to MEA in order to obtain axenic

isolates that were grouped based on their macro- and micro-morphological characters.

Fourteen endophytic fungi, including isolate B101, were recovered from B. simaruba

leaves. Isolate B101 was grown on cornmeal agar, malt extract agar, potato dextrose agar

(CMA, MEA, PDA; Difco™, Becton, Dickinson and Company, Sparks, Maryland, USA),

and V8 vegetable juice agar (V8A; V8 juice Campbell’, 160 mL; calcium carbonate

Sigma-Aldrich®, 2 g; agar, Difco™, 20 g; distilled water to final volume of 1,000 mL)

Acremonium camptosporum endophytic on Bursera simaruba (Mexico) ... 213

(Hanlin & Ulloa 1988). Four Petri dishes were prepared for each culture medium and

incubated at 25 + 1 °C for 28 d. Colony macroscopic attributes (e.g., color, odor, texture,

growth rate) for each tested medium were examined at low magnification with a Nikon

SMZ745T stereoscopic microscope (Nikon Instruments, Tokyo, Japan) after 7, 14, 21

and 28 d. Color designations follow the Methuen handbook (Kornerup & Wanscher

1978). Microscopic morphological characters (somatic and reproductive features,

hyphae, phialides, and conidia) of fungus B101 were studied by slide cultures on PDA

or mounted directly in water. Photographs were made using bright field contrast (BC),

dark field contrast (DC), and Nomarski differential interference contrast (DIC) with a

Nikon DS digital camera using NIS software on a Nikon Eclipse 80i microscope. Images

were processed with Adobe Photoshop CS6°. Also, the fungus was studied with a Jeol

5410LV scanning electron microscope according to Goh & Hanlin (1994).

DNA isolation, PCR amplification, sequencing, and phylogenetic analysis

Genomic DNA was extracted for molecular analyses from the endophyte fungus

B101 (Glenn et al. 1996). The nuclear 28S large subunit RNA gene (LSU) was amplified

with primers LROR and LR6 and sequenced with primers LROR, LR3, LR3R, LR5, and

LR6 (Vilgalys & Hester 1990, Rehner & Samuels 1995). The DNA was sequenced by the

USDA-Agricultural Research Service, South Atlantic Area Sequencing Facility (Athens,

GA, USA). The LSU sequence from B101 was compared with GenBank sequences

using the Geneious v7.1.8 Linnaeus Blast search feature. The compiled LSU dataset was

aligned using MUSCLE, also all within Geneious. The alignment was evaluated using

MEGA v.6.06 to determine the maximum likelihood nucleotide substitution model

having the best fit. Model TN93 (including estimated transition/transversion ratio,

proportion of invariable sites, and gamma distribution parameter) was used for PhyML

analysis to identify the maximum likelihood cladogram, which was assessed by 200

bootstrap replications. Model GTR with the same estimated parameters was used for

MrBayes analysis (10,000,000 chain length; 4 heated chains; 0.2 heated chain temp; 200

subsampling frequency; 0.125 burn-in length). PhyML and MrBayes assessments were

conducted within Geneious. Sequences from five species of Cosmospora/Nectria were

designated as the outgroup.

Fungal specimen

The Mexican endophytic fungus (B101) was deposited in the Coleccién de Hongos of

the Herbario Nacional de México, Universidad Nacional Aut6noma de México, Distrito

Federal, México (MEXU). In addition, a specimen from the tree from which B101 was

isolated was deposited in the Herbarium, University of California, Riverside CA, U.S.A.

(G.P. Schultz & R. Palestina # 1092/UCR 110695). The 28S rDNA sequence generated in

this study was deposited in GenBank.

Taxonomy

Acremonium camptosporum W. Gams, Cephal.-art. Schimmelp.: 57. 1971. Fics 1-4

MYCELIAL COLONIES in vitro growing 30-35 mm diam after 28 d on PDA at

25 °C, flat to slightly elevated in center, funiculose to floccose textured, white

(1A1) to yellowish white (1A2), reverse colored with pale yellow ochraceous to

214 ... Gonzalez & al.

Fic. 1. Acremonium camptosporum (MEXU 26354). A. obverse of colony

on potato dextrose agar after 28 d at 25 °C; B. reverse of same culture with

ochre-brown pigment.

Acremonium camptosporum endophytic on Bursera simaruba (Mexico) ... 215

dark yellow ochraceous central area. Cubical or elongated crystals numerous.

Odor not pronounced. SoMATIC HYPHAE septate, hyaline, smooth- and

thin-walled, slightly to highly convoluted, often anastomosed, 0.5-2.5 um

wide, plectonematogenous. CONIDIOPHORES often basitonously branched,

sporodochia not observed, sporulation abundant. PHIALIDEs straight or

slightly flexuous, 15-46 um long, 0.5-1 um wide at the base, tapering to

0.4-0.5 um at tip, without a chromophile base, collarette or periclinal thickening

at the apex, hyaline, thin- and smooth-walled, slightly constricted at basal

septum, orthophialides formed, prostrate phialides present, adelophialides and

schizophialides not observed. Conrp1A aseptate, hyaline, guttulate, smooth-

and thin-walled, heteropolar-bilateral, mostly plano-convex or asymmetrically

biconvex in front view, 1.5-2.5(-2.8) um long, 0.8-1.1 um wide, fusiform in

side view, sometimes slightly curved and basally apiculate, aggregated in small

slimy globose clusters, catenate conidia not seen. CHLAMYDOSPORES and

SEXUAL MORPH not observed.

MATERIAL EXAMINED: MEXICO. QUINTANA Roo: Isla Mujeres Municipality, El

Eden Ecological Reserve, 21°13’N 87°11’W, from leaves of Bursera simaruba (L.) Sarg.

(Burseraceae), May 2004, MC Gonzalez, AL Anaya (MEXU 26354; GenBank JQ811556).

Phylogenetic analysis & discussion

Maximum likelihood (ML) and Bayesian analyses support MEXU 26354

and the A. camptosporum isolates within the Clavicipitaceae s.l., with particular

association with Elaphocordyceps and Metacordyceps (FiG. 6). Bayesian support

was strong for this large clade, but there was no substantial support of this

relationship from the ML analysis. In general the Bayesian analysis was more

supportive of the major clades than was ML. The relationship between MEXU

26354 and the other A. camptosporum isolates was very well supported (Bayesian

posterior probability = 1; ML bootstrap = 100%). The LSU sequence of MEXU

26354 was 99.5-99.7% identical to the four A. camptosporum accessions.

According to Summerbell et al. (2011), Acremonium can be morphologically

characterized by its conidiophores that are mainly unbranched or poorly

basitonously branched and subglobose, obovate- ellipsoidal conidia, with

adelophialides usually absent. However, they conclude that because of the

relatively common plasticity of form in Acremonium and acremonioid species,

the distinctive LSU sequences for delineating the species phylogenetically,

precise morphological observation, and correct interpretation of the

morphological descriptions are all necessary.

Gams (1971) described the type specimen of Acremonium camptosporum

from Kiel-Kitzeberg, Germany; his description and drawings depict structures

that are similar with those produced by the Mexican collection MEXU 26354.

216 ... Gonzalez & al.

Fic. 2. Acremonium camptosporum (MEXU 26354). A, C, E: obverse of colonies on malt extract

agar, V-8 juice agar, and corn meal agar after 28 d at 25 °C; B, D, E reverse of same cultures.

Acremonium camptosporum endophytic on Bursera simaruba (Mexico) ... 217

{

}

;

Fic. 3. Acremonium camptosporum (MEXU 26354). A. phialides - arrow indicates one longer

and narrower; B. phialide slightly constricted at basal septum (arrow) and conidia clustered in

slimy head; C, D. orthophialide with basal septum (arrow); E. flexuous phialide with phialospore

attached at tip (arrow); E plectonematogenous sporulation, phialides unbranched and branched

(arrow). Scale bars: A = 10 um; B-E = 5 um; F = 20 um.

218 ... Gonzalez & al.

Fic. 4. Acremonium camptosporum (MEXU 26354). A. tiny prostrate hypha with subterminal

phialide and conidia clustered in subglobose slimy head (arrow); B. phialidic development

of curved phialoconidia; C. small hyaline curved conidia; D. guttulate conidia; E. aseptate

conidia; F. slightly curved and basically apiculate (arrow) and plano-convex conidiospores.

Scale bars: A, C = 10 um; B, E, F=5 um; D = 2 um.

Acremonium camptosporum endophytic on Bursera simaruba (Mexico) ... 219

Fic. 5. Acremonium camptosporum. A. dead mite attached to internal lateral wall of Petri dish

by A. camptosporum hyphae from edge of 6 wk old pure culture; B. detail of mite legs with

hyphae of A camptosporum; note a phialide with globose mass of conidia (arrow); C. dead mite

in advanced state of degradation by A. camptosporum;. D. cubical and irregular crystals.

As described by as Gams (1971), A. camptosporum is characterized by slow-

growing white colonies with a yellow ochre reverse, the presence of orthotropic

phialides, and small curved conidia arranged in slimy heads. The Mexican

specimen has the same micro-morphological characteristics and agrees with

220 ... Gonzalez & al.

the A. camptosporum description, particularly with the variable form of its

conidia (slightly curved and basally apiculate, heteropolar-bilateral, plano-

convex or asymmetrically biconvex) and the presence of characteristic crystals

(Fic. 5D). However, the Mexican material has conidia that are minutely

guttulate (sometimes with one or two conspicuous guttules) and slightly

smaller, with maximum measurement values corresponding to the minimum

values mentioned in Gams’ description (2.8-3.8 um long x 1.1-1.8 um wide).

Gams (1971) also noted the presence of sporodochial conidiomata in older

cultures—not observed in our cultures, although delimited higher sporulation

zones were noted in central area of colonies. Although the conidia of MEXU

26354 are smaller than those of the type description, based on our phenotypic

and phylogenetic analyses, we conclude that our material represents

A. camptosporum.

Gams (1971) mentioned that A. camptosporum shares some morphological

characters with Xenoacremonium recifei [= Acremonium recifei = Cephalosporium

recifei = Hyalopus furcatus = Hyalopus recifei], a tropical opportunistic

pathogen of humans characterized by aseptate, hyaline, ellipsoidal to fusiform

or reniform, slightly curved conidia, but X. recifei can be distinguished by its

shorter conidia without periclinal thickening, slower colony development, and

intensive pigmentation of the colony reverse.

Our results support a close phylogenetic relationship between Acremonium

camptosporum and the clade encompassing Ophiocordycipitaceae and

Clavicipitaceae s. str. (Sung et al. 2007) (Fic. 6). Interestingly, our study also

nests the A. camptosporum clade with the Elaphocordyceps clade. Data analysis

suggests that A. camptosporum is basal to Elaphocordyceps and other fungi with

asexual morphology in the Ophiocordycipitaceae. The Elaphocordyceps clade

contains the asexual genera Tolypocladium and Chaunopycnis. Tolypocladium

species typically produce smooth globose or cylindrical conidia on inflated-base

phialides with slightly tapering apex. They are parasites of the ectomycorrhizal

truffle-like genus Elaphomyces, entomopathogens of cicada nymphs buried

in soil, and scarab beetle larvae embedded in decaying wood and are also

isolated from soil (Gams 1971, Sung et al. 2007). Chaunopycnis species produce

minutely roughened globose or obovate conidia on cylindrical or inflated-base

phialides slightly tapering in the distal part, formed in conidiomata isolated

Fic. 6. LSU sequence cladogram (PhyML) showing the relationship of Acremonium camptosporum

to other taxa within the Clavicipitaceae sensu lato. Numbers above branches indicate the

Bayesian posterior probability support (>0.75) followed by the bootstrap support (>75%) from

200 maximum likelihood replicates.

Acremonium camptosporum endophytic on Bursera simaruba (Mexico) ... 221

4/96) Tolypocladium inflatum AB044645

4/86 Tolypocladium inflatum AF373286

Tolypociadium cylindrosporum AF245301

0.99/95) Elaphocordyceps ophioglossoides AB027367

Elaphocordyceps ophioglossoides JN941406

Elaphocordyceps jezoensis AB027365

72 1/100 y Elaphocordyceps capitata AB027364

Elaphocordyceps capitata JN941401

Elaphocordyceps inegoensis AB027368

Elaphocordyceps paradoxa JN941410

0.81/-- Elaphocordyceps paradoxa JN941411

4/91] f= Chaunopycnis pustulata AF373282

Chaunopycnis pustulata AF389190

1/984 Chaunopycnis alba AF373284

Chaunopycnis alba HM§95579

0.96/90 Myrothecium inundatum AF 193236

4/100 Myrothecium inundatum AY489731

Myrothecium gramineum AY283538

0.93/78 Myrothecium cinctum AY489710

Acremonium camptosporum HQ232007

Acremonium camptosporum HQ232009

A/24) Acremonium camptosporum HQ232011

Acremonium camptosporum HQ232010

Acremonium camptosporum MEXU 26354 JQ811556 g—

0.99/98 Epichioe amarillians U57680

Epichioe typhina U17396

Cordyceps gunnii HM119590

Paecilomyces carneus EF468843

1/-- Chamaeleomyces viridis HM635079

1/984 Chamaeleomyces granulomatis HM635077

Chamaeleomyces granulomatis JN990695

1/96 Torrubiella luteorostrata AF327380

Torrubiella luteorostrata AF327388

Atkinsonella hypoxylon U57087

Metacordyceps liangshanensis EF468814

Pochonia bulbillosa JQ780662

0.92/92 p= Metacordyceps tali GU979949

1/100 Metarhizium anisopliae HM142943

Metarhizium anisopliae AB027383

1/100

0.94/--

Nomuraea rileyi HQ165729

Metarhizium cylindrosporumHQ165717

Metarhizium cylindrosporumHQ165716

Cordyceps chlamydosporia DQ518758

Metarhizium cylindrosporumHQ165718

0.89/93) Torrubiella petchii EU369038

Torrubiella pruinosa EU369041

Beauveria bassiana AF245300

4/95 isaria farinosa JX481966

Beauveria bassiana JX481967

Beauveria bassiana JF429894

1/100 Cordyceps bassiana EF026006

Beauveria bassiana EU334674

0.88/— lsaria farinosa JF429895

Cordyceps militaris JN941384

Cordyceps militaris HQ591388

9.96/92) Cordyceps kyushuensis AY465959

Cordyceps militaris AY184966

0.77/76y Phytocordyceps ninchukispora EF468846

1/96] 1 Phytocordyceps ninchukispora EF468847

Coardyceps militaris JN941383

Torrubiella wailacei AY184967

Isaria fumosorosea EF429304

isaria farinosa JF415979

o.81/~ 1/90] isaria farinosa KC920582

Paecilomyces hepiali HM135171

Lecanicillium lecanii AY283556

0,75/—

ue Torrubiella piperis AY466442

0.98/96 Cordyceps pseudomilitaris AF327376

Cordyceps pseudomilitaris JN941394

4/90 Ascopolyporus philodendri AY886545

Hyperdermium bertonii AF242354

1/100 } Cosmospora coccinea AY489734

0.89/92 Nectria cosmariospora U17407

i= Casmospora henanensis GU075863

Cosmospora vililuscula GQ506003

Cosmospora gigas EF121869

0.0090

222 ... Gonzalez & al.

from soil, plant litter, crustose lichen thalli of Caloplaca regalis, and living

leaves of Kalmia latifolia (Gams 1980, Moller & Gams 1993, Bills et al. 2002).

The Mexican A. camptosporum strain (MEXU 26354) differs morphologically

and ecologically from those taxa. More robust statistical analyses still needed

for a higher-level phylogeny of A. camptosporum.

The endophyte strain of A. camptosporum (MEXU 26354) produces six

secondary metabolites of three different heterodimeric polyketide groups, one

of which is acremoxanthone E, a new compound that shows biological activity

against selected phytopathogens and human tumor cell lines (Meléndez-

Gonzalez et al. 2015).

Acremonium camptosporum is a rarely isolated fungus, previously reported

only from air in Germany (Gams 1971); soil in Czech Republic (Repova 1989),

Ivory Coast (Persiani et al. 1998), and Brazil (Prade 2007); and anthropogenic

substrates in Czech Republic (Vanova & Be¢vart 1998) and Poland (Blyskal

2009, 2015). Additional nucLSU sequences from leaf litter of Acacia

karroo soil in South Africa (CBS 757.69, CBS 677.74), from insects in Cuba

(CBS 835.91), and from soil nematodes in Germany (CBS 890.85) give a broad

view of A. camptosporum distribution and ecology. This is both the first record

of A. camptosporum from Mexico and the first record of the species as a leaf

endophyte.

Melanins function in protecting fungi from adverse environmental factors

(Gomez & Nosanchuk 2003). Some Sarocladium species have an ochre-brown

or dark grey-brown colony reverse due to melanin synthesis (Summerbell et al.

2011). The Mexican A. camptosporum strain produces yellow ochre to ochre-

brown pigments on the reverse of colonies cultured. The highest color intensity

was expressed on MEA, whereas it was colorless on CMA (Fig. 2B, F).

In a recent study on biodeterioration of cochineal dyed antique woolen

textiles, A. camptosporum caused significant degradation of wool fibers due

to its ability to utilize keratinous materials (Blyskal 2009, 2015). The keratin-

associated proteins and high glycine-tyrosine proteins of wool fibers are a

good nutritional resource for small arthropods (e.g., mites), and keratins for

fungi and other microorganisms. Interestingly, we observed here the apparent

degradation of a dead mite by the A. camptosporum MEXU 26354 endophytic

strain (Fic. 5A—-C).

The smaller somatic and reproductive structures of the A. camptosporum

endophytic strain from Bursera simaruba leaves of Yucatan Peninsula, along

with its apparent keratinophilic ability, suggest a possible adaptation to its

niche. More studies are necessary to clarify the ecology of this fungus.

Acremonium camptosporum endophytic on Bursera simaruba (Mexico) ... 223

Acknowledgments

The authors greatly thank the reviewers of the manuscript Charles W. Bacon,

James White, and Shaun Pennycook. This study was supported by Programa de

Apoyo a Proyectos de Investigacién e Innovacién Tecnoldgica of the Universidad

Nacional Auténoma de México (UNAM) to the Instituto de Ecologia, grant PAPIIT

IN230107-3. Some aspects of this work were financed by the Consejo Nacional de

Ciencia y Tecnologia (CONACyT 60502). The first author worked on this manuscript

during her sabbatical at University of Georgia with a DGAPA-PASPA grant. We thank

Carmen Loyola, Ma. Luisa Escobar Sanchez, and Rosa Ma. Picaso for assistance with

photography and scanning electron microscopy. We are very grateful to the Instituto de

Biologia, UNAM, University of Georgia, and the USDA-ARS Toxicology & Mycotoxin

Research Unit (Russell Research Center of Athens) for their support.

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MY COTAXON

January-March 2016—Volume 131, pp. 227-233

http://dx.doi.org/10.5248/131.227

Aspicilia, Lobothallia, and Rhizocarpon species

new for Turkey and Asia

KENAN YAZICI **& ALI ASLAN ”

‘Biology Department, Faculty of Science, Karadeniz Technical University,

61080, Trabzon, Turkey

?Atatiirk University, Kazim Karabekir Education Faculty, Biology Dept., Erzurum, Turkey

* CORRESPONDENCE TO: kcagri_1997@yahoo.com

AsstRAcT — Three lichen species, Aspicilia glomerulans, Lobothallia chadefaudiana, and

Rhizocarpon furfurosum, were identified as new to Turkey and Asia during a lichenological

survey in the Burdur and Mus regions (Turkey). Descriptions accompany notes on known

distribution, substrates, and chemistry and comparisons with morphologically similar taxa.

KEY worps — ascomycetes, biodiversity, Hymeneliaceae, Megasporaceae, Rhizocarpaceae

Introduction

Although many new lichen species have recently been recorded for Turkey

(Aptroot & Yazici 2012, Arslan et al. 2011, Yazici et al. 2010a, b, c, 2011a, b,

2012, Karagéz & Aslan 2012, Karago6z et al. 2011, Kinalioglu & Aptroot 2011,

Osyczka et al. 2011, Vondrak et al. 2012), more surveys are still needed of

unexplored regions in the country.

Rhizocarpon (Rhizocarpaceae) contains more than 200 crustose species

(Miadlikowska et al. 2014, McCarthy & Elix 2014), which are found on siliceous

rocks in montane and boreal habitats and (rarely) on substrates in wet or dry

(sub)tropical areas and hot-arid regions (McCarthy & Elix 2014). Aspicilia

(Hymeneliaceae) contains approximately 230 species (Nordin et al. 2010), while

Lobothallia (Megasporaceae) has 12 lichen taxa (Sharnoff 2014).

Thirty-eight taxa of Aspicilia, three taxa of Lobothallia, and 30 taxa of

Rhizocarpon have thus far been reported from Turkey. Of the approximately

1650 lichen taxa recorded for the country, only 37 lichenized fungi have been

228 ... Yazici & Aslan

reported from Burdur Province (Aslan & Yazici 2013, Cobanoglu 2005, Oztiirk

et al. 2005, Pisut & Guttova 2008, Senkardesler 2009, Yazici et al. 2013).

The present contribution results from fieldwork in the regions of Burdur,

southwestern Turkey, and Mus, eastern Turkey. We report here three new

records for Turkey and Asia.

Materials & methods

Specimens

Air-dried samples were examined with a Nikon SMZ1500 stereomicroscope and a

Nikon Eclipse 80i compound light microscope. Relevant keys were consulted (McCarthy

& Elix 2014, Clauzade & Roux 1985, Poelt 1970, Roux 1977, Smith et. al. 2009) for

the identifications. Vouchers are stored in the Herbarium of the Biology Department,

Karadeniz Technical University, Trabzon, Turkey (KTUB). The descriptions are based

on Turkish specimens.

Description of the study area

Areas visited were the Aglasun, Altinyayla, and Tefenni districts in Burdur and the

center and Bulanik and Varto districts in Mus. Burdur, which is very mountainous

and covered with mixed forests (Abies, Cedrus, Ficus, Fraxinus, Juniperus, Olea, Pinus,

Pistacia, Prunus), has a continental Mediterranean climate with cold snowy winters and

long dry and very hot summers. Mus province, which encompasses vast areas of grassy

meadows and steppe and high mountains with occasional to rare Quercus communities

and Salix trees, has a climate characterized by very cold snowy winters, and short hot

dry summers (Baytop & Denizci 1963, Akman 1999).

Taxonomy

Aspicilia glomerulans (Poelt) Poelt, Mitt. Bot. Staatssamml. Miinchen

8: 202 (1970) Fic. 1

Thallus crustose, to 7 cm diam, K-, areolate, with black hypothallus,

rounded, 0.5 mm thick, dark white or dark-grey, paler at the middle, dark

grey-brown towards the outer margin of the lobes; surface with numerous

isidia-like papillose branches, dispersed or aggregated on one areole or many

areolae; apothecia present or absent, 0.8-1 mm diam., single, rounded, disc flat,

concave later, black, edge flat, black, + rounded, disappearing later; ascospores

ellipsoid, 16-19 x 6-12 um, with granules; hymenium 130 um high, pale,

I+ blue; asci cylindrical, 8-spored, thick and contrasting tholus, strongly

regressed at maturity, I + blue; paraphyses barely in water, upper parts

moniliform and anastomosing in K.

EcoLocy—Associated species: Aspicilia cinerea, Candelariella vitellina,

Melanohalea infumata, Parmelina tiliacea, Physcia caesia, P. cernohorskyi,

Rhizoplaca chrysoleuca, R. melanophthalma, and Xanthoria elegans.

SPECIMEN EXAMINED — TURKEY. Burpur: ALTINyAYLA, between I[becik-Altinyayla

mainroad side, 36°58’07.19”N 29°26'17.70”E, 1348 m, on calcareous rock, 29.06.2012.

New lichen records for Turkey ... 229

Fic. 1. Aspicilia glomerulans, habitus

(top: with apothecia & papilla; left: with papilla; right: with apothecia & papilla).

Scales top,right = 1 mm; left = 1 cm

leg. K.Yazici. (KTUB-2427), Mus: VarRTO, Callidere village, roadside, 39°16’24.70”N

41°14’18.26”E, 1705 m, on calcareous rock, 01.07.2015, leg. K.Yazici. (KTUB-2445).

REMARKS—Aspicilia glomerulans, which is primarily sterile, grows on

calcareous rock. The species is morphologically similar to Koerberiella

wimmeriana, which differs by its grey-rose to grey thallus, cylindrical papillose

isidia, shorter pycnidiospores (4-5 x 1 um), larger apothecia (0.5-2 mm in

diam.), and larger ascospores (20-35 x 11-20 um) (Clauzade & Roux 1985,

Poelt 1970, Thiis & Schultz 2009). For additional details, see Poelt (1970).

Aspicilia glomerulans has previously been reported from Austria (Alps),

Greenland, North America, and Norway (Clauzade & Roux 1985, Poelt 1970).

This is a first record for Turkey and Asia.

230 ... Yazici & Aslan

Fic. 2. Lobothallia chadefaudiana, habitus (apothecia and thallus lobes). Scale = 1 mm

Lobothallia chadefaudiana (Cl. Roux) A. Nordin, Cl. Roux & Sohrabi, Bull. Soc.

Linn. Provence, num. spéc. 16: 216 (2012) Fic 2

Thallus crustose, pale white to grey white, 1.7- 3 cm in diam., squamules,

to 2 mm in diam.; surface with papillose, + pruinose, rounded, + lobed, spot

tests negative; cortex <85 um high, photobiont Trebouxia. Medulla <1300

um; apothecia to 2 mm diam., + immersed, in squamules in 1-2 groups,

irregular in shape, disc concave, flat later, + grey-blue, pruinose, K-, P-, C-;

N-, with paraphyses with gelatinous sheath at the top, K-; hymenium 80-95

um, colourless, without crystals, paraphyses 3-6 um thick, strongly coherent,

consistent, divided, green-brown, branched and anastomosing, I + blue later

red; hypothecium 35-55 um, colourless; asci clavate, thickened at the top,

80-95 x 20-30 um, 8-spored, I+ blue, later red. Ascospores 9-17 x 6-12 um.

EcoLocy—Associated species: Acarospora cervina, Lecanora dispersa,

Verrucaria nigrescens, and Diploschistes diacapsis.

SPECIMEN EXAMINED — TURKEY. Burpur: AGLASUN, 5 km to Camlidere village,

37°38'01.77’N 30°38’53.58”E, 1010 m, on calcareous rock, 17.07.2012, leg. K.Yazici.

(KTUB-2423), Mus: CENTER, 200 m to Alanici village, roadside, 38°39’15.52”N

41°21’10.55”E, 1709 m, on calcareous rock, 29.05.2015, leg. K.Yazici. (KTUB-2447).

New lichen records for Turkey ... 231

REMARKS— Lobothallia chadefaudiana mostly grows on calcareous rock. It is

morphologically similar to Aspicilia cernohorskyana, which is distinguished by

larger apothecia and narrower squamules (Clauzade & Roux 1985, Roux 1977).

For additional details, see Roux (1977).

Lobothallia chadefaudiana has previously been reported from Europe

(France, Austria, Greece, Spain; Clauzade & Roux 1985, Roux 1977, Roux et al.

2008). This is a first record for Turkey and Asia.

Fic. 3. Rhizocarpon furfurosum, habitus (with isidia). Scale = 1 mm

Rhizocarpon furfurosum H. Magn. & Poelt, Verh. Zool.-Bot. Ges. Wien

95-7110: 1955). Fig. 3

Thallus crustose, effuse, to 1.2-4 cm diam., continuous to rimose-areolate,

areoles to 1.2 mm diam., cracked, spreading, pale or dull-brown-grey to dark

grey-brown, prothallus + developed, covered with fragile isidia like blastidia

minutely warted at first; isidia 0.01-0.03 mm long, fragile, branched. Areoles

flat or + convex. Hypothallus black. Apothecia absent. Thallus K+ yellow, C-;

medulla K+ yellow, P+ yellow.

EcoLocy—Associated species: Acarospora cervina, Bellemerea cupreoatra,

Candelariella vitellina, Lecanora dispersa, Protoparmeliopsis murals,

Rhizocarpon geographicum, and Xanthoria elegans.

232 ... Yazici & Aslan

SPECIMEN EXAMINED — TURKEY. Burpur: TEFENNI, Seydiler village, 37°14’47.46”"N

29°48’24.83”E, 1191 m, on siliceous rock, 21.08.2013, leg. K. Yazici. (KTUB-2429), Mus:

BULANIK, 5 km to Arakonak village, roadside 39°04’08.79”N 42°07'48.39’E, 1602 m, on

calcareous rock, 30.06.2015, leg. K.Yazici. (KTUB-2446).

REMARKS— Rhizocarpon furfurosum, which occurs on metal-rich siliceous

rocks, is mostly sterile and is easy to overlook. For additional details, see Smith

et al. (2009).

Rhizocarpon furfurosum has previously been reported from Europe

(England, Germany, Scandinavia, the Czech Republic, Slovakia, Russia;

Guttova et al. 2013, Smith et al. 2009 Steinova et al. 2009). This is a first record

for Turkey and Asia.

Acknowledgements

We are grateful to Dr. Michele D. Piercey-Normore and Dr. Anna Guttova for

revisions and helpful comments on an earlier draft of this manuscript. We also thank

Dr. André Aptroot for his comments about the identification of the species. This study

was supported by TUBITAK (Projects 111T857 and 114Z892).

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MY COTAXON

January-March 2016—Volume 131, pp. 235-245

http://dx.doi.org/10.5248/131.235

New species of neotropical Rhodocollybia

JUAN L. MatTa™, CLARK L. OVREBO’,

TrmoTHy J. BARON? & KAREN W. HUGHES?

‘Biology Department, University of South Alabama,

5871 USA Dr. N, Room 124, Mobile, AL, 36688

*Department of Biology, University of Central Oklahoma,

100 North University Drive, Edmond, OK 73034

*Department of Biological Sciences, State University of New York — College Cortland,

PO. Box 2000, Cortland, NY 13045

‘Department of Ecology and Evolutionary Biology, University of Tennessee,

569 Dabney Hall, Knoxville, TN 37996

* CORRESPONDENCE TO: jmata@southalabama.edu

ABSTRACT —. Sequences from the internal transcribed spacer region of the ribosomes helped

circumscribe two new Rhodocollybia species for neotropical America: R. olivaceogrisea from

Costa Rica; and R. tenuipes from Dominican Republic and Puerto Rico. Both species are

phylogenetically related to R. butyracea. Rhodocollybia maculata represents a new report for

Dominican Republic.

Key worps — ITS, phylogeny, Omphalotaceae, Agaricomycetes

Introduction

The genus Rhodocollybia Singer (Omphalotaceae, Agaricomycetes) comprises

fleshy mushrooms with convex smooth pilei with crowded cream-colored

lamellae, striate and slightly twisted stipes, and a pinkish spore deposit (Antonin

& Noordeloos 2010). Basidiospores are at least partly, dextrinoid. However, not

all Rhodocollybia mushrooms explicitly exhibit this entire set of characters and

sometimes may be confused with closely related genera, such as Gymnopus

(Hughes et al. 2010). In addition to detailed morphological analyses, DNA

studies are very helpful determining identities of many mushrooms whose

species boundaries are difficult to resolve.

236 ... Mata & al.

Most reports for Rhodocollybia are from temperate regions (Antonin &

Noordeloos 2010, Cooper 2014, Halling 2009, Lee et al. 2014, Lennox 1979).

Studies by Halling (1989) and Mata et al. (2004) thus far provide the best

accounts for neotropical America.

A recent foray in Costa Rican oak-dominated forests confirmed a suspected

new species of Rhodocollybia. Previous studies of Caribbean material also

suggested new records. Here we propose two new neotropical species:

R. olivaceogrisea from Costa Rica and R. tenuipes from Dominican Republic

TABLE 1. Rhodocollybia and outgroup sequences used in the ITS phylogenetic analysis.

New sequences are set in bold font.

TAXON GENBANK VOUCHER REFERENCE

NO.

Lentinula raphanica (Murrill) AY016441 TFB 9156 Mata et al. 2001

J.L. Mata & R.H. Petersen

Gymnopus luxurians (Peck) Murrill KJ416241 TFB 14107 Petersen & Hughes 2014

R. amica J.L. Mata & Halling AF505754 TFB 9920 Mata et al. 2004

R. butyracea (Bull.) Lennox GU318386 TFB 13006 Hughes et al. 2010

KJ609163 SFC20120821-75 Lee et al. 2014

AY313290 TFB 8801 Mata et al. 2004

AY313293 TFB 7452 Mata et al. 2004

DQ444317 OKM27562 Mata et al. 2007

AY313292 TFB 8250 Mata et al. 2004

R. dotae J.L. Mata & Halling AF505758 REH 7007 Mata et al. 2004

R. laulaha Desjardin et al. GU369944 = SFSU: DED6393 Keirle et al. 2010

R. lignitilis J.L. Mata & Halling AF505753 REH 7907 Mata et al. 2004

R. maculata KT205402 TFB 11720 This paper

AY313296 TFB11045 Mata et al. 2004

AF505756 TFB 9605 Mata et al. 2004

GU947368 BRNM 707085 Antonin & Noordeloos 2010

GU947370 BRNM 699408 Antonin & Noordeloos 2010

GU947369 BRNM 714632 Antonin & Noordeloos 2010

R. olivaceogrisea KT205399 JLM 2175 This paper

KT205400 CLO 4368 This paper

R. pandipes Halling & J.L. Mata KT205401 JLM 2251 This paper

AY313295 TFB 9680 Mata et al. 2004

AF505752 TFB 11014 Mata et al. 2004

AY313294 TEB 7899 Mata et al. 2004

R. prolixa var. distorta (Fr.) AF505748 EFM 1403 Mata et al. 2004

Antonin et al.

R. purpurata (G. Stev.) J.A. Cooper KJ61902 PDD:95837 Cooper 2014

R. tenuipes AY313288 TFB 11707 Mata et al. 2004

R. tablensis J.L. Mata & Halling AF505755 EN2066 Mata et al. 2004

R. turpis (Halling) Halling AF505749 FB10077 Mata et al. 2004

R. unakensis (Murrill) Halling AY313298 TFB 10482 Mata et al. 2004

Rhodocollybia olivaceogrisea & R. tenuipes spp. nov. (neotropical America) ... 237

and Puerto Rico. Phylogenetic analysis nests both species in a clade with

R. butyracea. Rhodocollybia maculata is a new report for the Dominican

Republic, and it and R. tenuipes are the first Rhodocollybia records from this

country.

Materials & methods

Morphology

Basidiomata colors in capital alphanumeric codes in square brackets are from

Kornerup & Wanscher (1978) and small-cap codes are from Kelly (1965). Preparation

for tissues and basidiospores for microscopy observations and descriptive abbreviations

are as in Mata et al. (2004). Line drawings were generated using a drawing tube attached

to an Olympus CX41 microscope.

Phylogeny

DNA extraction and PCR protocols are as those used in Mata et al. (2004). Ribosomal

ITS-4 and ITS-5 primers were used to amplify the internal transcribed region (ITS)

of nuclear DNA (White et al. 1990). PCR products were purified with Qiaquick PCR

Purification Kit (Qiagen). Purified PCR products were sequenced at Macrogen (MD,

USA). Sequence files were first edited with Geneious R8 (Geneious 2014) and then

blasted against NCBI nucleotide databases. Selected Rhodocollybia sequences were

downloaded together with one of Lentinula raphanica and Gymnopus luxurians, which

were used as the outgroup (TABLE 1). The matrix was aligned with MAFFT (Katoh et

al. 2002). Maximum likelihood phylogenetic tree was calculated with PhyML (Guindon

& Gascuel 2003) using 1000 bootstrap replications and supported with posterior

probabilities using Mr. Bayes (Huelsenbeck & Ronquist 2001). All programs were run

with Geneious R8.

Abbreviations

Country abbreviations are as follow: Costa Rica (CRC), Czech Republic (CZR),

Dominican Republic (DOM), Italy (ITA), Korea (KOR), Mexico (MEX), New Zealand

(NZL), Sweden (SWE), and United States (USA). Herbarium acronyms where collections

are deposited are according to Thiers (2015).

Results

Phylogeny

ITS sequences of R. olivaceogrisea nest in a clade with those of R. butyracea

(a well-known species in temperate regions), but its closest relative appears

to be R. pandipes from Costa Rica (PLATE 1). Percent identity between these

two species is 95.39% based on the distance matrix. Sequences from both

R. olivaceogrisea collections are 99.73% identical, with one representing a

collection from the Talamanca Mountains and the other from the Central

Volcanic Range of Costa Rica. The R. tenuipes sequence from the Dominican

Republic also nests in the clade with R. olivaceogrisea and R. butyracea but is

238 ... Mata & al.

R. pandipes, CRC, AY313295

98 1.00] R. pandipes, CRC, AF505752

92/1.004'~" FR. pandipes, CRC, KT205401

R. pandipes, CRC, AY313294

99/1.0¢1_] R. olivaceogrisea, CRC, czas saa

R. olivaceogrisea, CRC, KT205400

R. purpurata, NZL, KJ461902 fous

400/1.0¢ R. fenuipes, DOM, AY313288

R. butyracea, MEX, AY313290

941.401 2 putyracea, KOR, KJ609163

84.14.00 R. butyracea, USA, GU318386

Bic R. butyracea, USA, AY313292

99/1.90¢4 /. Dutyracea, USA, DQ444317

R. butyracea, SWE, AY313293

R. dofae, CRC, AF505758

84/0.99 | R. maculata, ITA, GU947370

noe: se R. maculata, CZE, GU947369

- R. maculata, CZE, GU947368

96/1.00 R. maculata, USA, AF505756 |... co5,

93/4199 R. maculata, DOM, Sea 8.73%

R. maculata, USA, AY313296

R. turpis, (RC, AF505749

R. prolixa var. distorla, CRC, AF505748

100/1.00 7 R. Jaulaha, USA, GU369944

R, lignitilis, CRC, AF505753

R. unakensis, USA, AY313298

R. tablensis, CRC, AF505755

R. amica, CRC, AF505754

Gymnopus luxurians, USA, KJ416241

Lentinula raphanica, USA, AY016441

PiaTE 1. ITS maximum likelihood phylogeny for neotropical Rhodocollybia species. Branch

support is presented as: “bootstrap >80%”/“posterior probability”. Numbers after braces are percent

identity based on distance matrix. Scale bar is for number of substitutions per site.

weakly supported. This sequence was uploaded in GenBank as R. aff. pandipes

(Mata et al. 2004) and its phylogenetic placement was similar as in this study.

A sequence obtained from a Dominican Republic collection is 98.73% identical

to R. maculata from Oregon, USA. Although there are two subclades within

R. maculata, there is not enough data to draw vicariance conclusions, mainly

due to insufficient sampling. A newly generated sequence for R. pandipes

from the Central Volcanic Range in Costa Rica is added in the phylogeny to

document range extension.

Rhodocollybia olivaceogrisea & R. tenuipes spp. nov. (neotropical America) ... 239

Taxonomy

Rhodocollybia olivaceogrisea J.L. Mata & Ovrebo, sp. nov. PLATE 2

MycoBank MB 812967

Differs from other Rhodocollybia by its green to grayish colored pilei and from R.

pandipes by its olive to grayish stipes.

Type: Costa Rica, Province Cartago, County Oreamuno, District Potrero Cerrado,

vicinity of San Juan Chicua, at Volcan Irazu National Park, Sector Prusia, along El

Roble Trail, 9°57.92’N 83°52.29’W, 2950 m a.s.l., 5.Aug.2014, col. J.L. Mata JLM 2175

(Holotype, USAM-F00608; isotype, USJ; GenBank KT205399).

EryMo.oey: from Latin olivaceus for olive and griseus for gray.

Piteus (10-)35-45(-50) mm broad, broadly convex, to becoming almost

plane, shallowly umbonate; surface glabrous, dull or shiny, waxy or greasy to

the touch, when young greenish gray [3E3, 2E2, 3E2], olive brown [3C3] with

grayish hues, remaining dark olive [3F6] at umbo, grayish olive [3D3] when

hygrophanous, losing greenish color with age to drab grayish yellow [90 gy.y-91

d.gy.y]; margin decurved, finely crenulated, pale, translucent. FLEsH 2-4 mm

thick at center, translucent, watery gray, off-white when hygrophanous. Odor

pleasant; taste nondescript. LAMELLAE adnexed, deeply adnexed to free, close

(1-2/mm), 1.4-5 mm broad, white when young, in age becoming off-white to

cream [2A2-3A2], not discoloring; margin entire or uneven to irregularly wavy

(not serrate); two tiers of lamellulae of different lengths. StrpE (40-)80-100 x

4-7 mm at mid stipe, central, + equal to enlarged gradually towards base and

tapered towards apex, + flexing; surface glabrous, not ridged, drab olive above,

+ concolorous to pileus, and becoming olive brown, like pileus, or grayish

buff at base; base rounded; consistency very fragile; interior hollow. Whitish

mycelium scattered on stipe base.

BASIDIOSPORES 5.5-9 x 3.5-4.5 um (n = 40, x = 7.2 x 3.8 um, Q = 1.63-2.33

Qx = 1.88), ellipsoid in side view and profile, smooth, dextrinoid; wall thin.

PILEIPELLIS a cutis, composed of radially oriented hyphae, 2-6 um diam, with

occasional clusters of semi-erect terminal hyphae, 30-40 x 2-4 um, mostly

with obtuse endings, some with lobes. PILEUS TRAMA composed of radially

oriented, interwoven hyphae, 8-20 um, hyaline, thin-walled; refractive hyphae,

4-6 um, occasionally present. LAMELLAR TRAMA regular, hyphae 4-16 um

diam, + interwoven, thin-walled. HymMENium composed of basidioles, 15-20

x 3-6 um, and tetra-sterigmate basidia, 23-30 x 6-8 um, mostly club-shaped.

Pleurocystidia absent. Lamellar margin sterile. CHEILOCysTIDIA, 20-35 x 5-9

uum, club-shaped to cylindrical, with obtuse apex, frequently with diverticula;

abundant. STIPE CUTIS and trama parallel; epicutis hyphae 2-4 um in diam,

context hyphae 4-16 um in diam, thin-walled. Clamp connections present in

all hyphae.

240 ... Mata & al.

PLATE 2. Rhodocollybia olivaceogrisea. A) basidiomata (JLM 2175); B) basidiospores;

C) basidia and basidioles; D) cheilocystidia. Scale bar: B—D = 10 um.

Rhodocollybia olivaceogrisea & R. tenuipes spp. nov. (neotropical America) ... 241

EcoLoey clustered or scattered, some in cespitose bundles, in thick leaf

litter layer, under oak.

ADDITIONAL SPECIMENS EXAMINED: COSTA RICA, PROVINCE SAN JOSE, County Dota,

vicinity of San Gerardo, along road to San Gerardo, 500 m. S. of Interamerican Highway,

Km 80, 9°36’13”N 84°47’26’”W, 2.Jun.2004, col. C Ovrebo CLO 4368 (NY, USJ).

ComMENTARY- This species resembles R. butyracea in the smooth and greasy

aspect of the pileus, the swollen stipe, and spore dimensions. But it differs in its

gray-olive to brown-olive colors, and in Costa Rica no other Rhodocollybia has

these field characters. Its closest resemblance is to its Costa Rican sister taxon

R. pandipes, which is easily distinguished by its bent stipe that soon develops

reddish-colors. A tawny olive coloration is reported in pilei of North American

R. lentinoides (Peck) Halling, but its lamellae become distinctly serrated in time

(Halling 2009). The phylogenetically related R. tenuipes can be distinguished

from R. olivaceogrisea by its overall brown pileus colors and a slender stipe

that does not develop a strongly swollen base. Basidiomata of R. olivaceogrisea

do not develop any spots, a field character reported for R. turpis, R. dotae, and

R. laulaha [= R. lignitilis] in Costa Rica. Its dextrinoid spores set it apart from

R. amica and R. tablensis (Mata et al. 2004).

Rhodocollybia tenuipes J.L. Mata, TJ. Baroni & K.W. Hughes, sp. nov. PLATE 3

MycoBank MB 812968

Differs from R. butyracea by its slender and straight stipe with a striate surface, its less

red pileus colors, and its longer spores.

Type: Dominican Republic, Province Santiago, Las Placetas, near Los Montones Arribas,

vicinity of San José de los Matas, 19°14’N 70°53’W, 8.Jan.2003, col. EA Grand TFB 11707

(Holotype, TENN 59546; GenBank AY313288).

EryMo_oey: from Latin tenuis for thin, and pedes for foot.

Piteus 10-65 mm diam, convex to broadly convex, becoming broadly

campanulate or plane and then uplifted, umbonate, sometimes slightly

depressed at disc; surface glabrous, smooth, slightly hygrophanous, burnt

sienna [7D8], dull red-brown [5-6D5], cinnamon brown [6E6-7], paler on

some [near 5C5-6]; margin curved to uplifted or undulating, incised, cream.

FLESH watery cinnamon to cream, thin. Odor none or pungent-spicy, taste

unpleasant, bitter. LAMELLAE adnate to adnexed, crowded, 1-4 mm broad, light

orange [5A3], cream [5A2], pale cream [4A3] or creamy tan [4A3-5B3], now

cream [3A5-4A4]; margin entire, even, concolorous. STIPE 30-80 x 3-9 mm,

central, equal to flattened in some parts, somewhat twisted; surface glabrous

to pubescent, with striations running entire length, light orange [5A3], pale

cinnamon [5C4-5B3], whitish near apex; base slightly swollen in some; interior

hollow.

242 ... Mata & al.

PLATE 3. Rhodocollybia tenuipes. A) basidiomata (TJB 8803); B) basidiospores;

C) basidia and basidioles; D) cheilocystidia. Scale bars: A = 10 mm; B-D = 10 um

Rhodocollybia olivaceogrisea & R. tenuipes spp. nov. (neotropical America) ... 243

BASIDIOSPORES in mass cream [4A2-3]; 5.5-8.8 x 3-4.4 um (n = 4/70,

x = 6.8 x 3.4 um, Q = 1.64-2.33, Qx = 2.00), lacrymoid in side view, ellipsoid

in profile, hyaline, inamyloid or dextrinoid (pale reddish-brown to red-

brown); wall smooth, thin to slightly thickened in dextrinoid spores, retracted

endosporium in some. PILEIPELLIS a simple cutis; hyphae <5 um diam,

cylindrical, repent, radially oriented, somewhat interwoven and gelatinized;

wall thin. PILEUS TRAMA composed of interwoven hyphae, <20 um diam;

refractive hyphae present. LAMELLAR TRAMA irregular, hyphae <14-20 um

diam, long and cylindrical to short and barrel-shaped. Hymenrum composed

of basidia, 21-26 x 4-6 um, clavate, four-sterigmate, some dextrinoid; and

basidioles 16-24 x 3-5 um, clavate. Pleurocystidia absent. Lamellar margin

sterile. CHEILOCYSTIDIA 20-32 x 4-7 um, clavate to broadly clavate, or slender,

some flexuous, apically obtuse, nodulose to lobed; abundant; wall thin. STIPE

CUTIS and trama parallel; hyphae of epicutis, 2-6 um diam, loosely interwoven,

forming a mat, with terminal cells <50 x 5 um, long clavate, some lobed or

diverticulate, mostly prostrate, some semi-erect; hyphae of trama 6-16 um

diam, cylindrical; wall thin. Hyphae in all tissues inamyloid and with clamp

connections present.

EcoLocy cespitose on soil, well-decayed wood (like loam); in Pinus

vegetation.

ADDITIONAL SPECIMENS EXAMINED: DOMINICAN REPUBLIC, PROVINCE LA VEGA,

above Manabao, Cruz farm, 21.Nov.1999, coll. T Amstrong TA 265 (CORT 13982);

PROVINCE SAN PEDRO DE Maconris, Corrizal Cordillera, 18°52’43”N 69°37'26”"W,

14.Jan.2003, col. EA Grand, TFB 11725 (TENN 59554). PUERTO RICO, MuNICIPALITY

LUQUILLO, Ridge above Sabana chicken farm, 18°21’03”N 65°43’50’W, 8.Jun.1998, col.

TJ Baroni TJB 8803 (CORT 13983).

CoMMENTARY- Fruiting bodies of R. tenuipes are similar to R. butyracea in

color but have slender stipes. Spore dimensions are similar to American

reports for R. butyracea (Halling 2009) but larger than those cited for European

specimens (Antonin & Noordeloos 2010). Basidiomata of R. tenuipes lack the

bulbous bent stipe base so characteristic of R. pandipes, and colors are more

reddish-brown than are seen for R. pandipes. However, the spore and cystidia

dimensions are similar to R. pandipes. Rhodocollybia olivaceogrisea differs from

R. tenuipes by its overall olive gray-colored basidiomata. Rhodocollybia tenuipes

was collected under Pinus, a plant genus not indigenous to Costa Rica. DNA

extraction from vouchers TA 265 and TJB 8803 (and subsequent PCR intended

to obtain a more robust R. tenuipes clade) was unsuccessful.

Rhodocollybia maculata (Alb. & Schwein.) Singer, Schweiz. Z. Pilzk. 17: 71. 1939.

Basidiomata of Rhodocollybia maculata from the Dominican Republic

have pilei that become scrobiculate and cream-colored, lamellae that are

244 ... Mata & al.

adnexed, crowded, and cream-colored, and stipes with a slightly swollen

and bent base, collected under pine duff, or near roots of Pinus occidentalis.

Micromorphological characters are typical of R. maculata, with abundant

cheilocystidia 12-40 x 4-8 um, clavate to somewhat lobed, or slender and

flexuous, apically clavate, furcate or mucronate. Basidiospores 5.2-6.8 x

3.2-4.4 um (n = 3/55, x = 6.2 x 3.9 um, Q = 1.40-1.89, Qx = 1.56), short

lacrymoid in side view, broadly ellipsoid in profile view, hyaline, inamyloid or

dextrinoid (rosy brown to pale orange-brown); wall smooth, thin to slightly

thickened in dextrinoid spores.

SPECIMENS EXAMINED: DOMINICAN REPUBLIC, PROVINCE SANTIAGO, Las Placetas,

near Bao River, 19°14’N 70°53’W, 13.Jan.2003, col. EA Grand TFB 11720 (TENN

59804); Ebano Verde, trail from Arroyazo to Loma de la Sal, 19°03’03”N 70°32’46’W,

31.Aug.2003, col. O Perdomo, M Quirico, A Marmolejo, R Concepcién MQ 209 (DR

3020), MQ 212 (DR3021).

This represents a first report of Rhodocollybia maculata for Dominican Republic.

Acknowledgments

Juan L. Mata is thankful to Omar P. Perdomo (Instituto Tecnolégico, Dominican

Republic) for providing information on DR collections, and to CONAGEBIO for

providing collecting permit in Costa Rica. Funding for Clark L. Ovrebo was provided by

a grant No. 7341-02 to Roy E. Halling from the National Geographic Society Committee

of Research & Exploration. Field work in the Dominican Republic by Edward A. Grand,

Omar Perdomo, Tracy R. Armstrong, D. Jean Lodge, and Timothy J. Baroni was

made possible by a grant from the National Science Foundation, Biodiversity Surveys

and Inventories Program to the State University of New York, College at Cortland

(DEB-9525902 and DEB-0103621) and D. Jean Lodge of the USDA Forest Service,

Center for Forest Mycology Research, Forest Products Laboratory, in Puerto Rico who

provided additional logistical support while working in the DR. Thanks to Vladimir

Antonin (Moravian Museum, Czech Republic), Andrew Wilson (Chicago Botanic

Garden, USA) and Victor RM Coimbra (Universidade Federal de Pernambuco, Brazil)

for reviewing this manuscript.

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Clitopilus, Lyophyllum, Gerhardtia, Clitocybe, Hydnangium, Mycena, Rhodocollybia and

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243 p. + 30 color pl.

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basidiomycetous macrofungi and a record of five new species from Mt. Oseo in Korea.

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1102-1112. http://dx.doi.org/10.2307/3761671

Mata JL, Halling RE, Hughes KW, Petersen, RH. 2004. Rhodocollybia in neotropical montane

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with emphasis on the genus Gymnopus. Sydowia 58(2): 191-289.

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9(3): 1-22. http://dx.doi.org/10.2509/naf2014.009.003

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Methods and Applications. Academic Press, San Diego.

MY COTAXON

January-March 2016—Volume 131, pp. 247-253

http://dx.doi.org/10.5248/131.247

Two new Puccinia species on Melica (Poaceae) from USA

MEHRDAD ABBASI & MARY CATHERINE AIME

Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907, USA

* CORRESPONDENCE TO: mabbasi@purdue.edu

ABsTRACT—During a study of herbarium specimens of graminicolous rust fungi from

the USA deposited in the Arthur Fungarium (PUR), two previously undescribed species

infecting Melica spp. were encountered. Described as Puccinia melicae-porteri sp. nov. and

P. goldsbroughii sp. nov., they are compared to similar rust fungi on Melica spp. from around

the world. A key to the fourteen Puccinia species on Melica spp. is provided.

Key worps—mycobiota, Pucciniales, North America

Introduction

The genus Melica (Poaceae trib. Meliceae) comprises about 80 species

worldwide (Clayton & Renvoize 1986). Members of this genus are host to 12

Puccinia species. Cummins (1971) reported eight Puccinia species on Melica:

P. trebouxii Syd. & P. Syd., P graminis Pers., P coronata Corda s. lat.,

P. paradoxica Ricker, P. brachypodii G.H. Otth s. lat., P. schedonnardi Kellerm.

& Swingle, P poarum Nielsen, and P. abramoviana Lavrov. Four additional

species have subsequently been reported on Melica: Puccinia pygmaea Erikss.,

P. montanensis Ellis, P persistens Plowr., and P. recondita Dietel & Holw. (Arthur

1934, Tranzschel 1939, Lindquist 1982, Azbukina 1984, French 1989). Index of

Fungi (1970-2011) and Farr & Rossman (2015) cite no reports of other Puccinia

species on Melica. As part of a planned study of graminicolous rust fungi in

the USA, specimens of P. schedonnardi sensu Cummins (1971) on Melica spp.

deposited in the Arthur Fungarium (PUR) were revised. Our research revealed

two species that are morphologically distinct from all previously reported

Puccinia species on Melica and which we describe here.

248 ... Abbasi & Aime

Materials & methods

Herbarium specimens from PUR were used for this study. Microscopic

measurements and photomicrographs were made from material mounted in lactic acid

in glycerol (Kirk et al. 2008). At least 30 spores were measured from each specimen.

Photomicrographs were taken with a Nikon Digital camera mounted on a Nikon Eclipse

80i microscope. Terminology follows Cummins & Hiratsuka (2003). Spore stages are

indicated by conventional symbols: II = uredinia; II = telia.

Taxonomy

Puccinia melicae-porteri M. Abbasi & Aime, sp. nov. PL. 1

MycoBAank MB 814388

Differs from other Puccinia spp. on Melica by frequent teliospores with a conical apex or

a beak-like projection (tapering rostrum).

Type: USA, Colorado, Manitou, Garden of the Gods, on Melica porteri Scribn., 18 Oct.

1919, leg. Bethel, II+III (Holotype, PUR 21317, as Puccinia schedonnardi).

ErymMo.ocy: Named after its host species, Melica porteri.

Uredinia on stem and on adaxial leaf surface, elliptic or oblong, golden brown;

urediniospores ellipsoid or more or less globoid, 25.6-31.2 x 22.4-25.6 um,

wall pale brown, 1.6-2 um thick, echinulate, with 7-8 scattered germ pores

with slight to moderate internal ring and small pore cap. Telia mostly on

the stem (occasionally on adaxial leaf surface), narrowly oblong or linear

<2 cm long, exposed, semi-compact, and blackish, mostly surrounded by

ruptured epidermis; teliospores variable in shape, clavoid, oblong-clavoid or

fusiform, slightly constricted at septum, rounded, truncate, narrowly conical

or with one or (rarely) two beak-like projections (tapering rostrum) at apex,

35.2-61 x 15.2-27.2 um, walls smooth, chestnut brown at apex, lighter below,

1.5-2.5 um thick at sides, <10.5 um thick at apex (<20 um in spores with conical

apex), pedicels thick-walled, <49.5 um long. Mesospores present.

ADDITIONAL SPECIMENS EXAMINED [all labelled as Puccinia schedonnardi|:

On Melica porteri: USA, CoLorapo, Manitou, Oct. 1919, E. Bethel, III

(PUR 21319); Manitou, Garden of the Gods, Glen Eyrie, 27 Sept. 1919, Bethel, II+II

(PUR 21318).

The above combination of morphological features distinguishes P melicae-

porteri from all previously described Puccinia species on Melica spp. (Cummins

1971, Farr & Rossman 2015). The closest species to P melicae-porteri is

P. neocoronata, arust originally described on Piptochaetium and Stipa (both from

Poaceae trib. Stipeae). In addition to the host, Puccinia melicae-porteri differs

from P. neocoronata in having mostly caulicolous telia, more morphologically

diverse teliospores, and smaller urediniospores with paler walls.

Puccinia melicae-porteri & P. goldsbroughii spp. nov. (U.S.A.) ... 249

PLaTE 1. Puccinia melicae-porteri (PUR 21317, holotype): urediniospores and teliospores; teliospore

apex truncate, rounded, conical or with beak-like projection (arrows). Scale bars = 10 um.

250 ... Abbasi & Aime

Puccinia goldsbroughii M. Abbasi & Aime, sp. nov. Pie2

MycoBank MB 814389

Differs from Puccinia trebouxii in its smaller teliospores and thin-walled urediniospores

with scattered or bizonate germ pores.

Type: USA, Missouri, south of Pierpont, Missouri, on Melica nitens (Scribn.) Nutt.

ex Piper, 24 Jun. 1933, leg. WE. Maneval, II+III (Holotype, PUR 47968, as Puccinia

schedonnardi).

Erymo.oey: After Prof. Peter Goldsbrough, Department Head of Botany and Plant

Pathology, in recognition for his support of the Arthur Fungarium.

Uredinia cinnamon-brown, oblong, amphigenous, mostly on the adaxial

leaf surface and stems, sometimes arranged in short rows, first covered

by epidermis and later pulverulent, ruptured epidermis around sori is

conspicuous; urediniospores more or less globoid, ellipsoid or obovoid,

25.6-32 x 23.2-28 um, wall 1.6-2 um thick, densely echinulate, pale-brown,

germ pores 6-8, scattered or bizonate with small pore cap. Telia oblong or

linear, mostly on adaxial leaf surface and also on the stem, first covered by

epidermis, later exposed, semi-compact, blackish brown with a shining

appearance, teliospores mostly obovoid-clavoid or oblong, (28-)32-44(-49.6)

x 17.6-26.4 um, rounded, truncate, acuminate or obliquely acuminate at apex

and generally tapering downwards, wall smooth, 2-3.5 um at sides, and <12 um

at apex, clear chestnut brown, lighter towards the base and at apex especially in

acuminate spores, pedicels short (<35 um), mostly shorter than spore length,

dark brown below the attachment point to the spore, pale or yellowish brown

downward. Mesospores present, common in some telia.

ADDITIONAL SPECIMENS EXAMINED [all labelled as Puccinia schedonnardi|:

On Melica sp.: USA, INDIANA, Lafayette, 29 July 1899, Wm. Stuart, II+III

(PUR 21315); 18 July 1906, J.C. Arthur, II+III (PUR 21347); Lafayette, Elston, 31 Dec.

1896, Wm. Stuart, III (PUR 21348); 22 Oct. 1899, Stuart, II+II] (PUR 21314); Lafayette,

near Elston, 14 Oct. 1912, J.C. Arthur, II+II (PUR 21372); Lafayette, Bayles Mills, 3

Sept. 1915, J.C. Arthur & H.S. Jackson, II+HI (PUR 21360).

On Melica nitens: USA, INDIANA, Lafayette, Bayles Mills, 13 May 1903, J.C. Arthur,

II+III [III spores mostly germinated] (PUR 21313); 29 May 1903, J.C. Arthur, I

(PUR 21312); Mississippi, Lumberton, 12 Aug. 1921, L.E. Miles, II+III (PUR 45590).

The above combination of morphological features distinguishes P goldsbroughii

from all previously reported Puccinia species on Melica (Cummins 1971,

Farr & Rossman 2015). Puccinia trebouxii, which was originally described

from Samarkand (Uzbekistan), is the morphologically closest species to

P. goldsbroughii, but differs from P. goldsbroughii in its wider teliospores

(24-36 um) and thick-walled (< 4 um thick) urediniospores with occasionally

equatorial germ pores. On one of the P goldsbroughii paratypes (PUR 21313)

teliospores had mostly germinated in telia, but this phenomenon was not

observed on other examined material, including the holotype.

Puccinia melicae-porteri & P. goldsbroughii spp. nov. (U.S.A.) ... 251

y 4 Af 7 >>, a: f a

- et} ee . P

- ROE. Ox As

“a ot - \ = : a ENN tf . —_

PLATE 2. Puccinia goldsbroughii (PUR 47968, holotype): urediniospores, teliospores, and

mesospores; arrow shows dark-brown part of pedicel below attachment point. Scale bars = 10 um.

252 ... Abbasi & Aime

Key to the 14 Puccinia species on Melica

. Leliospores-verrucose i. 4c sense neaio es kws es ewe Ee Rolo LER o CBR AE eo P. paradoxica

HOS POTESPSHTO CIA Fo MON Moly ee ts tg ae ae Nips Mae igs Sh Meh ys PED ie BOE pe SES ge 2

LOH A*CR POSE es 2 4 22455 abil me hiya wh rye hea Wire a He Fra dia te Pea ga ts prada Ma Fes ez Hs Srna ti erage ahs Sevgte a hs fee 3

oN CLIGECOVELEC S62 UN huts LW hers AM Rens LM Re REN RRC REMEBER Bet T EME Se? 8 7

Teliospores with apical beak-like projection ................... P. melicae-porteri

» LEliOSPOTES-WALNOUE PROTEC OMe. xfer cee foes Eps coe ence Ree me ps ee Pace hs +

. Urediniospore pores exclusively eqtiatotial.. 0.4 saute sects teste tea P. graminis

. Urediniospore pores scattered, occasionally equatorial ....................004. 5

. Teliospores mostly broadly ellipsoid, broadly obovoid or more or less globoid

Se ee ee Lee ee eee een ee eee P. schedonnardi s. str.

. Teliospores mostly obovoid-clavoid or oblong.............. ce cece eee eee eee 6

= LEL@S POLES 32 =O pT osx. se cbs bee a be: Prada We deal a is Prada Me precios Hs Sraet a: Seager P. trebouxii

; Leligspotes-1776=26.4 windwides ss hots oaths hated Seite: watts ca P. goldsbroughii

rleliespotesswaith apical divitations., nck sok Ae ead Se ade petals ht P. coronata s. lat.

» Lehospores: without apical disitations .. so ase ean eae ie ae eee ge eee 8

Uredinianpataphysates..4 s5.S sates bets be she ete Me wettest Res uetNt Sates etd 9

-Urediniia-aparapliysate rs <c2ccs steno eu dtne 24 Reo ete oD weno Ed ete ah sil Ewen 8 ea 1d

#baraphiyses thick Walled: 8... ay ta cays oye + ae pepe ee P. brachypodii s. lat.

Paraplhiysesvami lorie ys thir Wallet tesa: bestt,a th; Praia th esta bh brats athe bred a seseteabe freeted dt 10

« Léliospores Mostly 22=32-um wide +.-< o45.05 aun yhone Sb oaw es P. montanensis

p Leliospores-mmosthy 1722: (iid WAGs. ead ade Bade en Bade: ross reed re P. pygmaea

SFE CUNTOS POLES VET RULCOGE? nx. 4: brads ats sraliya bs prodtya Me predate gre ae craee a P. abramoviana

JUredinivsporessechinulate.. 8.8 fect fi aecat ee tanat Geae eek Cee A ee A Med Ss i 12

. Urediniospore wall colorless or pale yellowish ...................44. P. poarum

sUrediniosporeavall biOwiisht 2.125 250«5.250659-0s ge sere sees pe cere eee 13

, Leltospores mostly 35-20 (nt long. fot Asa last Mie at 2a P recondita s. str.

. Teliospores mostly 34-46 um long ..... 0... eee eee eee P. persistens

knowledgments

The authors would like to thank Dr. Roger S. Peterson (St. John’s College, Santa Fe,

New Mexico, USA) and Dr. Alan Wood (ARC-Plant Protection Research Institute,

Stellenbosch, South Africa) for serving as pre-submission reviewers and Dr. Shaun

Lit

nnycook (Auckland, New Zealand) for nomenclatural review.

erature cited

Anon. 1970-2011. Index of fungi. CABI Bioscience, UK.

thur JC. 1934. Manual of the rusts in the United States and Canada. Lafayette, IN: Purdue

Research Foundation.

Puccinia melicae-porteri & P. goldsbroughii spp. nov. (U.S.A.) ... 253

Azbukina ZM. 1984. [Key to the rust fungi of the Soviet Far East]. Akademiya Nauk SSSR, Moscow.

Clayton WD, Renvoize SA. 1986. Genera Graminum: grasses of the world. Kew Bull., Addit. Ser.

XII HMSO, London.

Cummins GB. 1971. The rust fungi of cereals, grasses and bamboos. Springer-Verlag, New York.

Cummins GB, Hiratsuka Y. 2003. The illustrated genera of rust fungi. APS Press, St. Paul, Minnesota.

Farr DE, Rossman AY. 2015. Fungal databases, Systematic Mycology and Microbiology Laboratory,

ARS, USDA. http://nt.ars-grin.gov/fungaldatabases.

French AM. 1989. California plant disease host index. Calif. Dept. Food Agric., Sacramento.

Kirk PM, Cannon PF, Minter DW, Stalpers JA. 2008. Dictionary of the fungi.10th edition. CABI

Publishing, UK.

Lindquist JC. 1982. Royas de la Republica Argentina y zonas limitrofes. Inst. Nacional de Tecnologia

Agropecuaria.

Tranzschel VA. 1939. Conspectus Uredinalium URSS. Akademii Nauk SSSR, Moskva-Leningrad.

MYCOTAXON

January-March 2016—Volume 131, pp. 255-256

http://dx.doi.org/10.5248/131.255

Regional annotated mycobiotas

new to the Mycotaxon website

ABSTRACT—Mycotaxon is pleased to announce three new species distribution lists to our

‘web-list’ page covering Glomeromycota in Brazil (by Jobim, Oliveira & Goto), macromycetes

within Cistaceae-dominated ecosystems in Cyprus (by Loizides), and macrofungi of Mugla

province in Turkey (by Giingor, Solak, Alh, Isiloglu & Kalmis). This brings to 123 the

number of free access mycobiotas now available on the Mycotaxon website:

http://www.mycotaxon.com/resources/weblists.html

SOUTH AMERICA

Brazil

KHADIJA JOBIM, BRUNA IOHANNA SANTOS OLIVEIRA & BRUNO TOMIO GOTO.

Checklist of the Glomeromycota in the Brazilian Savanna. 13 p.

ABSTRACT— The Brazilian savanna (Cerrado) was the first Brazilian biome to be

surveyed for arbuscular mycorrhizal fungi (AMF) and currently comprises the

third Brazilian biome in species representation. This paper provides a checklist of

arbuscular mycorrhizal fungi (AMF) in the Cerrado. A total of 92 species of AMF

have been found in the Brazilian Cerrado over three decades of work conducted

in this biome. The results characterize the Cerrado as an important AMF reservoir

and show that rupestrian fields, one of several physiognomies of the cerrado, are

biologically promising

EUROPE

Cyprus

MicHaAEL Lorzipes. Macromycetes within Cistaceae-dominated ecosystems in

Cyprus. 33 p.

ABSTRACT—An inventory of macromycetes associated with Cistaceae plants,

including Cistus, Helianthemum, Tuberaria and Fumana species in Cyprus is

presented, following a ten-year survey between 2007 and 2016. One-hundred-and-

twenty-seven taxa are identified, sixty-five of which are reported for the first time from

Cyprus. Of these, some recently described or rarely reported species are noteworthy,

256 ... New regional mycobiotas online

such as Agaricus iesu-et-marthae, Astraeus telleriae, Fomitiporia rosmarini, Gymnopus

bisporus, Lepiota farinolens, Lepiota locquinii, Ombrophila rivulorum, Peziza

muscicola, Pholiota gallica, Tremella dactylobasidia and Xeromphalina cornui. ‘The

taxonomical problems associated with a number of insufficiently clarified taxa,

such as Clitocybe font-queri, Cortinarius caligatus, Leccinellum corsicum, Lyophyllum

fumosum, Peziza moseri, Peziza subviolacea, Plectania zugazae and Terfezia aphroditis

are discussed, and the role of Cistus communities in Mediterranean ecosystems is

evaluated, particularly in view of accelerated climate changes. Selected imagery and

notes about the fruiting season, host-plant, altitude and estimated abundance are

provided.

Mip-EAstT

Turkey

Ha.it GUNGOR, MEHMET HALtIt SOLAK, HAKAN ALLI, MUSTAFA IsILOGLU, ERBIL KALMIS.

Contributions to the macrofungal diversity of Mugla province (Turkey). 26 p.

ABSTRACT—This study was conducted on specimens of macrofungi collected from

Mugla province between 2005-2011. After field and laboratory studies, 211 taxa

belonging to 47 families and 2 classes were identified. Five of them—Cortinarius

biformis, Cortinarius percomis, Cortinarius renidens, Geastrum lageniforme, Hebeloma

aestivale—represent new records for Turkey.

MYCOTAXON

January-March 2016—Volume 131, pp. 257-260

http://dx.doi.org/10.5248/131.257

BOOK REVIEWS AND NOTICES:

ABSTRACT—Books reviewed include: FigELD GuipEs—'The essential guide to Rocky

Mountain mushrooms (Cripps, Evenson & Kuo 2016); LicHENs—Keys to lichens of

North America (Brodo 2016).

FIELD GUIDES

The essential guide to Rocky Mountain mushrooms by habitat. By C.L.

Cripps, V.S. Evenson & M. Kuo. 2016. University of Illinois Press, 1325 South

Oak Street, Champaign, IL 61820-6903 <www.press.uillinois.edu>. 272 p,

372 colour photos, ISBN 978-0-252-08146-0 US $29.95 (soft cover); ISBN

978-0-252-03996-6 $ 95.00 (hard cover).

The mushrooms of the Rocky Mountains from Montana to Colorado (USA)

southwards are covered in this lavishly illustrated book.

Unlike the usual guide, this book is organized by habitat, from the prairies

of the foothills through the extent of montane Ponderosa pine and subalpine

Spruce-Fir forests to the alpine high altitude vegetation. Each habitat is

introduced with a description of the main players, which includes plants,

animals, and fungi, followed by descriptions and photos of the characteristic

mushrooms of that particular habitat. The book is organized like a walk up the

mountains, but has also a chapter on burned ground and on snowbanks, with

their characteristic mycoflora. Snowbank fungi are unique to western North

America, and certainly deserve their own place in this book.

“Book reviews or books for consideration for coverage in this column should be sent to the Editor-

in-Chief at editor@mycotaxon.com or 6720 NW Skyline, Portland OR 97229 USA.

258 ... MYCOTAXON 131

The only other North American guide organized by habitat that I know of is

the one on Hawaiian fungi (Hemmes & Desjardin 2002). Special for the Rocky

Mountain book is the attention to organisms other than fungi.

Unlike the usual guides, this book does not have an extensive introduction

to fungi; one page on making spore prints is placed in the introduction, simple

keys are given at the end, and one page is devoted to eating mushrooms.

The index also serves as glossary, with terms explained with a referral to the

page where the term is used.

The approach to place the fungi so clearly in their environment—and to

show the interconnectedness between all organisms in a certain habitat—is

wonderful.

The emphasis is on the more conspicuous and easy to recognize species; one

will look in vain for an Inocybe species in this book, although many occur in the

Rocky Mountains. The descriptions cover macroscopical characters, and the

observations give information on similar species of the mountains.

One objection to the habitat approach is that of course some fungal species

occur in several habitats; to counter this, the inside of the front cover lists the

species per habitat and for each species also indicates in which other habitats it

might occur. Still, when one wants to compare Suillus species that occur in the

pine forests, one has to flip back and forth between habitats.

The quality of the photos bothers me - many photos are very flat, which

has probably to do with the printing process. Most photos of the species are

good and show all the characters of a species, while others do not include the

complete fruitbodies.

Some names still need to be updated - some examples: Plectania nannfeldtii

should be Donadinia nigrella (Carbone et al. 2013), and the more recent genus

for Pachylepyrium, Crassisporium (Matheny et al. 2015), is named in the

“Observations” as an option.

The approach presented in this book makes me want to see more books

like this, and I hope that the increasing knowledge of the North American

mycoflora will make this possible.

Carbone M, Agnello C, Alvarado P. 2013. Phylogenetic studies in the family Sarcosomataceae

(Ascomycota, Pezizales). Ascomycete.org 5 (1): 1-12.

Hemmes DE, Desjardin DE. 2002. Mushrooms of Hawai'i. An identification guide. Ten

Speed Press, Berkeley, Toronto.

Matheny PB, Moreau P-A, Vizzini A, Harrower E, De Haan A, Contu M, Curti M, 2015.

Crassisporium and Romagnesiella: two new genera of dark-spored Agaricales. Systematics

and Biodiversity 13: 28-41. http://dx.doi.org/10.1080/14772000.2014.967823

ELsE C. VELLINGA

861 Keeler Avenue, Berkeley CA 94708 USA

ecvellinga@comcast.net

Book Reviews ... 259

LICHENS

Keys to lichens of North America: revised and expanded. By I.M. Brodo,

with photographs by S.D. Sharnoff & S. Sharnoff and drawings by S. Laurie-

Bourque. 2016. Yale University Press, P.O. Box 209040, New Haven, CT 06520-

9040. <sales.press@yale.edu> ISBN 978-0-300-19573-6. 424 p., 13 color and 33 b/w

illustrations. $29.95.

When LICHENS OF NORTH AMERICA (Brodo et al. 2001) was published, it

quickly became a classic. The book gave a thorough introduction into the

world of lichens and provided keys to genera and many species. Beautiful color

photographs illustrated the treated species, and a number of additional species

were mentioned for comparison. All this made it an invaluable contribution to

lichenology in North America. Over the years, Brodo has received numerous

requests for a separate volume that could be used more practically as teaching

material in the lab and field. This newly revised KEys TO LICHENS OF NORTH

AMERICA is a response to those requests.

In this treatment, Brodo has greatly expanded and updated the original keys

found in LICHENS OF NorRTH America. After a brief preface explaining the

purpose of the present volume followed by acknowledgements, the Keys To

LICHENS OF NorRTH AMERICA dives directly into introductory identification

keys to genera and the following major groups: basidiolichens, fruticose lichens,

dwarf fruticose lichens, sterile crustose lichens, crustose perithecial lichens,

crustose script lichens, crustose disk lichens, squamulose lichens, umbilicate

lichens, jelly lichens, orange-yellow-yellow green foliose lichens, and non-

umbilicate, non-jelly, non-yellow foliose lichens. All genera as well as many

easy-to-identify species are separated in the group keys. Keys to genera and

major groups are followed by keys to species level where the genera are treated

in alphabetical order. Thus, more advanced lichenologists can turn directly

to the genus keys without having to wade through the introductory keys.

The original glossary, which contains clarifying drawings by Laurie-Bourque

and photographs by the Sharnoffs from LICHENS OF NORTH AMERICA, is also

included. Finally, references and an index to names of genera and species

complete the volume.

The layout is very clean and organized. Although I would prefer an indented

key because such a format fosters navigation and retracing earlier steps, the

bracket format keeps couplet components together and consumes far less space.

The keys are well written and contain abundant detail. For example, a spore is

not just “thick-walled” versus “thin-walled” but the key-step contains average

measurements that facilitate determination. While specialized terminology

260 ... MyCcoTAXON 131

cannot be completely avoided in any scientific discipline, Brodo has limited

his use of technical jargon, especially compared to some regional treatments

(e.g. Nash et al. 2002, 2004, 2007). These qualities extend this volume’s utility

to beginners and experts alike. The keys are of course not comprehensive;

including all North American taxa would require a volume more than twice

as thick, not to mention an enormous amount of work, since only about 42%

(2028 species) of the 4881 known species in North America (Esslinger 2015)

are treated. However, the most common and prominent species that users will

encounter are included. This makes the keys very useful as a first reference,

especially as a laboratory resource for university courses and workshops as

well as for amateur enthusiasts. Advanced lichenologists will find utility in

the general keys to reach genus level before seeking more comprehensive,

specialized monographs and regional treatments offering deeper taxonomic

resolution. A minor disadvantage of the KEYs TO LICHENS OF NORTH AMERICA

is that species are not described in detail. However, the volume is intended

as a companion for the original LICHENS OF NORTH AMERICA where detailed

descriptions, color photographs, distribution maps, and comparative notes are

provided for many of the mentioned species.

Overall, this volume of keys fully lives up to and complements LICHENS OF

NorTH AMERICA, and it is an essential identification tool for professional and

amateur lichenologists alike. Brodo invites comments, problems, and errors

in order to continually improve and update the keys. Keys TO LICHENS OF

NorTH AMERICA and potential updates will certainly boost the future of North

American lichenology.

Brodo IM, Sharnoff SD, Sharnoff S, 2001. Lichens of North America. Yale University

Press, New Haven and London.

Esslinger TL, 2015. A cumulative checklist for the lichen-forming, lichenicolous and allied

fungi of the continental United States and Canada. North Dakota State University:

(Posted 19 April 2015), Fargo, North Dakota. http://www.nsdu.edu/pubweb/~essling/

chcklst/chcklst7.htm

Nash III TH, Ryan BD, Gries C, Bungartz F, (eds.) 2002. Lichen flora of the greater

Sonoran desert region. Vol. 1. Lichens Unlimited, Arizona State University, Tempe,

Arizona, USA.

Nash II TH, Ryan BD, Diederich P, Gries C, Bungartz F, (eds.) 2004. Lichen flora of the

greater Sonoran desert region. Vol. 2. Lichens Unlimited, Arizona State University,

Tempe, Arizona, USA.

Nash III TH, Gries C, Bungartz F, (eds.) 2007. Lichen flora of the greater Sonoran desert

region. Vol. 3. Lichens Unlimited, Arizona State University, Tempe, Arizona, USA.

RIKKE REESE N2ZSBORG

University of California at Berkeley

Rikke. ReeseNaesborg@gmail.com

MY COTAXON

January-March 2016—Volume 131, pp. 261-262

http://dx.doi.org/10.5248/131.261

NOMENCLATURAL NOVELTIES AND TYPIFICATIONS

PROPOSED IN MYCOTAXON 131(1)

Agaricus columellatus (Long) R.M. Chapm., V.S. Evenson & S.T. Bates

(lectotypified) [MB809082], p. 105

Anacoronospora J.S. Monteiro, Gusmao & R.F. Castafieda [MB813219], p. 186

Anacoronospora diversiseptata J.S. Monteiro, Gusmao & R.E. Castaneda

[MB813220], p. 186

Annulohypoxylon bogoriense (Hohn.) Lar.N. Vassiljeva, S.L. Stephenson &

K.D. Hyde [MB801406], p. 63

Annulohypoxylon chiangmaiense Lar.N. Vassiljeva, S.L. Stephenson & K.D. Hyde

[MB801393], p. 64

Annulohypoxylon derelictum Lar.N. Vassiljeva, S.L. Stephenson & K.D. Hyde

[MB801394], p. 66

Annulohypoxylon dipterocarpi Lar.N. Vassiljeva, S.L. Stephenson & K.D. Hyde

[MB801395], p. 66

Annulohypoxylon maesaeense Lar.N. Vassiljeva, S.L. Stephenson & K.D. Hyde

[MB801396], p. 67

Annulohypoxylon microbovei Lar.N. Vassiljeva, S.L. Stephenson & K.D. Hyde

[MB801397], p. 68

Annulohypoxylon morisuspectum Lar.N. Vassiljeva, S.L. Stephenson & K.D. Hyde

[MB801398], p. 70

Annulohypoxylon neglectum Lar.N. Vassiljeva, S.L. Stephenson & K.D. Hyde

[MB801400], p. 70

Annulohypoxylon paratruncatum Lar.N. Vassiljeva, S.L. Stephenson & K.D. Hyde

[MB801401], p. 72

Annulohypoxylon planodiscum Lar.N. Vassiljeva, S.L. Stephenson & K.D. Hyde

[MB801402], p. 74

262 ... MYCOTAXON 131(1)

Annulohypoxylon pseudonitens Lar.N. Vassiljeva, S.L. Stephenson & K.D. Hyde

[MB801403], p. 74

Annulohypoxylon sordidum Lar.N. Vassiljeva, S.L. Stephenson & K.D. Hyde

[MB801404], p. 75

Aspergillus similanensis Dethoup [MB810782], p. 9

Dictyoaquaphila J.S. Monteiro, L.B. Conc., M.EO. Marques, Gusmao &

R.E. Castafieda [MB813243], p. 178

Dictyoaquaphila appendiculata J.S. Monteiro, L.B. Conc., M.F.O. Marques,

Gusmao & R.F. Castafieda [MB813244], p. 178

Fomitiporia deserticola Vlasak [MB813967], p. 198

Fomitiporia fissurata Vlasak [MB813966], p. 196

Gibellula clavispora Z.Q. Liang, Wan H. Chen & Y.E Han [MB810567], p. 115

Leptonia newlingii Largent & Bergemann [MB813886], p. 170

Leptonia sabulosa Largent & Bergemann [MB813883], p. 167

Leptonia subpanniculus Largent & Bergemann [MB813877], p. 160

Leptonia substricta Largent & Bergemann [MB813880], p. 163

Mirandina uncinata Fiuza, J.S. Monteiro, R.F. Castafeda & Gusmao

[MB813056], p. 143

Passalora caesalpiniicola Sh. Kumar & Raghv. Singh [MB812867], p. 26

Pleurothecium bicoloratum R.F. Castaneda, J.S. Monteiro, & Gusmao

[MB813040], p. 146

Puccinia goldsbroughii M. Abbasi & Aime [MB814389], p. 250

Puccinia melicae-porteri M. Abbasi & Aime [MB814388], p. 248

Ramularia hydrangeicola J.H. Park & H.D. Shin [MB813449], p. 97

Rhodocollybia olivaceogrisea J.L. Mata & Ovrebo [MB812967], p. 239

Rhodocollybia tenuipes J.L. Mata, T.J. Baroni & K.W. Hughes [MB812968], p. 241

Sarcogyne saphyniana A. Abbas, Nurtai & K. Knudsen, p. 136

Sporidesmiopsis pluriseptata J.S. Monteiro, Gusmao & R.F. Castaneda

[MB813041], p. 150

Sympodiosynnema J.W. Xia & X.G. Zhang [MB815649]], p. 46

Sympodiosynnema elegans J.W. Xia & X.G. Zhang [MB815650], p. 46

Ustulina pseudozonata Lar.N. Vassiljeva, S.L. Stephenson & K.D. Hyde

[MB801405], p. 80