Iv ... MYCOTAXON 131(4)

MYCOTAXON

VOLUME ONE HUNDRED THIRTY-ONE (4) — TABLE OF CONTENTS

COVER SECTION

TRV TOW CF Roe Ion tt dd th ced eh sg ele nics and eo eaed chee oe rede oi feel Vii

EP OVP TIC TIAUOLY 6 cece rie PAOD a sg de ac pede tattle gee Gorse bo ate i's astemae vy os ix

SUD ISSIOMPIOCEUUICS 0 Beck, Bin 5 Gass eshte ner AAO SE OR Sa Res IES, x

RESEARCH ARTICLES

Nawawia oviformis sp. nov. from China

JizE PENG, Dou CHANG, YING HUANG & ZE-FEN YU

Pezicula chiangraiensis sp. nov. from Thailand = AnusHa H. EKANAYAKA,

DINUSHANI A. DARANAGAMA, HIRAN A. ARIYAWANSA,

E.B. GARETH JONES, ALI H. BAKHALI & KEVIN D. HYDE

Abieticola koreana gen. et sp. nov., a griseofulvin-producing endophytic

xylariaceous ascomycete from Korea Hyanc-BurM LEE, HyE- YEON Moun,

THI THUONG THUONG NGUYEN, JIN-CHEOL KIM & JEFFREY K. STONE

Entalbostroma erumpens gen. et sp. nov. (Xylariaceae)

from Phormium in New Zealand

P.R. JOHNSTON, J.D. RoGERS, D. PARK & N.A. MARTIN

Podosporiopsis, a new genus of synnematous hyphomycetes

from China JIAN Ma, X1u-Guo ZHANG & RAFAEL FE, CASTANEDA-RU{Z

Distribution of Alternaria species among sections.

3. Sections Infectoriae and Pseudoalternaria

PHILIPP B. GANNIBAL & DANIEL P. LAWRENCE

Zasmidium oleae sp. nov. from China Bao-Ju Lr, XuE-WEN XIE,

YAN-XIA SHI, A-LI CHAI & YING-LAN GUO

Strigula sinoaustralis sp. nov. and three Strigula spp.

new to China SHU-HUuA JIANG, XIN-LI WEI & JIANG-CHUN WEI

Thelenella haradae sp. nov., a saxicolous lichen

from South Korea Joser P HALDA & JAE-SEOUN Hur

Notes on rust fungi in China 2. Two species of Coleosporium

on Compositae JING-XIN Jr, Q1 WANG,

ZHUANG LI, Yu Li & MAKOTO KAKISHIMA

Arthromoniliphora araucariae gen. & sp. nov.

from Brazilian pine SILVANA SANTOS DA SILVA,

Luis FERNANDO PASCHOLATI GUSMAO & RAFAEL F. CASTANEDA-RUIZ

735

739

749

765

773

781

791

795

805

811

821

OCTOBER-—DECEMBER 2016... V

Morphological and phylogenetic clarification of Peziza arvernensis,

P. pseudovesiculosa, P. pseudosylvestris, and P. domiciliana

ANGELA LANTIERI, GIANFRANCO MEDARDI & PABLO ALVARADO = 827

Podosporium bacilliforme sp. nov. and a first record of

Phaeoblastophora peckii from southern China Jin-YE WANG,

Kal ZHANG, CHUN-LING YANG, JI- WEN XIA, YING-RuI Ma,

JIAN-MEI GAo, XIANG-Yu LI, XtU-GUO ZHANG & YU-MEI Car 841

Apiosordaria hamata sp. nov. from lake sediment in China

BING Wu, JIAN-QING TIAN, LIN WANG,

JIAN-Kut Liu, Kevin D. HYDE & JING-ZU SUN 847

Discopycnothyrium palmae gen. & sp. nov. (Asterinaceae)

SINANG HONGSANAN, ALI H. BAHKALI, PUTARAK CHOMNUNTI,

JIAN-Kut Liu, JUN-Bo YANG, & KEVIN D. HypE 859

Two new records of Agaricus from Southwest China

Mao-QIANG HE, JIE CHEN & RuI-LIN ZHAO 871

Entoloma ochreoprunuloides from Italy, with notes on its

geographical distribution and allied species

FRANCESCO DOVANA, ALFREDO VIZZINI,

FABRIZIO BOCCARDO, MARCO MUCCIARELLI & MARCO CLERICUZIO 881

Phaeocollybia pakistanica sp. nov., the first representative

of the genus from Pakistan

JUNAID KHAN, HASSAN SHER & ABDUL NASIR KHALID 889

Mucor indicus isolated from the semiarid region of Brazil:

a first record for South America CarRLos A.F. DE SOUZA,

DioGo X. LIMA, RAFAEL J.V. DE OLIVEIRA,

LucraNa M.S. GURGEL & ANDRE L.C.M. DE A. SANTIAGO 897

Blodgettia saprophytica sp. nov. and Uberispora tropicalis,

new records from southern China CuuN-LING YANG,

Kat ZHANG, JIN- YE WANG, JI-WEN XIA, YING-RuI Ma,

JIAN-MEI Gao, X1u-GuoO ZHANG & ZHUANG Li 907

New record of Beauveria pseudobassiana from Morocco

ABDESSAMAD IMOULAN, YI LI,

WEN-JING WANG, ABDELLATIF EL MEZIANE & YI-JIAN YAO 913

New records of Graphis from Cameroon

with a key to African species of Graphis SANTOSH JOSHI,

D.K. Upret1, ENow ANDREW EGBE &JAE-SEOUN Hur 925

Revision of some Peziza collections in herb. TAAM (Tartu)

GIANFRANCO MEDARDI, ANGELA LANTIERI & GABRIELE CACIALLI 939

vi ... MYCOTAXON 131(4)

BOOK REVIEWS AND NOTICES LORELEI NORVELL 947

THE OUTER SPORES: MUSHROOMS OF HAIDA Gwall

(Kroeger, Kendrick, Ceska & Roberts; 2012)

RARE LICHENS OF OREGON

(Exeter, Glade & Loring; 2016)

LICHENS OF UTTAR PRADESH

(Nayaka & Upreti; 2013)

PLANT PATHOGENIC AND ENDOPHYTIC BOTRYOSPHAERIALES

KNOWN FROM CULTURE

(Phillips, Slippers, Groenewald & Crous, eds.; 2013)

A POLYPHASIC TAXONOMY OF DALDINIA (XYLARIACEAE)

(Stadler, Leessoe, Fournier, Decock,

Schmieschek, Tichy & Persoh; 2014)

HYPOCREALEAN LINEAGES OF INDUSTRIAL AND

PHYTOPATHOLOGICAL IMPORTANCE

(Lombard, Groenewald & Crous, eds.; 2015)

NOMENCLATURAL NOVELTIES AND TYPIFICATIONS

PROPOSED IN VOLUME 131(4) 961

RICHARD P. Korg (1925-1916): A Celebration

LORELEI L. NORVELL (ED.) 963

OCTOBER-DECEMBER 2016...

REVIEWERS — VOLUME ONE HUNDRED THIRTY-ONE (4)

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

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

prepared for this issue.

Najam ul Sehar Afshan

Hiran A. Ariyawansa

Andrew Armitage

Timothy J. Baroni

Uwe Braun

Matias J. Cafaro

Rafael F. Castaftieda-Ruiz

Giovanni Consiglio

Kanad Das

Pradeep K. Divakar

Jacques Fournier

Shouyu Guo

Bo Huang

Ze-Feng Jia

Santosh Joshi

Yu-Ming Ju

Samantha C. Karunarathna

Bryce Kendrick

Paul M. Kirk

De-Wei Li

Cristiano Losi

Robert Liicking

Sajeewa S.N. Maharachchikumbura

Eric H.C. McKenzie

Gabriel Moreno

Lorelei L. Norvell

Shaun R. Pennycook

Emmanuél Sérusiaux

José Ivanildo de Souza

Marc Stadler

Larissa Vasilyeva

Xiu-Guo Zhang

Ying Zhang

Qi Zhao

VII

vill ... MyCOTAXON 131(4)

PUBLICATION DATE FOR VOLUME ONE HUNDRED THIRTY-ONE (3)

MYCOTAXON for JULY-SEPTEMBER 2016 (I-x1I + 491-734)

was issued on October 18, 2016

OCTOBER-DECEMBER 2016... IX

FROM THE EDITOR-IN-CHIEF

RICHARD P. KorrF (1925-2016)—Our man in Ithaca was just too famous! When

assembling materials for MycotTaxon’s special issue (scheduled appropriately as

the final one in 2016), I encountered memorials, awards, over 400 manuscript titles

eponymous names, biographies including a surprisingly complete Wikipedia entry plus

three already published obituaries (not to mention plus Facebook blogs and tweets too

numerous to count)—most accompanied by full color photos capturing our favorite

curmudgeonss inherent greatness. How could his and Grégoire Hennebert’s precious

literary progeny possibly compete?

We can't. Fortunately this is not a competition (which Dick, in his own inimitable

fashion, would have won hands down) but a chance to honor a man with panache and

personality seemingly born to greatness. Thus we present photographs, long and short

vignettes, and quips that we hope will bring smiles in the wake of a mycological giant

who left us too soon.

THE EDITORIAL FINGER WRITES, AND HAVING WRIT - LIVES ON*—After flirting with

using a black cover for this issue dedicated to Dick followed by a vain attempt to find

a metallic gold that was bright instead of dull, we settled on the yellow-orange color

selected last January for 2016’s fourth issue. It was Noni, Dick’s daughter, who realized

that her dad’s celebration issue would display the identical color he selected in 1973 for

the first volume of Mycoraxon. How pleased (and amused) Dick would have been at

the coincidence.

WHAT DO WE DO WITH ‘INVALID’ HIGHER LEVEL TAXA? The MycoTaxon dictum that

all formal taxonomic names be italicized from subspecies to kingdom level sometimes

causes problems. There was a fair amount of editorial hand wringing in December

as to whether to distinguish the formal taxonomic subfamilies Xylarioideae and

Hypoxyloideae from phylogenetic clades by using italics. As the two subfamilies are

nomenclaturally invalid (Dennis proposed them in 1981 without the then-required

Latin diagnoses), we decided to present them in roman font in this issue's Lee & al. (pp.

749-764) and Johnston & al. (pp. 765-771).

METABOLICALLY INACTIVE CULTURES—Although it seems that the INTERNATIONAL

CODE OF NOMENCLATURE FOR ALGAE, FUNGI, AND PLANTS was only just revised, during

July 23-29, 2017, the 19" International Botanical Congress will ratify in China a new

“Shenzhen Code” to replace 2011’s Melbourne Code. As the current code’s Rec. 8B.3

(“When a culture is designated as a type, the status of the culture should be indicated,

including the phrase ‘permanently preserved in a metabolically inactive state’ or an

equivalent”) is expected to become a requirement, MycoTAXxoN now expects authors

who designate cultures as types of new taxa to cite their inactive metabolic status in

their type paragraphs.

*Adapted from Edward Fitzgerald’s translation of the Rubdaiyat of Omar Khayyam:

“The Moving Finger writes; and, having writ, / Moves on: nor all thy Piety nor Wit/

Shall lure it back to cancel half a Line,/ Nor all thy Tears wash out a Word of it?”

x ... MYCOTAXON 131(4)

SPEEDY PUBLICATION, ACCURACY, AND AUTHORS—Of late, we have received far too

many carelessly prepared submissions. Although we all too aware of the current delays

our authors experience between accession and final nomenclatural approval, we

nonetheless stress once again that all text and illustrations submitted to MycoTAXON

Must be error-free. Manuscripts with unacceptable formatting, incoherent text, and

spelling and grammatical errors will be returned to authors, with the returned revisions

placed at the end of the editorial review queue—needlessly delaying final publication.

Incredibly, three final submissions (12% of the total) intended for MycoTaxon

131(4) contained wrong illustrations, which authors did not think to check until AFTER

they received their PDF files. Such major errors should have been detected well before

final manuscript preparation; at the very least the corrected illustrations should have

been sent to the Editor-in-Chief prior to PDF conversion. Art files are not included in

the errata; rather than publish scientifically erroneous papers, a very unhappy editor

decided to replace the files at deadline and to access correction fees. Be warned!

MYCOTAXON 131(4) presents 24 papers by 103 authors (representing 17 countries) and

revised by 34 expert reviewers.

Within its pages are five new genera (Abieticola from South Korea, Arthromoniliphora

from Brazil, Discopycnothyrium from Thailand, Entalbostroma from New Zealand, and

Podosporiopsis from China) and 16 species new to science representing Abieticola and

Thelenella from South Korea; Alternaria from the U.S.A., Apiosordaria, Blodgettia,

Nawawia, Podosporiopsis, Podosporium, Strigula, and Zasmidium from China;

Arthromoniliphora from Brazil; Discopycnothyrium and Pezicula from Thailand;

Entalbostroma from New Zealand, and Phaeocollybia from Pakistan.

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

a paper dedicated to the “jubilee ofa flourishing 200-year old girl, Alternaria, that assigns

34 spp. to Alternaria sect. Infectoriae and 3 spp. to A. sect. Pseudoalternaria based on

morphological criteria. Two other papers work to clarify Peziza—one comparing four

troublesome species using both morphological and phylogenetic criteria and another

refining the determinations of 40 collections stored in the Mycological Herbarium of

Tartu (Estonia). Keys are provided for species of Apiosordaria worldwide, Mucor from

Brazil’s Caatinga biome, and Graphis from the African Palaeotropics.

Warm regards,

Lorelei L. Norvell (Editor-in-Chief)

16 January 2017

OCTOBER-DECEMBER 2016... XI

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Prospective MycoTaxon authors should download instructions PDE, review and

submission forms, and other helpful templates by clicking the ‘file download page’ link

on our INSTRUCTIONS TO AUTHORS page before preparing their manuscript. Below is a

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Mycotaxon publishes four quarterly issues per year. Both open access and

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MY COTAXON

October-December 2016—Volume 131, pp. 735-738

http://dx.doi.org/10.5248/131.735

Nawawia oviformis sp. nov. from China

JizE PENG’, Dou CHANG, YING HUANG & ZE-FEN YU*

Laboratory for Conservation and Utilization of Bio-resources, Key Laboratory

for Microbial Resources of the Ministry of Education, Yunnan University,

Kunming, Yunnan, 650091, P. R. China

*CORRESPONDENCE TO: zfyugm@hotmail.com

ABSTRACT—Nawawia oviformis, a new species collected from decaying leaves in China, is

described and illustrated. It differs from previously described Nawawia species in having

oviform conidia with 5 setulae, one of which is on the conidial apex.

Keyworps—freshwater fungi, hyphomycetes, taxonomy

Introduction

Nawawia was established by Marvanova (1980) based on Clavatospora

filiformis Nawawi (Nawawi 1973); it is characterized by septate, macronematous

conidiophores, terminal phialidic conidiogenous cells, and turbinate-

tetrahedral to obpyramidal unicellular hyaline conidia with a filiform

appendage at each blunt corner. Six Nawawia species have been described, four

from Malaysia (Crous et al. 2009, Goh et al. 2014, Kuthubutheen et al. 1992,

Nawawi 1973), one from South Africa (Hyde et al. 1996), and one from the

Russian Far East (Mel'nik & Hyde 2006).

In our survey of fungi from decaying leaves collected in a stream in China,

we isolated a species of Nawawia, which differed from the six described species

by its conidial shape.

Materials & methods

Submerged dicotyledonous leaves were collected from a stream in Damuling

Mountain of Gaosun County, Sichuan Province, China. Samples were preserved in zip-

‘These authors contributed these work equally

736 ... Peng, Chang & al.

PLATE 1. Nawawia oviformis (holotype, YMF 1.04361). A, B. Conidia viewed from side;

C. Conidium viewed from above; D. Conidia with conidiophore; E. Conidiophore; F. Conidiogenous

cells. Scale bars: A-C, F = 10 um; D, E = 50 um.

Nawawia oviformis sp. nov. (China) ... 737

locked plastic bags, marked, and later transported to the laboratory. By cutting several

2-4 x 2-4 cm fragments in the laboratory, the rotten leaves were spread on the surface

of CMA medium (20 g cornmeal, 18 g agar, 40 mg streptomycin, 30 mg ampicillin,

1000 ml distilled water) and incubated for about 10 days. Single conidia were isolated

using sterilized needles under a BX51 microscope, and cultivated on CMA in Petri

plates; morphological observations were made after incubation at 28°C for one week.

A metabolically inactive frozen pure culture was deposited in the Herbarium of the

Laboratory for Conservation and Utilization of Bio-resources, Yunnan University,

Kunming, Yunnan, PR. China (YMF; formerly Key Laboratory of Industrial

Microbiology and Fermentation Technology of Yunnan).

Taxonomy

Nawawia oviformis J. Peng & Z.F. Yu, sp. nov. PLATE 1

MycoBaAnk MB815592

Differs from other Nawawia spp. by its oviform conidia.

Type: PR China, Sichuan Province, Gaosun County, Damuling Mountain, 28°92’N

103°88’E, elev. 513 m, on submerged leaves of an unidentified dicotyledonous plant

in a stream, July 2015, Z.F. Yu (Holotype, metabolically inactive frozen culture YMF

1.04361).

EryMo_oey: Latin, oviformis referring to the egg-shaped conidia.

CoLonigs on CMA, reaching about 30 mm diameter after 10 days at 25°C.

Mycelium scanty superficial, mostly immersed composed of branched, septate,

hyaline hyphae. CoNIDIOPHORES arise from a small stromatic cushion,

cylindrical, single, unbranched, erect, robust, thick-walled, smooth, dark

brown, becoming paler towards the apex, 10-13-septate, 246-323 x 4-9 um.

CONIDIOGENOUS CELLS phialidic integrated, terminal, determinate, brown,

21-60 x 3.5-9 um. Conrp1A oviform to somewhat globose, slightly truncate

at the base, unicellular, smooth, hyaline, guttulate, 8-10.5 um diam. 13-20 x

11-15 um, with four equatorial filiform appendages and an apical filiform

appendage, all 3-10 um long.

Discussion

Among the previously described Nawawia species, N. dendroidea,

N. filiformis, N. nitida, and N. sasae-kurilensis have round-tetrahedral or

obpyramidal conidia that are distally 3-lobed and mostly with three apical

setulae, N. quadrisetulata has conidia that are distally 4—-5-lobed and mostly

with four apical setulae, and N. malaysiana has conidia that are distally

4-5-lobed and mostly with one basal and four apical setulae.

Acknowledgements

This work was jointly financed by National Basic Research Program of China

((973’Program: 2013CB127506) and National Natural Science Foundation Program

738 ... Peng, Chang & al.

of PR China (31260007, 31570023). We are very grateful to Prof. X.G. Zhang and

Dr. R.E Castafieda-Ruiz for critically reviewing the manuscript and providing helpful

suggestions to improve this paper.

Literature cited

Crous PW, Groenewald JZ, Lee SS. 2009. Fungal Planet 41 - Nawawia malaysiana. Persoonia 23:

194-195.

Goh TK, Lau WY, Teo KC. 2014. A new species of Nawawia from Malaysia, with a synopsis of the

genus. Mycotaxon 129: 109-118. http://dx.doi.org/ /10.5248/129.109

Hyde KD, Goh TK, Steinke T. 1996. Nawawia dendroidea, a new synnematous hyphomycete

from submerged Phragmites in South Africa. Mycological Research 100: 810-814.

http://dx.doi.org/10.1016/S0953-7562(96)80026-8

Kuthubutheen AJ, Liew GM, Nawawi A. 1992. Nawawia nitida sp. nov. (hyphomycetes) and

further records of Nawawia filiformis from Malaysia. Canadian Journal of Botany 70: 96-100.

http://dx.doi.org/10.1139/b92-013

Marvanova L. 1980. New or noteworthy aquatic hyphomycetes. Clavatospora, Heliscella,

Nawawia and Heliscina. Transactions of the British Mycological Society 75: 221-231.

http://dx.doi.org/10.1016/S0007-1536(80)80083-0

Mel'nik VA, Hyde KD. 2006. Nawawia sasae-kurilenses [sic] sp. nov. from the Russian Far East.

Mikologiya i Fitopatologiya 40: 411-414.

Nawawi A. 1973. Clavatospora filiformis sp. nov., an aquatic hyphomycete from Malaysia.

Transactions of the British Mycological Society 61: 390-393.

http://dx.doi.org/10.1016/S0007-1536(73)80163-9

MY COTAXON

October-December 2016—Volume 131, pp. 739-748

http://dx.doi.org/10.5248/131.739

Pezicula chiangraiensis sp. nov. from Thailand

ANUSHA H. EKANAYAKA?»”3, DINUSHANI A. DARANAGAMA®”4,

HirRAN A. ARIYAWANSA®, E. B. GARETH JONES‘,

AI H. BAKHALI® & KEVIN D. HyDE”°*

‘Center of Excellence in Fungal Research, and School of Science, Mae Fah Luang University,

Chiang Rai 57100, Thailand

? Key Laboratory for Plant Diversity and Biogeography of East Asia,

Kunming Institute of Botany, Chinese Academy of Sciences,

Kunming 650201, Yunnan, People’s Republic of China

°World Agro forestry Centre East and Central Asia Office,

132 Lanhei Road, Kunming 650201, Peoples Republic of China

‘State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences,

No. 3 Ist West Beichen Road, Chaoyang District Beijing 100101, China

°Guizhou Key Laboratory of Agricultural Biotechnology, Guizhou Academy of Agricultural Sciences,

Guiyang, 550006, Guizhou, Peoples Republic of China

°Department of Botany and Microbiology, College of Science, King Saud University,

PO. Box: 2455, Riyadh 1145, Saudi Arabia

* CORRESPONDENCE TO: kdhyde3@gmail.com

ABSTRACT—A sexual morph of a new species, Pezicula chiangraiensis, was collected on bark

of decaying wood in Chiang Rai Province, Northern Thailand. Morphologically it is closely

related to P. cinnamomea but differs by its ascospores having a gelatinous sheath; in culture it

produces a sporodochium-like asexual morph. Phylogenetic analysis of combined ITS, LSU,

and RPB2 sequence data confirmed that P chiangraiensis is distinct from other Pezicula spp.

The new species is described, illustrated, and compared with similar taxa.

KEY worDs—Cryptosporiopsis, Dermateaceae, discomycetes, phylogeny, taxonomy

Introduction

Pezicula Tul. & C. Tul. (Leotiomycetes, Helotiales, Dermateaceae), a wide-

spread discomycete genus typified by Peziza carpinea Pers., was established

by Tulasne & Tulasne (1865). Verkley (1999) accepted 26 species in Pezicula.

740 ... Ekanayaka & al.

Subsequently, two new species and nine new combinations have been added to

the genus (Johnston et al. 2014, Yuan & Verkley 2015, Zhong et al. 2001).

Pezicula species are characterized mainly by brightly coloured apothecia,

which occur on the bark of woody plants (Ooki et al. 2003). The apothecia

are frequently sessile, erumpent, and occasionally short-stalked with circular

discs (Verkley 1999). The disc, initially concave and surrounded by slightly

elevated margins, later becomes convex in most species (Verkley 1999). Most

Pezicula species have 8-spored, clavate, or cylindric-clavate inoperculate asci.

Ascospores vary in shape from broadly ellipsoid to elongately ellipsoid to

allantoid or fusoid (Ooki et al. 2003, Verkley 1999).

Cryptosporiopsis has been reported as the asexual morph of Pezicula

and 20 of the 26 Pezicula species accepted by Verkley (1999) are known to

have Cryptosporiopsis asexual morphs. Cryptosporiopsis species produce

sporodochial conidiomata, and most produce both macroconidia and

microconidia that form on different conidiophores (Verkley 1999).

Pezicula and Cryptosporiopsis species are generally known as plant

pathogens (Ooki et al. 2003). One Cryptosporiopsis species causes twig rot

of cherry (Koiwa & Yamada 1996), and C. corticola (Edgerton) Nannf., the

asexual morph of Pezicula corticola (C.A. Jorg.) Nannf., is reported to cause

bull’s-eye rot of Japanese pear. Pezicula cinnamomea (DC.) Sacc. causes stem

canker of apple and pear (Nitta et al. 2002). Pezicula species are also endophytes

(Kowalski & Kehr 1996). They play an important role in natural pruning that

helps to produce a clean bole by decaying and shedding of dead branches

(Kowalski & Kehr 1996). Species in this genus predominantly colonize woody

twigs, although some occur on herbaceous plants (Gené et al. 1990, Kowalski

& Bartnik 1995, Kowalski et al. 1998, Sankaran et al. 1995, Zhong et al. 2001).

Most Pezicula species have been recorded from temperate and boreal forests

of the northern hemisphere (Verkley 1999), with only a few species described

from the southern hemisphere (Sankaran et al. 1995, Zhuang & Korf 1988).

Here we provide detailed morphological descriptions of sexual and

asexual morphs of a new species, Pezicula chiangraiensis, and morphological

comparisons with other closely related taxa. We also provide a multi-gene

phylogenetic tree for Pezicula to infer the phylogenetic relationships of

P. chiangraiensis with other Pezicula spp.

Materials & methods

A collection of Pezicula was made in Chiang Rai Province, Northern Thailand, in

January 2015. Macroscopic and microscopic characters of the specimen were recorded.

A Motic SMZ-168 stereomicroscope was used to observe the structure of the apothecia.

Sections of apothecia were made by hand with a razor blade and mounted in water

Pezicula chiangraiensis sp. nov. (Thailand) ... 741

and preserved in lacto-glycerol. A Nikon ECLIPSE 80i compound microscope was

used to observe microscopic characters. Indian ink was added to detect the presence

of mucilaginous sheaths. The amyloidity of asci was tested using Meltzer’s reagent.

Photomicrography was carried out with a Canon 450D digital camera fitted to the

microscope. Measurements of apothecia, paraphyses, asci, and ascospores were made

from material mounted in water. Measurements were made with the Taro soft (R) Image

Frame Work v. 0.9.7 program and images used for figures were processed with Adobe

Photoshop CS6 software (Adobe Systems).

TABLE 1 Loramyces and Pezicula species used in the phylogenetic analysis.

New sequences are in bold.

GENBANK ACCESSION NO.

SPECIES CULTURE NO.

ITS SU. RPB2

L. macrosporus AFTOL-ID 913 JN033383 DQ470957 DQ470907

P. acericola CBS 239.97 KF376154 KR858884 KF376214

CBS 245.97 KF376153 KR858889 KF376213

P. aurantiaca CBS 201.46 KF376150 KR858893 KF376210

P. californiae CBS 124805 KR859104 KR858895 KR859332

CBS 124819 GU973504 GU973597 -

P. carpinea CBS 324.97 KF376156 KR858896 KF376160

CBS 921.96 KF376155 KR858898 KF376159

P. chiangraiensis MFLUCC 15-0170 KU310621 KU310622 KU310623

P. cinnamomea CBS 239.96 KF376102 KR858915 KF376165

CBS 625.96 KF376104 KR858943 KF376164

CBS 240.96 KF376105 KR858916 KF376163

CBS 626.96 KF376103 KR858944 KF376162

P. corni CBS 285.39 AF141182 - -

P. corticola CBS 259.31 AF141179 KR858956 -

CBS 260.31 KR859165 KR858957 -

P. corylina CBS 249.97 KF376106 KR858960 KF376161

P. diversispora CBS 185.50 KR859170 KR858962 -

CBS 282.47 KR859171 KR858963 -

P. eucrita CBS 656.96 KF376144 KR858977 KF376208

CBS 257.97 KR859177 KR858969 -

P. frangulae CBS 778.96 KF376151 KR859001 KF376212

CBS 100244 KF376152 KR858996 KF376211

P livida CBS 262.31 AF141180 = -

P. melanigena CBS 898.97 KR859211 KR859003 =

P. neosporulosa CBS 724.95 KR859231 KR859023 -

CBS 660.95 KR859228 KR859020 -

CBS 101.96 KR859223 KR859015 -

P. ocellata CBS 949.97 KF376149 KR859025 KF376215

P. pruinosa CBS 292.39 AF141188 KR859026 -

P. rubi CBS 253.97 KF376100 KR859042 KF376204

CBS 593.96 KF376101 KR859045 KF376203

P. sporulosa CBS 224.96 AF141172 KR859053 KF376201

CBS 225.96 KF376107 KR859054 KF376202

P. subcarnea CBS 203.46 AF141171 KR859059 -

742 ... Ekanayaka & al.

Observation of cultures and asexual morph

A pure culture was obtained from single spore isolation as described by Chomnunti

et al. (2014). Germinating spores were transferred to malt extract agar (MEA) and

incubated at 25 °C for one week. Cultural characteristics, such as mycelium colour,

shape and texture, were determined. After one week, hyphal tips were cut and

transferred to new media. The cultures were incubated at 25 °C under light for one

week and growth rate was measured. After one week, cultures on MEA were checked for

asexual structures. Conidiophores, conidiogenous cells and conidia were observed and

measured by phase contrast microscopy under 400x and 1000x optical magnifications.

The type specimen was deposited in the Mae Fah Luang University Herbarium, Chiang

Rai, Thailand (MFLU) and an ex-type culture in Mae Fah Luang University Culture

Collection (MFLUCC). A Faces of Fungi number was obtained as described in Jayasiri

et al. (2015).

DNA isolation, PCR, and sequencing

The fungal isolate was grown on potato dextrose agar (PDA) for 7-21 days at 25 °C.

Total genomic DNA was extracted as described by Thambugala et al. (2015). Polymerase

chain reactions (PCR) were carried out using three partial gene regions: ITS4 and ITS5

(White et al. 1990) for the internal transcribed spacer (ITS); LROR and LRS (Vilgalys &

Hester 1990) for the nuclear ribosomal large subunit (LSU); and fRPB2-5F and fRPB2-

7cR (Liu et al. 1999) for RNA polymerase IT (RPB2). The PCR mixtures (25 uL) contained

ddH,O (9.5 wL), PCR Master Mix (TIANGEN Co., China) (12.5 uL; 2x), DNA template

(1 wL), each primer (1 wL; 10 uM). Amplification conditions for all regions comprised

an initial denaturation step at 94 °C for 5 min and final extension step at 72 °C for 10

minutes. ITS amplification comprised 34 cycles of denaturation at 94 °C for 30 seconds,

annealing at 55 °C for 30 seconds and elongation at 72 °C for 1 minute. LSU and RPB2

amplification comprised 37 cycles of denaturation at 94 °C for 1 minute, annealing at 54

°C for 50 seconds, and elongation at 72 °C for 1 minute. The PCR products were viewed

on 1% agarose electrophoresis gels stained with ethidium bromide. The PCR products

were purified and sequenced at Invitrogen Biotechnology Co., Shanghai, China.

Sequence alignment and phylogenetic analysis

Newly generated sequences were subjected to a standard BLAST search of GenBank

for rough identification. Eighty-nine sequences belonging to three gene regions (ITS,

LSU, RPB2) from representative Pezicula species and the outgroup taxon Loramyces

macrosporus Ingold & B. Chapm. were downloaded from GenBank (TaBLE 1); these

sequences were published by Abeln et al. (2000), Chen et al. (2016), Spatafora et al.

(2006), and Verkley (1999). The newly generated sequences were deposited in GenBank

(TABLE 1). The consensus sequences for each gene were aligned using MAFFT v.

6.864b (http://mafft.cbrc.jp/alignment/server/index.html). The single alignment

was improved manually where necessary using BioEdit (Hall 2004). The single

gene alignments were concatenated into a combined dataset. Maximum likelihood

phylogenetic analyses were performed in CIPRES web portal (Miller et al. 2009) using

RAXxML-HPC Black Box (8.2.4) tool (Stamatakis 2006). The combined gene analysis

was carried out using default conditions. The best scoring tree was selected with a final

likelihood value of —7794.684804. The resultant tree was viewed with FigTree v.1.4.0

Pezicula chiangraiensis sp. nov. (Thailand) ... 743

(http://tree.bio.ed.ac.uk/software/figtree/). Maximum Likelihood bootstrap values 260%

are given near the nodes (Fie. 1).

Molecular phylogeny

A backbone tree for the genera Pezicula, Cryptosporiopsis, and Neofabraea

was generated using ITS sequence data (data not shown). Two basic clades,

the Pezicula-Cryptosporiopsis clade and the Neofabraea clade, were identified

in the backbone tree, and Pezicula chiangraiensis grouped with other Pezicula

species within the Pezicula—Cryptosporiopsis clade. A single gene tree for the

genus Pezicula was generated from ITS sequence data (data not shown). As

the single gene tree shows several taxonomic disagreements, a combined ITS,

LSU and RPB2 dataset comprising 34 strains of Pezicula species with Loramyces

macrosporus (CBS 235.53) as outgroup was generated to determine the exact

species placement of our strain (Fic. 1). The resulting tree supports our new

isolate in a distinct sister clade to Pezicula cinnamomea with strong bootstrap

support (93%).

Pezicula sporttosa CBS 225 96

Pezicufa sporulosa CBS 224 96

66 #9 Pezicula tivida CBS 262 31

re) I Pezieuta neosporulasa CBS 724 95

92K! Pezicute neosporutosa CBS 66095

Pezicula neosporulosa CBS 101 96

Pezicula cinnamomea CBS 626 96

Pezicula cinnamomea CBS 625 96

a Pezicula cinnamomea CBS 240 96

# Pexicula cinnamomea CRS 239 96

Pezicula chiangraiensis NIFLUCC 15-0470

95 _¢ Pezicuta rubi CBS 253 97

100 Pezicula rubi CBS $93 96

Pezicula corticola CBS 25¢

871 | Pezicuia eucrita CBS 257 97

(001 Pezicula eucrita CBS 656 96

100, Pezieule diversispora CBS 185 50

Pezicuta diversispora CBS 282 47

Pezicula aurantiaca CBS 201 46

Pezicula subcarnea CBS 203 46

Pezicula melanigena CBS 898 97

100) Pezicula acericola CBS 245 97

7 Pezicuta acericoke CBS 239 97

Pesicula pruinasa CBS 292 39

Peziciuta corni CBS 285 39

9O8

Pezicula ovellata CBS 949.97

fie Pezicula corticola CBS 260 31

1004 Pezicula frangulae CIS 100244

Pesicuta frangulae CBS 778 96

100, Pezicula californiae CBS 124819

Pezicula catiforniae CBS 124805

Pezicula corytina CBS 249 97

98 | Pezicula carpinea CBS 324.97

Pezicula carpinea CBS 921 96

Loramyces macresperus AFTOL ID 913

Fic. 1. The best scoring RAxML maximum likelihood phylogenetic tree based on a combined ITS,

LSU, and RPB2 sequence dataset. Strain/culture numbers are given following the taxon names.

The newly generated sequences are in bold. Bootstrap support values for maximum likelihood

>60% are given near the nodes. The tree is rooted with Loramyces macrosporus (CBS 235.53).

744 ... Ekanayaka & al.

Taxonomy

Pezicula chiangraiensis Ekanayaka & K.D. Hyde, sp. nov. PLATES 1, 2

INDEX FUNGORUM IF551789

Differs from other Pezicula species by possessing ascospores with a gelatinous sheath.

Type: Thailand, Chiang Rai Province, Kun Korn waterfall, 21 January 2015, A.H.

Ekanayaka (Holotype, MFLU 15-3566; ex-type culture, MFLUCC 15-0170; GenBank

KU310621, KU310622, KU310623).

EryMo_ocy: With reference to the province where the holotype was collected.

FACESOFFUNGI: FoF 01667

SEXUAL MORPH: SAPROBIC on dead stems. APOTHECIA 800-870 x 230-280 um

(x = 826 x 253 um, n = 10), arising singly or in small groups, sessile, slightly

erumpent from the substrate, subsphaerical, urceolate, bright yellow when

fresh, brownish yellow when dried. Disc convex. RECEPTACLE yellow to light

brown when fresh. ECTAL EXCIPULUM variably thick, composed of large thin-

walled yellow cells in a textura angularis. MEDULLARY EXCIPULUM thin, not

clearly distinguishable, composed of vertically arranged rows of yellow cells

in a textura prismatica. HyMENIUM hyaline. PaRAPHYSES 1.8-2.1 um diam.

(x = 2.0 um, n = 20), numerous, filiform, obtuse, septate, branched, slightly

enlarged at the apex. Asci 90-120 x 15-25 um (x = 108 x 19 um, n = 30),

8-spored, unitunicate, cylindric-clavate, short pedicellate, arising from croziers

with conical and non-amyloid apices. Ascospores 18-22 x 8-10 um (x = 20 x

9 um, n = 40), partially biseriate, lower spores uniseriate, hyaline to grey,

fusoid, gelatinous sheath present; immature spores non-septate and filled with

guttules; mature spores 3-septate, aguttulate, and smooth-walled.

ASEXUAL MORPH: CONIDIOMATA sporodochium-like, produced in 7-day-old

cultures, gregarious, dark brown. CONIDIOPHORES 11-12 x 5-6 (x = 11.5 x

5.8 um, n = 20), branched near the base, septate, smooth-walled, composed

various size cells. CONIDIOGENOUS CELLS holoblastic, cylindrical, smooth,

hyaline. Conrp1a 23-29 x 11-12 um (x = 25.2 x 11.2 um, n = 20), cylindrical,

straight, smooth with rounded apex and slightly truncate base, multi-guttulate

or filled with granular cytoplasm, hyaline and aseptate when immature, pale

brown and 1-3-transversely septate at maturity.

CULTURE CHARACTERISTICS: Ascospores germinating on MEA within 24 h.

Colonies on MEA plates at 25-27 °C reaching 2 cm diameter within 1 week,

circular, flat or effuse, with diffuse edge, medium honey-brown, grey aerial

mycelium surrounded by pink mycelia, mycelium composed of 2-3 um wide,

septate, branched, smooth, hyaline hyphae, reverse dark brown to black.

Notes—Pezicula cinnamomea is morphologically similar to P chiangraiensis

in having apothecia, paraphyses, asci, and ascospores of a similar size and

Pezicula chiangraiensis sp. nov. (Thailand) ... 745

PiaTE 1. Pezicula chiangraiensis (holotype, MFLU 15-3566), sexual morph: a. Substrate;

b. Ascomata on wood; c. Ascoma; d. Cross section of ascoma; e. Vertical section of ascomal margin;

f. Septate paraphyses; g-j. Short pedicellate asci; k. Germinated ascospore; |. Apex of mature ascus;

m. Ascospore with gelatinous sheath; n—q. Fusoid ascospores. Scale bars: b = 500 um; c, e = 100 um;

d = 300 um; f, k = 50 um; g-j = 40 um; |, m = 20 um; n-q = 10 um.

shape, but differs by its pale luteous to cinnamon or red-brown apothecia, its

transversely and longitudinally septate spores without gelatinous sheaths, its

faster growth rate, and absence of diffusing pigment in culture (Ooki et al. 2003,

746 ... Ekanayaka & al.

PLATE 2. Pezicula chiangraiensis (MFLUCC 15-0170), asexual morph in culture: a. 7-day-old MEA

colony from above; b. 7-day-old MEA colony from below; c. Conidiomata; d. Conidioma; e. Septate

and branched hyphae; f. Fragment of small sporodochium-like conidioma; g, h. Different stages of

conidiogenesis; i-l. Conidia at different stages. Scale bars: e = 50 um; f, j-1 = 20 um; g, h = 30 um;

i=10um.

Verkley 1999). Pezicula aesculea Kirschst. differs from P chiangraiensis

by its larger apothecia, asci, and ascospores; P. amoena Tul. & C. Tul. by its

dark brown, thick-walled ascospores; P. heterochroma Verkley, P. frangulae

(Pers.) Fuckel, P rubi (Lib.) Niessl, P puberula (EJ. Durand) Verkley,

P. crataegicola (E.J. Durand) J.W. Groves, P. myrtillina (P. Karst.) P. Karst., and

P. grovesii Wehm. by their short, stout stalked apothecia; P linda Korf and

Pezicula chiangraiensis sp. nov. (Thailand) ... 747

P. sepium (Desm.) Dennis by their asci with an amyloid ring; P tasmanica

W.Y. Zhuang & Korf by its positive reaction with Meltzer reagent after

pretreating with KOH; P hamamelidis J.W. Groves & Seaver by its broader

apothecial base that is entirely hidden under the bark of its host substrate; and

P. melastomatis Rehm by its smaller asci and ascospores (Verkley 1999). The

asexual morphs of most Pezicula species produce both macro and microconidia

(Verkley 1999), but microconidia were not observed in P. chiangraiensis.

Acknowledgments

This work was supported by the International Research Group Program (IRG-14-27),

Deanship of Scientific Research, King Saud University, Saudi Arabia. Anusha H.

Ekanayaka is grateful to Dr. Peter R. Johnston for valuable suggestions, to Dr. Eric

McKenzie and Dr. Qi Zhao for presubmission reviews, and to Mr. W. Ekanayaka

(deceased), Mrs. C. Ekanayaka, and Mr. A. Surasinghe for their valuable support and

encouragement.

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MY COTAXON

October-December 2016—Volume 131, pp. 749-764

http://dx.doi.org/10.5248/131.749

Abieticola koreana gen. et sp. nov., a griseofulvin-producing

endophytic xylariaceous ascomycete from Korea

HYANG BurRM LEE?*, HYE YEON Mun?”, THI THUONG THUONG NGUYEN’,

JIN-CHEOL KIM? & JEFFREY K. STONE?

‘College of Agriculture & Life Sciences, Chonnam National University,

Gwangju 61186, Republic of Korea

*Fungal Resources Research Division, Nakdonggang National Institute of Biological Resources,

Sangju 37242, Republic of Korea

°Department of Botany & Plant Pathology, Cordley Hall 2082, Oregon State University,

Corvallis, OR 97331-2902, USA

*CORRESPONDENCE TO: hblee@jnu.ac.kr

ABSTRACT—A new genus and species. Abieticola koreana, is described. This xylariaceous

fungus was isolated from the inner bark of a Manchurian fir (Abies holophylla) in Korea.

Phylogenetic analyses based on the sequences of four gene regions—ITS1-5.8S-ITS2, 28S,

6-tubulin, and rpb2—were used to confirm this new genus and species.

Key worDs—Ascomycota, anamorph, multigene, Xylarioideae, Xylariaceae

Introduction

The Xylariaceae is a large family of ascomycetes comprising approximately

85 genera and at least 1,340 species that are distributed worldwide and exhibit

exceptional diversity in the tropics (Whalley 1996, Velmurugan et al. 2013,

Stadler et al. 2014). It has been estimated that the Xylariaceae contains 10,000

undescribed species (Stadler 2011, Richardson et al. 2014). Recent phylogenetic

studies (Tang et al. 2009, Daranagama et al. 2015) using combined ITS, LSU,

rpb2, and $-tubulin sequences, suggested that Xylariaceae has two major

lineages, Xylarioideae and Hypoxyloideae.

To date, 11 genera of Xylariaceae have been shown to occur as endophytes

of various plants (Pazoutova et al. 2013), including firs, palms, orchids,

bromeliads, aroids, ferns, and rainforest trees (Dreyfuss & Petrini 1984, Bayman

750 ... Lee & al.

et al. 1998; San Martin et al. 2001; Park et al. 2005). Recently, endophytes have

been predominantly investigated because of their ability to produce either new

or interesting secondary metabolites and for their potential use in bioenergy

applications (Bayman et al. 1997, Isaka et al. 2000, Boonphong et al. 2001,

Wiyakrutta et al. 2004, Park et al. 2005, Liu et al. 2007).

During screening for organisms that produce antifungal agents, an

endophytic fungus designated as FO010, was isolated from the inner bark of

Abies holophylla (Manchurian fir), a tree that is widespread in Korea (Park

et al. 2005). Initially, the isolate was provisionally identified as an unknown

species of Xylaria and was shown to produce two antifungal metabolites,

dechlorogriseofulvin and griseofulvin (Park et al. 2005). However, further

investigation suggested that FO010 differs from species classified in Xylaria

based on both morphological details of the anamorph and on molecular

phylogenetic analyses.

The objectives of this study were to image the ultrastructure of the conidial

state of FO010 by scanning electron microscopy (SEM), for comparison of its

mode of conidiogenesis with that of other Xylariaceae, and to investigate its

phylogenetic relationship to other xylariaceous fungi based on a multigene

analysis, including the nuclear ribosomal ITS1-5.8S-ITS2 and 28S subunit,

rpb2, and 6-tubulin gene sequences. As a result of this study, a new genus

Abieticola and species A. koreana are proposed here to accommodate this novel

xylariaceous fungus.

Materials & methods

Fungal isolation

Samples of Abies holophylla bark were collected from a garden in Daejeon, Korea,

placed in zip-lock bags, stored in a refrigerator, and then used for the isolation of

endophytic fungi within 72 h after sampling. Samples were cleaned under running

tap water and then air-dried. The samples were surface sterilised by immersion in

70% ethanol for 1 min and a 3.5% sodium hypochlorite solution for 5 min. Next, the

surface-sterilized samples were washed in sterile water three times to remove the surface

sterilization agents (Wiyakrutta et al. 2004). The samples were then cut into small pieces

and placed on cornmeal malt extract agar (17.0 g of cornmeal, 20.0 g of malt extract,

2.0 g of yeast extract, and 20 g agar in 1.0 L of distilled water) supplemented with

chloramphenicol (50 mg/mL) (Sigma, St. Louis, MO, USA), and then incubated at 25°C

for up to 3 weeks. Individual fungal strains were transferred to potato dextrose agar

(PDA, Bacto Potato Dextrose Dehydrated, Becton Dickinson, USA) and incubated at

25°C for at least 2 weeks. After checking for purity, each fungal culture was transferred

to a fresh agar plate. Stock cultures of test species were grown on MEA at 25°C and used

as inocula. The basal medium consisted of 2% malt extract agar (MEA, Difco) at pH 5.5.

The water activity (a,) of the basal MEA medium was 0.999, which was modified to 0.95,

0.98, and 0.995 by addition of KCl and glucose. The a, of media was measured using

Abieticola koreana gen. & sp. nov. (Korea) ... 751

a Novasina ICII (Novasina AG, Zurich, Switzerland). The growth of the fungus was

determined by transferring mycelial plugs (5 mm diameter) from the growing margin

of stock cultures in Petri dishes (90 x 15 mm). After inoculation, plates with the same a,

were sealed in polyethylene bags and incubated at 20, 25, and 30°C in darkness for up

to 10 days. Mycelial growth was recorded periodically by measuring the diameter of the

colony in two directions at right angles to each other.

Ten strains, FO010-1 to FO010-10, were derived from isolate FO010. Dried MEA

cultures were conserved in Chonnam National University Fungal Collection, Gwangju,

Korea (CNUFC), and live cultures were conserved in CNUFC and in the Korean

Collection for Type Cultures, Daejeon, Korea (KCTC).

Ultrastructure of isolate FO010

The micromorphological features of strain FO010-1 were investigated by light

microscopy (LM) with a Nikon Labophot 2 Microscope (Nikon, Tokyo, Japan) with

differential interference contrast optics. For scanning electron microscopy (SEM),

samples were fixed in 2.5% paraformaldehyde-glutaraldehyde in 0.1 M phosphate buffer

(pH 7.2) for 2 h, post-fixed with 1% OsO, in the same buffer for 1 h, dehydrated in

graded ethanol, substituted by isoamyl acetate and then critical-point dried in CO,,.

Finally, the sample was sputter-coated with gold (SC502; Polaron, West Sussex, UK)

and observed with a SEM515 scanning electron microscope (Phillips, Eindhoven, the

Netherlands) and an S-3500N low vacuum scanning microscope (Hitachi Science

Systems Ltd., Tokyo, Japan).

DNA extraction and PCR amplification

Strains FO010-1 and FO010-2 were grown on PDA, covered with cellophane at 25°C

for 4-5 days. Genomic DNA was extracted using the HiGene™ Genomic DNA Prep

Kit for fungi (Biofact Co., Daejeon, Korea). The target regions, ribosomal ITS1-5.8S-

ITS2, 28S subunit, B-tubulin, and rpb2 (RNA polymerase II subunit 2) were amplified

by PCR (TABLE 1) in 20 uL reaction mixtures containing 2 uL of genomic DNA, 1.5 uL

of each primer (5 pM), 14 uL of demineralised sterile water, and 1 uL of PCR premix

TABLE 1. Primers and annealing conditions used in the molecular analysis.

ANNEALING TEMP.

Locus PRIMER SEQUENCE (5’-3’) (°C) REFERENCE

te LR5F GCTATCCTGAGGGAAAC

55 (30 sec) Vilgalys &

LROR ACCCGCTGAACTTAAGC Hester 1990

B-tubulin BL

a GGTAACCAAATCGGTGCTGCTTTC 59 (Limin) Sang et al.

2010

Bt2b ACCCTCAGTGTAGTGACCCTTGGC

FESSDNS ITS1 T TAGGTGAACCT

SORES LGC ECETCCeS 55 (30 sec) White et al.

ITS4 TCCTCCGCTTATTGATATGC 1990

rpb2 RPB2-5F_Eur GAYGAYCGKGAYCAYTTC 50-72 (1 min), Hibbett 2006;

ramping 0.3°C Houbraken et

RPB2-7CR_Eur = CCCATRGCYTGYTTRCCCAT per sec al. 2011

752... Lee & al.

(Bioneer, Daejeon, Korea) using the following fungal-specific primer sets: ITS1 and

ITS4 for ITS1-5.8S-ITS2, LROR and LRSF for 28S (White et al. 1990), Bt2a and Bt2b for

6-tubulin (Sang et al. 2010), and RpB2-5F-Eur and rpB2-7cr-Eur for RpB2 (Hibbett 2006,

Houbraken et al. 2011).

DNA sequencing and phylogenetic analyses

Amplified PCR products of strains FO010-1 and F0010-2 were electrophoresed on

a 0.75% agarose gel. The amplicons were purified with the AccuPrep PCR Purification

Kit (Bioneer). The purified PCR products were sequenced using an ABI 3700 automated

DNA sequencer (Applied Biosystems Inc., USA). The four gene sequences generated

were initially aligned with other sequences from GenBank using Clustal X v. 2.0 (Larkin

et al. 2007) with a gap opening penalty of 10.0 and a gap extension penalty of 0.05.

BioEdit v. 7.2.5 (Hall 1999) was used to manually exclude ambiguous and uninformative

variable sites. The alignment was manually refined using MEGA v. 6.0 (Tamura et al.

2013). The ITS, 28S, B-tubulin, and rpb2 gene sequences of F0010-1 and F0010-2 were

deposited in GenBank (see TABLE 2).

TABLE 2. Sequences used in the phylogenetic multigene analysis.

GENBANK ACCESSION NO.

TAXON SAMPLE

ITS LSU rpb2 6-tubulin

Abieticola EML-F0010-1 JN977612 JQ014618 KP792128 KP792126

koreana (T)

A. koreana EML-F0010-2 KP835547 KP835546 KP792129 KP792127

Annulohypoxylon CBS123834 DQ631935 DQ840061 DQ631960 DQ840095

moriforme

var. microdiscum

A. stygium MFLUCC 12-0826 —_KJ940870 KJ940869 KJ940868 KJ940867

Anthostomella brabeji CBS 110128 EU552098 EU552098 ~ _

A. proteae CBS 110127 EU552101 EU552101 — x:

Astrocystis MFLUCC 14-0174. KP297404 KP340545 KP340532 KP406615

concavispora

A. mirabilis HAST 94070803 GU322448 — GQ844835 GQ495941

A. sublimbata HAST 89032207 GU322447 — GQ844834 GQ495940

Collodiscula japonica CBS 124266 JF440974 — — JF440974

Daldinia concentrica CBS 113277 AY616683 ~ KC977274 —

D. decipiens CBS 122879 JX658441 — - AY951694

D. loculata BCRC 34117 EF026145 — AY951698 —

Discoxylaria JDR 169 GU322433 _ GQ844819 GQ487710

myrmecophila

Entoleuca mammata JDR 100 GU300072 _ GQ844782 GQ470230

Euepixylon JDR 261 GU292821 _ GQ844774 GQ470224

sphaeriostomum

Hypoxylon fendleri MFLUCC 12-0816 = KM017563 KMO017565 KMO017566 KM017564

Abieticola koreana gen. & sp. nov. (Korea) ... 753

H. fragiforme MUCL 51264 KM186294 KM186295 KM186296 KM186293

H. lenormandii MFLUCC 13-0120 KM039135 KM039136 KM039137 KMO039138

H. monticulosum MFLUCC 12-0818 | KM052716 KM052717 KM052719 KM052718

Kretzschmaria HAST 89062903 GU300079 — GQ844792 GQ478214

guyanensis

Lunatiannulus MFLUCC 14-0014 KP297398 KP340540 KP340526 KP406609

irregularis

Nemania diffusa FR AT-113 DQ658238 DQ840073 DG631947 DQ840088,

N. serpens FR AT-114 DQ631942 DQ840075 DQ631948 DQ840086

Podosordaria WSP 176 GU324762 _ GQ853039 GQ844840

mexicana

P muli WSP 167 GU324761 — GQ853038 GQ844839

Poronia pileiformis WSP 88113001 GU324760 _ GQ853037 GQ502720

Rhopalostroma lekae MFLUCC 13-0123 KJ472428 KJ472427 KJ472429 —

Rosellinia HAST 89112602 EF026118 — GQ844778 EF025604

lamprostoma

R. merrillii HAST 89112601 GU300071 — GQ844781 GQ470229

R. necatrix HAST 89062904 EF026117 — GQ844779 EF025603

R. sancta-cruciana HAST 89062903 GU292824 — GQ844777 GQ470227

Rostrohypoxylon CBS 119137 DQ631943 DQ840069 DQ631954 DQ840097

terebratum

Ruwenzoria MUCL 51394 GU053568 — — —

pseudoannulata

Stilbohypoxylon YMJ 89091608 EF026120 -- GQ853021 EF025606

quisquiliarum

Thamnomyces MUCL 51396 FN428828 — — —

camerunensis

Xylaria adscendens JDR 865 GU322432 — GQ844818 GQ487709

X. bambusicola WSP 205 EF026123 — GQ844802 AY951762

X. hypoxylon CBS 122620 AM993141 KM186301 KM186302 KM186300

CBS Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands, BCRC Bioresource Collection and

Research Centre, Taiwan, HAST Herbarium, Research Centre for Biodiversity, Academia Sinica, Taipei,

JDR Herbarium of Jack D. Rogers, MFLCC Mae Fah Luang University Culture Collection, Chiang Rai,

Thailand, MUCL Mycotheque de l'Université Catholique de Louvain, Germany, YMJ Herbarium of

Yu Ming Ju, WSP Washington State University, USA, EML Environmental Microbiology Laboratory

Fungarium, Chonnam National University, Gwangju, South Korea; T ex-holotype strain.

Maximum-likelihood (ML) (Felsenstein 1985) algorithms were applied using the

general time reversible (GTR) model under assumption of the gamma distribution

to accommodate substitution rates at the remaining sites and the starting tree was

obtained via neighbor-joining with nearest neighbors. For the Maximum Parsimony

analysis (MP), heuristic search method of tree bisection-reconnection (TBR) branch

swapping was used. A neighbor-joining (NJ) analysis was calculated from the Kimura

two-parameter model (Kimura 1980) with uniform rates as rate among sites (Saitou

& Nei 1987). Bootstrap analyses were performed with 1000 resampled datasets using

MEGA 6.05 (Tamura et al. 2013).

795A ... Lee & al.

Stilbohypoxylon quisquiliarum YMJ 89091608

Kretzschmaria guyanensis HAST 89062903

Xylaria adscendens JDR 865

Xylaria bambusicola WSP 205

Xylaria hypoxylon CBS122620

Nemania diffusa FRAT-113

Nemania serpens FR AT-114

Astrocystis mirabilis HAST 94070803

Astrocystis sublimbata HAST 89032207

Astrocystis concavispora MFLUCC 14-0174

Discoxylaria myrmecophila JDR 169

Colfodiscula japonica CBS 124266

Lunatiannulus irregularis MFLUCC 14-0014

Rosellinia sancta-cruciana HAST89062903

Rosellinia necatrix HAST 89062904

a Rosellinia lamprostoma HAST 89112602

Euepixyion sphaeriostomum JDR 261

[ Entoleuca mammata JDR 100

99 Rosellinia merrillii HAST 89112601

Podosordaria muli WSP 167

Podosordaria mexicana WSP 176

Poronia pileiformis WSP 88113001

Abieticola koreana EML-F0010-1

Abieticola koreana EML-F0010-2

aevooeuel|Ax seapioe|AX

Anthosiomella brabeji CBS 110128

Anthostomella proteae CBS 110127

Daldinia loculata BCRC 34117

Daldinia decipiens CBS 122879

Daldinia concentrica CBS 113277

Ruwenzoria pseudoannulata MUCL 51394

Thamnomyces camerunensis MUCL 51396

Rhopalostroma lekae MFLUCC 13-0123

Annulohypoxylon moriforme var. microdiscum CBS123834

Rostrohypoxylon terebratum CBS 119137

Annulohypoxylon stygium MFLUCC 12-0826

Hypoxyion fendleri MFLUCC 12-0816

Hypoxylon lenormandii MFLUCC 13-0120

Hypoxylon monticulosum MFLUCC 12-0818

Hypoxylon fragiforme MUCL51264

seaoeelAX seopiojAxodAY

Fic. 1. Phylogenetic position of Abieticola koreana EML-F0010-1 and EML-F0010-2 and

relationships among Xylariaceae based on multigene analysis (ITS, 28S, rpb2, and $-tubulin).

Hypoxylon fragiforme was used as outgroup. The scale bar represents one substitution per 100

nucleotides. The trees were constructed based on Maximum Likelihood (ML). Phylogenetic

classification by Daranagama et al. (2015).

Results

Phylogenetic analysis

FO010-1 and F0010-2 clustered within a clade including Podosordaria and

Poronia species, but formed a separate monophyletic group in maximum-

Abieticola koreana gen. & sp. nov. (Korea) ... 755

likelihood (ML) trees. BLASTn search revealed that the ITS sequence of

FO010-1 strain (JN977612) was 96.5% identical with Pod. mexicana WSP

176 (GU324762; 409 of 424 bp), 97.2% identical with Pod. muli WSP 167

(GU324761; 412 of 424 bp), and 96.5% identical with Por. pileiformis WSP

88113001 (GU324760; 409 of 424 bp). However, the sequences of F0010-1

were <95% similar to those of several isolates known to be species of Xylaria

(FJ612908; EU716506; AY315407; EU716509). In addition, the BLASTn search

results for the 28S rDNA sequence of strain F0010-1 (JQ014618) was 98.1%

homologous with Xylariaceae sp. 1175 (FJ425703; 527 of 537 bp), 97.8%

homologous with Xylaria sp. DIS99a (DQ327620; 715 of 731 bp), and 97.6%

homologous with Xylaria sp. DIS255j (DQ327627; 721 of 739 bp).

To further elucidate the phylogenetic status of isolate FO010, sequence data

for additional genes, rpb2, and 6-tubulin were analysed. The BLAST results for

the rpb2 and £-tubulin gene sequences indicated that the fungus is somewhat

closely related to genera such as Podosordaria and Poronia, but showing identity

values of only 76.6-81.0% with Pod. mexicana WSP 176, 83.0-83.9% with Pod.

muli WSP 167, and 73.2-82.2% with Por. pileiformis WSP 88113001. Based on

the sequence data and the structure of the conidial anamorph, isolate FO010

appeared to constitute a new genus and species, forming a distinct lineage

within the Xylarioideae clade, sister to the Podosordaria/Poronia lineage

(Fret).

Taxonomy

Abieticola Hyang B. Lee, gen. nov.

MycoBank MB 811702

Differs from Poronia by its slightly curved conidia, and its shorter conidiogenous cells.

TYPE SPECIES: Abieticola koreana Hyang B. Lee

ErymMo.oey: Abieticola, meaning “Abies-inhabiting’, referring to the host genus.

A coremioid, synnematous conidial anamorph produced on artificial

media is characterized by small clubs with white heads consisting of coiled

conidiogenous hyphae and synnemata that resemble those of Poronia species

but differ in bearing slightly curved conidia from shorter conidiogenous cells,

some of which bear three conidia.

Abieticola koreana Hyang B. Lee, sp. nov. FIGS 2, 3

MycoBAnk MB 811703

Differs from Poronia spp. by its slightly curved conidia, and its shorter conidiogenous

cells that sometimes bear 3 conidia.

Type: Republic of Korea, Daejeon, Yuseong-gu, endophytic in inner bark of Abies

holophylla Maxim. (Pinaceae), August 2006 (CNUFC EML-F0010-1 [preserved as

756 ... Lee & al.

metabolically inactive frozen culture]; ex-type cultures, KCTC 10629BP, CNUFC

EML-F0010-1; GenBank JN977612, JQ014618, KP792128, KP792126).

EryMo.oey: koreana, referring to the country of isolation, Korea.

STROMATA frequently developed on PDA medium, resembling small clubs with

white heads, <20.5 mm tall and with blackish brown exudates oozing out along

the lower stalk. Substrate mycelia forming in abundance, but aerial mycelia

poorly produced. SyNNEMATA generally erect but curving in age, 8.0-20.5

(av. 13.15 + 1.4) x 0.9-1.4mm, with knotted hyphae of variable thickness (mostly

1-1.8 um), pigmented brown to yellow in age; functioning as conidiophores.

ConlipiA | or 2 (sometimes 3) in number, produced laterally on mycelia and

detaching with distinct scars, 2.5-4.0 x 1.0-1.5 (av. 3.0 x 1.5) um on PDA,

Fic. 2 (above). Abieticola koreana (F0010-1): macromorphological culture characteristics.

A, B: PDA containing 5% wild dog dung culture at 27°C for 1 month (A) and 2 months (B - plate

refrigerated for 2 days and then moved to RT to induce fruiting); C, D: PDA culture at 18°C for 1

month (C) and 50 days (D); E: MEA culture with abundant mycelia and aerial club-like synnemata

with long whitish heads and yellowish bases; F: culture on 5% dung medium at 27°C for 50 days;

G: culture on PDA containing 5% dung at 27°C for 50 days; H: culture on MEA medium at 27°C

for 1 month; I and J: culture on PDA containing 5% dung at 27°C for 50 days; K: old fruit bodies

producing whitish powdery conidia and hairy mycelia (yellow arrow) in age on PDA, L: culture

on PDA containing 5% dung at 27°C for 2 months (plate refrigerated for 2 days and then moved

to RT to induce fruiting).

Fic. 3 (right). Abieticola koreana (F0010-1): micromorphological culture characteristics. A: fruit

bodies of the vase-shaped structure on PDA; B: magnified picture of the round head shown in A;

C: slender, long, tangled thread-like mycelia with conidia, D: magnified picture of numerous conidia

Abieticola koreana gen. & sp. nov. (Korea) ... 757

on mycelia shown in C, E: magnified pictures of anamorphic conidia in different forms including

elliptical, or oblong to banana-like (solid arrow) shown in C and D, F: conidia and normal mycelia

with small knots (solid arrow) and flat mycelia (dotted arrow), G: conidia (solid arrow) and scar

(dotted arrow) formed on mycelia, H: conidiation in cluster (solid arrow) on mycelia.

758 ... Lee & al.

hyaline, bases brown to yellow, tips white to grey; morphologically variable,

Poronia-like, ellipsoid or fusoid to ovoid, often slightly curved. No teleomorph

found on artificial media. COLONIES on PDA 25 mm diam. after two weeks at

25°C, yellow to brown in age.

ADDITIONAL STRAINS: REPUBLIC OF KOREA, DAEJEON, Yuseong-gu, endophytic in

inner bark of Abies holophylla, August 2006 (CNUFC EML-F0010-2 to F0010-10).

DISTRIBUTION: Known only from the type locality in South Korea.

Growth

Colonies attained an average diameter of 25 mm on MEA medium after

two weeks incubation at 25°C (the optimum temperature for growth). The

medium became pigmented yellow to brown with age. Mycelial growth rates

were determined under different water activity conditions (0.90-0.999 a, ;

Fia. 4. Growth of strain F0010-1 was maximal at 0.999 a /25°C, with a similar

growth rate at 0.98 a , but with almost no mycelial growth at 0.90 a, regardless

of temperature. At 0.95 a, the strain showed moderate growth on medium

amended with glucose, but no growth on medium amended with KCI solution.

The strain showed almost no mycelial growth at lowered water availability level

(0.90 a) regardless of temperature.

Mycelial growth (diameter, mm)

0.999 0.995 0.98 0.95

Water activity (a,,)

Fic. 4. Abieticola koreana (F0010-1): effect of water activity (a,) on mycelial growth on PDA.

The water activity levels were adjusted using two different solutes, KCl (left) and glucose (right).

Abieticola koreana gen. & sp. nov. (Korea) ... 759

Discussion

Several attempts were made to induce a teleomorph of Abieticola koreana

on a variety of artificial media (including dung in some media because some

xylariaceous fungi are coprophilous), but our attempts were unsuccessful.

The conidiomata that formed in culture on artificial media were tested for

germination in solution, but no spores germinated within 3 days.

Anamorphs of Xylariaceae differ in their modes of conidiogenesis and the

complexity of the conidiophores, and these characters have previously been

employed to define different form-genera (Hsieh et al. 2005). No teleomorphic

structures were observed here on various artificial media including dung-

containing media.

Although many related genera produce anamorphs on developing

stromata, few detailed ultrastructural studies of these anamorphs have

been published. The anamorphs of genera such as Calceomyces, Daldinia,

Hypoxylon, Rhopalostroma, Thamnomyces, and Thuemenella (“Hypoxyloideae’)

have Nodulisporium-like anamorphs (Stadler et al. 2013); and Xylocoremium

flabelliforme has been reported as the anamorph of Xylaria cubensis (Rogers

1984, Hsieh et al. 2005). The ultrastructure of the stromata and conidia and

mode of conidiogenesis in A. koreana could be used to distinguish it from

similar xylariaceous anamorphs. The mode of conidiogenesis on the surface of

the synnemata of A. koreana was examined by SEM and found similar to other

Xylariaceae (including those in Podosordaria, Poronia, and Xylaria), but the

morphology of the synnemata, conidiomata, and conidia differed from those

of the related genera.

Conidia formed by A. koreana resembled those of Pod. leporina, but differed

slightly in that they were produced by geniculate conidiogenous cells; the

elongated tips of the synnemata resembled those of Por. ingii in being slightly

shorter but differed in the ellipsoid and subglobose conidia (Koehn & Cole

1975, Ribes & Negrin 2011). According to Rogers et al. (1998), Podosordaria

has Geniculosporium-like anamorphs, while Poronia has Lindquistia-like

anamorphs. Podosordaria, with a typically stalked stroma with a subglobose

head, differs from Poronia, with a flattened disc (Krug & Cain 1974).

Koehn & Cole (1975) compared the ultrastructure of Pod. leporina and

Por. oedipus suggesting that Pod. leporina should be returned to Poronia. The

morphology and molecular phylogenetic position of A. koreana also differed

from these two genera.

Podosordaria and Poronia are closely related genera containing species

that have been considered close relatives of Xylaria but differ by their capitate

stromata and coprophilous nature (Deepna & Manimohan 2012). Neighbor-

joining (NJ) and maximum parsimony (MP) analyses of four DNA loci (ITS,

760 ... Lee & al.

28S, B-tubulin, rpb2) performed in this study produced tree patterns almost

identical to ML (not shown). Three ITS-based phylogenetic analyses of Xylaria

and related genera proved to be practical for taxonomic determination of

intra-specific relationships in highly morphologically variable fungi like the

Xylariaceae (Spatafora & Blackwell 1993). Nevertheless, studies based on the ITS

marker encountered major issues when attempting to determine phylogenetic

relationships with other Xylaria species because of its highly variable nature

(Hsieh et al. 2010). In addition, the number of Xylaria-related sequences in

GenBank is limited.

Nevertheless, it is very important to note the phylogenetic status and

relatedness of Abieticola among xylariaceous fungi because until now no

intermediate group related to Poronia and Podosordaria has been reported.

Results revealed that Abieticola is a clade within the intermediate group on

phylogenetic trees such as ML, NJ, and MP.

Most Xylaria species (‘Xylarioideae’) and some segregates like Sarcocylon

have elongate stromata; interestingly, Daldinia asphalatum (‘Hypoxyloideae’)

also produce elongate stromata. The sporulating structures and attachment

patterns of A. koreana resembled those of Poronia spp. (Lindquistia-like) and

Xylaria spp. (Geniculosporium-like), but differed from them in having broader

conidiogenous cells and slightly curved conidia.

The presence or absence of specific chemicals can, in many cases, be

linked with taxonomic position, which may prove invaluable in determining

associations between species, species groups, and genera (Whalley & Edwards

1995). The placement of the FO010-1 and F0010-2 strains in the Xylarioideae

clade constructed by Daranagama et al. (2015) suggests that other members of

the clade should be investigated for ability to produce griseofulvin.

Endophytic fungi are organisms that live inside plant tissue for at least part

of their life cycle without causing any disease symptoms (Petrini 1991, Schulz

& Boyle 2005). It is interesting that Xylaria species isolated from eastern white

pine (Pinus strobus) needles and lowbush blueberry (Vaccinium angustifolium)

stems has been reported to produce griseofulvin (Richardson et al. 2014). Liu

et al. (2007) reported that an endophytic Xylaria sp. isolated from Ginkgo biloba

had broad antimicrobial activity. Quang & Bach (2008) also reported that

ergosta-4,6,8(14),22-tetraen-3-one produced bya Xylaria species from Vietnam

can inhibit nitric oxide production. Several metabolites, including linoleic

acid, linoleic acid methyl ester, and 4-hydroxyscytalone, were isolated from a

methanolic extract of another Xylaria species (Jang et al. 2009). A xylariaceous

coprophilous fungus, Pod. tulasnei, was shown to produce tulasnein,

a new metabolite with strong antimicrobial activity and weak cytotoxic and

phytotoxic activities (Ridderbusch et al. 2004). Poronia punctata has been

Abieticola koreana gen. & sp. nov. (Korea) ... 761

shown to produce a range of biologically active sesquiterpenes (Poyser et al.

1985, Anderson et al. 1988). Stadler et al. (2014) noted that fungal secondary

metabolite profiles can have taxonomic value beyond the species rank and

even coincide with phylogenetic data. Kuhnert et al. (2014) indicated that the

biosynthetic pathways of diverse compounds derived from fungi have evolved

independently during the evolution of fungi. More studies on the molecular

phylogeny and ultrastructure of diverse groups belonging to the Xylarioideae

clade, which are related to the new genus and species, are needed.

Acknowledgements

This work was supported by the project on the survey and discovery of Korean

indigenous species of the National Institute of Biological Resources (NIBR) under the

Ministry of Environment, Republic of Korea. Authors are grateful to Prof. Marc Stadler

(Helmholtz-Zentrum fiir Infektionsforschung GmbH, Germany), Dr. Yu-Ming Ju

(Institute of Plant and Microbial Biology, Taiwan), and Dr. Paul M. Kirk (Royal Botanic

Gardens Kew, U. K.) for the kind reviews and comments.

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MY COTAXON

October-December 2016—Volume 131, pp. 765-771

http://dx.doi.org/10.5248/131.765

Entalbostroma erumpens gen. et sp. nov. (Xylariaceae)

from Phormium in New Zealand

P.R. JOHNSTON’, J.D. ROGERS’, D. PARK! & N.A. MARTIN?

"Landcare Research, Private Bag 92170, Auckland 1142, New Zealand

2 Department of Plant Pathology, Washington State University,

Pullman, WA 99164-6430, USA

" CORRESPONDENCE TO: johnstonp@landcareresearch.co.nz

ABSTRACT—A xylariaceous fungus on the dead leaves of Phormium tenax and P. cookianum

is described as a new genus and species, Entalbostroma erumpens. Phylogenetically

E. erumpens is close to Xylaria amphithele. The sexual state resembles Kretzschmariella in

general morphology, but the two genera are phylogenetically distant. The asexual state can

be used to distinguish the genera morphologically, Entalbostroma having an anamorph with

typical xylariaceous conidia, while Kretzschmariella has Mirandina-like conidia, unknown in

any other xylariaceous taxon.

Key worps— phylogeny, Xylariales, Xylarioideae

Introduction

The generic taxonomy of the Xylariaceae remains uncertain. Phylogenetic

studies have shown that few of the long recognised, morphologically based

genera form well resolved, monophyletic clades, most being polyphyletic,

paraphyletic, or with poorly resolved relationships in DNA based phylogenetic

studies (Hsieh et al. 2010, Pazoutova et al. 2010, U’Ren et al. 2016) As discussed

by U’Ren et al. (2016), resolution of phylogenetically robust genera within the

Xylariaceae requires not only additional genes but also more complete taxon

sampling.

In this paper we fill a gap in the taxon sampling of these fungi by

describing a morphologically unusual xylariaceous fungus from leaves of

the monocotyledonous Phormium spp. (Asparagales, Hemerocallidaceae)

as a new genus and species. Morphologically the sexual state is similar to

766 ... Johnston & al.

Kretzschmariella Ju & Rogers 1994); however newly obtained DNA sequences

for Kretzschmariella culmorum show the two taxa to be phylogenetically distant.

Materials & methods

Specimens were collected during general surveys of fungi associated with the native

plants of New Zealand.

Ascospore masses were removed aseptically from perithecia in the holotype specimen

PDD 107494 and spotted onto SMEA medium (Kenerley & Rogers 1976). Germinated

ascospores were transferred to 2% oatmeal agar (OA, Difco), Leonian’s medium (Booth

1971), and half strength potato dextrose agar (PDA, Difco). Asci and ascospores from

dried specimens and conidia from pure cultures were examined in water or 5% KOH

with Melzer’s reagent.

For DNA sequencing, mycelium was harvested from the cultures, dried, then ground

in 400 uL lysis buffer (Qiagen, USA) with a plastic pestle followed by incubation for

2 hr at 55°C Then 220 uL lysed solution was loaded into the QIAxtractor Robot (Qiagen,

USA) and DNA extraction performed with a Qiagen DX reagent pack and tissue

extraction protocol. DNA sequences for ITS, B-tubulin, and RPB2 were generated using

the amplification primers from Hsieh et al. (2010). In addition, DNA was extracted from

the hymenium of a fruit body from PDD 107494, prior to the specimen being dried for

storage, and an ITS sequence generated.

The DNA sequences were concatenated and a phylogenetic analysis performed

using our newly generated sequences together with selected taxa from U’Ren et al.

(2016) and previously unpublished sequences from a culture grown from a collection

of Kretzschmariella culmorum (Guadeloupe, on culm of bamboo, coll. J. Vivant,

1 Nov 1992, sent by H.W. Ravenel (#2505) to WSP; culture stored at The Institute

of Plant and Microbial Biology, Academia Sinica). An initial analysis showed that

Entalbostroma erumpens was within the Xylaria ‘HY clade of U’Ren et al. (2016). The

taxa selected were those that represented the diversity across that clade, together with

representative taxa from the other clades recognised within the Xylarioideae by U’Ren

et al. Poronia pileiformis and Podosordaria mexicana were used as the outgroup. The

sequences were aligned using MAFFT as implemented in Geneious R9 (Kearse et al.

2012). A Bayesian phylogenetic tree was generated using MrBayes 3.2 (Ronquist et al.

2012) with gaps treated as missing data, applying the GITR+I+G model for all three

genes, the same model selected for all genes using the Akaike information criterion

method in MrModeltest 2.3 (Nylander 2004). The data set was run with four chains for

10 million generations, trees sampled every 1000 generations with a burn-in of 25%.

Bayesian posterior probabilities were obtained from 50% majority rule consensus trees.

Taxonomy

Entalbostroma J.D. Rogers & PR. Johnst., gen. nov.

MycoBAnk MB817225

Differs from Kretzschmariella in having typical xylariaceous conidia.

Type: Entalbostroma erumpens J.D. Rogers & P.R. Johnst.

EtyMo.oey: For the white entostroma.

Entalbostroma erumpens gen. et sp. nov. (New Zealand) ... 767

FiGurE 1 Entalbostroma erumpens (A-C, holotype, PDD 107494; D, E, I-K, PDD 107550;

F-H, ICMP 21152). A, B, fruiting bodies erumpent from leaf, variable in shape. C, fruiting body

in section showing white internal tissue and continuous narrow black outer layer. D, erumpent,

partly exposed fruiting body showing barely differentiated ostioles. E, fruiting body from D with

upper part removed to show internal structure; F, detail of surface of culture on PDA, darkened

structures, and powdery layer of conidia in places. G, conidia from PDA culture. H, conidiogenous

cells from PDA culture. I, ascus apex in KOH and Melzer’s reagent. J, ascospores with gelatinous

sheath and small apical gelatinous cap. K, ascospores with spore-length germ slit. Scale bars:

A-F = 1mm; G-K = 10 um.

Stromata pulvinate to applanopulvinate, orbicular to elliptical to irregular,

solitary to gregarious, erumpent becoming superficial; surface blackish,

interior perithecium-bearing tissue white. Perithecia more or less spherical;

768 ... Johnston & al.

ostioles ill defined. Ascus apical ring inverted hat-shaped, bluing in Melzer’s

iodine reagent. Ascospores dark brown, with germ slit on flattened side,

a hyaline gelatinous sheath. Paraphysate.

In culture, conidiophores forming a palisade-like layer of short, cylindric

cells with conidia produced holoblastically on several scars near apex. Conidia

hyaline, smooth, ellipsoid with narrowed flattened bases.

Entalbostroma erumpens J.D. Rogers & P.R. Johnst., sp. nov. FIG. 1

MycoBank MB817226

Differs from Xylaria amphithele by its initially immersed, then erumpent, non-stipitate

ascomata, its non-erumpent perithecia, and its ascospores being surrounded by a

gelatinous sheath.

Type: New Zealand: Auckland: North Piha, Laird Thomson Track, on dead leaf

Phormium tenax J.R. Forst. & G. Forst., 28 Aug 2015, coll. N.A. Martin (Holotype,

PDD 107494; isotype, WSP 72775; ex type culture, ICMP 21152; GenBank KX258204,

KX258205, KX258206).

EryMo_oey: For the erumpent nature of the ascomata from leaf tissue.

Stromata pulvinate to applanopulvinate, orbicular to elliptical to irregular,

solitary to gregarious, erumpent becoming superficial, 2-10 mm long x 1-3

mm broad x 1-2 mm thick; surface blackish, interior perithecium-bearing

tissue white. Perithecia more or less spherical, 0.2-0.4 mm diam. Ostioles ill

defined. Asci short-stipitate, 120-146 x 8 um total length. Ascus apical ring

inverted hat-shaped, bluing in Melzer’s iodine reagent, ca. 4.5 um high, 2.7 um

broad. Ascospores dark brown, ellipsoid-inequilateral, 16-19 x 8-8.8 um, with

long germ slit on flattened side, with a hyaline gelatinous sheath that is often

broader on the flattened side, and often with a small gelatinous cap at one end.

Paraphysate.

Cultures from multiple ascospores on OA under 12 hr fluorescent light and

12 hr dark white to pinkish, covering 9 cm diam plate in 2-3 wk. Surface at

first plane, appressed and smooth after 4-6 wk showing small areas of raised

pimples. Pimples becoming enlarged and blackened, hollow. Conidiophores

forming a palisade-like layer of short, cylindric cells with conidia produced

holoblastically on several scars near apex. Conidia hyaline, smooth, ellipsoid

with narrowed flattened bases indicating point of attachment to conidiogenous

cell, (3-)4—5(-7) x 2-2.5 um.

FIGURE 2. Bayesian phylogenetic tree based on concatenated ITS, B-tubulin, and RPB2 sequences,

Bayesian posterior probabilities >0.95 on edges. Taxa selected from U’Ren et al. (2016) are labelled

with voucher numbers; Podosordaria mexicana and Poronia pileiformis were selected as outgroup.

Clade designations follow U’Ren et al. (2016). Entalbostroma erumpens sequences are deposited

in GenBank as KX258204, KX258205, and KX258206; those from Kretzschmariella culmorum as

KX430043, KX430045, and KX430046.

Entalbostroma erumpens gen. et sp. nov. (New Zealand) ... 769

Kretzschmaria deusta CBS 826.72

Kretzschmaria neocaledonica HAST 94031003

1 Kretzschmaria clavus JDR 114

Kretzschmaria pavimentosa JDR 109

Kretzschmaria sandvicensis JDR 113

{ Kretzschmaria guyanensis HAST 89062903

Kretzschmaria megalospora JDR 229

Xylaria cranioides HAST 226

Xylaria sp. ARIZ FL1777

Xylaria tuberoides HAST 475

Xylaria microceras HAST 414

0.97 Xylaria muscula HAST 520

Penzigia cantareirensis HAST 526

Xylaria alboareolata HAST 453 HY Clade

Xylaria coccophora HAST 786

Xylaria oligotoma HAST 784

Xylaria venustula HAST 88113002

0.99

ae Xylaria sp. genotype 262 isolate FLO224

Xylaria sp. genotype 425 isolate FL1042

1 Entalbostroma erumpens ICMP 21152

nae i Xylaria sp. 6 HMH 2010f

Xylaria amphithele HAST 529

1 Xylaria sicula f. major HAST 90071613

Xylaria arbuscula HAST 89041211

Xylaria venosula HAST 94080508

Xylaria striata HAST 304

0.98 Xylaria bambusicola WSP 205

Xylaria meliacearum JDR 148

Xylaria intraflava HAST 725

Xylaria ochraceostroma HAST 401 TE Clade

Xylaria cirrata HAST 664

Xylaria brunneovinosa HAST 720

0.99 Xylaria schweinitzii HAST 92092023

Xylaria telfairif HAST 90081901

Xylaria plebeja HAST 91122401

Xylaria allantoidea HAST 94042903

1 Xylaria berteri JDR 256

Xylaria castorea PDD 47417

Xylaria feejeensis JDR 180

1 Xylaria frustulosa HAST 92092010 PO Clade

1 Astrocystis mirabilis HAST 94070803

Kreizschmariella culmorum Candoussau 5281

Xylaria badia HAST 95070101

Stilbohypoxylon elaeicola JDR 173

Xylaria culleniae JDR 189

Xylaria curta HAST 92092022

Discoxylaria myrmecophila JDR 169

0.97 Xylaria palmicola PDD 47440

Nemania serpens HAST 235 NR Clade

0.99 Rosellinia necatrix HAST 89062904

Podosordaria mexicana WSP 176

Poronia pileiformis WSP 88113001

770 ... Johnston & al.

ADDITIONAL SPECIMENS EXAMINED: NEW ZEALAND: NorTHLAND: Poor Knights

Islands, on dead leaves of Phormium tenax, 3 Sep 1984, coll. R.E. Beever (PDD 46231).

AUCKLAND: Piha South, Lookout Track, on dead leaves of Phormium tenax, 28 Aug

2015, coll. N.A. Martin (PDD 107553);. Muriwai, Quarry Track, on dead leaves of

Phormium tenax, 21 July 2015, coll. N.A. Martin (PDD 107552); Muriwai, Maori Bay

Track, on dead leaves of Phormium tenax, 21 Aug 2015, coll. N.A. Martin (PDD 107550);

Waitakere Ranges, Nihotupu Dam, on dead leaves of Phormium tenax, 4 Nov 1960,

coll. J.M. Dingley (PDD 42824). Mrp CANTERBURY: Bealey Valley, on dead leaves of

Phormium cookianum Le Jol. (as P. colensoi Hook. f.), 11 Mar 1983, coll. E. Horak (PDD

93072). STEWART IsLAND: Doughboy Bay, on dead leaves of Phormium sp., 29 Apr 2002,

coll. PR. Johnston, S. Whitton, R. Leschen (PDD 107551).

Phylogeny

The ITS sequences from the fruit body from the PDD 107494 fungarium

specimen and from cultures derived from that specimen (ICMP 21152) were

identical. The DNA sequences from ICMP 21152 have been deposited in

GenBank as KX258204, KX258205, and KX258206.

Entalbostroma erumpens formed a strongly supported sister relationship

with Xylaria amphithele and Xylaria sp. 6 sensu Hsieh et al. (2010) within

the ‘HY’ clade of U’Ren et al. (2016) (Fic 2). Morphologically it is divergent

from X. amphithele (San Martin Gonzalez & Rogers 1989), but it shares a leaf-

inhabiting ecology with both this species and Xylaria sp. 6 (Hsieh et al. 2010).

Kretzschmariella is phylogenetically distant, positioned within the ‘PO’ clade

of U’Ren et al. (2016).

Discussion

The morphology of the sexual state of Entalbostroma recalls Kretzschmariella,

both genera having erumpent ascomata with a thin black crust and white soft

entostromatic tissue surrounding the perithecia. The two genera differ in their

anamorph, our new fungus having a reduced Geniculosporium-like anamorph

in culture (typical of species of Xylaria and Kretzschmaria), rather than the

Mirandina-like conidial state of Kretzschmariella (Ju & Rogers 1994). This

reinforces earlier studies showing that the morphology of the anamorph may be

phylogenetically informative among these fungi. Inclusion of morphologically

divergent taxa such as Entalbostroma will be necessary for future phylogenetic

resolution of generic level clades within the Xylariaceae, and for recognition of

the characters that can be used to recognise those clades morphologically.

Entalbostroma erumpens is geographically widespread on dead, partly

decomposed leaves of Phormium spp. in New Zealand, and appears to be quite

common on its host.

Entalbostroma erumpens gen. et sp. nov. (New Zealand) ... 771

Acknowledgments

Yu-Ming Ju (IPMB, Academia Sinica) is thanked for providing unpublished

sequences for Kretzschmariella culmorum. Jacques Fournier and Yu-Ming Ju are

thanked for reviewing the manuscript and suggesting improvements. This research was

supported through the Landcare Research Systematics Portfolio with funding from the

Science and Innovation Group of the New Zealand Ministry of Business, Innovation

and Employment, and by Dept. of Plant Pathology, Washington State University.

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http://dx.doi.org/10.1016/j.ympev.2016.02.010

MY COTAXON

October-December 2016— Volume 131, pp. 773-780

http://dx.doi.org/10.5248/131.773

Podosporiopsis, anew genus of synnematous hyphomycetes

from China

JIAN Ma’, X1u-Guo ZHANG? & RAFAEL F. CASTANEDA-RUiz?

‘College of Agronomy, Jiangxi Agricultural University, Nanchang, 330045, China

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

3 Instituto de Investigaciones Fundamentales en Agricultura Tropical Alejandro de Humboldt’

(INIFAT), Académico Titular de la “Academia de Ciencias de Cuba”,

Calle 1 Esq. 2, Santiago de Las Vegas, C. Habana, Cuba, C.P. 17200

*CORRESPONDENCE TO: majian821210@163.com

ABSTRACT—Podosporiopsis gen. nov. is proposed. It is characterized by distinct unbranched

conidiophores forming distinct synnematal conidiomata with emergent monotretic,

integrated, terminal, determinate or percurrently extending conidiogenous cells that produce

solitary, obclavate, distoseptate conidia. Podosporiopsis sinensis sp. nov. (the type species) and

P. obclavata sp. nov., both collected on dead branches of unidentified plants in China, are

described and illustrated. A key to Podosporiopsis and similar genera is provided.

KEY worps—anamorphic fungi, asexual morph, taxonomy

Introduction

Lushan Mountain is a world heritage site in Jiangxi Province, China, south

of the Yangtze River and northwest of Poyang Lake. It covers 302 km’ and

is protected by an outlying buffer zone of 500 km’. However, its mycobiota,

especially of saprobic microfungi, are poorly known. During our continuing

mycological surveys (2012-15) in this region, two novel synnematous

hyphomycetes were encountered on dead branches. Close examination showed

significant differences from previously described hyphomycetes, and a new

genus, Podosporiopsis, is erected for them.

Materials & methods

At the end of rainy season, samples of dead branches were collected from

humid or riparian environments, and processed as described by Ma et al.

774 ... Ma, Zhang & Castafieda-Ruiz

(2012). Conidia and conidiophores were measured and photographed using

a Nikon Eclipse E200 with 100x (oil immersion) objectives and SmartV550Dc

digital camera. Adobe Photoshop 7.0 was used to process images into plates,

with backgrounds replaced for esthetic reasons. The specimens studied

are deposited in the Plant Pathology Herbarium, Jiangxi Agricultural

University, Nanchang, China (HJAUP), and the Mycological Herbarium,

Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

(HMAS; http://hmas.im.ac.cn).

Taxonomy

Podosporiopsis Jian Ma, X.G. Zhang & R.F. Castafieda, gen. nov.

INDEX FUNGORUM IF 551862

Differs from Podosporium by its distoseptate conidia.

TYPE SPECIES: Podosporiopsis sinensis Jian Ma et al.

EryMoLoey: refers to its similarity to Podosporium.

CONIDIOMATA synnematous, solitary, erect, cylindrical, indeterminate,

dematiaceous. CONIDIOPHORES synnematous, unbranched, _ septate,

dematiaceous. CONIDIOGENOUS CELLS diverging from the synnematal axis,

monotretic, integrated, terminal, determinate or with percurrent extensions.

Conidial secession schizolytic. Conip1a solitary, dry, acrogenous, distoseptate.

Podosporiopsis sinensis Jian Ma, X.G. Zhang & R.F. Castafieda, sp.nov. —_ Fics 1, 2

INDEX FUNGORUM IF 551863

Differs from Podosporium spp. by its distoseptate conidia.

Type: China, Jiangxi Province: Lushan Mountain, on dead branches of an unidentified

broadleaf tree, 8 November 2014, J. Ma (Holotype, HHAUP M0279-1; isotype, HMAS

245604).

EryMoLoecy: refers to the country in which the fungus was collected.

Cotontgs effuse on the natural substratum, dark brown to black, hairy.

Mycelium partly superficial, partly immersed in the substratum. CONIDIOMATA

synnematous, solitary, erect, cylindrical, dark brown to black, becoming

narrower toward the apex, up to 1000 um high, 100-120 um wide at the often

swollen base, fertile region with more than half the length of the synnemata.

CONIDIOPHORES distinct, synnematous, cylindrical, unbranched, septate,

smooth, brown to dark brown, up to 1000 um long, 6-10 um wide, diverging

laterally and terminally. CoNIDIOGENOUS CELLS monotretic, integrated,

terminal, cylindrical, smooth, brown, determinate, or sometimes with 1-2

cylindrical percurrent extensions, 12-27 x 6-8.5 um. Conidial secession

schizolytic. Conrp1 solitary, dry, acrogenous, smooth, straight or curved,

Podosporiopsis gen. & spp. nov. (China) ... 775

Fic. 1. Podosporiopsis sinensis (holotype, HJAUP M0279-1): A-C. Synnemata with conidiophores

and conidia; D, E. Conidia. Scale bars: A~C = 100 um; D, E = 40 um.

obclavate, 10-14-distoseptate, pale brown to brown, 65-97 um long, 10-15 um

thick at the widest part, tapering to 3-4.5 um at the apex, 5-7 um wide at the

truncate base.

Podosporiopsis obclavata Jian Ma, X.G. Zhang & R.F. Castafieda, sp. nov. Fics 3, 4

INDEX FUNGORUM IF 551864

Differs from Podosporiopsis sinensis by its longer and narrower, 8-20-distoseptate

conidia with the truncate base narrower than the apex.

Type: China, Jiangxi Province: Lushan Mountain, on dead branches of an unidentified

broadleaf tree, 8 November 2014, J. Ma (Holotype, HHAUP M0279-2; isotype, HMAS

245605).

EryMo_oey: obclavata, referring to the obclavate conidia of this fungus.

Cotontgs effuse on the natural substratum, dark brown to black, hairy.

Mycelium partly superficial, partly immersed in the substratum. CONIDIOMATA

synnematous, solitary, erect, cylindrical, dark brown to black, becoming

narrower toward the apex, <1420 um tall, 65-170 um diam. at the often

776 ... Ma, Zhang & Castafieda-Ruiz

Fic. 2. Podosporiopsis sinensis (holotype, HJAUP M0279-1): A-C. Conidiophores with terminal,

monotretic conidiogenous cells; D-F. Conidiophores with terminal conidia and 0-1 percurrent

extension. Scale bars = 40 um.

swollen base, fertile region with more than half the length of the synnemata.

CONIDIOPHORES distinct, synnematous, cylindrical, unbranched, septate,

smooth, brown to dark brown, <1400 um long, 5-7.5 um diam., diverging

Podosporiopsis gen. & spp. nov. (China) ... 777

Fic. 3. Podosporiopsis obclavata (holotype, HJAUP M0279-2): A-C. Synnemata with conidiophores

and conidia; D, E. Conidia. Scale bars: A = 100 um; B, C = 80 um; D, E = 40 um.

laterally and terminally. CoNIDIOGENOUS CELLS monotretic, integrated,

terminal, cylindrical, smooth, brown to pale brown, determinate or sometimes

with 1-2 cylindrical percurrent extensions, 20-40 x 5-6.5 um. Conidial

secession schizolytic. Conip1A solitary, dry, acrogenous, smooth, straight or

curved, obclavate, 8-20-distoseptate, pale brown, 95-160 um long, 8-11 um

diam. at the widest part, tapering to 3.5-5.5 um at the apex and 2-4 um at the

truncate base.

Discussion

Podosporiopsis sinensis and P. obclavata are unique in having distinct

unbranched conidiophores forming synnematous conidiomata, and solitary

obclavate distoseptate conidia seceding schizolytically from monotretic,

integrated, terminal, determinate or percurrently extending conidiogenous

cells diverging from the axis of the synnemata. We initially thought of assigning

them to the genus Podosporium Schwein. (Schweinitz 1832), but the conidia in

Podosporium are euseptate rather than distoseptate, and the addition of these

species would have significantly broadened its generic concept. Furthermore,

conidial septation (euseptate vs distoseptate) has often been used as a generic

character to divide otherwise morphologically similar fungi (e.g., Ellisembia

vs Sporidesmium, Linkosia vs Stanjehughesia, Sporidesmiella vs Repetophragma,

778 ... Ma, Zhang & Castafieda-Ruiz

Fic. 4. Podosporiopsis obclavata (holotype, HJAUP M0279-2): A. Conidiophore with developing

conidium; B, C. Conidiophores with terminal conidia, conidiogenous cells, and percurrent

extensions; D. Conidiophores with terminal, monotretic conidiogenous cells and percurrent

extensions. Scale bars = 40 um.

Solicorynespora vs Corynespora, and Parablastocatena vs Blastocatena) and

provides a narrow generic concept (Seifert et al. 2011; Zhang et al. 2012).

Such distinctions also help avoid confusion and complexity in a number of

hyphomycete genera and facilitate the morphological division of larger genera

into smaller units (Subramanian 1992; McKenzie 1995; Wu & Zhuang 2005).

For these reasons, we propose the new genus Podosporiopsis to accommodate

P. sinensis and P. obclavata.

Podosporiopsis resembles Dendrographium Massee (Massee 1892),

Exosporium Link (Link 1809), Corynespora Gtssow (Glssow 1906), and

Solicorynespora R.F. Castaheda & W.B. Kendr. (Castafeda-Ruiz & Kendrick

1990) in possessing distinct conidiophores with tretic conidiogenesis.

However, in Dendrographium and Exosporium conidiogenesis is polytretic, in

contrast to the monotretic conidiogenesis of Podosporiopsis. Conidiogenesis

Podosporiopsis gen. & spp. nov. (China) ... 779

in Corynespora and Solicorynespora is the same as in Podosporiopsis, but

Podosporiopsis has synnematous conidiomata not found in Corynespora and

Solicorynespora.

Several other genera including Gangliostilbe Subram. & Vittal (Vittal

1976), Novozymia W.P. Wu (Wu & Zhuang 2005), Neosporidesmium Mercado

& J. Mena (Mercado Sierra & Mena Portales 1988), Raizadenia S.L. Srivast.

(Srivastava 1981), and Annellophragmia Subram. (Subramanian 1963) have

morphological characters similar to those of Podosporiopsis. However,

conidiogenesis is polyblastic sympodial in Annellophragmia, monoblastic with

percurrent extension in Novozymia, and monoblastic but producing euseptate

conidia in Gangliostilbe, all of which differ from the monotretic conidiogenesis

of Podosporiopsis. Conidia in Neosporidesmium and Raizadenia are distoseptate

but conidiogenesis is monoblastic, an obvious difference from Podosporiopsis.

Key to Podosporiopsis and morphologically similar genera

1. Coenidiogenous cells enteroblastic :..4 bce sda 4 ences oon ten sn ee le 2

f. Gonidiogenousscellsiiolablastices nok Sanat Att Aneel Aiea neat As ettt Aeet An: 7

PEC Onidiosenious- cell spol VTHOELE cts & axesube fst fj Paul Cj ececth geese patos pe nce Ge ne Eos 3

2yGonidiogenous cella mGnomehicis AT on wT ok yl Steal Bhatt ek call ahs 4

3. Conidiophores divergent from synnematal capitulum,

conidia with an non-darkened hilum ...................... Dendrographium

3. Conidiophores mononematous or divergent from stipe and synnematal capitulum,

conidia with a conspicuously darkened hilum ................... Exosporium

4 Conidiophores morionemMatous «0.6 5) pe Dae eek Bare seey rere Dae been wr hha ot eto 5

AV GMI CIOPMOTESSyAINERIALOUR: 7 ate eRe Re Sa Re ea Me Rr Me ee 6

DUG ON Iga MSL OSEPLALEs 125 Gaote ena uac te Pine adeaht-oeedeade owes sate aie ase nate ahtne worth Corynespora

PLC OBICIA GUIS CL ALG: Ws or Sia Od sen Oa id en a Hato bh a Hodes la ta besa be ret a sarge a Solicorynespora

GAC Oiicia GISLOSEPLAtes ng hehe hater pte arse oe Bere! rater Podosporiopsis

GAGonidia- CUS plates. Hts Bede Beit 2 hcwiies« Mecwites« Meewiiose Whom bp fens s heontens Podosporium

7. Conidiogenous cells: polyblastic «wi... nee connie tana eeeee came es Annellophragmia

Fe Gonidiosenois.cellsmOno blastic” so. ps .ecbea ce sce bencer a ecben.erwcacbencera Seb qe se Geese e qeac shark 8

8. Conidiophores divergent from stipe and synnematal capitulum ................ 9

8. Conidiophores divergent from synnematal capitulum .....................00. 10

9. Conidiogenous cells with annellidic percurrent proliferation .......... Novozymia

9. Conidiogenous cells determinate or with lageniform, ampulliform,

ordohiform proliteratOney At... 2eten teams tan ean atta is Neosporidesmium

IG s@onidia-cdistoseptate te. m eae eod Cet ok eet ad er tod wees ad whe od ow, Raizadenia

LO “Conidia’ euseptatery div. 2.iy 1h teen Be cing te cme te eet he gis Be tts Gangliostilbe

780 ... Ma, Zhang & Castafieda-Ruiz

Acknowledgments

The authors express gratitude to Dr. W.B. Kendrick and Dr. De-Wei Li for serving

as pre-submission reviewers, to Dr. Shaun Pennycook for nomenclatural review, and to

Dr. Lorelei L. Norvell for editorial review. This project was supported by the National

Natural Science Foundation of China (No. 31360011).

Literature cited

Castaneda-Ruiz RF, Kendrick B. 1990. Conidial fungi from Cuba: II. Univ. Waterloo Biol. Ser. 33:

1-61.

Giissow HT. 1906. Uber eine neue Krankheit an Gurken in England (Corynespora mazei, Giissow

gen. et sp. nov.). Z. Pflanzenkrankh. 16: 10-13.

Link HE 1809. Observationes in ordines plantarum naturales. Dissertatio I. Mag. Ges. Naturf.

Freunde Berlin 3: 3-42.

Ma J, Zhang YD, Ma LG, Castafieda-Ruiz RF, Zhang XG. 2012. Three new species of Sporidesmiella

from southern China. Mycoscience 53: 187-193. http://dx.doi.org/10.1007/s10267-011-0152-1

Massee GE. 1892. Notes on exotic fungi in the Royal Herbarium, Kew. Grevillea 21: 1-6.

McKenzie EHC. 1995. Dematiaceous hyphomycetes on Pandanaceae. 5. Sporidesmium sensu lato.

Mycotaxon 56: 9-29.

Mercado Sierra A, Mena Portales J. 1988. Nuevos o raros hifomicetes de Cuba. VI. Neosporidesmium,

nuevo genero sinematico. Acta Bot. Cub. 59: 1-6.

Schweinitz LD von. 1832. Synopsis fungorum in America boreali media degentium. Trans. Am.

phil. Soc. 4: 141-316.

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

Biodiversity Series 9. 997 p.

Srivastava SL. 1981. Raizadenia: a new genus of hyphomycetes from India. Indian Phytopath.

34: 334-336.

Subramanian CV. 1963. On Arthrobotryum coonoorense. Proc. Indian Acad. Sci. B 58: 348-350.

Subramanian CV. 1992. A reassessment of Sporidesmium (hyphomycetes) and some related taxa.

Proc. Indian. Natn. Sci. Acad. B 58: 179-190.

Vittal BPR. 1976 [“1975”]. Gangliostilbe indica, a new synnematous hyphomycete from India.

Kavaka 3: 69-71.

Wu WP, Zhuang WY. 2005. Sporidesmium, Endophragmiella and related genera from China. Fungal

Divers. Res. Ser. 15. 351 p.

Zhang YD, Ma J, Ma LG, Zhang XG. 2012. Parablastocatena tetracerae gen. et sp. nov.

and Corynesporella licualae sp. nov. from Hainan, China. Mycoscience 53: 381-385.

http://dx.doi.org/10.1007/s10267-011-0171-y

MY COTAXON

October-December 2016—Volume 131, pp. 781-790

http://dx.doi.org/10.5248/131.781

Distribution of Alternaria species among sections. 3.

Sections Infectoriae and Pseudoalternaria

PHILIPP B. GANNIBAL™ & DANIEL P. LAWRENCE?

‘Laboratory of Mycology and Phytopathology, All-Russian Institute of Plant Protection,

Shosse Podbelskogo 3, Saint Petersburg, 196608, Russia

? Department of Plant Pathology, University of California,

One Shields Avenue, Davis, CA 95616, USA

* CORRESPONDENCE TO: phbgannibal@yandex.ru

ABsTRACT—Morphological examination of material conforming to the morphological

description of Alternaria sect. Infectoriae (but not phylogenetically characterized) allowed

the inclusion of nine additional species in this section, which thus encompasses 34 species.

Alternaria sect. Pseudoalternaria consists of two species and one misidentified isolate that

represents a novel taxon, described here as Alternaria parvicaespitosa. Emended descriptions

of A. sect. Infectoriae and A. sect. Pseudoalternaria are presented, and their species are listed.

Key worps—Alternaria infectoria, Alternaria rosae, Lewia

This article is dedicated to the jubilee of the flourishing 200-year-old girl, Alternaria.

Introduction

Alternaria Nees 1816 is a large pleomorphic genus that comprises

approximately 280 distinguishable species (Simmons 2007). A large series of

morphological and phylogenetic studies have attempted to better resolve the

phylogeny of Alternaria and other alternarioid hyphomycetes by utilizing more

than ten different genomic loci (Pryor & Bigelow 2003; Hong et al. 2005; Runa

et al. 2009; Lawrence et al. 2012, 2013, 2014; Woudenberg et al. 2013, 2014;

Armitage et al. 2015). Several well-supported lineages (phylogenetic species-

groups) have been revealed within the alternarioid hyphomycetes and have led

to several taxonomic novelties. The genus Alternaria was recently divided into

eight taxonomic sections by Lawrence et al. (2013) followed by the elevation

782 ... Gannibal & Lawrence

of 19 additional clades by Woudenberg et al. (2013, 2014), Grum-Grzhimaylo

et al. (2016), and Lawrence et al. (2016), thereby bringing the total number of

sections of Alternaria to 27.

All works support the monophyly of two morphologically peculiar

Alternaria groups, two of several groups having members with catenate, small-

spored conidia. Recently these groups were elevated to sectional status, as

A. sect. Infectoriae and A. sect. Pseudoalternaria.

Twenty-five species were indicated as members of sect. Infectoriae by

Lawrence et al. (2013). Woudenberg et al. (2013) added two additional species

to this clade, bringing the total number of recognized species to 27. Lawrence

et al. (2013) also mentioned three species (A. peglionii, A. dianthicola, and

A. photistica) that cannot be included in sect. Infectoriae. The sequenced

strain of A. peglionii (CBS 103.26) was not the type, and this species was

previously listed as incertae sedis (Simmons 2007). The sequenced strain of

A. dianthicola was not designated by E.G. Simmons as a representative strain.

When a representative strain of A. dianthicola was utilized it clustered distantly

to sect. Infectoriae (Woudenberg et al. 2013). Also Lawrence et al. (2013) and

Woudenberg et al. (2013) used the same strain of A. photistica (EGS 35.172), but

it was obtained from different sources. Conclusions by Woudenberg et al. (2013)

based on molecular and morphological data placed A. photistica in sect. Panax

and A. dianthicola in sect. Dianthicola. Ariyawansa et al. (2015) described a new

species, A. murispora, in section Infectoriae based on ascomatal morphology

and DNA analysis; no living culture or conidial state of this species is known.

Ariyawansa et al. (2015) also noted that the type isolate of Xenobotryosphaeria

calamagrostidis (CBS 303.71) clustered in sect. Infectoriae (albeit with low

support) and only the sexual state was produced in culture and clearly

differed from species within Alternaria sect. Infectoriae. The authenticity of

X. calamagrostidis remains unresolved and would benefit from a re-examination

of morphological and molecular characters for this peculiar isolate/taxon.

The number of species recovered by phylogenetic species recognition

approaches can differ from that revealed by morphological analysis. Very likely

a morphological concept favors splitting. For instance, phylogenetic analyses of

three genomic regions from 39 infectoria species-group isolates (Andersen et al.

2009) were unable to provide strong support for species-level designations for

most isolates. No correlations were found among phylogenetic data, metabolite

production, morphological identity, proascoma formation, ability to grow

at 37°C, and host or geographic origin. Gannibal & Yli-Mattila (2007), who

utilized a set of 52 infectoria species-group isolates (including representative

strains of eight morphospecies), were unable to divide the isolates into distinct

groups based on data from molecular fingerprinting.

Alternaria sect. Infectoriae & A. sect. Pseudoalternaria ... 783

The main discriminating morphological features of many Alternaria sect.

Infectoriae species are small conidia (in culture usually no longer than 60 um)

and long secondary conidiophores (Lawrence et al. 2013; Woudenberg et al.

2013). Ten species have a known sexual state (A. alternarina, A. daucicaulis,

A. ethzedia, A. hordeiaustralica, A. hordeicola, A. infectoria, A. intercepta,

A. murispora, A. scrophulariae, and A. viburni). Some strains are capable of

producing proascoma in axenic culture, suggesting that members of this

group, at least in part, are homothallic (Andersen et al. 2009). In general

fungi in this section are common. Usually they exist as saprobes showing no

substrate preference. This group produces reliable chemotaxonomic markers

(Christensen et al. 2005; Andersen et al. 2002, 2009). Some authors have shown

that strains of this group do not produce mycotoxins generally made by other

small-spored Alternaria sections (e.g., sect. Alternaria) (Andersen et al. 2002).

Other studies revealed the ability of some species to produce alternariol and

monomethyl ether (Oviedo et al. 2013), which probably is common for other

Alternaria sections.

Lawrence et al. (2014) assigned two species, sister to A. sect. Infectoriae, to

a new genus, “Pseudoalternaria” nom. inval. This placement became untenable

after all alternarioid hyphomycetes were collected under the single generic

name Alternaria (Woudenberg et al. 2013). The thorough review by Lawrence

et al. (2016) resurrected “Pseudoalternaria” as a new section, Alternaria sect.

Pseudoalternaria, typified by a new species, Alternaria arrhenatheri.

Section Pseudoalternaria currently consists of two described species,

A. arrhenatheri D.P. Lawr. et al. and A. rosae E.G. Simmons & C.F. Hill, and

one misidentified isolate (X1272, isolated from blueberry and identified as

A. rosae by Zhu & Xiao 2015) but which represents a third/new species (based

on molecular and morphological data), described here as A. parvicaespitosa.

Ongoing molecular studies will likely identify additional species and novel

lineages within Alternaria. However, living cultures are not known for several

morphospecies. Additionally some herbaria materials are recalcitrant to DNA

isolation and/or devoid of conidia. Fortunately, the morphological diversity

of Alternaria has been well studied (Simmons 2007). Classical comparative

morphological studies are useful for establishing phylogenetic affiliation. This

work is a continuation of the series started with the manuscripts on sections

Porri and Alternaria (Gannibal 2015, 2016). The aim of the present work is

to detect among phylogenetically unexamined Alternaria species those species

that fit the current morphological circumscription of sections Infectoriae and

Pseudoalternaria and to emend the morphological concept of these sections.

784. ... Gannibal & Lawrence

Materials & methods

The morphology of almost all Alternaria species with legitimate names was

analyzed with regard to conformity with criteria of Alternaria sections Infectoriae and

Pseudoalternaria. The morphological assessment was based on descriptions made by

Simmons (2007) or using original diagnoses and illustrations for species described after

2007. Descriptions of three species included in sect. Infectoriae were found in Roberts

(2008: A. roseogrisea), Roberts et al. (2010: A. cerasidanica), and Ariyawansa et al.

(2015: A. murispora). Strain X1272 was cultured on potato carrot agar (PCA) at 24°C

under light/dark cycle (12/12 h) for 7-10 days to promote sporulation in order to

describe this as a new taxon; the holotype specimen (a metabolically inactive dried PCA

culture) was conserved in the Mycological Herbarium, All-Russian Institute of Plant

Protection, Saint Petersburg, Russia (LEP).

Results & discussion

The evaluation of all available descriptions of Alternaria species allowed

for the inclusion of nine additional species in sect. Infectoriae, two of which

have no known living isolates. The diversity of this section consists of 34

morphologically recognizable species. Recircumscription of the misidentified

isolate, A. rosae strain X1272 (Zhu & Xiao 2015), allowed for the addition of

one species to sect. Pseudoalternaria bringing the total number of species in

this clade to three.

Alternaria parvicaespitosa Gannibal & D.P. Lawr., sp. nov. FIGS 1, 2

MycoBAank MB 816666

Differs from Alternaria rosae by its shorter conidial chains (non-branched parts) and

narrower conidia with fewer transverse septa.

Type: USA, California, Tulare, harvested blueberry fruit from a commercial fruit-

packing center, 6 June 2013, X.Q. Zhu & C.L. Xiao X1272 (Holotype, LEP 014858, a

metabolically inactive dried culture).

EryMo_oey: parvi (Latin), small; caespitosa (Latin), bushy.

Sporulation on PCA occurs primarily in discrete bushy clumps. Conidiophores

arise as series in close proximity to one another from submerged or (more

often) aerial hyphae. Aerial conidiogenous hyphae are hyaline filiform or thick

colored conidiophore-like and may be positioned vertically or horizontally.

Most conidiophores remain relatively short, 35-70 x 3-4 um. They are

simple or sometimes produce two branches. Each branch is multigeniculate

(1-6 genua near conidiogenous loci). The conidiogenous sites produce short

chains of conidia (<3-4 between branching points) which usually have one

or a few branches due to the production of several conidiogenous sites on

an apical secondary conidiophore or (very rarely) due to the formation of a

lateral secondary conidiophore. Aged clumps of conidia developed on a single

conidiophore may comprise <20 conidia.

Alternaria sect. Infectoriae & A. sect. Pseudoalternaria ... 785

50 um

————

Fic. 1. Alternaria parvicaespitosa (ex holotype, LEP 014858): sporulation pattern.

Fic. 2. Alternaria parvicaespitosa (ex holotype, LEP 014858): conidia and conidiophores.

786 ... Gannibal & Lawrence

Both juvenile and mature conidia are ovoid or elliptical. Small portion of

conidia is obclavate. Maximum size range of mature conidia is 10-25 x 7-12 um.

Conidia have 1-3(-4) transverse septa and at most one longiseptum in 1-2 of

the transverse segments. Rarely conidia have no longisepta. Most conidia have

no secondary conidiophores; but any conidium, including 1-celled units and

conidia with relatively large conidial bodies, can produce an apical secondary

conidiophore (a single cell ca 3 x 3 um) or a geniculate extension 5-28 um

long with 2-5 conidiogenous sites. Rarely apical secondary conidiophores are

branched. Lateral secondary conidiophores appear rarely and when present

are short. Conidia and conidiophores are concolorous, medium brown. The

conidium outer wall is smooth or (in the largest conidia) slightly punctulate.

Alternaria species in sections Infectoriae and Pseudoalternaria are listed in

TABLE l.

Herein we present emended and somewhat expanded versions of the

descriptions of Alternaria sect. Infectoriae by Woudenberg et al. (2013) and of

A. sect. Pseudoalternaria by Lawrence et al. (2016). Characters are quantified

when appropriate, and examples are provided of species whose quantified

characters either depart from the section norm or conform to it.

Alternaria sect. Infectoriae Woudenb. & Crous, Stud. Mycol. 75: 194. 2013; emend.

Gannibal & Lawrence

On PCA or weak potato dextrose agar (WPDA) primary conidiophores arise

from the agar surface or from vegetative aerial hyphae. Primary conidiophores

are solitary or aggregated, cylindrically straight or geniculate, simple or

branched, short to long, septate, brown, 20-150 x 3-4 um, with one or several

(2-6) conidiogenous loci (pores) at the apex and throughout its length. Conidial

chains are moderately long to long, simple or (usually) branched. Branching is

produced by the occurrence of several conidiogenous loci on apical secondary

conidiophores or (rarely: in some species completely absent) lateral secondary

conidiophores.

Young conidia are ovoid, broadly ovoid, ellipsoid or subellipsoid, erostrate

or with short to long secondary conidiophore at the apex. Mature conidia

are obclavate, ovoid, ellipsoid or subellipsoid; small or moderate in size; with

(1-)4-7(-10) transverse and no, one or a few longitudinal septa; slightly

constricted near some transverse septa. Size of conidia is 30-60 x 6-15 um

(except conidia with long secondary conidiophores); sometimes a large number

of conidia remain small (12-30 x 5-10 um). Several species (e.g., A. triticina)

can produce relatively larger conidia, <60 x 25 um, not including secondary

Alternaria sect. Infectoriae & A. sect. Pseudoalternaria ... 787

TABLE 1. List of Alternaria spp. assigned to A. sect. Infectoriae and

A. sect. Pseudoalternaria.

Names with bracketed annotations = species with phylogenetic data (in the cited references).

Unannotated names = species with reliable living cultures and herbarium specimens.

Names with asterisk = species for which no known living isolates are available.

Section Infectoriae

Ces ee ees

pe Seis es Ba

peo

eT ee

Be CBE BO ee ies De

. alternarina E.G. Simmons (Lawrence et al. 2013, 2014)

. arbusti E.G. Simmons (Lawrence et al. 2013, 2014)

astragali Wangeline & E.G. Simmons

. caespitosa (de Hoog & C. Rubio) Woudenb. & Crous (Woudenberg et al. 2013; Grum-Grzhimaylo et al.

2015)

californica E.G. Simmons & S.T. Koike (Lawrence et al. 2013, 2014)

cerasi Potebnia [*]

cerasidanica R.G. Roberts

daucicaulis E.G. Simmons (Lawrence et al. 2013, 2014)

ethzedia E.G. Simmons (Pryor & Bigelow 2003; Hong et al. 2005; Lawrence et al. 2013; Woudenberg et al.

2013; Grum-Grzhimaylo et al. 2015)

frumenti E.G. Simmons & C.E Hill (Lawrence et al. 2013, 2014)

geophila Dasz.

graminicola E.G. Simmons (Lawrence et al. 2013, 2014)

hordeiaustralica E.G. Simmons & Alcorn (Lawrence et al. 2013, 2014)

hordeicola E.G. Simmons & Kosiak (Lawrence et al. 2013, 2014)

humuli E.G. Simmons (Lawrence et al. 2013, 2014)

incomplexa E.G. Simmons (Lawrence et al. 2013, 2014)

infectoria E.G. Simmons (Pryor & Bigelow 2003; Lawrence et al. 2013; Woudenberg et al. 2013; Grum-

Grzhimaylo et al. 2015)

intercepta E.G. Simmons (Lawrence et al. 2013, 2014)

iridiaustralis E.G. Simmons, Alcorn & C.F. Hill

litorea (Pivkin & Zvereva) E.G. Simmons [*]

merytae E.G. Simmons (Lawrence et al. 2013, 2014)

metachromatica E.G. Simmons (Hong et al. 2005; Lawrence et al. 2013, 2014)

murispora Ariyaw. & K.D. Hyde [*] (Ariyawansa et al. 2015)

novae-zelandiae E.G. Simmons (Lawrence et al. 2013, 2014)

oregonensis E.G. Simmons (Hong et al. 2005; Lawrence et al. 2013; Woudenberg et al. 2013; Grum-

Grzhimaylo et al. 2015)

roseogrisea R.G. Roberts

scrophulariae (Desm.) Rossman & W.C. Allen (Hong et al. 2005; Lawrence et al. 2013; Woudenberg et al.

2013; Grum-Grzhimaylo et al. 2015)

seleniiphila Wangeline & E.G. Simmons

slovaca (Svob.-Pol., L. Chmel & Bojan.) Woudenb. & Crous (Woudenberg et al. 2013; Grum-Grzhimaylo

et al. 2015)

. triticimaculans E.G. Simmons & Perellé (Lawrence et al. 2013, 2014)

. triticina Prasada & Prabhu (Pryor & Bigelow 2003; Lawrence et al. 2013)

. vaccinii E.G. Simmons

. ventricosa R.G. Roberts (Lawrence et al. 2013, 2014)

viburni E.G. Simmons (Lawrence et al. 2013, 2014)

Section Pseudoalternaria

. arrhenatheri D.P. Lawr., Rotondo & Gannibal (Lawrence et al. 2014, 2016)

. rosae E.G. Simmons & C.F. Hill (Lawrence et al. 2013, 2014)

. parvicaespitosa Gannibal & D.P. Lawr. (Zhu & Xiao 2015, as “A. rosae”)

788 ... Gannibal & Lawrence

conidiophores. Some mature conidia remain erostrate or produce secondary

conidiophores. Apical secondary conidiophores are short or long, simple or

branched, usually geniculate with one or (often) a few (<5-7) conidiogenous

loci over its entire length. Approximately 30% of mature conidia produce at

least 4 transverse septa in the conidium body terminating in a chain that is

ovoid or ellipsoid and has no secondary conidiophores, beak or conidiogenous

loci on apical cell. At least 10% of mature conidia produce apical secondary

conidiophores that are longer than conidial body and exceed 30 um (often

reaching 50-100+ um). Long apical conidiophores having only one transverse

septum and no longitudinal septa are sometimes produced by very small

conidia. Some conidia have short solitary lateral secondary conidiophores

with one or two conidiogenous loci. Conidia are pale brown, yellowish to dark

brown; the spore wall is smooth or conspicuously ornamented.

Ascomata on natural substrata are erumpent, almost superficial or submerged

into host cortex, scattered, subglobose to sphaeroid with a short blunt beak,

dark, smooth, thin-walled at maturity. Asci subcylindrical, straight, curved or

muriform, usually 8-spored. Mature ascospores are ellipsoid, 5-7 transverse

septa, forming one or two longitudinal septa through each central or sometimes

through apical transverse segments, conspicuously constricted at median

septum and slightly so at two other transepta, brown.

Colony color on rich media like PDA, PSA, DRYES or Czapek-Dox agar, is

light: white, delicate rose, light gray or pale olivaceous. Colonies usually do not

sporulate in the dark, especially on rich media. Sporulation is usually copious

on PCA or V8 agar medium, intensive light or near UV light (when grown in

glass Petri dishes).

Alternaria sect. Pseudoalternaria D.P. Lawr., Rotondo & Gannibal, Mycol. Progr.

15:3 [11 of 22]. 2016; emend. Gannibal & Lawrence

On PCA or WPDA sporulation aggregated, appearing granular to the

naked eye. Mycelium subhyaline to light brown; hyphae smooth, branched,

septate, 3.8-5 um wide. Primary conidiophores aggregated on agar surface or

developing from arachnoid aerial hyphae, simple or branched, septate, smooth,

medium brown, 8.7-37.5 x 3.7-5 um (M = 18.1 x 4.8 um), simple with single

apical pore. Secondary conidiophores short to long, simple to multi-geniculate

with one to many conidiogenous loci. Conidia 10-32.5 x 4.7-10 um (M = 16.8

x 7.3 um), mostly catenulate, ellipsoid to obclavate, medium brown to golden

brown, 3-4 transverse septa, 1-2 longitudinal septa, smooth, may produce a

secondary conidiophore (false beak).

Alternaria sect. Infectoriae & A. sect. Pseudoalternaria ... 789

Acknowledgments

The authors are grateful to Dr. Hiran Ariyawansa (Guizhou Academy of Agricultural

Sciences, Guizhou University, Guiyang, China) and Dr. Andrew Armitage (Genetics

and Crop Improvement, East Mailing Research, UK) for presubmission review. This

work was supported by Russian Science Foundation (project #14-26-00067).

Literature cited

Andersen B, Kroger E, Roberts RG. 2002. Chemical and morphological segregation of Alternaria

arborescens, A. infectoria and A. tenuissima species-group. Mycol. Res. 106(2): 170-182.

http://dx.doi.org/10.1017/S0953756201005263

Andersen B, Sorensen EL, Nielsen KF, van den Ende BG, de Hoog S. 2009. A polyphasic approach

to the taxonomy of the Alternaria infectoria species-group. Fungal. Genet. Biol. 46: 642-656.

http://dx.doi.org/10.1016/j.fgb.2009.05.005

Ariyawansa HA, Thambugala KM Manamgoda DS, Jayawardena R, Camporesi E, Boonmee S,

Wanasinghe DN, Phookamsak R, Hongsanan S, Singtripop C, Chukeatirote E, Kang J-C, Jones

EBG, Hyde KD. 2015. Towards a natural classification and backbone tree for Pleosporaceae.

Fungal Diversity 71(1): 85-139. http://dx.doi.org/10.1007/s13225-015-0323-z

Armitage AD, Barbara DJ, Harrison RJ, Lane CR, Sreenivasaprasad S, Woodhall JW, Clarcson

JP. 2015. Discrete lineages within Alternaria alternata species group: Identification using

new highly variable loci and support from morphological characters. Fungal Biol. 119(11):

994-1006. http://dx.doi.org/10.1016/j.funbio.2015.06.012

Christensen KB, Van Klink JW, Weavers RT, Larsen TO, Andersen B, Phipps RK. 2005. Novel

chemotaxonomic markers of the Alternaria infectoria species-group. J. Agric. Food Chem.

53(24): 9431-9435. http://dx.doi.org/10.1021/jf0513213

Gannibal PhB. 2015. Distribution of Alternaria species among sections. 1. Section Porri. Mycotaxon

130(1): 207-213. http://dx.doi.org/10.5248/130.207

Gannibal PhB. 2016 [“2015”]. Distribution of Alternaria species among sections. 2. Section

Alternaria. Mycotaxon 130(4): 941-949. http://dx.doi.org/10.5248/130.941

Gannibal PhB, Yli-Mattila T. 2007. Morphological and UP-PCR analyses and design of a PCR assay

for differentiation of Alternaria infectoria species-group. Mikologiya i Fitopatologiya 41(4):

313-922,

Grum-Grzhimaylo AA, Georgieva ML, Bondarenko SA, Debets AJM, Bilanenko EN. 2016. On the

diversity of fungi from soda soils. Fungal Diversity 76(1): 27-74.

http://dx.doi.org/10.1007/s13225-015-0320-2

Hong SG, Cramer RA, Lawrence CB, Pryor BM. 2005. Alt a 1 allergen homologs from Alternaria

and related taxa: analysis of phylogenetic content and secondary structure. Fungal Genet. Biol.

42: 119-129. http://dx.doi.org/10.1016/j.fgb.2004.10.009

Lawrence DP, Park MS, Pryor BM. 2012. Nimbya and Embellisia revisited, with nov.

comb. for Alternaria celosiae and A. perpunctulata. Mycol. Progress 11(3): 799-815.

http://dx.doi.org/10.1007/s11557-011-0793-7

Lawrence DP, Gannibal PhB, Peever TL, Pryor BM. 2013. The sections of Alternaria: formalizing

species-group concepts. Mycologia 105(3): 530-546. http://dx.doi.org/10.3852/12-249

Lawrence DP, Gannibal PhB, Dugan FM, Pryor BM. 2014. Characterization of Alternaria isolates

from the infectoria species-group and a new taxon from Arrhenatherum, Pseudoalternaria

arrhenatheria sp. nov. Mycol. Progress 13(2): 257-276.

http://dx.doi.org/10.1007/s11557-013-0910-x

Lawrence DP, Rotondo F, Gannibal PhB. 2016. Biodiversity and taxonomy of the pleomorphic

genus Alternaria. Mycol. Progress 15(1):3 1-22. http://dx.doi.org/10.1007/s11557-015-1144-x

790 ... Gannibal & Lawrence

Oviedo MS, Sturm ME, Reynoso MM, Chulze SN, Ramirez ML. 2013. Toxigenic profile and

AFLP variability of Alternaria alternata and Alternaria infectoria occurring on wheat. Braz. J.

Microbiol. 44(2): 447-455. http://dx.doi.org/10.1590/S1517-83822013000200017

Pryor BM, Bigelow DM. 2003. Molecular characterization of Embellisia and Nimbya species and

their relationship to Alternaria, Ulocladium and Stemphylium. Mycologia 95: 1141-1154.

https:/doi.org/10.2307/3761916

Roberts RG. 2008. Alternaria roseogrisea, a new species from achenes of Helianthus annuus

(sunflower). Mycotaxon 103: 21-26.

Roberts RG, Reymond ST, Andersen B. 2010. Alternaria cerasidanica sp. nov., isolated in Denmark

from drupes of Prunus avium. Mycotaxon 111: 175-182. https:/doi.org/10.5248/111.175

Runa F, Park MS, Pryor BM. 2009. Ulocladium systematics revisited: phylogeny and taxonomic

status. Mycol. Progress 8: 35-47. http://dx.doi.org/10.1007/s11557-008-0576-y

Simmons EG. 2007. Alternaria: an identification manual. Utrecht: CBS. 775 p.

Woudenberg JHC, Groenewald JZ, Binder M, Crous PW. 2013. Alternaria redefined. Studies in

Mycology 75: 171-212. http://dx.doi.org/10.3114/sim0015

Woudenberg JHC, Truter M, Groenewald JZ, Crous PW. 2014. Large-spored Alternaria pathogens

in section Porri disentangled. Studies in Mycology 79: 1-47.

http://dx.doi.org/10.1016/j.simyco.2014.07.003

Zhu XQ, Xiao CL. 2015. Phylogenetic, morphological, and pathogenic characterization of

Alternaria species associated with fruit rot of blueberry in California. Phytopathology 105:

1555-1567. http://dx.doi.org/10.1094/PHY TO-05-15-0122-R

MYCOTAXON

October-December 2016— Volume 131, pp. 791-794

http://dx.doi.org/10.5248/131.791

Zasmidium oleae sp. nov. from China

Bao-Ju L1’*, XUE-WEN XIE, YAN-XIA SHI’, A-LI CHAI’ & YING-LAN Guo?*

‘Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences,

Beijing 100081, PR. China.

° Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR. China

* CORRESPONDENCE TO: guoyl@im.ac.cn

ABSTRACT—A new species, Zasmidium oleae on Olea tsoongii is described, illustrated and

discussed. The type specimen is deposited in HMAS.

Key worps—cercosporoid fungi, Mycosphaerellaceae, plant pathogen

Introduction

The genus Zasmidium, established by Fries (1849), was considered a

monotypic genus until recently. Based on molecular sequence analyses,

this genus is now used for cercosporoid fungi in the Mycosphaerellaceae

morphologically characterized by verruculose superficial hyphae in vivo

(rarely lacking); smooth to verruculose conidiophores; conspicuous, somewhat

thickened and darkened, planate conidiogenous loci; and solitary or catenate

conidia with thin to somewhat thickened, smooth to verruculose walls and

somewhat thickened and darkened planate hila (Braun et al. 2013). Zasmidium

oleae, a new species on Olea tsoongii collected in Hainan Province, China,

is described, illustrated, and compared with other cercosporoid species on

Oleaceae. The type specimen is deposited in the Herbarium Mycologium,

Chinese Academy of Sciences, Beijing, China (HMAS).

Taxonomy

Zasmidium oleae Y.L. Guo, X.W. Xie & B.J. Li, sp. nov. FIG. 1

FUNGAL NAME FN570159

Differs from Passalora amurensis by its shorter, smooth, 3-4-septate conidia.

*These authors contributed equally to this work

792 ... Li, Xie & al.

Fic. 1. Zasmidium oleae (HMAS 245707, holotype).

a. Conidia; b. Conidiophores; c. Stroma. Bar = 25 um.

Type: China, Hainan Province, Wanning, on living leaves of Olea tsoongii (Merr.) PS.

Green (Oleaceae), 10 April 2011, coll. Guo Ying-lan 380 (Holotype, HMAS 245707).

ErymMo oey: Derived from the host genus Olea.

Leaf spots amphigenous, circular to angular, 2-8 mm diam., often confluent,

red-brown to dark brown with a yellow to pale olivaceous-brown halo on the

upper surface, pale brown with yellow to pale olivaceous-brown halo on the

lower surface. Caespituli mainly hypogenous. Mycelium internal. Stromata

Zasmidium oleae sp. nov. (China) ... 793

substomatal, globose, pale brown to brown, 25-50 um diam. Conidiophores

densely fasciculate, pale olivaceous-brown to olivaceous-brown, uniform

in color, irregular in width, straight to slightly curved, not branched,

geniculate, thin-walled, smooth, conical to conically truncate at the apex,

0-2-septate, mostly aseptate, 6.7-20(-48) x 4-5.3 um. Conidiogenous cells

integrated, terminal or mostly reduced to conidiogenous cells, 6.7-20 um

long. Conidiogenous loci conspicuous, thickened, darkened, 1.3-2.5 um wide.

Conidia formed singly, cylindrical, brown, thick-walled, verruculose, straight

to variously curved, rounded at the apex, rounded to short obconically truncate

at the base, indistinctly 3- to multiseptate, 40-128 x 4.0-5.8 um, hila thickened

and darkened, 1.3-2.5 um wide.

Discussion

Based on the description in Chupp (1954), Cercospora lumbricoides Turconi

& Maffei on Fraxinus sp. (Oleaceae) is very similar to Z. oleae but differs by its

brown, longer conidiophores (30-60 x 4-6 um) and longer conidia (80-200

x 4-6 um). However, the generic affinity of C. lumbricoides is quite unclear

(the type material has not been traced and the species is known only from the

original description) (Turconi & Maffei 1915).

Passalora amurensis (Ziling) H.D. Shin & U. Braun on Syringa amurensis

Rupr. (Oleaceae), also morphologically similar to Z. oleae, differs in having

pale olivaceous, 3-4-septate, shorter, and smooth conidia (35-70 x 4.0-5.5 um;

Shin & Braun 1996).

Although superficial verruculose mycelium, common in most Zasmidium

species, is lacking on Olea tsoongii, assignment of the new species to Zasmidium

is nevertheless justified. Several comparable species with distinctly verruculose

conidia have recently been allocated to Zasmidium based on molecular data

that support their affinities to this genus.

Acknowledgments

This work was supported by the Project for Fundamental Research on Science

and Technology, Ministry of Science and Technology of China (No. 2013FY110400);

CAAS Agricultural Science and Technology Innovation Program and funded by Key

Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of

Agriculture, China. We are grateful to Dr. U. Braun and Prof. S.Y. Guo for reviewing the

manuscript and to Ms. X.F. Zhu for inking the line drawing.

Literature cited

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://dx.doi.org/10.5598/imafungus.2013.04.02.12

79A< ... Li, Xie & al.

Chupp C. 1954. A monograph of the fungus genus Cercospora. Ithaca, New York.

Fries EM. 1849. Summa vegetabilium Scandinaviae. Sectio posterior. Typographia Academica,

Uppsala.

Shin HD, Braun U. 1996. Notes on Korean Cercosporae and allied genera (II). Mycotaxon 58:

157-166.

Turconi M, Maffei L. 2015. Note micologiche e fitopatologiche. I. Cercospora lumbricoides n. sp.

sul Frassino e Nectria Castilloae n. sp. sulla Castilloa elastica, nel Messico. I. Steganosporium

Kosaroffii n. sp. sul Gelso, in Bulgaria. Atti dell’ Istituto Botanico dell’ Universita di Pavia,

Serie 2, 12: 329-336.

MY COTAXON

October-December 2016— Volume 131, pp. 795-803

http://dx.doi.org/10.5248/131.795

Strigula sinoaustralis sp. nov.

and three Strigula spp. new to China

SHU-HUA JIANG »?, XIN-LI WEI ! & JIANG-CHUN WEI"?

'State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences,

Beijing 100101, China

? University of Chinese Academy of Sciences, Beijing 100049, China

* CORRESPONDENCE TO: weijc2004@126.com

ABSTRACT—A new foliicolous lichen is described from South China. Strigula sinoaustralis is

most similar to S. concreta in ascospore dimensions but differs by its white-punctate thallus

with entire margins and its longer asci. An analysis of its relationships based on molecular

phylogeny is given. Strigula antillarum, S. laureriformis, and S. prasina are reported as new

to China.

KEY WORDS—new species, taxonomy, phylogenetic analysis, lichenized Ascomycota

Introduction

Strigula Fr. (Strigulaceae) is a genus of lichenized fungi containing about 70

species (Hyde et al. 2013). Seventeen Strigula species (twelve foliicolous; five

corticolous or saxicolous) have been recorded from China (Santesson 1952, Wei

& Jiang 1991, Aptroot & Seaward 1999, Aptroot & Sipman 2001, Aptroot 2003,

Aptroot et al. 2003, Licking 2008). During our ongoing studies of lichenized

fungi in China, we found material representing an undescribed Strigula species

and three Strigula species new to China, which are reported here.

Materials & methods

Lichen collections

Most Strigula specimens in this study were collected from Guangxi, Hainan, Yunnan,

and Guangdong provinces and are preserved in the Fungarium of the Institute of

Microbiology, Chinese Academy of Sciences, Beijing, China (HMAS-L); one additional

collection from Xizang is preserved in the Herbarium of Kunming Institute of Botany,

Chinese Academy of Sciences, Kunming, China (KUN).

796 ... Jiang, Wei & Wei

Phenotypic analysis

A Leica M125 dissecting microscope was used for the morphological studies and a

Zeiss Axioscope2 compound microscope with a Zeiss Axio Imager A2 for the anatomical

studies. Sections were studied in tap water and photographed with an AxioCam MRc5

camera. Thin-layer chromatography (TLC) (Culberson & Kristinsson 1970, Culberson

1972, Orange et al. 2001) was used to detect lichen substances.

TABLE 1. Specimens of Strigula spp. and Falciformispora outgroup used in the

phylogenetic analysis

SPECIES VOUCHER SPECIMEN GENBANK No.

S. antillarum HMAS-L 0137209 KX216696*

HMAS-L 0137208 KX216697*

HMAS-L 0137211 KX216702*

S. prasina HMAS-L 0137213 KX216700*

HMAS-L 0137212 KX216701*

S. smaragdula KoLRI016344 KF553663

LFF016344 KF553664

S. sinoaustralis HMAS-L0137203 (T) KX216699*

HMAS-L0137204 KX216698*

E. senegalensis 1P614.60 KP132365

EF. tompkinsii IP559.60 KP132366

* = sequences newly generated for this study by the authors

DNA extraction, amplification, and sequencing

Seven fresh specimens representing three different Strigula species reported from

China were chosen for DNA extraction (TABLE 1). The extraction procedure followed

a modified CTAB method (Rogers & Bendich 1988). The nuclear ribosomal RNA gene

region, including the internal transcribed spacer (ITS), was amplified through PCR

using the primer pair ITS5 and ITS4 (White et al. 1990). Reactions were carried out in

25 ul reaction volume including 1 ul DNA, 1 ul each primer (10 uM), 2 ul dNTP (2.5 mM

each), 2.5 ul amplification buffer (10x, 25 mM MgCl, contained), 0.5 ul Taq polymerase

(rTaq DNA Polymerase, 5 U/ul), 17 ul ddH,O. Cycling parameters were set to an initial

denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 s,

annealing at 52°C for 30 s, extension at 72°C for 50 s, and a final extension at 72°C for 10

min. The new sequences generated for this study were deposited in GenBank (TABLE 1).

Phylogenetic analysis

Eleven sequences including seven sequences were newly generated for this study

(TABLE 1) were aligned with the program MAFFT (Katoh 2002); two other accessions of

Strigula smaragdula Fr. were retrieved from GenBank (Jayalal et al. 2013). Due to lack of

ITS sequences of other genera in Strigulales, two species, Falciformispora tompkinsii (El-

Ani) S.A. Ahmed et al. and Falciformispora senegalensis (Segretain et al.) S.A. Ahmed et

al., belonging to Pleosporales (the family most closely related to Strigulales), were chosen

Strigula sinoaustralis sp. nov. (China) ... 797

100 [© Strigula antillarum KX216696

® Siri guila antilarum KX216697

Strigula antillarum KX216702

Strigula smaragdula KF553663

Strigula smaragdula KF553664

2. Strigula prasina KX216700

e Strigula prasina KX216701

®*Swrigula sinoaustralis sp.nov. KX216698

® SHigula sinoaustralis sp.nov. 1-X216699 (T)

Falciformispora tompkinsii KP132366

Falciformispora senegalensis KP132365

0.05

Fic. 1. The Neighbor-Joining tree based on nrDNA ITS sequences. Samples marked with '®’ were

sequenced by the authors; others were downloaded from GenBank. Genetic distance scale = 0.05

changes per site. Numbers above each node represents bootstrap support values >50%.

as outgroup. The alignment was subjected to a Neighbor-Joining analysis involving 1000

replicates with MEGA (Tamura et al. 2011), using the p-distance model.

Results

The best-scoring Neighbor-Joining tree, based on the eleven ITS sequences

(481 bp), is shown in Fic. 1. Within the well-supported (bootstrap = 100%)

monophyletic four-species clade of Strigula, the new species S. sinoaustralis and

the Chinese material of S. antillarum and S. prasina each formed a separate

well-supported branch (bootstraps = 100%).

Taxonomy

Strigula sinoaustralis S.H. Jiang, X.L. Wei & J.C. Wei, sp. nov. PL. 1

FUNGAL NAMES FN570267

Differs from Strigula concreta by its white-punctate thallus with entire margins and its

longer asci.

Type: China. Guangxi Province, Shangsi County, Shiwan mountain forest park, along

the way to Yingkesong and Jiulongsong, 21°54’16”N 107°54’09”E, alt. 271 m, on living

leaves, 26/V/2015, Xin-Li Wei & Jiao-Hong Wang GX20150342 (Holotype, HMAS-L

0137203; GenBank KX216699).

Erymo toey: The epithet sinoaustralis refers to South China.

798 ... Jiang, Wei & Wei

THALLUuS subcuticular, dispersed into rounded patches with entire margins,

without forming distinct lobes, 2-11 mm across and 25-35 um thick; surface

smooth, grayish green, often minutely white-punctate. PHOTOBIONT a

Cephaleuros sp., composed of angular-rounded cells, 8-14 x 4-6 um.

PERITHECIA exposed, almost conical, basal part somewhat broadly

spreading, 0.25-0.4 mm diam. and 100-180 um tall, black. ExcrpuLum

prosoplectenchymatous, 10-18 um thick, brown; INVOLUCRELLUM 22-40 um

thick, carbonaceous, black; PARAPHySES unbranched. Asci cylindrical or

almost thread like, numerous and dense, 72.5-92.5 x 4-5 um. ASCOSPORES

8 per ascus, uniseriate, ellipsoid, 1-septate, usually with 1-2 oil droplets per cell

when fresh, distinctly constricted at the septum and sometimes broken into

parts (fragmented) outside the asci, 8-12 x 2.5-3 um (4-6 um long for each

fragment), 3-4 times as long as broad.

PycnipIA usually rather few, wart-shaped, 0.05-0.15 mm diam.; two types

present: one type 0.1-0.15 mm diam. producing macroconidia that are bacillar,

0-1-septate, 4-6 x 1.8-2.3 um, with a mucilaginous appendage at one or both

ends, 5-10 um long; the second type 0.05-0.1 mm diam., rather few, and often

empty. MICROCONIDIA not seen.

CHEMISTRY. No substances detected by TLC.

ECOLOGY & SUBSTRATE. Found in humid, partly exposed habitats on living

leaves of bamboo.

ADDITIONAL SPECIMENS EXAMINED: CHINA. GUANGDONG PROVINCE, Shixing

County, Chebaling national nature reserve, along the way to Xianrendong Village,

24°44’7”N 114°12’31”E, alt. 486 m, on living leaves, 15/V/2015, X.L. Wei & J.H. Wang

GD2015042-8 (HMAS-L 0137206). GUANGXI PROVINCE, Shangsi County, Shiwan

mountain forest park, along the Shitou River, 21°54’3”N 107°54’26’E, alt. 342 m, on

living leaves, 25/V/2015, X.L. Wei & J.H. Wang GX20150265 (HMAS-L 0137204;

GenBank KX216698), GX20150387 (HMAS-L 0137205).

REMARKS: Two other Strigula species with carbonized perithecia develop

similarly white-punctate thalli: S. lacericola PM. McCarthy, which can be

distinguished by its shallowly lobate thallus margins and very narrow (5-7

times longer than broad) ascospores that do not separate either within or

outside the asci (McCarthy 2009) and S. albomaculata Sérus., which has

longer asci (120-140 x 5-8 um) and larger ascospores (18-22 x 5-6 um) and

macroconidia (8-8.5 x 1.5-2 um) (Aptroot et al. 1997).

Anatomically, the new species is most closely related to S. concreta (Fée)

R. Sant. Both share the same perithecial anatomy and small, uniseriate

ascospores that fragment outside the asci, but they differ in thallus morphology

and ascus dimensions: S. concreta lacks white dots on the thallus and has

lobulate thallus margins and shorter asci (30-70 x 4-6 um; Santesson 1952).

Strigula sinoaustralis sp. nov. (China) ... 799

PLaTE 1. Strigula sinoaustralis. A. Thallus with perithecia (holotype, HMAS-L 0137203);

B. Macroconidia, with appendage at one or both ends (HMAS-L 0137204); C. Ascus, with eight

uniseriate ascospores (holotype, HMAS-L 0137203); D, E. Ascospores, with distinct constriction

at septum and breaking into parts outside the asci (holotype, HMAS-L 0137203). Scale bars:

A = 300 um; B-E = 10 um.

The new species also resembles S. nitidula Mont., which is distinguished by

a very thin (8-15 um) marginally effuse bright metallic green thallus usually

bordered by a narrow black line (Licking 2008).

Strigula microspora Licking can be separated from S. sinoaustralis by its

thallus with a white central part surrounded by crenulate or shortly lobulate

margins, ascospores that do not fragment outside the asci, and larger

macroconidia (8-10 x 2-2.5 um; Liicking 1991, 2008).

Strigula sinoaustralis cannot be confused with any species of the S. smaragdula

group, which is characterized by broader asci and larger biseriate ascospores

(Santesson 1952, Liicking 2008). Besides the morphological differences, the

new species is also supported by the phylogenetic analysis (see p. 797).

800 ... Jiang, Wei & Wei

Strigula antillarum (Fée) Miill. Arg., Bot. Jahrb. Syst. 6: 379 (1885). PL. 2A-E

THALLUS often medium green, 0.5-7.5 mm diam.

PERITHECIA exposed, immersed at the base, not sharply delimited from the

thallus, not easily found in the specimens bearing pycnidia. Asct 55-87.5 x

8-12 um. AscosporEs 8 per ascus, biseriate, fusiform, with acute ends, 17.5-25

x 4-7.5 um.

PycnipiA producing macroconidia aggregated. MAcroconrpi1A bacillar,

1-septate, 12.5-20 x 3-5 um.

CuHEMiIstTRY. No substances detected by TLC.

SPECIMENS EXAMINED: CHINA. GUANGXI PROVINCE, Nanning, Long’an County,

Longhu mountain natural reserve, 22°57’42”N 107°37’40’E, alt. 147 m, on living

leaves, 1/XII/2015, S.H. Jiang GX201511110 (HMAS-L 0137209; GenBank KX216696),

GX201511112 (HMAS-L 0137208; GenBank KX216697), GX201511114 (HMAS-L

0137207). HAINAN PROVINCE, Dongfang city, Nanlang village E’xian Ling, 19°00°18”N

109°04’09’E, alt. 160 m, on living leaves, 13/XII/2014, J.H. Wang & R.D. Liu HN2014376

(HMAS-L 0130571), HN2014354 (HMAS-L 0130622); 19°00’07”N 109°04’20’E, alt.

126 m, J.H. Wang & R.D. Liu HN2014393 (HMAS-L 0130573). YUNNAN PROVINCE,

Xishuangbanna, Mengla County, tropical botanical garden of Chinese Academy of

sciences, East area, 21°55’39”N 101°15’52”E, alt. 560 m, on living leaves, 18/X1/2015, X.L.

Wei & S.H. Jiang XTBG2015051 (HMAS-L 0137211; GenBank KX216702); Lvshilin,

21°54’35’N 101°16’52”E, alt. 626 m, 18/XI/2015, X.L. Wei & S.H. Jiang XTBG2015119

(HMAS-L 0137210).

REMARKS: Strigula antillarum is characterized by its aggregated and confluent

pycnidia in the center of the thallus. This species is anatomically similar to

S. smaragdula, which is distinguished by its thallus-covered perithecia and

dispersed pycnidia (Licking 2008).

Strigula laureriformis Aptroot & Licking, Biblioth. Lichenol. 97: 131 (2008).

PL. 2BG

THALLUS growing on bark, bright green to white green.

PyYCNIDIA immersed-erumpent, wart-shaped, covered by thallus, with

ostiole exposed outside. Conrp1A ellipsoid, 1-septate, hyaline, 7.5-10 x 2.5-4 um,

sometimes with appendage 15-25 um long.

PERITHECIA not seen.

CHEMISTRY. No substances detected by TLC.

SPECIMENS EXAMINED: CHINA. XIZANG PROVINCE, Motuo County, alt. 1000 m, on

living leaves, 1982, Y.G. Su 2532 (KUN 7537).

REMARKS: Strigula laureriformis (very typical of the genus; Aptroot et al. 2008)

does not produce perithecia and is easily recognized by its corticolous habit,

bright white green thallus, and simple 1-septate conidia.

Strigula sinoaustralis sp. nov. (China) ... 801

PLATE 2. Strigula antillarum. A. Thallus with pycnidia (HMAS-L 0137209); B. Ascospores,

fusiform, with acute ends, not breaking into parts outside the asci (HMAS-L 0137211); C, D. Asci,

with eight biseriate ascospores (HMAS-L 0137211); E. Macroconidia (HMAS-L 0137209). Strigula

laureriformis (KUN 7537). FE. Thallus with pycnidia; G. Macroconidia. Strigula prasina (HMAS-L

0137213). H. Hypophyllous thallus; I. Asci, with irregularly arranged ascospores; J. Ascospores,

fusiform-ellipsoid, 1-septate, with slight constriction at septum and not breaking into parts outside

the asci. Scale bars: A = 200 um; B-E = 10 um, F = 800 um, G = 20 um, H = 300 um, I, J = 10 um.

802 ... Jiang, Wei & Wei

Strigula prasina Miil. Arg., Flora 68: 343 (1885). PL. 2H-J

THALLUS hypophyllous, subcuticular, dispersed into patches, pale green.

PERITHECIA basally immersed, hemispherical, black. PARAPHysEs slightly

branched. Asci dense and compact, cylindrical, 52.5-70 x 7.5-10 um.

AscosporEs 8 per ascus, biseriate to irregularly arranged or occasionally

uniseriate, fusiform-ellipsoid, 1-septate, with slight constriction at septum,

12-17 x 4-5 um.

PYCNIDIA not seen.

CHEMISTRY. No substances detected by TLC.

SPECIMENS EXAMINED: CHINA. HAINAN PROVINCE, Ledong County, Jianfengling, on

living leaves, 2009, J.C. Wei 046F1 (HMAS-L 0137213; GenBank KX216700), 046F2

(HMAS-L 0137212; GenBank KX216701).

REMARKS: Strigula prasina is typically a hypophyllous species with a thallus

morphology rather similar to that of S. concreta. However, S. concreta is

typically epiphyllous and has distinctly smaller ascospores, often broken into

parts (Santesson 1952). The other hypophyllous species, S. janeirensis (Mull.

Arg.) Licking, mainly differs in its thinner thallus, larger perithecia, and much

larger ascospores that easily fragment within the asci (Roux & Sérusiaux 1995,

Liicking 2008).

Acknowledgements

This project was supported by Ministry of Science and Technology of China

(2013FY110400, 2014FY120100). Sincere thanks to Dr. Robert Liicking and Prof.

Emmanuél Sérusiaux for reviewing the manuscript and providing references during

our studies. The authors are indebted to Dr. Patrick McCarthy and Prof. L.S. Wang for

lending us specimens. We thank Ms. H. Deng for giving considerable assistance during

the studies in HMAS-L.

Literature cited

Aptroot A. 2003. Pyrenocarpous lichens and related non-lichenized ascomycetes from Taiwan.

Journal of the Hattori Botanical Laboratory 93: 155-173.

Aptroot A, Seaward MRD. 1999. Annotated checklist of Hong Kong lichens. Tropical Bryology

17: 57-101.

Aptroot A, Sipman HJM. 2001. New Hong Kong lichens, ascomycetes and lichenicolous fungi.

Journal of the Hattori Botanical Laboratory 91: 317-343.

Aptroot A, Diederich P, Sérusiaux E, Sipman HJM. 1997. New or interesting lichens and

lichenicolous fungi from Papua New Guinea. Bibliotheca Lichenologica 64: 186-188.

Aptroot A, Ferraro LI, Lai MJ, Sipman HJM, Sparrius LB. 2003. Foliicolous lichens and their

lichenicolous ascomycetes from Yunnan and Taiwan. Mycotaxon 88: 41-47.

Aptroot A, Licking R, Sipman HJM, Umania L, Chaves JL. 2008. Pyrenocarpous lichens with

bitunicate asci. Bibliotheca Lichenologica 97. 162 p.

Strigula sinoaustralis sp. nov. (China) ... 803

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(1):

113-125. http://dx.doi.org/10.1016/0021-9673(72)80013-X

Culberson CF, Kristinsson H. 1970. A standardized method for the identification of lichen products.

Journal of Chromatography 46: 85-93. http://dx.doi.org/10.1016/S0021-9673(00)83967-9

Hyde KD, Jones EBG, Liu JK, Ariyawansa H, Boehm E, Boonmee §, et al. 2013. Families of

Dothideomycetes. Fungal Diversity 63: 1-313. http://dx.doi.org/10.1007/s13225-013-0263-4

Jayalal U, Oh SO, Liicking R, Joshi S, Kim JA, Park JS, Hur JS. 2013. Contributions to the foliicolous

lichens flora of South Korea. Mycobiology 41: 202-209.

http://dx.doi.org/10.5941/MYCO.2013.41.4.202

Katoh K, Misawa K, Kuma K, Miyata T. 2002. MAFFT: a novel method for rapid multiple

sequence alignment based on fast Fourier transform. Nucleic Acids Research 30: 3059-3066.

http://dx.doi.org/10.1093/nar/gkf436

Liicking R. 1991. Neue Arten foliikoler Flechten aus Costa Rica, Zentralamerika. Nova Hedwigia

52: 267-304.

Licking R. 2008. Foliicolous lichenized fungi. Flora Neotropica 103. 866 p.

McCarthy PM. 2009. Strigulaceae. Flora of Australia 57: 570-601.

Orange A, James PW, White FJ. 2001. Microchemical methods for the identification of lichens.

British Lichen Society, London.

Rogers SO, Bendich AJ. 1988. Extraction of DNA from plant tissues. 1-10, in: SB Gelvin & RA

Schilperoort (eds). Plant Molecular Biology Manual A6. Boston: Kluwer Academic Publishers.

Roux C, Sérusiaux E. 1995. Présence d’'appendices mucoides sur les ascospores de Raciborskiella

janeirensis (Mull. Arg.) R. Sant. Bulletin de la Société Linnéenne de Provence 46: 91-94.

Santesson R. 1952. Foliicolous lichens I. A revision of the taxonomy of the obligately foliicolous,

lichenized fungi. Symbolae Botanicae Upsalienses 12(1). 590 p.

Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. Mega5: molecular evolutionary

genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony

methods. Molecular Biology and Evolution 28: 2731-2739.

http://dx.doi.org/10.1093/molbev/msr121

Wei JC, Jiang YM. 1991. Some foliicolous lichens in Xishuangbanna, China. 201-216, in: D

Galloway (ed.). Systematics, Conservation and Ecology of Tropical Lichens, Systematics

Association Special Volume no. 42. Clarendon Press, Oxford.

White TJ, Bruns TD, 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. Academic Press, New York.

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

MY COTAXON

October-December 2016— Volume 131, pp. 805-809

http://dx.doi.org/10.5248/131.805

Thelenella haradae sp. nov.,

a saxicolous lichen from South Korea

JOSEF P. HALDA’ & JAE-SEOUN HuR”*

'Muzeum a galerie Orlickych hor, Jiraskova 2, 516 01 Rychnov nad KnéZnou, Czech Republic

*Korean Lichen Research Institute, Sunchon National University, Suncheon, 57922, Korea

“CORRESPONDENCE TO: jshur1@sunchon.ac.kr

ABSTRACT—A new species of saxicolous lichen is described, and named as Thelenella

haradae. It occurs on exposed granite rocks of Mt. Daebang, Gyeongnam Prov., southern

maritime Korea, and is similar to T. brasiliensis but differs in having wider ascospores

(25-30 x 13-17 um) and completely immersed ascomata.

Key worps—East Asia, lichenized ascomycetes, biogeography, taxonomy

Introduction

During examination of pyrenocarpus lichens deposited in KoLRI herbarium

(Sunchon Nationbal University), a new species of Thelenella from Korea was

discovered.

The genus Thelenella Nyl. (Thelenellaceae) consists of 35 corticolous,

foliicolous, muscicolous, or saxicolous taxa and is characterized by a crustose

thallus with a chlorococcoid photobiont and immersed ascomata usually

lacking an involucrellum (Mayrhofer 1987, Morse 2016). Most saxicolous

Thelenella species are limited to maritime or oceanic climates and non-

calcareous substrata (with one exception, T! calcicola C.A. Morse; Morse 2016).

Thelenella modesta (Nyl.) Nyl. has recently been reported from Korea (Aptroot

& Moon 2014). We now present a second record for the country, here described

as Thelenella haradae.

Materials & methods

The specimen was collected from southern part of Korea in 2011 and deposited

in the lichen herbarium of the Korean Lichen Research Institute, Suncheon, Korea

806 ... Halda & Hur

(KoLRI). The specimen was examined morphologically and sections were made by

razor blade under an Olympus SZX-7 stereomicroscope. The thin hand-cut sections of

thallus and ascomata were mounted in tap water and examined anatomically using an

Olympus BX50 compound microscope with DIC optics connected to a Canon 5D digital

camera. All measurements were made in water, but sections of ascomata or pycnidia

were mounted in 10% aqueous solution of KOH for observing asci and ascospores in

more detail. Ascospores were measured, and mean length and width values + standard

deviations and mean length/width ratio were calculated. The methodology for colour

spot reaction tests and thin layer chromatography (TLC) followed Orange et al. (2010).

Taxonomy

Thelenella haradae J. Halda, Xin Y. Wang, J.A. Ryu & Hur, sp. nov. PL.1

MycoBank MB819398

Differs from Thelenella brasieliensis by its completely immersed ascomata and wider

ascospores.

Type: South Korea, Gyeongnam Prov., Namhae County, Mt. Daebang, 34°51’00”N

127°59’26’E, alt. 87 m, on vertical face of an exposed granite rock, 29 April 2011,

X.Y. Wang & J.A. Ryu 110271 (Holotype, KoLRI 13485).

EryMoLocy: The new species is named in honor of the well-known Japanese

lichenologist Dr. H. Harada for his numerous contributions to our knowledge of

pyrenocarpous lichens in East Asia.

THALLUS lichenized, epilithic, 2-5 mm diam., 400-600 um thick, gray to gray-

green, smooth, + glossy, verrucose to subsquamulose, thick, with prominent

brown hypothallus. I-, KI-, composed mostly of oblong to spherical hyphae

(ca. 5-8 um diam.) and partly of linear hyphae (especially in the basal parts).

EPINECRAL LAYER well-developed, ca. 20 um thick, hyaline, composed

of horizontally flattened hyphal remnants. CorTICAL LAYER 60-80 um

thick, plectenchymatous, composed of hyphae 1.5-2 um diam., colourless.

PHOTOBIONT LAYER continuous, 40-60 um thick. MEDULLA often present,

white, 40-100 um high. PHoToBIonrT cells of trebouxioid green algae mostly

7-10 um diam., usually in vertically oriented clusters.

ASCOMATA perithecioid (pseudothecial), oval to pyriform, 350-400 um

tall, 300-350 um broad, without involucrellum, completely immersed in

thalline warts; the warts 0.5-1 mm across, usually with several perithecia,

dome-shaped to hemispherical, the same colour as surrounding thallus,

colourless to brown in upper parts around ostiola. OsTIoLuM not visible on

PLaTE 1 (right). Thelenella haradae (holotype, KoLRI 13485). A, thalline warts with ascomata;

B, subsquamulose thallus; C, cross section of thalline wart with ascoma; D, cross section of

thallus with upper epinecral layer, cortical layer, and medulla; E, cross section of ascoma showing

perithecial wall structure, hamathecium, asci with ascospores, and anastomosing paraphysoids

Thelenella haradae sp. nov. (Korea) ... 807

(mounted in water); F, cross section of ascoma with detail of ostiolum; G, H, young and mature

ascospores; I, apical part of non-amyloid ascus with a young ascospore. Scale bars: A = 1000 um;

B = 500 um; C, E, F = 100 um; D = 50 um; G-I = 10 um.

808 ... Halda & Hur

the thallus surface, pale brown in upper part. ExcipuLuM grayish to brownish

in the upper parts, hyaline below, 25-30 um thick in sides, 40-50 um at base;

subhymenium concave above, 20-30 um thick at base. HYMENIUM 300-320 um

high and 250-280 um wide. All hymenial elements I-, KI-. PERrpHyses absent,

PSEUDOPARAPHYSES septate, branched and anastomosing to form a network,

even in thickness (1 um). Ascr clavate, fissitunicate; endotunica universally

thick, lacking prominent ocular chamber, cylindrical, 8-spored, 80-100 x

12-15 um. Ascus apices thickened, non-amyloid, bilabiate (for terminology see

Eriksson 1981).

ASCOSPORES uniseriate, rarely biseriate in the ascus, (22—)25-30(-33) x

(10-)13-17(-17) um [means = 27.2 + 2.5 x 14.5 + 1.8 um (n = 56); 1/w ratio =

1.9], broadly ellipsoidal to oval, colourless at first, dark in maturity, muriform

(with 7 transverse and 2-3 longitudinal septa, 25-35 visible cells), lacking

perispore.

Pycnrp1A not seen in thalline warts and thallus sections.

ECOLOGY & DISTRIBUTION: Thelenella haradae is known only from the type

locality, Mt. Daebang, on an island off the southern coastline of the Korean

Peninsula, where it grows at low elevation on the vertical surface of exposed

rock (granite), together with Xanthoparmelia sp.

ComMENTSs— Thelenella luridella (Nyl.) H. Mayrhofer, known from Japan

(Harada 1999), differs from T: haradae by its colourless ascospores, its black

perithecial ostioles visible on thalline warts, the presence of pycnidia, and the

lack of a hypothallus. Thelenella melanospora Etayo & H. Mayrhofer, described

from Spain (Etayo & Mayrhofer 2003), differs by its smaller (18-22 x 9-12

um) ascospores, its paler coloured hypothallus, and its different substratum

(smooth bark of shrubs or small trees). Thelenella fernandeziana (Zahlbr.) H.

Mayrhofer, described from Juan Fernandez Islands (Mayrhofer 1987), differs by

its ascomata with dark pigmented ostiolum and its longer (30-45 x 15-22 um)

ascospores; and T. brasiliensis (Mull. Arg.) Vain., known from tropical South

and Central America, Africa, and China, differs by its narrower ascospores

(20-32 x 9-13um) and its emergent ascomata with a pale brown ostiolum

(Mayrhofer 1987).

Acknowledgments

This work was supported by Sunchon National University Research Fund in 2016.

Authors are grateful to Dr. Santosh Joshi (CSIR-National Botanical Research Institute,

India) and Prof. Pradeep K. Divakar (Universidad Complutense de Madrid, Spain) for

their valuable comments on the manuscript.

Thelenella haradae sp. nov. (Korea) ... 809

Literature cited

Aptroot A, Moon KH. 2014. 114 new reports of microlichens from Korea, including the description

of five new species, show that the microlichen flora is predominantly Eurasian. Herzogia 27:

347-365. http://dx.doi.org/10.13158/heia.27.2.2014.347

Eriksson O. 1981. The families of bitunicate ascomycetes. Opera Botanica 60: 1-209.

Etayo J, Mayrhofer H. 2003. Thelenella melanospora (Thelenellaceae, lichenized ascomycetes), a new

species from the Mediterranean region. Nova Hedwigia 77: 109-114.

http://dx.doi.org/10.1127/0029-5035/2003/0077-0109

Harada H. 1999. Thelenella luridella (lichenized Ascomycota, Thelenellaceae), newly found in Japan.

Journal of the Natural History Museum and Institute, Chiba 5(2): 91-95.

Mayrhofer H. 1987. Monographie der Flechtengattung Thelenella. Bibliotheca Lichenologica 26.

106 p.

Morse CA. 2016. Two new species of Thelenella and new reports from the Great Plains of central

North America, with a worldwide key to the genus. Opuscula Philolichenum 15: 22-36.

Orange A, James PW, White FJ. 2010. Microchemical methods for the identification of lichens. 2"¢

edition. British Lichen Society. 101 p.

MY COTAXON

October-December 2016—Volume 131, pp. 811-820

http://dx.doi.org/10.5248/131.811

Notes on rust fungi in China 2.

Two species of Coleosporium on Compositae

JING-XIN Jr?, QI WANG 1, ZHUANG L1?, Yu L1* & MAKOTO KAKISHIMA *3"

' Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi,

Jilin Agricultural University, Changchun, Jilin 130118 China

College of Plant Pathology, Shandong Agricultural University, Taian 271000 China

> University of Tsukuba, Tsukuba, Ibaraki 305-8572 Japan

* CORRESPONDENCE TO: kakishima.makoto.ga@u.tsukuba.ac.jp

AssTrRACT—During investigations of rust fungi in northeast China, specimens of

Coleosporium on various Compositae were collected. Based on comparative morphology and

molecular phylogenetic analysis specimens on Hieracium, Parasenecio, and Senecio were

identified as C. neocacaliae, and those on Ligularia, Saussurea, and Synurus as C. saussureae.

Hieracium umbellatum is a new host for C. neocacaliae, and Saussurea odontolepis and

S. pulchella are new hosts for C. saussureae. Coleosporium synuricola is treated as a synonym

of C. saussureae.

Key worps—Pucciniomycetes, rust flora, taxonomy, Uredinales

Introduction

The rust genus Coleosporium is characterized by 1-celled teliospores that

are produced in a 1-layered crust under host epidermal cells (Cummins &

Hiratsuka 2003). The teliospores germinate without dormancy and produce

4-celled internal basidia. About 240 species have been described (data from

MycoBank) and most of the species have a heteroecious life cycle producing

spermogonia and aecia on needles of pines and uredinia and telia on various

angiosperms (Kaneko 1981). In China about 45 species have been reported

(Miura 1928, Ito 1938, Tai 1979, Cao & Li 1999, You et al. 2010), but their

taxonomy and host plants are not well circumscribed and life cycles are still

not investigated.

During investigations of rust fungi in Jilin Province, northeast China, we

collected rust specimens on species of Compositae (Hieracium, Ligularia,

812 ... ji&al.

Parasenecio, Saussurea, Senecio, Synurus). These rust fungi have been referred

to the genus Coleosporium, but species identifications are sometimes confused

because of morphological similarities among species reported on these host

plants. After morphological examinations and phylogenetic analyses, specimens

on Hieracium, Parasenecio, and Senecio were identified as C. neocacaliae and

those on Ligularia, Synurus, and Saussurea were identified as C. saussureae.

Hieracium umbellatum L. was recorded as a new host plant for C. neocacaliae

and Saussurea odontolepis (Herder) Sch.-Bip. ex Herder and S. pulchella

(Fisch.) Fisch. were recorded as new hosts for C. saussureae. ‘The identity of

Coleosporium synuricola Y. Xue & L.P. Shao, described on Synurus deltoides

(Aiton) Nakai, was clarified, and it was shown to be identical to C. saussureae.

Materials & methods

Specimens

Specimens on Compositae were collected mostly in the vicinity of Changbai

Mountain, Jilin Province, China, from 2013 to 2015. Some specimens deposited

in the Herbarium of Mycology, Engineering Research Center of Chinese Ministry

of Education for Edible and Medicinal Fungi, Jilin Agricultural University, China

(HMJAU) were also examined. Their host plants were Hieracium umbellatum, Ligularia

fischeri (Ledeb.) Turcz., Parasenecio hastatus (L.) H. Koyama (= Cacalia hastata L.),

Saussurea odontolepis, S. pulchella, S. umbrosa Kom., Senecio nemorensis L., and Synurus

deltoides. The specimens used for morphological observations and molecular analysis

were deposited in HMJAU.

Morphological observations

Light microscopy (LM) was used to examine morphological characters including

the size and shape of sori and spores. Spores or thin-sections of sori from specimens

were mounted in a drop of lactophenol solution on glass slides for LM. Approximately

20 spores from each specimen were randomly chosen and the length, width, and wall

thickness of spores were measured using Leica LAS X software attached to a Leica

DM2000 microscope (Leica, Germany).

The surface features of spores were examined by scanning electron microscopy

(SEM). For SEM, samples obtained from dry specimens were attached to specimen

holders by double-sided adhesive tape and coated with platinum-palladium using a

Hitachi MC1000 Ion Sputter Coater and examined with a Hitachi SU8010 SEM operated

at 5-7 kV.

DNA extraction and sequencing

The total genomic DNA was extracted from about 200 spores obtained from a

single uredinium or telium. Spores were crushed between two sterilized glass slides

and suspended in 30 ul extraction buffer [10 mM Tris-HCl pH 8.3, 1.5 mM MgCl,

50 mM KCl, 0.01% sodium dodecyl sulfate (SDS), 0.01% Proteinase K], and the

suspensions were incubated at 37°C for 1 hour and 95°C for 10 min, followed by a

4°C soak (Suyama et al. 1996, Virtudazo et al. 2001). From the crude extract, 5-7 ul

Notes on Coleosporium neocacaliae & C. saussureae (China) ... 813

1,00/94 __ ©. S@ussureae [Soussurea odontolepis] HMJAUS8O9:

C. saussureae [Soussurea puichelia] HMJAU8161 | C. saussureae

0.99/68 C. synuri [Synurus deltoides} HMJAU8179

C. neacacatiae [Hieraciumum bellatum] HMJAU8237

1.0099) © neocacatiae [Hieraciumum betlatum] HMJAU8238 7

C. neocacaliae

C. neocacaliae [Senecio nemaresis] HMJAU80S8

0.92/90! ©. cacatiae [Syneilesis patmata] JF273972

C. plectranthi [Peritla frutescens] EFO95711

C. plumeriae [Plumeria sp.] KF879087

0.80/58 C. plumeriae [Pkimeria sp.] GU145555

Uredo sp. [Verbesina sp.] EU851161

C. asterum [TDB1465] AF522165

1.00/97 C. asterum [Solidago virgaurea) AB847107

C. solidaginis [Solidago sp.] DQ354559

C. asterum [Sofidage sp.] HQ317530

C. asterum [TOB1464] AF522164

0.97/61

0.98/71

C. elephantopi [Etephantopus mollis] EU851163

C. phellodendri (Phetlodendron amurense]KP017566

C, senecionis [Senecio sp.) KJ716348

C. phellodendri [Pheitodendron amurense]KP017566

C. senecionis (DB 1719] AY512840

C. campanutae [Campanula sp.] KP017565

C. tussilaginis [Senecio sp.] KT199395

0.98/60 | © tussilaginis [Tussitago farfara] AF426242

C, cacafiae [Adenostyles glabra] AF426243

C. evodiae [Tetradium glabrifotium] KP017567

C. zanthoxyli (Zanthoxylum sp.] KPO17568

C. phellodendri [Zanthoxylum ailanthoides] AB639023

Chrysomyxa arctostaphyli [Picea sp.] EF561640

FiGurE 1. Phylogenetic tree of Coleosporium. Bayesian 0.5 majority-rule consensus tree based on

the 28S RNA gene. Chrysomyxa arctostaphyli was used as outgroup. Values on the branches indicate

maximum likelihood bootstrap values / Bayesian posterior probabilities. Bootstrap values <75%

and Bayesian posterior probabilities <0.75 are not shown on the branches.

samples were used directly for each polymerase chain reaction (PCR). The rDNA-28S

region was amplified using primers NL1 (5’-GCATATCAATAAGCGGAGGAAAAG-3’) and

NL4 (5’-GGTCCGTGTTTCAAGACGG-3’) (O'Donnell 1993). The PCR amplifications were

performed in 50 ul of mixture containing 5 ul of template DNA, 200 un of each primer,

25 ul of Premix Taq™ (TaKaRa Taq™ Version 2.0 plus dye) (TaKaRa, Tokyo, Japan),

and 18 ul of ddH,O. Cycling conditions for amplification consisted of 94°C for 5 min,

followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and

extension at 72°C for 1 min, and a final extension at 72°C for 10 min. PCR products

were separated on 1% agarose gels containing Nucleic Acid Stain (Beijing Dinggou

Changsheng Biotechnology Co. Ltd.) and purified using the TaKaRa MiniBEST Agarose

Gel DNA Exaction Kit Ver.4.0. Purified PCR products were cloned in pEASY -T1

Cloning Vector (Transgene Biotech, Beijing, China) and then transferred into Trans1-T1

phage, resistant chemically competent cell according to the manufacturer's instructions.

The positive clones were sequenced by Sangon Biotech Co., Shanghai, China. All data

sequenced in this experiment were deposited at GenBank (KX344988- 344993)

Phylogenetic analysis

The sequence data were manually aligned using BioEdit ver. 7.0.9 (Hall 1999).

To make molecular comparison, 23 sequences were retrieved from GenBank based on

host species and genus (FIG. 1). Multiple alignments were performed using MEGA6.

The final dataset contained sequences from 29 specimens with a length of 610 bp, which

included 120 parsimony-informative characters.

814 ... ji &al.

Phylogenetic trees were constructed with a sequence of Chrysomyxa arctostaphyli

Dietel as the outgroup. Topologies were constructed based on maximum likelihood

(ML) analyses using RaxmalGUI1.5b1. Bayesian Markov chain Monte Carlo (MCMC)

analyses were performed using MrBayes ver. 3.1.2 (Huelsenbeck & Ronquist 2001).

In ML and Bayesian analyses, the best-fit substitution models were estimated using

Modeltest ver. 3.7 (Posada & Crandall 1998), and K81uf+I+G was selected as the best

evolutionary model.

Results & discussion

Specimens on Hieracium, Parasenecio, and Senecio

Specimens on H. umbellatum, P. hastatus, and S. nemorensis were

morphologically similar and their uredinial and telial characteristics were

identical to the descriptions of C. neocacaliae (Kaneko 1981, Azbukina 2005).

Molecular phylogenetic analysis of the rDNA 28S regions based on ML and

Bayesian inference placed the specimens on H. umbellatum (HMJAU8237 and

HMJAU8238) and S. nemorensis (HMJAU8098) in the same lineage (Fic. 1).

GenBank data of a rust from Korea recorded as C. cacaliae (DC.) G.H. Otth on

Syneilesis palmata (Thunb.) Maxim. [= Cacalia thunbergii Nakai] (JF273971)

FiGurRE 2. Coleosporium neocacaliae on Hieracium umbellatum. A. Telia on lower leaf surface.

B. A vertical section of a telium. C. Vertical section of uredinium. D. Urediniospores. Scale bars:

A =1cm; B-D = 20 um.

Notes on Coleosporium neocacaliae & C. saussureae (China) ... 815

was also included in this lineage. However, this plant was reported as a host of

C. neocacaliae (Kaneko 1981) and it is phylogenetically distinct from C. cacaliae

from Europe (Fic. 1, AF426243). Based on these specimens a description of

C. neocacaliae is provided.

FiGurRE 3. Coleosporium neocacaliae. A. Urediniospore on Hieracium umbellatum. B. Uredinium

on H. umbellatum. C. Surface of urediniospore on H. umbellatum. D. Urediniospore on Senecio

nemorensis. Scale bars: A, D = 5 um; B = 50 um; C = 2 um.

Coleosporium neocacaliae Saho, Trans. Mycol. Soc. Japan 7: 59, 1966. Figs 2, 3

Uredinia hypophyllous, scattered, orange-yellow, pulverulent, globoid;

urediniospores ellipsoid, broad-ellipsoid, 16.0-34.5 x 13.0-21.5 um (av. 24.5

x 16.5 um), walls uniformly thick 0.3-1.5 um (av. 1.0 um), verrucose with a

smooth reticulum-like spot. Telia hypophyllous, scattered, rounded, orange-

red; one-celled teliospores cylindrical, clavate or oblong-ellipsoid, 44-82

x 16-27 um excluding gelatinous apical layer, four-celled internal basidia

60-107 x 21-34 um, longitudinally or obliquely septate, mature teliospores or

basidia arranged in a single layer, gelatinous apical layer 8-10 um thick, wall

thin, hyaline.

SPECIMENS EXAMINED — CHINA, JILIN PROVINCE:

on Hieracium umbellatum: Changbai Mt., stage II, 29 July 2015 (HMJAU8237; GenBank

KX344992); stage III, 23 September 2015 (HMJAU8238; GenBank KX344993);

816... Ji &al.

on Parasenecio hastatus: Lushuihe, Baishan, stage II, 21 July 2002 (HMJAU8077, 8078,

8079); Changbai Mt., 29 July 2015, (HMJAU8263);

on Senecio nemorensis: Changbai Mt., stage 11, 23 July 2002 (HMJAU8098; GenBank

KX344991).

Coleosporium neocacaliae has been reported only on species of Cacalia and

Senecio from the Russian Far East, China, Korea, and Japan (Tai 1979, Kaneko

1981, Hiratsuka et al. 1992, Azbukina 2005), which makes H. umbellatum a

new host plant for C. neocacaliae. This species differs in telial morphology from

C. cacaliae and C. parvisporum S. Kaneko reported on species of Cacalia and

Senecio (Kaneko 1981). The 4-celled basidia of C. parvisporum are smaller than

those of C. neocacaliae. The 4-celled basidia of C. cacaliae have sterile cells

at their base, but those of C. neocacaliae have no sterile cell. Our molecular

analysis also confirms a phylogenetic difference between C. neocacaliae and C.

cacaliae (Fic. 1).

Specimens on Ligularia, Saussurea, and Synurus

Specimens on Ligularia fischeri, Saussurea odontolepis, S. pulchella, S.

umbrosa, and Synurus deltoides were morphologically similar and uredinial

and/or telial characteristics were identical with the descriptions of C. saussureae

(Kaneko 1981, Azbukina 2005). Our DNA sequence analyses also supported

specimens on Saussurea pulchella (HMJAU8161), S. umbrosa (HMJAU8091)

and Synurus deltoides (HMJAU8179) in the same linage (Fic. 1). Therefore,

these specimens were identified as C. saussureae. Rust on Synurus deltoides

was reported as C. synuricola (Xue & Shao 1995, Azbukina 2005). However,

we observed no morphological difference between specimens on Ligularia

and Saussurea and so consider C. synuricola a synonym of C. saussureae.

This synonymy is also supported by our molecular analysis. Based on these

specimens a description is provided.

Coleosporium saussureae Thiim., Bull. Soc. Imp. Nat. Moscow 55: 212, 1880.

Fics 4, 5

= Coleosporium. synuricola Y. Xue & L.P. Shao, Acta Mycol. Sinica 14: 248, 1995.

Uredinia hypophyllous, scattered, rounded, pulverulent, orange-yellow;

urediniospores ellipsoid, oblong-ellipsoid or subglobose 21.5-32.5 x 13.0-

20.0 um (av. 26.0 x 16.0 um), verrucose, with a smooth reticulum-like spot,

walls uniformly 0.5-2.0 um (av. 1.0 um) thick. Telia hypophyllous, scattered,

rounded, orange-red; one-celled teliospores obovoid to cylindrical, 48.5-88.5 x

18.0-30.0 um (av. 68.5 x 23.5 um) excluding gelatinous apical layer, four-celled

internal basidia 73.5-96.0 x 18.0-27.0 um (av. 81.5 x 22.5 um), longitudinally

or obliquely septate, mature teliospores or basidia arranged in a single layer,

adhering among teliospores, gelatinous apical layer 9.5-32.5 um thick, wall

Notes on Coleosporium neocacaliae & C. saussureae (China) ... 817

Ficure 4. Coleosporium saussureae. A. Telia on lower leaf surface of Synurus deltoides. B. Telia on

lower leaf surface of Saussurea pulchella. C. Basidiospores with smooth surface on Synurus deltoides.

D. Urediniospores on Saussurea umbrosa. E. Teliospores on Saussurea pulchella. F. Teliospores on

Synurus deltoides. Scale bars: A, B = 1 cm; C-F = 20 um.

thin, hyaline; basidiospores ellipsoid, 23.0-29.5 x 15.0-18.5 um (av. 25.5 x 16.5

um), wall thin, hyaline.

SPECIMENS EXAMINED — CHINA, JILIN PROVINCE:

on Ligularia fischeri: Yanbian, stages IJ, II, July 2015 (HMJAU8186);

on Saussurea odontolepis: Changbai Mt., stages II, IIH, 26 August 2014 (HMJAU8088);

27 August 2014 (HMJAU8089);

on S. pulchella: Changbai Mt., stage I], 29 July 2015 (HMJAU8161: GenBank

KX344988); stages II, III, 15 September 2013 (HMJAU8143); stage II, III, 16 September

2013 (HMJAU8163); stage II, III, 24 August 2014 (HMJAU8086); stage II, 24 August

2014 (HMJAU8087, 8090); stage II, 23 September 2015 (HMJAU8266, 8267); Yanbian,

818 ... Ji & al.

FicureE 5. Coleosporium saussureae. A. Urediniospore on Saussurea umbrosa. B. Urediniospore

on S. odontolepis. C. Urediniospore on S. pulchella. D. Uredinium on S. pulchella. Scale bars:

A-C =5 um; D=50 um.

stage II, IH, 30 July 2015 (HMJAU8165); stage I, III, 24 September 2015 (HMJAU8265);

Yanji, stage HI, 15 September 2013 (HMJAU8162, 8163, 8264); stage II, 17 September

2013 (HMJAU8 146);

on S. umbrosa: Changchun, stage II, III, 11 September 2014 (HMJAU8091; GenBank

KX344991);

on Synurus deltoides: Changbai Mt., stage II, 23 September 2015 (HMJAU8179;

GenBank KX344989).

Coleosporium saussureae has been reported on Ligularia and Saussurea

species and is known from the Russian Far East, China, Korea, Taiwan, and

Japan (Kaneko 1981, Hiratsuka et al. 1992, Azbukina 2005). Coleosporium

pedunculatum S. Kaneko was also described on species of Saussurea, but its

teliospores have long sterile cells at their base, whereas C. saussureae has no

sterile cell (Kaneko 1981). No sterile cells were observed in specimens on

Ligularia, Saussurea, and Synurus. Therefore, we identified them as C. saussureae.

Notes on Coleosporium neocacaliae & C. saussureae (China) ... 819

Ito (1938) and Tai (1979) listed Saussurea odontolepis and S. pulchella as host

plants of C. saussureae. Later, Kaneko (1981) segregated C. pedunculatum

from C. saussureae and treated them as host plants of C. pedunculatum, but

specimens on these plants had no sterile cells on the teliospore bases and so

were identified as C. saussureae. Therefore, these two plant species are new

hosts for C. saussureae as revised by Kaneko (1981).

Geographical distribution of C. saussureae differs from that of C. pedunculatum

due to the distributional differences of their aecial host species of Pinus (Kaneko

1981). Coleosporium pedunculatum is found mainly in warm regions, whereas

C. saussureae occurs in alpine and mountainous regions, reflecting distribution

of the aecial hosts. We collected specimens from Changbai Mt., an alpine (cold)

region in China. Therefore, this identification of specimens is also supported

by their distribution.

Coleosporium ligulariae Thiim. and C. zhuangii C.M. Tian & C.J You have

been recorded on species of Ligularia from China (You et al. 2010). These species

were reported to differ slightly in size and urediniospore surface structure from

C. saussureae, but they are morphologically similar to each other. Comparative

morphological studies of these species are needed to clarify their relationships.

Coleosporium synuricola [= Uredo synuri Azbukina] was described based

on a specimen collected on Synurus deltoides in Heilongjiang Province, China,

which is geographically close to Jilin Province (Kaneko 1981, Xue & Shao

1995, Azbukina 2005). This species is morphologically similar to C. saussureae

as reported by Kaneko (1981) and Xue & Shao (1995). We confirmed

morphological and phylogenetic identity between them based on the specimen

on S. deltoides. Therefore, we treat C. synuricola as a synonym of C. saussureae.

Acknowledgments

This work was financed by the National Natural Science Foundation of China

(No. 31470646). We wish to thank Dr. E.H.C. McKenzie (Landcare Research, Auckland,

New Zealand) and Dr. L.N. Vasilyeva (Institute of Biology and Soil Science, Far East

Branch of the Russian Academy of Sciences, Vladivostok, Russia) for critical reading

of the manuscript and suggestions. We express our thanks to Dr. B. Qu, Shenyang

Agricultural University for identification of plant specimens.

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Fungi, vol. 5. Dal’nauka, Vladivostok. 616 p. (In Russian)

Cao Z, Li Z. 1999. Rust fungi of Qinling Mountains. China Forestry Publishing House, Beijing.

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820 ... Ji & al.

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Ito S. 1938. Mycological flora of Japan, vol. 2, no 2. Yokendo, Tokyo.

Kaneko S. 1981. The species of Coleosporium, the causes of pine needle rusts, in the Japanese

Archipelago. Report of Tottori Mycological Institute 19. 159 p.

Miura M. 1928. Flora of Manchuria and east Mongolia II. Cryptogams, Fungi.

Minamimanshutetsudo, Dalian.

O'Donnell K. 1993. Fusarium and its near relatives. 225-233, in: DR Reynolds, JW Taylor (eds).

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from a fossil pollen of Abies spp. from Pleistocene peat. Genes & Genetic Systems 71: 145-149.

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MY COTAXON

October-December 2016— Volume 131, pp. 821-826

http://dx.doi.org/10.5248/131.821

Arthromoniliphora araucariae gen. & sp. nov.

from Brazilian pine

SILVANA SANTOS DA SILVA’, LUIS FERNANDO PASCHOLATI GUSMAO'*

& RAFAEL F. CASTANEDA-RUIZ?

‘Departamento de Ciéncias Biologicas, Universidade Estadual de Feira de Santana,

44031-460, Feira de Santana, BA, Brazil

?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: lgusmao@uefs.br

ABSTRACT—A new genus and species, Arthromoniliphora araucariae, is described and

illustrated. This fungus is distinguished by differentiated hyaline conidiophores that are

branched above the globose base and robustly cylindrical below, moniliform and subulate

toward the apex. The conidia are thallic-arthric, cylindrical, bacilliform to cuneate, and

hyaline and occur in unbranched chains formed by disarticulation of the moniliform,

subulate conidiophore branches.

Key worps—Araucaria angustifolia, asexual fungi, conidial fungi, taxonomy

Introduction

During a survey of hyphomycetes associated with plant litter in Floresta

Nacional de Sao Francisco de Paula, Rio Grande do Sul State, Brazil, an

interesting fungus was collected. This fungus shows remarkable differences

from all previously described genera (Seifert et al. 2011) and is therefore

described as a new genus and species.

Material & methods

Samples of litter of Araucaria angustifolia (Brazilian pine; Parana pine) were placed

in paper bags. In the laboratory the samples 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 (Castafeda-Ruiz 2005). Mounts were prepared in PVL (polyvinyl

822 ... Silva, Gusmao & Castafieda-Ruiz

alcohol, lactic acid, and phenol). 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 specimen is deposited in the Herbarium

of Universidade Estadual de Feira de Santana, Bahia, Brazil (HUEFS).

Taxonomy

Arthromoniliphora S.S. Silva, Gusmao & R.F. Castafieda, gen. nov.

MycoBAank MB 813427

Differs from Arthrographis and Geotrichum by its moniliform, closely fasciculate

conidiophores and its conidia with a highly variable number of septa.

TYPE SPECIES: Arthromoniliphora araucariae S.S. Silva et al.

EryMoLoecy: Greek Arthro-, meaning jointed (referring to thallic disarticulation) +

-monili- Latin, meaning cylindrical but contracted at regular intervals + -phora referring

to the conidiophores.

Conidial fungi. CoLoNres on the natural substratum effuse, white.

CONIDIOPHORES differentiated, single, densely fasciculate, branched,

moniliform, septate, hyaline. CONIDIOGENOUS HYPHAE moniliform, subulate,

holothallic, discrete, determinate. CONIDIAL SECESSION schizolytic. CONIDIA

thallic-arthric, in unbranched chains, unpigmented, unicellular or multiseptate,

cylindrical, doliiform or cuneate, forming by disarticulation of the terminal

moniliform conidiogenous conidiophore branches.

Arthromoniliphora araucariae S.S. Silva, Gusmao & R.F. Castafieda, sp. nov.

MycoBank MB 813428 FIGS 1-3

Differs from all Arthrographis and Geotrichum spp. by its densely fasciculate

conidiophores that are moniliform and cylindrical below and subulate above, and its

cylindrical, unicellular to multiseptate conidia.

Type: Brazil, Rio Grande do Sul State, Sao Francisco de Paula, Floresta Nacional de

Sao Francisco de Paula, 29°25’S 50°23’W, alt. 838 m, on decaying twig of Araucaria

angustifolia (Bertol.) Kuntze (Araucariaceae), 10.1II 2015, S.S. Silva (Holotype: HUEFS

216008).

EryMo_oey: Latin araucariae, meaning growth on an Araucaria species.

CoLonliEs on the natural substratum effuse, hairy, white. Mycelium superficial

and immersed. Hyphae septate, branched, smooth, hyaline, 3-5 um diam,

sometimes forming a rudimentary globulose stroma. CONIDIOPHORES

distinct, single, erect, compact fasciculate, branched just at one level near the

base, moniliform, subulate toward apex of each branch, hyaline, multiseptate,

60-250 x 5-6 um. CONIDIOGENOUS BRANCHES moniliform, subulate,

holothallic, multiseptate, hyaline, discrete, determinate, 38-100 x 2.5-4 um.

CONIDIAL SECESSION schizolytic. Conrp1a thallic-arthric, in unbranched

chains, hyaline, 0-36-septate, cylindrical, doliiform or cuneate, truncate at

Arthromoniliphora araucariae gen. & sp. nov. (Brazil) ... 823

Fic. 1. Arthromoniliphora araucariae (ex holotype, HUEFS 216008). A, B. Conidiophores,

coniiogenous branches, and conidia. C. Rudimentary stroma and basal part of conidiophores.

the ends, sometimes truncate at the base and obtuse at the apex, 3.5-92 x

2.5-4 um forming by disarticulation of the terminal, subuliform, moniliform

conidiogenous branches of the conidiophores

824 ... Silva, Gusmao & Castafieda-Ruiz

10 pom

—

rrrrrr?yYYT”TvTTSet

Fic. 2. Arthromoniliphora araucariae (ex holotype, HUEFS 216008).

Conidial chains and conidia.

CoMMENTS— The genera Arthrographis G. Cochet ex Sigler & J.W. Carmich., Geomyces

Traaen, and Geotrichum Link superficially resemble Arthromoniliphora in their

hyaline thallic-arthric conidia produced by disarticulation of fertile hyphae.

In Arthrographis, however, conidiophores are multi-branched, producing

Arthromoniliphora araucariae gen. & sp. nov. (Brazil) ... 825

Fic. 3. Arthromoniliphora araucariae (ex holotype, HUEFS 216008).

A. Conidiophores, conidiogenous branches, and conidia. B. Conidia.

826 ... Silva, Gusmao & Castafieda-Ruiz

conidia that are globose, cylindrical, Y-shaped or irregular (usually discoid),

rounded or subtruncate at the ends after disarticulation (Sigler & Carmichael

1976, Giraldo et al 2014). Geomyces has branched hyaline conidiophores,

but the conidia have separating cells and the conidial secession is rhexolytic.

Geotrichum is characterized by undifferentiated conidiophores, and after

disarticulation of the fertile hyphae the conidia are unicellular, cylindrical,

Y-shaped to irregular, sometimes forming chains, or slimy or yeast-like (Cole &

Kendrick 1969, Sigler & Carmichael 1976, Hoog et al. 2011, Seifert et al. 2011).

Thedgonia B. Sutton (Sutton, 1973) is somewhat similar to Arthromoniliphora,

but its conidiogenous cells were described as polyblastic and sympodial and

its conidia as blastocatenate, cylindrical, and 0-3-septate by Sutton (1973),

while Seifert et al. (2011) described the conidial ontogeny as thallic-arthric.

The Thedgonia conidiophores are branched and cylindrical but not moniliform

and subulate as in Arthromoniliphora

Acknowledgments

The authors express their sincere gratitude to Dr. De-Wei Li and Prof 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 “Programa de Pesquisa em Biodiversidade do Semiarido (PPBio Semiarido -CNPq/

MCTI) for supporting this study. 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

Castafieda Ruiz RF. 2005. Metodologia en el estudio de los hongos anamorfos. 182-183 in:

Anais do V Congresso Latino Americano de Micologia. Brasilia.

Cole GT, Kendrick WB. 1969. Conidium ontogeny in hyphomycetes. The arthrospores of

Oidiodendron and Geotrichum, and the endoarthospores of Sporendonema. Canadian Journal

of Botany 47: 1773-1780. https://doi.org/10.1139/b69-255

Giraldo A, Gené J, Sutton DA, Madrid H, Cano J, Crous PW, Guarro J, .2014. Phylogenetic

circumscription of Arthrographis (Eremomycetaceae, Dothideomycetes) Persoonia 32: 102-114.

https://doi.org/10.3767/003158514X680207

Hoog GS de, Guarro J, Gené J, Figueras MJ.2011. Atlas of clinical fungi, electronic version 3.1. CBS-

KNAW Fungal Biodiversity Centre, Utrecht, Netherlands

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

Biodiversity Series 9. 997 p.

Sigler L, Carmichael JW. 1976. Taxonomy of Malbranchea and some other hyphomycetes with

arthroconidia. Mycotaxon 4: 349-388.

Sutton BC. 1973. Some hyphomycetes with holoblastic sympodial conidiogenous cells. Transactions

of the British Mycological Society 61: 417-429.

https://doi.org/10.1016/S0007-1536(73)80112-3

MY COTAXON

October-December 2016— Volume 131, pp. 827-839

http://dx.doi.org/10.5248/131.827

Morphological and phylogenetic clarification of

Peziza arvernensis, P. pseudovesiculosa,

P. pseudosylvestris, and P. domiciliana

ANGELA LANTIERI? , GIANFRANCO MEDARDI? & PABLO ALVARADO?

'Via Novaluce 38, I-95030 Tremestieri Etneo (Catania), Italy

°Via Giuseppe Mazzini 21, I-25086 Rezzato (Brescia), Italy

4ALVALAB, La Rochela 47, 39012 Santander, Spain

* CORRESPONDENCE TO: angelalantieri@gmail.com

ABSTRACT—The concepts of the morphologically similar species Peziza arvernensis,

P. pseudovesiculosa, P. pseudosylvestris, and P. domiciliana are revisited in light of new

morphological and molecular data from freshly collected and herbarium specimens.

Phylogenetic inference based on ITS nrDNA sequences suggests that P arvernensis,

P. pseudovesiculosa, and P. pseudosylvestris are monophyletic and that P. domiciliana represents

an independent taxon.

Key worps—Pezizales, taxonomy

Introduction

The distinction between Peziza arvernensis (subject of previous

phylogenetic studies by Norman & Egger 1999 and Hansen et al. 2001, 2002),

P. pseudovesiculosa |= Aleuria amplissima Boud.], P. pseudosylvestris |= Galactinia

pseudosylvestris], and P. domiciliana is controversial because macro- and

microscopic morphological characters seem insufficient to unambiguously

define these taxa. Moreover, despite our several requests for samples of

P. pseudovesiculosa and P. pseudosylvestris to institutional and private herbaria,

we were able to obtain and examine only a few collections under these names.

Furthermore, P pseudovesiculosa and P. pseudosylvestris are inadequately

described (Gamundi 1975; Donadini 1977, 1978, 1979; Gamundi & Giaiotti

1998; Baiano et al. 2000; Moyne 2008). The two species seem to differ from

828 ... Lantieri, Medardi & Alvarado

P. arvernensis primarily in the thickness of the layers of the excipulum, while

all other characters overlap among the three species. Some authors (Hohmeyer

1986, Baiano et al. 2000) considered them to represent separate species, based

on the thickness of the excipulum layers and a few other macroscopical and

ecological features. The abundant observations reported by Donadini (1977,

1978, 1979, 1981) do not contribute significantly to a better delimitation of

these taxa, because the characters he used are not decisive.

In the present work, we obtained new morphological and molecular data to

clarify the phylogenetic position of these taxa, including: (1) Boudier’s original

material of Aleuria amplissima (PC 0084256, PC 0084257, PC 0084258),

analyzed also by Donadini (1977) when he proposed the replacement name

P pseudovesiculosa; (2) original specimens described as Galactinia

pseudosylvestris by Gamundi (1975); and (3) modern Italian specimens

representing P. domiciliana Cooke.

Materials & methods

Material studied

The study was carried out on both fresh and dried samples (including holotypes)

from the following herbaria—AMB, HMA, K, PC, MCVE, SIENA, TAAM, UPS—as

well as from several private collections. The thirteen herbarium specimens selected for

DNA extraction are listed in TABLE 1.

TABLE 1. Newly sequenced specimens of Aleuria, Galactinia, and Peziza used in

phylogenetic analysis

SPECIES COUNTRY COLLECTOR & HERBARIUM NO. ITS SEQUENCE

A. amplissima France E. Boudier PC 0084257 KC832902

France E. Boudier PC 0084258 KC832903

G. pseudosylvestris Argentina —_‘- I. Gamundi (UPS) F-570657 KC832900

Chile I. Gamundi (UPS) F-570665 KC832899

P. arvernensis Belgium herb. D. Tanchaud DT 26051301 KP125489

Italy herb. F. Padovan FP 84 KC832897

Italy herb. A. Lantieri AL 0406055; K(M) 134792 KC832896

P. domiciliana Italy herb. G. Medardi AL 11030266; MCVE 16472 KC832906

Italy herb. G. Medardi GM 27058844 KC832907

Italy herb. F. Padovan FP 2014 KC832904

P. pseudovesiculosa Italy herb. G. Medardi GM 16100447 KC832901

Italy herb. G. Medardi GM 22099945 KC832898

Italy herb. G. Medardi GM 28100346 KC832905

Clarification of four Peziza species ... 829

SPECIMENS EXAMINED:

Peziza arvernensis: BELGIUM: VALLONIA, Foret de Soignes Brabant Wallon, on the

ground in a deciduous wood, 26.05.2013, leg. & det. D. Tanchaud (DT 26051301;

GenBank KP125489). DENMARK: ZEALAND, Stenholdtsvang, Grib Skov, on sandy

soil, 14.08.1965, leg. P.M. Petersen, det. H. Dissing (TAAM 187941). ENGLAND:

HEREFORDSHIRE, Ross-on-Wye, on sandy soil, 19.06.1977, leg. & det. W.D. Graddon

(K(M) 42798); SuRREY, Sheepleas, West Horsley, on the ground in a deciduous wood

with Fagus, 12.10.1981 (K(M) 50729); West Sussex, Madehurst, on the ground under

deciduous trees, 2.07.1978, leg. D.A. Reid (K(M) 50718). ESTONIA: Tartu Co.,

along the road to Tallinn, on the ground in Fagus litter, 13.05.2007, leg. L. Tedersoo,

det. K. Hansen (TAAM 192338). ITALY: ABRUZZO, Prato di Camposecco, near woody

residue, 10.10.1984 (HMA 3754); Teramo, Intermesoli, Fonte Novello, Pietracamela, on

the ground in a Fagus wood, 20.06.2004 (HMA 6216); BAsILIcaTA, Potenza, Picerno,

Monti li Foi, on the ground in a Fagus wood near fallen wood, 24.10.1998, leg. & det.

G. Medardi (MCVE 14131); CaLasrtia, Reggio Calabria, Molochio, on the ground in

a mixed deciduous wood (mostly Fagus), 11.11.1988, leg. & det. F Padovan (FP 159);

Gambarie, near woody residue in a Fagus wood, 18.10.2003, leg. & det. G. Medardi

(MCVE 16614); near woody residue in a Fagus wood, 6.06.2010, leg. & det. B. Recchia

(BR 7386); CAMPANIA, Salerno, Camalonga, 11.06.2008, leg. G. Ruocco, det. L. Pecoraro

& P. Leonardi (SIENA 6546); EM1L1A-ROMAGNA, Reggio Emilia, Bergogno, Casina, on

the ground in a Fagus wood, 28.04.1991, leg. & det. G. Simonini (MCVE 7282); Lazio,

Terni, Trevi del Lazio, 20.07.1986 (HMA 4128); Roma, Cervara di Roma, 30.07.1986

(HMA 4127, as Peziza repanda); PIEMONTE, Vercelli, Alagna Valsesia, near woody

residue in a mixed deciduous wood, 25.08.1995, leg. & det. P.G. Jamoni (PGJ 931, as

Peziza pseudosylvestris); StcILy, Catania, Maletto, Parco dell’Etna, near woody residue

in a Fagus wood, 2.05.2004, leg. & det. A. Lantieri (AL); Messina, Cesard, Parco dei

Nebrodi, on the ground in a mixed deciduous wood, 4.06.2005, leg. & det. A. Lantieri

(K(M) 134792, AL 0406055; GenBank KC832896); TRENTINO-ALTO ADIGE, Belluno,

Monte Palmina, Canale d’Agordo, on the ground, 24.08.1996, leg. & det. E. Bizio

(MCVE 10690); Tuscany, Pisa, Calambrone, on sandy soil near Cistus, Juniperus and

Ammophila in marine environment, 26.04.1996, leg. & det. G. Medardi (MCVE 11261);

VENETO, Belluno, Campon, Bosco del Cansiglio, on the ground near Fagus, 25.07.1988,

leg. & det. F Padovan (FP 84; GenBank KC832897); Padola, on the ground, 21.08.2012,

leg. & det. A. Testoni (GM 21081216); Tambre, Pian Osteria, Loc. Canaie, under

deciduous, 7.07.2001, leg. G. Robich, det. E. Campo (MCVE 15980); Vicenza, Recoaro,

Malga Chempele, under deciduous, 5.06.2004, leg. & det. G. Medardi (MCVE 16797).

SCOTLAND: HIGHLAND, Inner Hebrides, Isle of Skye, Armadale Castle, on sandy soil,

24.05.1982, leg. & det. R.W.G. Dennis (K(M) 50726); on sandy soil, 27.09.1983, leg. &

det. R.W.G. Dennis (K(M) 50731); on sandy soil, 15.05.1984, leg. & det. R.W.G. Dennis

(K(M) 50730); PERTH AND Kinross, Birks of Aberfeldy, under deciduous, 2.08.2010,

leg. J. & D. Bailey, det. B.M. Spooner (K(M) 166578). SWEDEN: NARKE, Lanna, on the

ground, 12.07.1981, leg. & det. L. Sodeberg (UPS F-640159).

Peziza domiciliana: ITALY: ABRUZzzoO, Aquila, Chiarino, near woody residue of Fagus,

24.05.2000 (HMA 12); Provvidenza, 2.10.2000 (HMA 519); CALABRIA, Reggio Calabria,

Gambarie, near woody residue of Fagus, 7.11.1988, leg. & det. A. Para (AMB 000236,

as Peziza micropus); on the ground under Fagus, 9.12.2000, leg. & det. G. Medardi

(MCVE 15807); LomBarRpIA, Brescia, Ceto, on sandy soil under mixed deciduous

trees, 27.05.1988, leg. & det. G. Medardi (GM 27058844; GenBank KC832907);

VENETO, Belluno, near woody residue of Fagus, 15.04.1993, leg. & det. EF Padovan (FP

2014; GenBank KC832904), Padova, Monselice, on the ground under deciduous trees,

830 ... Lantieri, Medardi & Alvarado

5.03.1997, leg. & det. G. Medardi (MCVE 12412); SiciLy, Siracusa, R.N.O. Vendicari,

on sandy soil in a mixed wood, 11.03.2002, leg. & det. G. Medardi (MCVE 16472, AL

11030266; GenBank KC832906).

Peziza pseudovesiculosa: ITALY: EMILIA-ROMAGNA, Reggio Emilia, Marola, on

the ground under Fagus, 23.05.1997, leg. G. Baiano, det. D. Garofoli (MCVE 12816);

LOMBARDIA, Brescia, Manerba del Garda, 22.09.1999, leg. & det. G. Medardi (GM

22099945; GenBank KC832898); Calvagese, on the ground under Castanea, 4.11.2000,

leg. & det. G. Medardi (MCVE 15497); on the ground in a mixed deciduous wood

(mostly Carpinus and Alnus), also near burnt residue, 28.10.2003, leg. & det. G. Medardi

(GM 28100346; GenBank KC832905); Puegnago del Garda, under mixed deciduous

trees,16.10.2004, leg. & det. G. Medardi (GM 16100447; GenBank KC832901);

PIEMONTE, Alessandria, Vignole Borbera, fraz. Variano Superiore, under deciduous

trees, 22.04.2009, leg. & det. M. Carbone (MCVE 24022); Tuscany, Siena, Riserva di

Cornocchia, 26.05.2010, leg. H. Danielli, L. Pecoraro & E. Salerni, det. L. Pecoraro & E.

Salerni (SIENA 6547); VENETO, Belluno, Falcade, Caviola, near Fagus, 15.08.1984, leg.

& det. E. Bizio (EB 15088407).

Aleuria amplissima: FRANCE: Parts, fle-de-France, Val d’Oise, Bois de Beauchamp et

Forét P’'Isle-Adam, on sandy soil in the forest, s.d., leg. & det. E. Boudier (PC 0084256,

holotype); on sandy soil in the forest, 04.1889, leg. & det. E. Boudier (PC 0084258;

GenBank KC832903); on sandy soil in the forest, 04.1902, leg. & det. E. Boudier (PC

0084257; GenBank KC832902).

Galactinia pseudosylvestris: ARGENTINA: TIERRA DEL FuEGO, Ushuaia, on the

ground in a deforested area, 21.01.1940, leg. & det. I. Gamundi (UPS F-570656

[containing 6 collections]; UPS F-570657 [containing 4 collections]; GenBank

KC832900). CHILE: TIERRA DEL FUEGO, Magallanes y Antarctica Chilena, Rio Rubens,

on the ground in a Nothofagus pumilio forest, 11.01.1941, leg. & det. I. Gamundi (UPS

F-570666 [containing 10 collections]); Canal Whiteside, Puerto Yartou, on the ground

in a Nothofagus pumilio forest, 12.02.1941, leg. & det. I. Gamundi (UPS F-570665

[containing 4 collections]; GenBank KC832899).

Morphological studies

Measurements and descriptions of microscopic characters were made using material

mounted in water, rehydrated when necessary with 5% KOH. Melzer’s reagent and

Cotton blue in lactic acid were also used. An Optika BK 1301 microscope with 40x or

100x (immersion oil) objectives was used to study and photograph the morphological

features. Spore dimensions were calculated by measuring 50 mature spores.

DNA isolation, PCR, and sequencing techniques

Total DNA was extracted from dried specimens (TABLE 1) following a CTAB-based

procedure. 600 pl of 2X CTAB-buffer was added to a small piece of ascoma and blended

with the aid of a micropestle. It was then incubated at 65°C for 15 min; a volume of

chloroform: isoamylalcohol (24:1) was added; and the mixture was gently shaken to

emulsify both phases and centrifuged at 13,000 g.

One volume of isopropanol was added to the recovered supernatant, and the mixture

was centrifuged again at 13,000 g for 15 min. The resulting pellet was washed in 70%

ethanol, centrifuged for 2 min, dried, and finally eluted in 200 ul ddH,O.

PCR amplifications each comprised a 45 ul working volume to which 1-2 ul of

template DNA was added. Primer pairs ITS1F-ITS4, ITS1F-ITS2, or ITS3-ITS4 White

Clarification of four Peziza species ... 831

et al. (1990), and Gardes & Bruns (1993) were used to amplify nrDNA the ITS1 and IT2

(or ITS1+2) regions. Amplification protocols were: 95°C initiation for 5 min; 35 cycles

at 95°C for 45 s, 54°C for 30 s, and 72°C for 45 s; and a final step at 72°C for 10 min.

PCR products were checked in 1% agarose gel prior to purification and sequencing with

primers ITS1F or ITS4.

Chromatograms were visually checked in MEGA5 (Tamura et al. 2011) to detect

ambiguous nucleotides and sequencing problems. Consensus sequences were placed in

GenBank (TABLE 1). Newly generated sequences and their closest relatives in public

databases were incorporated into the alignment produced by Hansen et al. (2002)

stored in TreeBase (M1221), and only slight manual modifications were needed to

accommodate insertions. Final analyses included 902 positions, of which 316 were

parsimony-informative. A maximum parsimony (MP) search was performed in

PAUP*4.0b10 (Swofford 2001) using standard conditions (2000 bootstrap replications,

TBR swapping algorithm, 50 sequence additions per replicate, MULTREES not in

effect). 50% majority-rule consensus tree bootstrap values were annotated in the best-

scoring phylogram evaluated after a maximum-likelihood score analysis of all trees

recovered. Finally, a Bayesian analysis was performed in MrBayes 3.1 (Ronquist &

Huelsenbeck 2003), where the best-scoring model in MrModeltest 2.3 (Nylander 2004)

was simulated. Six chains were employed with temperature set to 0.2, sampling every

100th generation until convergence parameters were met after 2,150,000 generations.

Results & discussion

The analyses were congruent with each other in the suggested topology (Fic.

1) and similar to the phylogenetic relationships already reported for this group

(Hansen et al. 2002, Medardi et al. 2012).

Our results imply P pseudovesiculosa [= A. amplissima Boud.] and

P. pseudosylvestris as closely related to P arvernensis within a monophyletic

clade, sister to those formed by P. varia (Hedw.) Alb. & Schwein. and by

P. fimeti (Fuckel) E.C. Hansen and related taxa. Within this monophyletic

clade, the ambiguity of the taxonomic relationships among the different

lineages could be subject to conflicting interpretations. We choose to consider

them all as synonyms of P arvernensis (the oldest available name) because of

their insufficient genetic differences, the absence of diagnostic morphological

features, and the similarly high intraspecific variability seen in the sister species

P. varia. This synonymy can be summarised as:

Peziza arvernensis Roze & Boud., Bull. Soc. Bot. Fr. 26 (Suppl.): LX XVI. 1879.

= Peziza pseudosylvestris (Gamundi) Alessio, Micol. Ital. 4(2): 21. 1975.

= Galactinia pseudosylvestris Gamundi, Fl. Cript. Tierra del Fuego 10(3): 37. 1975.

= Peziza pseudovesiculosa Donadini, Bull. Soc. Linn. Provence 30: 60. 1977.

= Aleuria amplissima Boud., Hist. Class. Discom. Eur.: 44. 1907, nom. illeg.

(non A. amplissima (Fr.) Pat. 1904).

= Peziza amplissima Sacc. & Traverso, Syll. Fung. 20: 309. 1911, nom. illeg.

(non P. amplissima Fr. 1849).

= Galactinia amplissima Svréek, Ceska Mykol. 16: 110. 1962.

832 ... Lantieri, Medardi & Alvarado

AF491629 Peziza ampelina

1.00/100; AF491627 Peziza subcitrina

AF491628 Peziza subcitrina

KC832899 Galactinia pseudosylvestris

KC832900 Galactinia pseudosylvestris

' KC832901 Peziza pseudovesiculosa

4.00/74 ‘ KC832902 Aleuria amplissima

KC832903 Aleuria amplissima

AF491580 Peziza arvernensis

AF491579 Peziza arvernensis

JQ654489 Peziza fimeti

ALV3919 Peziza arvernensis

KC832898 Peziza pseudovesiculosa

0.98/87

0.98/78

| | KC832897 Peziza arvernensis

KC832896 Peziza arvernensis

| | JF908538 Peziza varia

1.00/98

JF908569 Peziza arvernensis

AF491581 Peziza arvernensis

AF491583 Peziza arvernensis

AF491582 Peziza arvernensis

AF491584 Peziza arvernensis

AF491585 Peziza arvernensis

AF491577 Peziza arvemensis

-00/97 ' AF491578 Peziza arvermensis

1.00/83 AF491587 Peziza sp. (C)

AF491586 Peziza sp. (C)

JF908560 Peziza varia

AF491554 Peziza varia

AF491553 Peziza varia

1.00/99} AF491550 Peziza varia

AF491552 Peziza varia

AF491551 Peziza varia

AF491544 Peziza varia

AF491545 Peziza varia

AF491549 Peziza varia

1.00/100} AF491546 Peziza varia

AF491548 Peziza varia

AF491547 Peziza varia

AF491555 Peziza varia

AF491556 Peziza varia

Lf AF491557 Peziza varia

00/100 * AF491558 Peziza varia

AF491559 Peziza varia

AF491565 Peziza varia

AF491561 Peziza varia

AF491562 Peziza varia

AF491568 Peziza varia

1.00/93) } AF491567 Peziza varia

1100/73 AF491569 Peziza varia

AF491570 Peziza varia

AF491563 Peziza varia

|| AF491564 Peziza varia

AF491566 Peziza varia

AF491560 Peziza varia

~ KC832904 Peziza cf. domiciliana

AF491573 Peziza echinispora

1.00/100} AF491574 Peziza echinispora

KC832905 Peziza pseudovesiculosa

AF491575 Peziza echinispora

AF491571 Peziza sp.

1.00/100' AF491572 Peziza sp.

-00/100

Clarification of four Peziza species ... 833

AF491610 Peziza alcis

AF491611 Peziza alcis

AF491612 Peziza alcis

AF491613 Peziza sp. (F)

AF491608 Peziza sp. (E)

1.00/100 ' AF491607 Peziza sp. (E)

AF491588 Peziza ampliata

AF491589 Peziza ampliata

AF491590 Peziza ampliata

AF491592 Peziza ampliata

~ AF491591 Peziza ampliata

AF491614 Peziza domiciliana

AF491593 Peziza fimeti

AF491594 Peziza fimeti

AF491595 Peziza fimeti

AF491596 Peziza fimeti

AF491597 Peziza fimeti

AF491604 Peziza fimeti

0.99/100 1.00 ia AF491605 Peziza fimeti

* AF491606 Peziza fimeti

7 AF491602 Peziza sp. (H)

AF491603 Peziza sp. (I)

| AF491598 Peziza fimeti

AF491600 Peziza fimeti

AF491601 Peziza fimeti

AF491599 Peziza fimeti

~ AF491609 Peziza sp. (B)

AF491617 Peziza sp. (A)

son

1.00/100 1.00/100

AF491615 Peziza sp. (A)

AF491616 Peziza sp. (A)

AF491618 Peziza sp. (G)

L_{ AF491620 Peziza nivalis

1.00/100 - AF491619 Peziza nivalis

KC832906 Peziza domiciliana

oon KC832907 Peziza domiciliana

0.99/100 | JF908561 Peziza domiciliana

——~ AF491621 Peziza ammophila

1.00/100 —- AF491622 Peziza ammophila

| AF491623 Peziza vesiculosa

AF491624 Peziza vesiculosa

1.00/100 | AF491625 Peziza vesiculosa

AF491626 Peziza vesiculosa

1.00/100

0.1

Fic. 1. Consensus ITS phylogram of Peziza s. str. obtained in MrBayes 3.1. Bayesian posterior

probabilities and maximum parsimony bootstrap proportions are presented for nodes significantly

supported by at least one of these analyses. Our new sequences are set in bold font.

TABLES 2-5 summarise morphological data for P arvernensis, P. pseudo-

vesiculosa, P. pseudosylvestris, and P. domiciliana, and make comparisons

between data from the literature and from our study.

834 ... Lantieri, Medardi & Alvarado

TABLE 2. Peziza arvernensis: comparison of cited and new morphological data

CHARACTER ROZE & BOUDIER (1879) CURRENT STUDY

APOTHECIUM — Regularly cup-shaped to Often rather deep cup-shaped, tending to open in

undulate, fragile, rather thick, age, fragile, rather thick, sessile

subsessile

HyMENIUM Ochraceous-ferruginous with + Pale brown, brown-beige, sometimes pale tobacco,

olivaceous tints at times with reddish-violaceous or fawn tints

RECEPTACLE Ochraceous-ferruginous with Concolorous with the hymenium or greyish

olivaceous tints, slightly ochraceous-fawn with rust tints, tending to lighten

furfuraceous. Base with white when dried. Smooth to slightly furfuraceous

tomentum

ASCOSPORES 16-18 x 8-9 um, finely warted 16-19(-20) x 9-10(-11) tm, finely warted

ASCUS (not reported) 270-280 x 15-16 um

PARAPHYSES Clavate above, septate, slightly Clavate, 6-8 ttm above; subcylindrical,

coloured, with several oil 3-4 um below; septate

droplets

EXCIPULUM (not reported) SH: text. globulosa-angularis, cells 13-24 um diam.

ME: 3-layered (Upper & Lower layers: text.

globulosa, cells 35-90 um diam;

Intermediate: text. intricata, hyphae 5-10 um diam

EE: text. intricata, hyphae 5-10 um diam,

mixed with globose cells 15-20 um diam

HABITAT Ground, also between mosseson —_ Ground, also sandy soil; marine to mountain

one thatch environment, under deciduous trees (Fagus,

Quercus), at times also near burnt residue or on

woody remnants

SH = Subhymenium; ME = Medullary excipulum; EE = Ectal excipulum

The microscopical analyses of Aleuria amplissima revealed spores measuring

14—16(-18) x 8-9 um, finely warted, with multiple small internal guttules

and asci measuring 270-300 x 14-16 um. We observed an ectal excipulum

composed of septate hyphae 9-12 um in diam. arranged in a textura intricata,

more or less perpendicular to the hymenium and parallel to one another,

instead of the textura globulosa-angularis reported by Donadini (1977, 1978,

1979) and Baiano et al. (2000). These characters are remarkably similar to those

observed in several P. arvernensis collections: our observations of more or less

finely warted spores of 16-19(-20) x 9-10(-11) um and asci 270-280 x 15-

16 um) agreed with those by Korf (1985). We also noticed a similar excipular

structure and spore measurements in original material of A. amplissima (PC

0084256, PC 0084257, PC 0084258) and in G. pseudosylvestris.

Since the type material of G. pseudosylvestris in LPS was not available for

study, other specimens collected and identified by Gamundi were analyzed

Clarification of four Peziza species ... 835

TABLE 3. Peziza pseudovesiculosa: comparison of cited and new morphological data

CHARACTER DoNADINI (1977) CURRENT STUDY

depenenncrane Cup-shaped, rarely opened, fragile, Cup-shaped, tending to open in age,

thick, sessile or nearly so fragile, + thick, sessile or nearly so

+ Pal nb ,

HyMENIUM Ochraceous to ochraceous-brown a brown Ate

with some reddish tints

Paler than the hymenium, + Concolorous with the hymenium,

RECEPTACLE

furfuraceous furfuraceous

ASCOSPORES 15-18 x 8-10 um, finely warted 16-19 x 9-10(-11) wm, finely warted

ASscus 240-270 x 14-16 um 270-280 x 15-16 um

Cylindrical, 3-4 um below; Subcylindrical, 3-4 tum below;

PARAPHYSES

clavate, 5-10 um above; septate clavate, 6-8 tum above; septate

EXCIPULUM SH: text. globulosa-angularis, SH: text. globulosa-angularis,

cells 7-15 x 15-20 um cells 10-25 pm diam.

ME: 3-layered (Upper: text. globulosa- ME: 3-layered (Upper: text. globulosa,

angularis, cells 15-90(-130) um diam.; cells 30-80 tum diam;

Intermediate: text. intricata & porrecta, Intermediate: text. intricata,

hyphae 6-12 um diam; Lower: text. hyphae 5-10 um diam;

globulosa, cells 30-80 um diam.) Lower: as first layer)

EE: text. globulosa, cells 15-25 um diam. _ EE: text. intricata, hyphae 5-10 um diam,

with protruding hyphae 7-17 um diam. mixed with globose cells 12-17 um diam.

HABITAT Humus, woody remnants, burn residue Humus, woody remnants, burn residue

SH = Subhymenium; ME = Medullary excipulum; EE = Ectal excipulum

(UPS-F570656, UPS-F570665, UPS-F570666, UPS-F570657). These samples

showed spores measuring (15—)16—19(—19.5) x 8.5-11 um, with punctiform

warts ca. 0.5 x 0.5 um and asci <280-320 x 12-14 um. Gamundi (1975)

argued that G. pseudosylvestris could be distinguished from P arvernensis by

its thicker excipulum, especially the upper and lower layers of the medullar

excipulum. Development of more or less wide cells in the excipulum and of

moniliform or thread-like paraphyses seems, however, to be correlated with a

more or less high level of humidity, as stated by Kullman (1995) and Hansen

et al. (2002). Nonetheless, we observed an average thickness ca. 1500-1600

um in all rehydrated samples, in line with the measurements observed in

“typical” P arvernensis. We conclude that this species does not represent an

independent lineage at the species level, but should be considered a synonym

of P. arvernensis.

Most authors mentioned that P arvernensis sensu stricto is typically

associated with Fagus sylvatica L. (fruiting on soil, on litter, or near woody

836 ... Lantieri, Medardi & Alvarado

TABLE 4. Peziza pseudosylvestris: comparison of cited and new morphological data

CHARACTER GAMUNDI 1975; DONADINI 1978 CURRENT STUDY

APOTHECIUM Cup-shaped, then more opened, _

subsessile to stalked

HyYMENIUM Pale ochraceous, pale hazel, 7

ochraceous to olivaceous

RECEPTACLE Concolorous to paler than the hymenium, iF

furfuraceous

ASCOSPORES 14.1-18.2 (-19.2) x 7.2-9.6 um, (15-)16-19 (-19.5) x 8.5-11 um,

finely warted finely warted

Ascus 240-355 x 9.6-14.4 um 280-320 x 12-14 um

PARAPHYSES Thread-shaped, 2.4-4.3 tum large, septate, Subcylindrical, 3-4 um below;

containing ochraceous granules clavate, 6-7 um above; septate

EXCIPULUM SH: text. globulosa, SH: text. globulosa-angularis,

cells 8.4-15.6 um diam. cells <20 um diam.

ME: 3-layered (Upper: text. globulosa, cells ME: 3-layered (Upper: text. globulosa-

50-120 pm diam; Intermediate: text. angularis, cells up to 80 um diam;

intricata, hyphae 4.8-9.6 tum diam; Lower: Intermediate: text. intricata, hyphae

text. globulosa, cells 30-96 um diam.) 8-10 um large; Lower: as first layer)

EE: text. intricata, hyphae 6-9.6 um diam EE: text. intricata, hyphae <5 um diam,

mixed with globose cells 15-20 um diam.

HABITAT Ground, near woody residuals of Nothofagus —

SH = Subhymenium; ME = Medullary excipulum; EE = Ectal excipulum

remnants), but our observations show the habitat of P. arvernensis (both sensu

stricto and sensu lato) to be quite variable: in addition to Nothofagus, some

specimens have been collected under Castanea (MCVE 15497), Carpinus,

Alnus, and other mixed deciduous trees in a floodplain environment (GM

28100346); Quercus ilex L. (AL 0406055); typical Mediterranean vegetation

near Cistus, Juniperus, and Ammophila in a marine retro-dunal environment

(MCVE 11261); and on ground with burn residues (MCVE 15497).

Peziza arvernensis sensu lato may also be confused with P. domiciliana,

which has very similar features: a light umber-brown hymenial surface, paler

(whitish-ochraceous to sub-concolorous) outer surface, spores measuring 15—17

(—20) x 7—9(—10) um that are both smooth and finely warted even in the same

apothecium when ejected from the asci, a similar flesh architecture, and a

substrate of soil, loam, or decaying wood. Sequences of two P. domiciliana

collections (GM 27058844, AL 11030266) seem to match a sequence of another

P. domiciliana stored in GenBank (JF908561; Garbellotto et al., unpubl.) that

represents a lineage distinct from P arvernensis (Fic. 1), thus supporting

Clarification of four Peziza species ...

TABLE 5. Peziza domiciliana: comparison of cited and new morphological data

837

CHARACTER COOKE 1877 CURRENT STUDY

APOTHECIUM Quite regularly cup-shaped, Cup-shaped, tending to open in age, fragile,

then cochleate and contorted, rather thick, sessile or nearly so

fragile

HyMENIUM Hues ranging from white to Amber-brown, honey coloured

amethystine violet;

sometimes rose-tinged

RECEPTACLE Concolorous Paler than the hymenium,

whitish-ochraceous

ASCOSPORES 11-13 x 6-7 um, 15-17(-20) x 7-9(-10) pm,

ornamentation not described smooth to finely warted

ASCUS Not reported 270-300 x 13-15 um

PARAPHYSES Not reported Subcylindrical 2--3 tum below,

clavate 5--6 tum above, septate

EXCIPULUM Not reported SH: text. globulosa-angularis,

cells <10-15 um diam.

ME: 3-layers:

Upper & lower: text. globulosa, cells <50 um diam;

Intermediate: text. intricata, hyphae 5-10 um diam

EE: text. intricata, hyphae <5-10 um diam

HABITAT In a garden Sandy or clay soil, loam, sawdust or other woody

residue: also in cellars or caves

SH = Subhymenium; ME = Medullary excipulum; EE = Ectal excipulum

recognition of P domiciliana as an independent species in accordance with

Hansen et al. (2002). However, it should be noted that the sample of P. domiciliana

sequenced by Hansen et al. (2002; AF491614) seems different from JF908561

and those sequenced in the present work and more closely related to the lineage

formed by Peziza ampliata Pers., P. fimeti, and Peziza alcis Harmaja.

Hansen et al. (2002) noted that both P domiciliana and P. arvernensis could

be confused with P. varia. However, the spores in P varia were observed by

Medardi et al. (2012) as slightly smaller (14-16(-17.5) x 9-11(—12) um), and

the ornamentation in P varia, very difficult to observe with a conventional light

microscope in either surface or outline view, is readily visible in P domiciliana and

P. arvernensis. Nevertheless, one of our collections determined morphologically

as P. domiciliana (FP 2014) fell instead in the clade of P. varia.

Acknowledgments

We wish to thank Prof. D.H. Pfister (Massachusetts, USA), who checked the English of

the text; Prof. Pfister, Prof. Gabriel Moreno (Spain), and Dott. C. Losi (Italy) for critically

838 ... Lantieri, Medardi & Alvarado

reviewing the manuscript; the curators of the institutional Herbaria AMB, HMA, K, PC,

MCVE, SIENA, TAAM, and UPS for arranging loans of material; and E. Bizio, P.G. Jamoni,

FE Padovan, B. Recchia, and A. Testoni for putting their collections at our disposal. We

want to express a particular gratitude to Dr. B. Buyck (PC) and Dr. S. Ryman (UPS) for

granting permission to carry out molecular analysis on their samples.

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MY COTAXON

October-December 2016— Volume 131, pp. 841-846

http://dx.doi.org/10.5248/131.841

Podosporium bacilliforme sp. nov. and

a first record of Phaeoblastophora peckii

from southern China

JIn- YE WANG’, Kat ZHANG’, CHUN-LING YANG}, JI- WEN X1A3, YING-RuI Ma3,

JIAN-MEI GAO, XIANG-YU LB, X1U-GUO ZHANG} & YU-MEI CAI”

‘College of Animal Science and Veterinary Medicine, Shandong Agricultural University,

Sino-German Cooperation Research Centre for Zoonosis of Animal Origin Shandong Province,

Taian, Shandong, China

?Department of Landscaping, Shandong Yingcai University, Jinan 250104, China

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

*CORRESPONDENCE TO: sdau613@163.com, caiyum@163.com

ABSTRACT—Podosporium bacilliforme is described and illustrated from specimens collected

on dead branches in Guangxi Province. The fungus is characterized by black synnematal

conidiomata and monotretic clavate conidiogenous cells producing solitary elongate-

bacilliform phragmoconidia. Phaeoblastophora peckii is recorded for the first time in China.

Key worps—conidial fungi, taxonomy

Introduction

Decaying wood in the tropical and subtropical forests of southern China

supports a high level of fungal diversity, and several wood-inhabiting

macrofungi and microfungi have been recently discovered there (Gao et al.

2015; Ma et al. 2011, 2014, 2015, 2016; Ren et al. 2012; Xia et al. 201 4a, b, c;

Zhang et al. 2008, 2011). During our ongoing surveys of saprobic fungi

associated with woody debris in Guangxi and Hainan provinces, two interesting

hyphomycetes were collected on dead branches. From morphological

and developmental characteristics, we determined that one of these is an

undescribed synnematous species of Podosporium, while the other represents a

first record of Phaeoblastophora peckii for China.

842 ... Wang & al.

Material & methods

Samples were processed, examined and photographed following the methods

described in Xia et al. (2014b). Adobe Photoshop 7.0 was used to process the photographs

into compound images, with backgrounds modified for esthetic reasons. In view of our

failure to obtain cultures of this species, no molecular data are presented here. The

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).

Podosporium bacilliforme J.Y. Wang, Yu M. Cai & X.G. Zhang, sp. nov. FIG. 1

MycoBank MB 819468

Differs from all other Podosporium spp. by its larger bacilliform or elongated bacillifom

conidia with many more eusepta.

Type: China, Guangxi Province: Tianyang county, Baiyao village, on dead branches of

unidentified bamboo, 15 Nov. 2015, J.Y. Wang (Holotype, HSAUP H10119; isotype,

HMAS 245637).

EryMo_oey: From the Latin bacilliforme, referring to the conidial shape.

Co.toniEs on the natural substratum effuse, brown. Mycelium mostly immersed,

composed of septate, pale brown, flexuous branched hyphae. SYNNEMATA erect,

cylindrical, black, solitary or in groups of 2-3, scattered, 470-720 um long,

14-45 um wide, up to 70 um wide at the apical swollen region, expanded at the

base. CONIDIOPHORES erect, arranged in synnemata, brown, smooth, septate,

sometimes branched at the apex. CONIDIOGENOUS CELLS monotretic, clavate or

cylindrical, integrated and discrete, terminal, determinate, pale brown, 5-8 x

3.5-4.9 um. Conip1a solitary, dry, smooth, bacilliform or elongate-bacilliform,

base truncate, apex rounded, brown to dark brown, 77-310 x 4.5-6.0 um,

9-43-euseptate, slightly constricted at the septa.

COMMENTS—Podosporium was erected by Schweinitz (1832) and typified

by P. rigidum Schwein. The genus is characterized by subulate or cylindrical,

darkly pigmented synnemata with monotretic, percurrent conidiogenous cells

scattered along their length (Ellis 1971). More than 60 Podosporium species

have been described (Index Fungorum 2016).

Podosporium bacilliforme bears some resemblance to P elongatum J.L.

Chen & Tzean, P. etheldoidgeae Crous et al., and P. nilgirense (Subram.) M.B.

Ellis in conidial shape. However, the conidia of P elongatum (62-188 x

6-10 um, 8-21-septate; Chen & Tzean 1993), P. etheldoidgeae (32-85 x 6-9 um,

4-12-septate; Crous et al. 1995) and P. nilgirense (32-50 x 7-9 um, 4-6-septate;

Fic. 1. Podosporium bacilliforme (holotype, HSAUP H10119): A, B. Synnematous conidiomata,

conidiogenous cells, and conidia. C, D. Conidia. E. Conidiogenous cells with conidia.

E Conidiogenous cells.

843

Podosporium bacilliforme sp. nov. (China) ...

844 ... Wang & al.

Ellis 1976) are much smaller and have many fewer septa than those in

P. bacilliforme. Moreover, the terminal conidiogenous cells in P. bacilliforme

clearly separate it from P etheldoidgeae, which is not acropleurogenous.

@9 00

Sse

Va)

20um

Fic. 2. Phaeoblastophora peckii (HSAUP H7519): A. Conidiophores, conidiogenous cells, and

conidia. B. Conidia. C. Conidiogenous cells and conidia.

Phaeoblastophora peckii (Sacc. & P. Syd.) Partr. & Morgan-Jones,

Mycotaxon 83: 342 (2002)

Cotonigs effuse, dark brown to black. MyceLium partly superficial,

immersed, composed of branched, septate, brown to dark brown, smooth,

FIG. 2

Podosporium bacilliforme sp. nov. (China) ... 845

3-7 um wide hyphae. ConrpiopHores single, distinct, arising as lateral

branches, ascending, erect, straight or slightly flexuous, unbranched, cylindrical,

inflated toward the extreme apex, brown to dark brown, slightly darker

in the distal part, smooth, <280 um long, 4.5-8 um wide. CONIDIOGENOUS

CELLS monoblastic or mostly polyblastic, integrated, terminal or intercalary,

cylindrical, broadly clavate to capitate. Conip1A acropleurogenous, simple or

branched acropetal chains, dry, unicellular, smooth, sub-globose to ellipsoidal,

dark brown, generally paler when young, usually narrowly truncate at one or

both ends, unthickened at the ends, sub-globose 5-7 um diam, ellipsoidal,

7-13 X 5.5-7 um.

SPECIMEN EXAMINED: CHINA, HAINAN PROVINCE: Diaoluoshan, on dead branches of

unidentified tree, 10 May 2014, X.Y. Li (HSAUP H7519).

ComMENtTS—Phaeoblastophora peckii was originally described from America.

Our specimen fits well with the description of the type material (Partridge &

Morgan-Jones 2002), although the conidiophores of the Chinese specimen are

slightly longer (280 um vs 200 um). Therefore, we believe they are the same

species.

Acknowledgments

The authors express gratitude to Dr. Bryce Kendrick and Dr. Rafael F. Castafeda-

Ruiz 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, 31200013) and the Ministry of Science and

Technology of the People’s Republic of China (Nos. 2006FY 120100).

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new record of Bahusutrabeeja angularis from southern China. Mycotaxon 130(3): 821-825.

http://dx.doi.org/10.5248/130.821

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Ma J, Xia JW, Zhang XG, Castafieda-Ruiz RF. 2014. Arachnophora dinghuensis and Websteromyces

inaequale sp. nov., and two new records of anamorphic fungi from dead branches of broad-

leaved trees in China. Mycoscience 55: 329-335. http://dx.doi.org/10.1016/j.myc.2013.11.007

846 ... Wang & al.

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genus of hyphomycetes from Hainan, China, with relationship to Chaetothyriales. Mycological

Progress 14: 77 [5 p.]. http://dx.doi.org/10.1007/s11557-015-1071-x

Ma YR, Xia JW, Gao JM, Li Z, Zhang XG. 2016. Anacacumisporium, a new genus based on

morphology and molecular analyses from Hainan, China. Cryptogamie, Mycologie 37(1):

45-59. http://dx.doi.org/10.7872/crym/v37.iss1.2016.45

Partridge EC, Morgan-Jones G. 2002. Notes on hyphomycetes LXX XVIII: New genera in which

to classify Alysidium resinae and Pycnostysanus azaleae, with a consideration of Sorocybe.

Mycotaxon 83: 335-352.

Ren SC, Ma J, Ma LG, Zhang YD, Zhang XG. 2012. Sativumoides and Cladosporiopsis,

two new genera of hyphomycetes from China. Mycological Research 11: 443-448.

http://dx.doi.org/10.1007/s11557-011-0759-9

Schweinitz LD von. 1832. Synopsis fungorum in America boreali media degentium. Transactions

of the American Philosophical Society 4(2): 141-316.

Xia JW, Ma LG, Ma J, Zhang XG. 2014a [“2013”]. Two new species of Spadicoides from southern

China. Mycotaxon 126: 55-60. http://dx.doi.org/10.5248/126.55

Xia JW, Ma LG, Castafieda-Ruiz RF, Zhang XG. 2014b. Minimelanolocus bicolorata sp. nov.,

Paradendryphiopsis elegans sp. nov. and Corynesporella bannaense sp. nov. from southern

China. Mycoscience 55: 299-307. http://dx.doi.org/10.1016/j.myc.2013.11.003

Xia JW, Ma LG, Castafeda-Ruiz RF, Zhang XG. 2014c. A new species of Sporidesmiopsis and three

new records of other dematiaceous hyphomycetes from southern China. Nova Hedwigia

98(1-2): 103-111. http://dx.doi.org/10.1127/0029-5035/2013/0145

Zhang K, Ma J, Zhang XG. 2008. Two new species of Corynespora from Hainan, China. Mycotaxon

104: 159-163.

Zhang YD, Ma J, Ma LG, Zhang XG. 2011 [“2010”]. A new species of Podosporium and a new

record from southern China. Mycotaxon 114: 401-405. http://dx.doi.org/10.5248/114.401

MY COTAXON

October-December 2016—Volume 131, pp. 847-857

http://dx.doi.org/10.5248/131.847

Apiosordaria hamata sp. nov. from lake sediment in China

BING Wt’, JIAN-QING TIAN’, LIN WANG’,

Jran-Kui Liu’, Kevin D. HYDE”? & JING-ZuU SUN’?

‘State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences,

No. 3 Park 1, Beichen West Road, Chaoyang District, Beijing 100101, China

? Centre of Excellence in Fungal Research, Chiang Rai 57100, Thailand

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

* CORRESPONDENCE TO: sunjz@im.ac.cn;

ABSTRACT—A new species, Apiosordaria hamata, is described and illustrated from sediment

collected from Donghu Lake, Wuhan, Hubei Province, China. Apiosordaria hamata is

characterized by its cleistothecial ascomata with agglutinated hairs, a thin peridium, clavate

asci, and two-celled, biseriate, ovoid ascospores. However, it differs from other Apiosordaria

species in having hooked spines on the upper cell of the ascospores and lacking germ pores.

Combined ITS and LSU sequence analyses using maximum likelihood and Bayesian analysis

nest A. hamata in the Apiosordaria clade. Both morphological and molecular analyses support

this fungus as a new species of Apiosordaria. A key to Apiosordaria species is provided.

Key worps—phylogenetic analyses, soil fungi, Lasiosphaeriaceae, Sordariales, taxonomy

Introduction

Arx & Gams (1967) introduced the genus Apiosordaria Arx & W. Gams

(Lasiosphaeriaceae, Sordariales) based on the type species Pleurage verruculosa

C.N. Jensen [= A. verruculosa]. Krug et al. (1983) synonymised Echinopodospora

and Lacunospora under Apiosordaria based on morphological similarities. In

Apiosordaria ascospores are usually two-celled, composed of a dark upper cell

and a lower hyaline cell; the upper cell is ellipsoidal to subglobose, with walls

ornamented with numerous broad or narrow pits; the lower cell is triangular to

cylindrical and possessing a minute hyaline terminal appendage (Arx & Gams

1967). In addition, the presence of spines, warts, or pits on the upper ascospore

cell is used as a principal character that differentiates this genus from related

848 ... Wu &al.

genera in the Lasiosphaeriaceae that also produce two-celled ascospores (e.g.,

Cercophora, Podospora, Triangularia, Zopfiella; Garcia et al. 2003).

Apiosordaria currently comprises 23 species with a worldwide distribution

in garden soils (Stchigel et al. 2000), field soils (Jensen 1912), grassland soils

(Warcup 1951a), and forest soils (Warcup 1951b). The genus is also found in

dung (Krug et al. 1983) and decaying wood (Badura & Badurowa 1964). Some

species have also been found associated with plant material (Rostrup et al.

1916) and the insect Plecia nearctica (Kish et al. 1974).

During a study of the fungal diversity of wetlands in China, an interesting

Apiosordaria species was isolated. Morphological characteristics distinguishing

this taxon from all other species in the genus (Krug et al. 1983, Guarro & Cano

1988, Stchigel et al. 2000) are here supported by phylogenetic analyses. In this

paper, the new species is introduced, described, and illustrated, and compared

with the most similar species.

Materials & methods

Collection, isolation, and morphological examination

Soil samples were collected from Donghu Lake (30°33’N 114°23’E) in the

northeastern outskirts of Wuchang, Wuhan City, Hubei, China. Donghu Lake, situated

on the alluvial plain in the middle basin of the Changjiang (Yangtze) River, has a total

surface area of 32 km? and lies 21.4 m (20.8-21.7 m) above sea level. The lake is usually

3-4 m deep with a maximum of 4.5 m, and the flat muddy lake bottom is sparsely covered

with hydrophytes. The area is dominated by a damp subtropical monsoon climate, with

a temperature range of 12.9-21.5 °C and annual precipitation of ca. 1040 mm. Sediment

samples were collected along the bank of the lake at depths of 0-40 cm. The samples

were sealed in plastic bags, placed in a 4 °C car refrigerator, and transported to the

laboratory for processing.

A 10 g sediment subsample was homogenized, serially diluted in sterile distilled

water, shaken, and 200 ul plated onto % strength potato dextrose agar (PDA) containing

100 mg! streptomycin and 100 mg!’ chloromycetin. The plates were kept at 25 °C for 1

week, after which fungi with different colony morphologies were selected and grown on

new PDA plates with antibiotics, on oatmeal agar (OA), potato carrot agar (PCA, potato

20 g, carrot 20 g, agar 20 g) and malt extract agar (MEA). Isolates were identified based

on morphological characteristics after 10 days incubation at 25 °C.

Microscopic characteristics of the new species of Apiosordaria were observed with

a Nikon Ellipse 80i light microscope equipped with differential interference contrast

(DIC) optics and a FEI] Quanta 200 scanning electron microscope (SEM). Ex-type and

isotype cultures are deposited in the China General Microbiological Culture Collection

Center (CGMCC), Beijing, China.

DNA sequencing & phylogenetic analysis

After 10 days growth on PDA, ca. 0.2 g of mycelium was collected with a sterile

scalpel and placed into a 1.5 ml centrifuge tube. Genomic DNA was extracted with

Apiosordaria hamata sp. nov. (China) ... 849

TABLE 1. Sources and sequences used in the phylogenetic analysis.

TAXON

Apiosordaria backusii [=

Triangularia backusii]

A. hamata

A. longicaudata

A. nigeriensis

A. tetraspora

A. verruculosa

var. maritima

Apodus deciduus

A. oryzae

Bombardia bombarda

Bombardioidea anartia

Cercophora areolata

C. sparsa

C. sulphurella

Cladorrhinum sp.

Immersiella caudata

I. immersa

Jugulospora rotula

Lasiosphaeria glabrata

L. lanuginosa

L. ovina

L. rugulosa

Podospora cupiformis

P. curvicolla

P. intestinacea

Schizothecium aloides

S. fimbriatum

S. glutinans

Strattonia carbonaria

Triangularia mangenotii

T. tanzaniensis

Xylaria hypoxylon

Zopfiella tabulata

Zygopleurage zygospora

COLLECTION NO.*

CBS 106.77

CGMCC 3.15230

CGMCC 3.15231

CBS390.84

FMR 6363

CBS 363.84

F-152365

CBS 550.66

CBS 506.70

CBS 376.74

AFTOL 967

HHB99-1

UAMH7495

JF00229

SMH2531

MJ-2014

SMH3298

SMH2589

ATCC38359

TL4529

SMH3277

CBS 958.72

SMH4438

CBS 246.71

IFO 8548

CBS 113106

CBS 879.72

CBS 144.54

CBS 134.83

ATCC 34567

ATCC 38847

TRTC51981

AFTOL-ID 51

CBS 230.78

SMH4219

ORIGIN

Sandy soil, Japan

Wetland soil, China

Soil, Japan

Garden soil, Nigeria

Soil, Thailand

Ethanol-pasteurized soil,

Spain

Ethanol-pasteurized soil,

Spain

Moose dung

Branch, Panama

Decaying wood, Germany

Kobus defassa dung

Rabbit dung

Horse dung

Soil, Netherlands

Horse dung

Arctostaphylos uva-ursi,

Switzerland

Burned soil, Japan

Soil, Japan

Downed rotting wood

Porcupine dung, Canada

Dung of cow, USA

GENBANK ACCESSION NO.

ITS

GQ922520

KP878306

KP878307

AJ458184

AY681199

AY681200

AY587911

AY587913

KJ572182

AY587914

AY587920

AY587925

AY587932

AY999125

AY999122

AY999121

AY999120

AY999115

AY999116

DQ491487

AY999132

LSU

KP878304

KP878305

FR692340

FR692341

AY346258

FR692345

AY681165

AY681166

DQ470970

AY346264

AY587936

AY587937

AY587938

AY436407

AY436408

AY346287

AY436410

AY587943

AY587946

AY587952

AY999102

AY999099

AY999104

AY999097

AY999092

AY999093

AY346302

AY346303

AY780081

AY544648

AY999105

AY346306

* ATCC, American Type Culture Collection, USA; AFTOL, Assembling the Fungal Tree of Life, USA; CBS,

Centraalbureau voor Schimmelcultures, Netherlands; CGMCC, China General Microbiological

Culture Collection Center, China; HHB, Field Museum of Natural History, Chicago, Illinois;

IFO, Institute for Fermentation, Osaka; SMH, Sabine M. Huhndorf; TL, Thomas Leessoe; UAMH,

University of Alberta Microfungus Collection and Herbarium.

850 ... Wu &al.

a simple and rapid ‘thermolysis’ method (Zhang et al. 2010). Partial 28S rDNA and

complete ITS+5.8S rDNA were amplified using fungal specific primers LROR/LR7

(Vilgalys & Hester 1990) and ITS5/ITS4 (White et al. 1990), respectively.

All PCR amplifications were carried out using a Biometra T-professional

thermocycler. The reaction mixture included 5 ul of 10x Takara Bio PCR buffer, 2 ul of

25 mM MgCl, (1 uM final concentration), 4 ul of 2.5 mM deoxynucleotide triphosphate

mix (final concentration, 250 uM each; Takara Bio, Japan), 2 ul of 10 uM primers each

(final concentration, 0.25 uM; Sangon Biotech, China), 1 ul Sigma deionized formamide,

and 0.4 ul Taq polymerase. The amplification was carried out as follows: 95 °C for 3

min; followed by 35 cycles at 95 °C for 1 min, 54 °C for 45 s, and 72 °C for 90 s; and

a final extension for 10 min at 72 °C. After the PCR products were separated in 1%

(w/v) agarose gels, they were stained with 0.4% GoldView nucleic acid gel stain (SBS

Genetech) and viewed and photographed on a Gel imaging system (Bio-Rad).

The internal transcribed spacer (ITS) region and large-subunit rRNA (LSU) gene

data sets were constructed with sequences of Apiosordaria hamata (isolates CGMCC

3.15230, CGMCC 3.15231) and reference sequences from related taxa retrieved from

GenBank (TaBLE 1). All sequences were aligned with ClustalX v1.83 (Thompson et

al. 1997), and the alignment was manually adjusted to allow maximum alignment and

minimize gaps. Maximum likelihood (ML) and Bayesian analysis were used to estimate

phylogenetic relationships of A. hamata with its related taxa, with Xylaria hypoxylon (L.)

Grev. as the outgroup taxon.

Maximum Likelihood analysis was conducted in RaxmlGUI v1.3 (Silvestro &

Michalak 2012) with the GTRGAMMAI model, 1000 bootstrap replicates. While

Bayesian analysis was performed in a likelihood framework as implemented by MrBayes

v3.0b4 software package to reconstruct phylogenetic trees (Huelsenbeck & Ronquist

2001), the best-fit evolutionary model was determined by MrModeltest v2.3 (Posada &

Crandall 1998, Nylander 2004) through comparing different nested DNA substitution

models in a hierarchical hypothesis-testing framework. The best-fit model determined

by MrModeltest was the “GTR+1+G’, lsetnst = 6, rates = invgamma; prset statefreqpr =

dirichlet (1, 1, 1, 1) for Bayesian analyses. Multiple Bayesian searches using Metropolis-

coupled Markov chain Monte Carlo sampling were conducted using one cold and three

heated Markov chains in the analysis. Bayesian analysis was run for 645,000 generations,

with trees sampled every 100 generations. The first 1290 trees, representing the burn-in

phase, were discarded. To estimate posterior probabilities (PP) of recovered branches

(Larget & Simon 1999) 50% majority rule consensus trees were created from the

remaining trees using PAUP.

Results

Molecular phylogeny

Nucleotide sequence blasts of the NCBI-nrDNA database showed that the

new fungus, Apiosordaria hamata, exhibited 98% LSU sequence similarity with

Cercophora sp. isolate SMH3200 (AY780055) (query cover = 99%, identities

= 1274/1295, gaps = 2/1295), 97% similarity with Arnium arizonense isolate

18211-c (S) (KF557668) (query cover = 100%, identities = 1252/1297, gaps

Apiosordaria hamata sp. nov. (China) ... 851

1.00/100);- Lasiosphaeria lanuginosa SMH3277

Lasiosphaeria ovina CBS958.72

Lasiosphaeria rugulosa SMH4438

1.00/78 Lasiosphaeria glabrata TL4529

Cercophora sparsa JF00229

Cercophora areolata UAMH7495

Zoptiella tabufata CBS230.78

1.00/*| Triangularia mangenotii ATCC 38847

Apodus oryzae CBS376.74

0.98/87 Cercophora sulphurella SMH2531

Bombardioidea anartia HHB99-1

4.00/100 Bombardia bombarda AFTOL967

1.00/100, Apiosordaria hamata CGMCC 3.15230"

“160 Apiosordaria hamata CGMCGC 3.15231

Cladorrhinum sp. MJ-2014

Apiosordaria verruculosa F-152365

Apiosordaria nigeriensis FMR 6363

*#/98, Apiosordaria longicaudata CBS390.84

Apiosordaria tetraspora CBS363.84

Apiosordaria verruculosa var. maritima CBS550.66

Apiosordaria backusii CBS106.77

1.00/97 Apodus deciduus CBS506.70

0.96/90 Podospora intestinacea CBS113106

1.00/100 Zygopleurage zygospora SMH4219

Podospora curvicolla |FO 8548

*/93 Podospora cupiformis CBS246.71

Triangularia tanzaniensis TRTC51981

*/64 Schizothecium fimbriatum CBS144.54

1.00/100 Schizothecium aloides CBS897.72

Schizothecium giutinans CBS134.83

0.9541 00 rp Jugulospora rotula ATCC38359

Strattonia carbonaria ATCC34567

Immersiella caudata SMH3298

1.00/95 © Immersiella immersa SMH2589

Xylaria hypoxylon AFTOL-ID51

1.00/*

“167

0.99/86

“(57

1.00/95

0.03

Fic. 1. Maximum likelihood (ML) tree from PAUP analysis and Bayesian tree by MrBayes based on

LSU and ITS sequences of Apiosordaria and GenBank data from related species. Bayesian posterior

probabilities >0.95 and ML bootstrap support values > 50% are shown on the upper branches.

= 3/1297), and 97% similarity with Arnium arizonense isolate 724 (UPS)

(KF557669) (query cover = 99%, identities = 1250/1295, gaps = 3/1295) in

family Lasiosphaeriaceae. ITS sequence results indicated a 92% similarity

with Cladorrhinum sp. MJ-2014 (KJ572182) (Lasiosphaeriaceae) (query cover

= 100%, identities = 498/544, gaps = 10/544). We therefore included related

reference sequences in Lasiosphaeriaceae in our phylogenetic analyses.

The LSU and ITS combined gene datasets were analyzed using maximum

likelihood (ML) and Bayesian analysis (Fic. 1). The phylogenetic trees derived

from ML and Bayesian analysis gave similar results; the Apiosordaria hamata

852 ... Wu &al.

strains (CGMCC 3.15230 and CGMCC 3.15231) formed a well-supported

clade with A. backusii (L.H. Huang) Guarro [= Triangularia backusii] (CBS

106.77), A. longicaudata (Furuya & Udagawa) J.C. Krug et al. (CBS390.84),

A. nigeriensis Stchigel & Guarro (FMR 6363), A. tetraspora J.C. Krug et al.

(CBS 363.84), A. verruculosa (C.N. Jensen) Arx & W. Gams (F-152365),

A. verruculosa var. maritima (Apinis & Chesters) Arx & W. Gams (CBS 550.66),

and Cladorrhinum sp. (MJ-2014) within Lasiosphaeriaceae (Bayesian posterior

probability = 1; ML bootstrap value = 95%). However, the two A. hamata strains

formed a separate clade with high support (Bayesian posterior probability = 1;

ML bootstrap value = 100%).

Taxonomy

Apiosordaria hamata B. Wu, K.D. Hyde, Jing Z. Sun & Xing Z. Liu, sp.nov. Fra. 2

MycoBAnk MB 812280

Differs from related Apiosordaria species by lacking germ pores in the ascospores and by

having hooked spines on the upper cell of the ascospores.

Type: China, Hubei, Wuhan, Donghu Lake, isolated from sediment, July 2009, B. Wu

(Holotype, HMAS 246231; ex-type cultures, CGMCC 3.15230, C@MCC 3.15231).

EryMo.ocy: hamata refers to the hooked spines on the upper cell of the ascospores.

FACES OF FUNGI NUMBER: FoF 00663

ASCOMATA superficial, scattered, cleistothecial, dark brown, globose,

overall 475-490 x 480-525 um. PERIDIUM 4-6-layered, up to 4-5 um wide,

translucent, brownish orange to brown, comprising cells arranged in a textura

angularis. PARAPHYSES hyaline, filiform, septate, unbranched, 1.8-2.2 um wide.

Asci 8-spored, clavate, broadly rounded at the apex, 420-450 x 90-100 um,

pedicel without conspicuous apical structures, evanescent, 90-150 um long.

Ascosporss biseriate, clavate, hyaline, 1-celled when young and becoming

two-celled with the formation of a transverse septum, warted wall, 26-31 x

22-26 um; upper cell obovoid, truncate at the base and with a slightly acuminate

apex, brown, thick-walled, uniformly ornamented with numerous 2-3 um

diam (x = 2.4 um, n = 30) hooked spines, lacking a germ pore; lower cell sub-

hyaline, conical and smooth, 4-7 x 12-15 um (x = 5.2 x 13.1 um, n = 30) long.

ASEXUAL MORPH: Undetermined.

Fic. 2. Apiosordaria hamata (holotype, HMAS 246231). A. colony on PCA; B, ascomata;

C. immature ascoma; D, E. mature ascomata; F. immature ascus; G-I. mature asci in cotton blue;

J. ascospores; K. ascospore with warted coat; L. ascospore with smooth coat; M, O. ascospores

with hooked spines (SEM); N. ascoma (SEM). Scale bars: B = 1 mm; C, D = 100 um; E = 50 um;

F-I, K, L = 10 um; J = 20 um.

Apiosordaria hamata sp. nov. (China) ... 853

‘

WD Mag _ HV_ Det Spot. —————20.0um

12:01:55 PM 20.1 mm 2736x 15.0 kVETD 3.0

WD 2) WD Mag! HV | Det Spot 10.0um:

11:59:34 AN) 20.0 mm 345x 15.0 kVETD 3.0 -40:08 AM 20.0 mm 3068x 15.0 kKVETD 3.0

854 ... Wu &al.

Discussion

Apiosordaria hamata can be recognized by its spiny ascospores in the clavate

asci and is supported in the phylogeny. Its ascospores are similar in size to

A. nigeriensis (24-32 x 20-23 um), A. jamaicensis (B.M. Robison) Krug et al.

(24-30 x 17-23 um), A. sacchari (B.M. Robison) Krug et al. (33-40 x 25-31

um), and A. spinosa (Cailleux) Krug et al. (25-32 x 18-21 um), all of which

also have spinulose to spiny ascospores (Krug et al. 1983). However, A. hamata

is distinct in having hooked spines on the upper cell of the ascospores and

in lacking germ pores, which Meléndez-Howell (1970) observed in her SEM

studies of previously described Apiosordaria species.

Phylogenetic analysis supported the two isolates of A. hamata in a subclade

with A. nigeriensis, A. verruculosa, and Cladorrhinum sp. (asexual morph of

Apiosordaria) with >50% bootstrap ML value but separated from the other

Apiosordaria species. Although A. hamata is morphologically similar to

A. nigeriensis, they are separated on the ITS and LSU phylogenetic tree

(Frc. 1), which clusters A. hamata in a monophyletic group with seven species

of Apiosordaria. Here, morphology coupled with molecular analysis support

this fungus as a new species in Apiosordaria (Lasiosphaeriaceae).

Key to species of Apiosordaria

if, “ACT Pas OTRO RCLAV ALC Ae yi ah Steal bk ead Pace tered chert edeeal Fae e Beste Mite Ape 2

Mi Ascr Bspored, clavate Or cyl rical solo NOP 2 et or spat his Sigg hye Sieger Sih uer ER gr sis 5

2. Ascomata globose, upper ascospore cell verrucose, 18-22 x 14-17 um;

lower ascospore cell 4-6 X 5-6 UM 0... eee eee eee eee A. effusa

2, Ascomata pyr wornysOstiolates. Wat an. was wn wane «a vedo ee wee ore Rose 3

3. Upper ascospore cell spinulose or warty, 16-21 x 13-15 um;

lower ascospore cell 6-10 x 9-19 Um «1.0... eee eee eee ee A. verruculosa

SWIPE asc OSPOES Cell Cle, tn MF Ben. c, hreeconneh het nacg ens aeea ist pon asses gee Anessa 4

4, Lower ascospore cell 10-14 X 7.5-9 UM 11... .. ee eee eee eee eee A. longicaudata

4, Lower ascospore cell 4—7.5(-10) x 6-8(-11) um... eee eee eee A. tetraspora

5. Ascospore wall with broad pits;

ornamentation appearing as spines or irregular warts .................0000. 6

5. Ascospore wall with narrow pits; ornamentation appearing as pits ............. 18

6. Upper ascospore cell polygonal, five-angled in side view,

ornamented with longitudinal ribs, 10-12 x 8-9 um ............ A. striatispora

6. Upperascospore-cell-more or-less ellipsoidal 5 ech) eead pod ooetak: wertade ehoaes 7

PEAS COUIALOS 1G ARG: Atlee ea Mel oa See ary Ae tS galt Me tt Me tyne cele Me oe Re ese 8

7. Ascoma smooth or with another type of vestiture, non-ostiolate;

ascospores overall typically larger than 14uum................ 0... eee ee eee 11

Apiosordaria hamata sp. nov. (China) ... 855

8. Ascoma lacking agglutinated hairs;

upper ascospore cell 10-12.5 x 6-8 um with flat-topped spines;

lower cell 4-6 um long; asexual morph absent ................ A. yaeyamensis

§.ASEOMA: SVE AS SIL ALCON ALG’ 5. Pax soa: fax, cate ap ae BEC fe ene PG vas PA need Be oe a faces pe E

9. Upper ascospore cell 15-19 x 10-12.5 um; lower cell 5-6.5 um long;

asexual MOLPI GHEY SOSPOLIE sx Bente tes Bacdhine Badel an Sioues op ads ong ee A. microcarpa

> Upper-ascospore-celDlarger ony otc eh hcg cos By enn eran te Hin eihey eine x Berne eA ns 10

10. Ascoma pyriform; upper ascospore cell pitted, 27-34 x 18-23 um ..... A. backusii

10. Upper ascospore cell uniformly ornamented with numerous rounded warts,

23-28(-30) x 18-22(-23) um; lower ascospore cell 1-5(-6) um long;

used Ter pM Abseny . eye Seis. hore ke eg ete hte Seen yee tse ee A. hispanica

11. Ascospores with a granulose to wart-like ornamentation;

lower ascospore cell 4-5 um long; asexual morph absent ........ A. tuberculata

11. Ascospores with spine-like ornamentation or flat-topped ridges;

lower ascospore:cell longer ...5 don <xa% ge u eh ee hres elas 4 eee Metage o 12

12. Asci cylindrical and curved; upper ascospore cell 16-20(-22) x 12-15 um,

lower cell frequently transversely septate, 12-20 um long;

asexual morphs Cladorrhinum and Chrysosporium ............ A. vermicularis

12. Asci clavate; upper ascospore cell larger; lower cell non-septate............... 13

13. Ascospore ornamentation 2-5.5 um long,

upper cell (24—)25-32 x 18-21(-22) um, lower cell 4-8 um long;

sexual morph Chrysosporium (when present) ............--0--e eee A. spinosa

13. Ascospore ornamentation shorter (<3 tum); lower cell longer ................. 14

14. Upper ascospore cell 33-40 x 25-31 um; lower cell 14-20 um long;

ASEM AOL PM ADSSME yg octet tr ate, in ae Ue aae wee ean een seeaen ate A. sacchari

14. Upper and lower ascospore cells shorter ............ 0. eee e eee e eee e ence 15

15 Ascospore ornamentation irregular flat-topped ridges forming an incomplete

reticulum, upper cell 28.5-33 x 17-19.5 um; lower cell ca. 8-10 um long;

asexual MOP CHPVSOSPOTEAIA® fo .5) she utans. 3 os alten eae teh eockecs, earktle ens A. terrestris

LS wesCOSPOre: OLN AMIE talon Spin ert ees sy a8 Sa ol Note oh! Bote ol! Mate oh eb Aiea ats 16

16. Upper ascospore cell without germ pore, 26-31 x 22-26 um, uniformly

ornamented with hooked spines of 2-3 um diam; lower cell

sub-hyaline, conical, smooth, 12-15 um long; asexual morph absent A. hamata

1G: Wppedscospore cellowitlhyCerm Ores sre Fo aga tw deat seat h ao S peg t oraiphgd prt hald ors 17

17. Upper ascospore cell 24-32 x 20-23 um, with a dark area surrounding

the germ pore and ornamented with spines <7 um long;

bower ceule7—WOstirin Ln Se ia porte Sree eck oe sa red ata taper eral atl i A. nigeriensis

17. Upper ascospore cell 24-30 x 17-23(-26) um, ornamented with

spines <3 um long; lower cell ca. 11-18 um long;

asexual morph Chrysosporium (when present) .............+--- A. jamaicensis

856 ... Wu &al.

18. Ascoma glabrous; ascospores overall (16-)18-21(-23) x 10-12 um, with rugose

flanges; lower ascospore cell 2.5-5 um long; asexual morph absent ... A. rugosa

[8Ascoma with agelutinated: Has... mt rl mel ok eal ek el ae ok coat ot, coals fet 19

19. Upper ascospore cell regularly pitted, 20-25 x 12-15 um, lacking flanges;

lower ascospore cell 4—5 um long; asexual morph Cladorrhinum ..... A. vestita

19" Upper-aseospore cellicovered: withismallwarts<. x... Stal oo Seas .-o Seles cep Leeds ot Pace 20

20. Upper ascospore cell 27-34 x 18-23 um, walls ornamented with shallow pits;

lower ascospore cell 11-17 x 10-12 um long ................ A. tenuilacunata

20 ,Upperascospore cellismaller var. cosets iis ant. slec ates hel anes Aet o's Mae ane ane AS one 21

21. Asexual morph Chrysosporium-like;

upper ascospore cell 20-24 x 15-18 um... eee eee A. otanii

DAL ASE RUAN OEP a DSCINE 4.5 spf in race coke EME nt hk to, asset ccatateeinet NE eer hee nat oft 22

22. Upper ascospore cell 25-27(-29) x (22—)23-27 um, uniformly ornamented

with small warts; lower cell (1-) 4-6 um long, slightly warted ....... A. globosa

22. Upper ascospore cell 19-24 x 8-10 um, ornamented with small warts;

lower cell 3:5-4.5-* 3=4 um. bg eh hea ae cates oa eee tee ee bees A. antarctica

Acknowledgments

This research was jointed supported by the Natural Science Foundation of China

(No. 31600024 and 41101238). The authors thank Professors Xue-Wei Wang (Institute

of Microbiology, Chinese Academy of Sciences) and Yu-Cheng Dai (Beijing Forestry

University, China) for their kind suggestions and valuable comments in identification;

they are also grateful to Dr. Sajeewa S.N. Maharachchikumbura (Guizhou Academy of

Agricultural Sciences) and Dr. Eric H.C. McKenzie (Landcare Research, New Zealand)

for presubmission reviews.

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MY COTAXON

October-December 2016—Volume 131, pp. 859-869

http://dx.doi.org/10.5248/131.859

Discopycnothyrium palmae gen. & sp. nov. (Asterinaceae)

SINANG HONGSANAN?”, ALI H. BAHKALI?, PUTARAK CHOMNUNTI ””,

J1AN-Kut Liu*’, JUN-BoO YANG* & KEVIN D. HyDE*”?

"Institute of Excellence in Fungal Research, Mae Fah Luang University,

Chiang Rai, 57100, Thailand

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

° Department of Botany and Microbiology, College of Sciences, King Saud University,

Riyadh, KSA

* Germplasm Bank of Wild Species in Southwest China, Kunming Institute of Botany,

Chinese Academy of Science, Kunming 650201, Yunnan, China

* CORRESPONDENCE TO: putarak.cho@mfu.ac.th

ABSTRACT—A new genus and species, Discopycnothyrium palmae, is described from palms

(Arecaceae) in Narathiwat Province, southern Thailand, and is known only from its asexual

morph. The genus is characterized by circular pycnothyria with darkened cells around the

central ostiole, variably long hyphae at the margin, and pycnothyriospores that are 1-septate

with the septum between a larger brown upper cell and hyaline lower cell. LSU gene sequence

analysis clustered D. palmae in the Asterinaceae clade and supported (59% ML and 0.88 PP

support) the taxon as phylogenetically distinct from other Asterinaceae. Morphological and

phylogenetic differences confirm the new genus, here introduced and illustrated.

Key worps—Asterinales, foliar epiphytes, taxonomy

Introduction

The family Asterinaceae was introduced by Hansford (1946), with the

generic type Asterina Lév. (Miller & von Arx 1962, Luttrell 1973, Eriksson

1981, Hosagoudar 2004, Kirk et al. 2008, Hofmann 2010, Hyde et al. 2013,

Hongsanan et al. 2014). The family comprises biotrophs found on living plant

leaves and is distributed worldwide (Kirk et al. 2001, Barr & Huhndorf 2001,

Taylor et al, Hofmann et al. 2010, Hyde et al. 2013, Hongsanan et al. 2014).

While most previous studies focused on the morphology, Hofmann (2010)

provided the first molecular data of Asterinaceae that supported its phylogenetic

860 ... Hongsanan & al.

placement in Dothideomycetes and which were included in the phylogenetic tree

representing Asterinales sensu stricto (Hyde et al. 2013, 2016, Wijayawardene

et al. 2014). The order comprises a single family Asterinaceae, which contains

17 genera that are differentiated using both morphological and phylogenetic

characters (Hongsanan et al. 2014). The asexual morph in the family can be

either coelomycetous (Asterostomella) or hyphomycetous (Asterina), although

classification of the asexual morphs needs further clarification (Hyde et al. 2013,

Hongsanan et al. 2014). Redescription of the asexual morphs of Asterinaceae

(Hofmann 2010, Hosagoudar 2010) and key to genera by Hosagoudar (2010)

were based mainly on morphology and host association

In this study, we introduce a new genus Discopycnothyrium and its type

species D. palmae based on phylogeny, morphology, and host association.

Discopycnothyrium is an asexual morph genus that somewhat resembles

Asterostomula and Prillieuxina in its circular pycnothyria with radially

arranged cells. However, Asterostomula and Prillieuxina differ in having

pycnothyria with star-like or irregular openings and brown pycnothyriospores,

while pycnothyria in Discopycnothyrium have a central ostiole and 1-septate

pycnothyriospores comprising brown upper cells and hyaline lower cells. The

placement of Discopycnothyrium in Asterinaceae is also supported by rDNA

sequence analysis.

Materials & methods

An Asterinaceae-like specimen was collected from Narathiwat Province in the

southern part of Thailand. Morphological characters were observed under Nikon 80i

stereo and compound microscopes. Measurements were determined using Tarosoft (R)

Image Frame Work (v. 0.9.7). We attempted to isolate the specimen by using a single

spore technique (Chomnunti et al. 2011, 2014), but the spores did not grow in culture.

The fresh specimen was therefore dried in silica gel, and DNA was extracted directly

from pycnothyria. Type material was conserved in the Herbarium, Center of Excellence

in Fungal Research, Mae Fah Luang University, Chiang Rai, Thailand (MFLU), and the

Herbarium, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming,

China (KUN).

Genomic DNA was extracted from dried pycnothyria using the E.Z.N.A° Forensic

Genomic DNA Extraction Kit (OMEGA Bio-tek Norcross GA 2013). Individual

pycnothyria were placed into 1.5 ml sterilized tubes and kept in -20 °C overnight, after

which DNA was extracted following the manufacturer's instructions.

Universal primers LR5 and LROR were successfully used to amplify the 28S rDNA

(LSU) region. The PCR thermal cycle program was: an initial cycle at 94°C for 3

min; 40 cycles of denaturation (94°C for 30 s), annealing (54°C for 40 s), elongation

(72°C for 1 min); final extension (72°C for 10 min). After visualization on 1% agarose

electrophoresis gels stained with ethidium bromide, the PCR products were sent to

Discopycnothyrium palmae gen. & sp. nov. (Thailand) ... 861

TABLE 1. Taxa used in the phylogenetic analysis: specimen codes and

GenBank accession numbers (LSU)

SPECIES

Apiosporina collinsii

Asterina cestricola

Asterina fuchsiae

Asterina phenacis

Asterina siphocampyli

Asterina sp.

Asterina weinmanniae

Asterina zanthoxyli

Discopycnothyrium palmae

Asterotexis cucurbitacearum

Chaetothyriothecium elegans

Coleroa robertiani

Fusicladium africanum

Fusicladium pini

Gibbera conferta

Gloniopsis praelonga

Gloniopsis subrugosa

Hysterium angustatum

Hysterobrevium smilacis

Lembosia albersii

Lichenothelia convexa

Microthyrium microscopicum

Natipusilla limonensis

Natipusilla naponensis

Oedohysterium insidens

Oedohysterium sinense

Protoventuria barriae

Psiloglonium araucanum

Psiloglonium clavisporum

Psiloglonium simulans

Rhytidhysteron rufulum

Sympoventuria capensis

Tyrannosorus pinicola

Venturia inaequalis

Veronaeopsis simplex

Zeloasperisporium siamense

VOUCHER/CULTURE

CBS 118973

TH 591

TH 590

TH589

ppMP 1324

MFLU13-0619

TH 592

TH 561

MFU13-0485

PMA M-0141224

CPC 21375

CBS 458.64

CPC 12828

CBS 463.82

CBS 191.53

CBS 112415

CBS:123346

CBS 236,34

CBS 114601

MFLU13-0377

L1608

CBS 115976

L_AF286_1A

PE3_2b

L_AF217_1A

CBS:238.34

CBS:123345

CBS 300.93

CBS:112412

CBS:123338

CBS:206.34

AFTOL-ID 2109

CPC 12840

CBS 120136

AFTOL-ID 1235

CBS 815.69

CBS 588.66

IFRDCC 2194

"LSU: 28s rDNA (Newly generated sequence in bold)

GENBANK NO.

GU301798

GU586215

GU586216

GU586217

HQ701140

KM386978

GU586218

GU586219

KM386979

HQ610510

KF268420

JQ036231

EU035423

EU035436

GU301814

FJ161173

FJ161210

FJ161180

FJ161174

KM386982

KC015085

GU301846

HM196370

JX474862

HM196371

FJ161142

FJ161209

JQ036232

FJ161172

FJ161197

FJ161178

FJ469672

DQ885904

KF156104

DQ470974

GU301878

EU041877

JQ036228

862 ... Hongsanan & al.

Majorbio Co., Ltd. (China) for purification and sequencing. The sequence generated for

Discopycnothyrium palmae was deposited in GenBank (KM386979).

Other LSU sequence data in this study were obtained from GenBank to compile the

dataset (TABLE 1), which represents Asterinaceae, Hysteriaceae Chevall., Microthyriaceae

Sacc., Mytilinidiaceae Kirschst., Natipusillaceae Raja et al., Sympoventuriaceae Y. Zhang

ter et al., and Venturiaceae E. Mill. & Arx ex M.E. Barr following the publications

by Schoch et al. (2009), Hyde et al. (2013), Boonmee et al. (2014), Hongsanan et al.

(2014), and Wijayawardene et al. (2014). Lichenothelia convexa Henssen was selected as

outgroup. Alignments were performed automatically using BioEdit v 7.1.9 and Clustal

X 2.0.11 (Thompson et al. 1997) and improved manually when necessary (Hall 2004).

Maximum likelihood analysis was performed in RAxML with raxmlGUIVv.0.9b2

(Silvestro & Michalak 2012); the search strategy was set to rapid bootstrapping and

carried out using GTRGAMMAI model of nucleotide substitution for a small dataset

to account for rate heterogeneity. The nucleotide substitution model was selected with

MrModel test 2.2 (Nylander 2008); the number of replicates was automatically inferred

using the stopping criterion (Pattengale et al. 2009). The evolution model was performed

with MrModeltest 2.2 (Nylander 2008). Posterior probabilities (PP) (Rannala & Yang

1996, Zhaxybayeva & Gogarten 2002) were determined by Markov Chain Monte

Carlo sampling (MCMC) in MrBayes (v. 3.0b4; Huelsenbeck & Ronquist 2001). Six

simultaneous Markov chains ran for 1,000,000 generations, with trees sampled every

100th generation. 10,000 trees were obtained; the first 2000 trees, representing the burn-

in phase were discarded, and the remaining 8000 trees were used to calculate posterior

probabilities; the default convergence criterion was set at 0.001 (Cai et al. 2006, 2008).

Phylogenetic trees were viewed in TreeView (v. 1.6.6; Page 2001). Maximum likelihood

bootstrap values higher than 50% are given as the first set of numbers above the nodes

(Fic. 2). Bayesian posterior probabilities (BYPP) equal or higher than 0.9 are given as

the second set of numbers above the nodes (Fic. 2).

Taxonomy

Discopycnothyrium Hongsanan & K.D. Hyde, gen. nov.

INDEX FUNGORUM IF551024

Differs from other genera in Asterinaceae by its pycnothyria with darker cells surrounding

a rounded central ostiole, and 1-septate pycnothyriospores with a hyaline lower cell.

TYPE SPECIES: Discopycnothyrium palmae Hongsanan & K.D. Hyde

EryMoLoecy: from Latin disco meaning “circular” or “plate-like”, and pycnothyrium

referring to the technical term for the type of fructification.

FACES OF FuNGI FoFoo0569

Foliar epiphytes. SUPERFICIAL MYCELIUM aseptate, pale brown to brown.

SEXUAL MORPH: undetermined. ASEXUAL MORPH: PYCNOTHYRIUM superficial

on the host surface, circular, walls comprising radially arranged cells, dark

brown, with central ostiole surrounded by dark cells. PERIpIuM poorly

Discopycnothyrium palmae gen. & sp. nov. (Thailand) ... 863

Fic 1. Discopycnothyrium palmae (MFLU 13-0485, holotype). A, B. Pycnothyria on surface of

host. C. Pycnothyrium when viewed in squash mounts. D. Pycnothyriospore arrangement in

pycnothyrium. E. Vertical section through pycnothyrium. F. Upper wall of pycnothyrium when

viewed in squash mounts. G. Darkened cells around ostiole. H. Aseptate hyphae without appressoria.

I, J. Conidiogenous cells giving rise to pycnothyriospores. K, M-O. Mature pycnothyriospore.

L. Immature pycnothyriospore. Bars: C-E = 100 um; K = 20 um; D, F-J, L-O = 10 um.

developed at the base. CONIDIOGENOUS CELLS holoblastic, cylindrical, hyaline.

PYCNOTHYRIOSPORES ovoid or broadly clavate, widest and rounded near the

apex and tapering towards lower end, 1-septate near the tapering base, upper

cell pale brown to brown, lower cell hyaline.

Discopycnothyrium palmae Hongsanan & K.D. Hyde, sp. nov. FIG. 1

INDEX FUNGORUM IF551023

Differs from other species in Asterinaceae by its darker brown cells surrounding a

rounded central ostiole, and its 1-septate pycnothyriospores with a hyaline lower cell.

864 ... Hongsanan & al.

Type: Thailand, Narathiwat Province, on the branches of palm (Arecaceae), September

2013, K.D. Hyde (holotype MFLU 13-0485; isotype KUN; GenBank KM386979).

Erymo ocy: Referring to the host palm on which the fungus was found.

FACES OF FuNGI FoFo0570

Foliar epiphytes on surface of palm leaves (Arecaceae). SUPERFICIAL MYCELIUM

1-2 um diam (x = 2 um, n = 10), aseptate, pale brown to brown, dark brown at

the margin, lacking appressoria and setae.

SEXUAL MORPH: Undetermined.

ASEXUAL MORPH: PYCNOTHYRIUM 162-195 um diam (x = 187 um, n = 10),

superficial on host surface, easily removed, mostly solitary, circular, hyphae of

different lengths at the margin, wall comprising radial cells, dark brown, with

central ostiole, and with darker cells around the central ostiole. PERIDIUM poorly

developed at the base. PPEUDOPARAPHYSES not observed. CONIDIOPHORES not

observed. CONIDIOGENOUS CELLS 6-8 x 2-3 um (x = 8 x 3 um, n=5), holoblastic

in cavity of pycnothyria, cylindrical, hyaline, smooth. PYCNOTHYRIOSPORES

18-22 x 12-15 um (x = 20 x 13 um, n = 10), ovoid or broadly clavate, widest

and rounded near the apex and tapering towards the lower end, 1-septate near

the tapering base, not constricted at the septum, upper cell with 2 layers when

immature, outer layer disappearing at maturity, pale brown in upper cell when

immature and dark brown at maturity with smooth-walls, lower cell always

hyaline, smooth-walled or sometimes rough.

Notes: Discopycnothyrium palmae is most similar to Asterostomula loranthi

Theiss. by having a superficial mycelium without appressoria and setae and

pycnothyrium walls composed of radial cells, but A. loranthi differs in the stellate

dehiscence of the pycnothyrium centers and its unicellular pycnothyriospores

(Hosagoudar 2010).

The new species also somewhat resembles the asexual morph of Prillieuxina,

which also has circular pycnothyria and dark pycnothyriospores with a single

septum, and a similar host association (Hofmann 2010), with P calami (Syd.

& P. Syd.) R.W. Ryan and P. saginata (Syd. & P. Syd.) R.W. Ryan also described

from arecaceous hosts. Prillieuxina differs from Discopycnothyrium by its

circular pycnothyria with X- or Y-shaped fissures at maturity and its 1-septate

pycnothyriospores with a brown (not hyaline) lower cell (Hongsanan et al.

2014).

Allothyrium marcgraviae Syd. also has circular pycnothyria but differs from

D. palmae by the star-like fissures of its pycnothyrial opening and its multi-

septate ascospores (Hongsanan et al. 2014). The LSU sequence analyses cluster

D. palmae in the clade of Asterinaceae but in its own lineage, with 59% ML and

0.88 PP support (Fic. 2), confirming it as a new genus.

Discopycnothyrium palmae gen. & sp. nov. (Thailand) ... 865

65091 terobrevium smilacis C&S 114601

530.96 tenum angustatum C&S 236.34

77,097, Psilogionium clavisporum C&8S.1233338

‘Psiloglonium areucanum C&8S.112412

; Psiloglonium simulans C8S:206.34 Hysteriaceae

FAG 71.0 loniopsis subrugosa C8S-12346

ess loniopsis praelonga CBS 112415

70 $6] 10001 Osdohystenum sinense C8S12345

Oedohystenum insidens C8S.236.34

Phytidhysteron rufulum AFTOLAD 2109

Asterotexis cucurbitacearum PMA M-0141224

Asterina fuchsiae TH $90

Asterina weinmanniae TH $82

Astenna sp. MFLU13-0619

6301

‘Asterina zanthoxyli TH $61 5 5

a i raxyl Asterinales sensu stricto

sai Asterina cesvicola TH $91

oe Asterina siphocampyli ppMP 1324

7 “- Asterina phenacis TH $9

Discopycnothyrium palmae MFU13-04S

Lembosia albersii MFLU13-0377

6191 $30.1 Chaetothyriothecium elegans CPC 21375

Microthyrium microscopicum C&S 115976

Zeloasperisporium siamense IFROCC 2194

1000.1 ,Natipusilla limonensis t_A=206_1A

1000.1 Natipusilla limonensis PE3_2 Hatipusillaceae

Natipusilla naponensis L_AF217_1A

Microthyriaceae

63! Tyrannosors pinicola AFTOLAD 1235

S40.93 Gibbere conferta CBS 191.53

$101 Ventuna inaequalis C&S 815.69 F

dogs | [— Calera robertiani Cas 45864 Venturiaceae

Apiosponna collinsii CBS 118873

590.91 “ Protoventunia bamiae CBS 30093

61 4000.1 )Sympoventuna capensis CPC 12640

1920 Sympoventuns capensis CBS 120136

sr! Fusicladium africanum CPC 12628

ane Fusicladium pini C&S 463.62

Veronaeopsis simplex C&S $33.66

Lichenothelia convexa Li60s

Sympoventuriaceze

Fic 2. RAxML maximum likelihood phylogenetic tree based on sequence analysis of LSU dataset.

The first set of numbers above the nodes are RAxML values >50%; the second set are Bayesian

posterior probabilities 20.9. Strain numbers are indicated after species names. The new species

introduced is in red bold, other types are in black bold.

Molecular phylogeny

LSU sequence data from six families (Hysteriaceae, Microthyriaceae,

Mytilinidiaceae, Natipusillaceae, Sympoventuriaceae, Venturiaceae) were

included in the phylogenetic analysis (Fic. 2). The Asterinaceae clade includes

six strains of Asterina: A. cestricola (R.W. Ryan) Hosag. & T.K. Abraham,

A. fuchsiae Syd., A. phenacis Syd., A. siphocampyli Syd., A. weinmanniae Syd.,

and A. zanthoxyli W. Yamam. The new taxon, Discopycnothyrium palmae,

forms a moderately supported (59% ML, 0.88 PP) clade sister to the other

Asterina species, supporting D. palmae as clearly separate from other species

in Asterinaceae. Moreover, Lembosia albersii Henn. (MFLU13-0377) forms a

well-supported clade basal to the other genera in Asterinaceae with high (99%

ML, 1.0 PP) bootstrap support.

The Microthyriaceae clade comprises two strains: Chaetothyriothecium

elegans Hongsanan & K.D. Hyde and Microthyrium microscopicum Desm.,

866 ... Hongsanan & al.

also with high (99% ML, 1.0 PP) bootstrap support. The Natipusillaceae clade

includes three strains of freshwater fungi: Natipusilla limonensis A. Ferrer

et al. (two strains) and N. naponensis A. Ferrer et al. with 100% ML and

1.0 PP support; although they are phylogenetically related to Zeloasperisporium

siamense Hongsanan et al., there is no morphological similarity between

Natipusilla and Zeloasperisporium, so these should be regarded as tentatively

placed lineages.

The Venturiaceae clade ) with high bootstrap support of 91% ML, 1.0 PP)

includes six representative strains—Apiosporina collinsii(Schwein.) Hohn.,

Coleroa robertiani (Fr.) E. Mull., Gibbera conferta (Fr.) Petr., Protoventuria

barriae Carris & A.P. Poole, Tyrannosorus pinicola (Petrini & PJ. Fisher)

Unter. & Malloch, and Venturia inaequalis (Cooke) G. Winter.The clade of

Venturiaceae—and is closely (66% ML support) related to the Sympoventuriaceae

clade with five strains: Sympoventuria capensis Crous & Seifert (two strains),

Fusicladium africanum Crous, F. pini Crous & de Hoog, and Veronaeopsis

simplex(Papendorf) Arzanlou & Crous.

Discussion

Discopycnothyrium (type species D. palmae) is introduced as a new genus

in the family Asterinaceae (characterised by a superficial pycnothyrium

comprising radiating cells) that differs from other genera in having circular

pycnothyria with variably long marginal hyphae, darkened cells around a

central ostiole, and pycnothyriospores that are widest near the apex, taper

towards lower end, and have one septum near the tapering base that separates

the larger pale brown to brown upper cell from the smaller hyaline basal cell.

There are limited sequence data for taxa in Asterinaceae because members of

this family are mostly obligate parasites/biotrophs, and pure cultures are difficult

to obtain; therefore direct DNA extraction and sequencing must be obtained

from specimen. However, sequence data from Asterina species do support the

Asterinaceae within Dothideomycetes (Hofmann 2010, Wu et al. 2011, Hyde

et al. 2013, Boonmee et al. 2014, Hongsanan et al. 2014, Wijayawardene et

al. 2014). Although phylogenetic LSU analyses of Discopycnothyrium cluster

D. palmae within the Asterinaceae clade, it is distinctly separate from other

genera in that clade. Bootstrap values are not high because of low number of

populations representing this group, and further study is needed to clarify the

divergence of the species. Based on

Nonetheless, both morphology and phylogeny supports the new taxon

as a species in a new genus within Asterinaceae, which we introduce here as

Discopycnothyrium.

Discopycnothyrium palmae gen. & sp. nov. (Thailand) ... 867

Acknowledgments

The authors extend their sincere appreciations to the Deanship of Scientific Research

at King Saud University for its funding this Prolific Research group (PRG-1436-9). We

thank the reviewers Hiran Ariyawansa (Guizhou Academy of Agricultural Sciences,

Guizhou University) and Ying Zhang (Institute of Microbiology, Beijing Forestry

University) for improving this manuscript.

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MY COTAXON

October-December 2016— Volume 131, pp. 871-880

http://dx.doi.org/10.5248/131.871

Two new records of Agaricus from Southwest China

Mao-QIANG HE?”, JIE CHEN?* & RuI-LIN ZHAO'™*

'State Key Laboratory of Mycology, Institute of Microbiology,

Chinese Academy of Sciences, Beijing 100101, China

*Department of Entomology and Plant Pathology, Faculty of Agriculture,

Chiang Mai University, Chiang Mai 50200, Thailand

°Center of Excellence in Fungal Research & *School of Science,

Mae Fah Luang University, Chiang Rai 57100, Thailand

°College of Life Sciences, University of Chinese Academy of Sciences,

Huairou District, Beijing 100408, China

* CORRESPONDENCE TO: Zhaoruilin@gmail.com

ABSTRACT—Two specimens of A. brunneosquamulosus (from China and Thailand) and four

specimens of A. duplocingulatus (from China) were identified and studied by morphological

and ITS sequence analyses. They are presented here with descriptions, line drawings, and

color photographs as new records for China. Our additional molecular sampling of A.

duplocingulatus has revealed intraspecific variability within this species.

KEY WORDS—Agaricaceae, rDNA, taxonomy

Introduction

Agaricus L. is a well-known genus containing species with a high nutritional

and medicinal value, such as the famous cultivated mushrooms A. bisporus

(J.E. Lange) Imbach and A. subrufescens Peck. Zhao et al. (2011) recognized

386 species in this cosmopolitan genus, a number that has subsequently risen

to over 400 with the discovery of numerous novel taxa from Asia, Australia,

and Europe (Chen et al. 2012, Gui et al. 2015, Lebel 2013, Lebel & Syme 2012,

Li et al. 2014, Parra 2013, Zhao et al. 2012). Eight sections were traditionally

recognized in the genus, long classified based on temperate species (Challen et

al. 2003; Kerrigan et al. 2005; Kerrigan et al. 2008; Parra 2008, 2013). However,

following a phylogenetic study of samples from both temperate and tropical

872 ... He, Chen & Zhao

regions by Zhao et al. (2011), eleven new lineages comprising tropical or

subtropical taxa were revealed.

One new clade determined by Zhao et al. (2011) has been phylogenetically

reconstructed and identified as corresponding with Agaricus sect. Brunneopicti

Heinem. (Chen et al. 2015). The section has been delimited to accommodate

species that combine the following morphological characters: punctiform

squamulose remnants of the universal veil or brownish larger squamules on

pileus surface; a double or complex annulus with cortinate fibrils on the under

side; context when bruised discoloring faint to strong yellow, orange, rufescent,

brownish rufescent, or red; odor of bitter almond, phenol, or solvent; a positive

KOH reaction, and a negative or (rarely) weakly positive Schaffer’s reaction

(Chen et al. 2015). The section, which has a strictly palaeotropical distribution,

contains 16 species.

Southwestern China is known as home to glacial refugia and plays an

important role in postglacial biological recolonization. In order to reveal the

species spreading patterns and biodiversity of Agaricus, since 2010 we have

conducted a series of investigations in this area during the July-August rainy

season. As one result, two representatives of A. sect. Brunneopicti—Agaricus

brunneosquamulosus and A. duplocingulatus—are newly recorded from China.

Materials & methods

Morphological study

Specimens were collected in Yunnan Province, China, during the rainy season.

Photographs were taken immediately in the field. Each specimen was wrapped in

aluminum foil or kept separate in a plastic collection box. Macroscopic characters were

recorded and chemical tests were performed immediately after the specimens reached

the laboratory according to Largent (1986). The specimens were dried completely at

70°C in a food drier, sealed in plastic bags, and deposited in the herbarium of Southwest

Forestry University, Kunming, China (SWFC), with duplicates in Herbarium,

Mycologicum Academiae Sinicae, Beijing, China (HMAS). An additional specimen

from Thailand (deposited in HMAS) was included in the study.

Following the methodology described by Largent (1986), microscopical features

(basidiospores, basidia, cystidia, pileipellis, annulus hyphae) of each dried specimen

were examined. At least 20 measurements were made to calculate the dimensions

presented as follows: X = the means + SDs of length x width, Q = basidiospore length :

width ratio, and Q. = the mean + SD of Q values.

DNA extraction, PCR, and sequencing

DNA was extracted from the dried fruiting bodies using the E.Z.N.A. Forensic

DNA Extraction Kit (D3591-01, Omega Bio-Tek). The internal transcribed spacer (ITS)

regions were amplified with the primers ITS4 and ITS5 following the protocol of White

et al. (1990). DNA sequencing was performed in commercial biotechnology company.

Agaricus species new for China... 873

TABLE 1. Agaricus specimens included in the phylogenetic analyses.

SPECIES

A. sp. 1

A, sp. 2

A. sp. 3

. bingensis

A. niveogranulatus

A. chiangmaiensis

A. brunneopunctatus

A. toluenolens

sp. 4

sordidocarpus

subsaharianus

sp. 5

Si hat ge

megacysidiatus

A. sp. 6

A. brunneosquamulosus

A. cf. inoxydabilis

A. duplocingulatus

SAMPLE

C3182

LD2011027

ZRLwxh3115

ADK1992

C3155

LD2011025

LD2011023

LD2011024

PYP009

NTS113

ADK2564

CA911

CA926

NTT117

LD201237

ADK4732

CA800

LD2012168

LD2012179

NTS115

NTS116

NTO19

NTT118

LD201238

ZRL3005

ZRL4017

LD2012105

ZRL133

ZRL2013266

LAPAFI1

ZRL3064

CA903

LD2012177

LD201218

LD201233

LD201274

LD201275

ZRL3031

ZRL3041

NTT34

ZRL2013328

ZRL2012267

ZRL2012120

ZRL2013348

ORIGIN

Togo

Thailand

China

Bénin

Togo

Thailand

Bénin

Thailand

Burkina-Faso

Thailand

China

Togo

Thailand

China

ITS SEQUENCE

KJ540955

KR812336

KJ540954

KJ540950

KJ540957

KJ540958

KJ540959

KJ540960

JF514513

JF514518

KJ540947

KJ540948

JF514534

KJ540946

JF440300

JE727826

KF305947

KF305946

KC971098

JF514532

JE727844

KJ540970

KJ540969

KR869107

JF691549

KJ540968

KR812337

KR812340

JF727841

KR869108

KJ540962

KJ540961

KJ540967

KJ540965

KJ540963

KJ540964

JF691550

KR869109

JF514536

KR812341

KR812339

KR812338

KR812342

874 ... He, Chen & Zhao

Phylogenetic analyses

Our original sequences plus those retrieved from GenBank (TaBLE.1) were first

aligned using ClustalX 2.0 (Thompson et al. 1997) and then manually adjusted in

BioEdit v.7.0.4 (Hall 2007). Bayesian analysis was performed with MrBayes 3.1.2

(Ronquist & Huelsenbeck 2003). One million generations using a GIR+I+G model

nucleotide substitution detected by MrModeltest 2.2 (Nylander 2004) were run for

six Markov chains and sampled every 100th generation resulting in 10,000 trees.

Maximum Parsimony analysis was performed using PAUP*4.0b10 (Swofford 2004).

One thousand heuristic searches were conducted with random sequence addition,

tree bisection-reconnection (TBR) branch swapping, and gaps treated as missing data.

Parsimony bootstrap values were obtained from 1000 bootstrap replicates, with starting

trees obtained via stepwise addition, random sequence addition, and max-trees set to

1,000,000.

1.0/100 7m ©3182 Agaricus sp. |

1/99 LD2011027 Agaricus sp. 2

ZRLwxh3115 Agaricus sp. 3

1.0/99 - ADK1992 A. bingensis

C3115 A. bingensis

10/77 o.q¢af” LD2011025 A. niveogranulatus

1 ooo PoP D201 1023 A. niveogramulatus

- LD2011024 A. niveogramiatus

0.9/8] CA954 PY P009 A. niveogranuatis

1.0/94 NTS113 A. chiangmaiensis T

-/59 ADK2564 A. bpuleopeeaees

1.0/100 -CA9I1 A. foluenolens T

CA926 A. toluenolens

NTT117 Agaricus sp. 4

1.0/92 LD201237 A. sordidocarpus

ADK4732 A. subsaharianus T

CA800 Agaricus sp. 5

LD2012168 4. megacystidiatus

10/100 LD2012179 A. megacystidiatus

- NTS11S A. megacystidiatus

NTS 116 4. megacystidiatus

NTO19 Agaricus sp. 6

NTT1L18 A. brunneosquamulosus

LD201238 A. brameosquamulosus

10/79 ZRL3005 A. breemeosquamulosus

-/59 Pl ZRL4017 A. briemeasquanndosus

1.0/96 1 LD2012105 4. dbrunneosquamulosus T

15) a ZRL133 A. brunneosquamilosus

ZRL2013266 A. brinneosquamulosus

LAPAFI A. cf. inoxydabilis

ZRL3064 A. duplocingulatus

1.0/100 CA903 A. duplocingulatus

LD2012177 4. duplocingulatus

LD201218 A. duplocingudlatus

1.09/99 LD201233 A. duplocingulatus

LD201274 A. duplocingulatus

LD201275 A. duplocingulatus

ZRL3031 A. duplocingudatus

ZRL3041 A. duplocingulatus

NTT34 A. duplocingulatus

ZRL2013328 A. duplocingulatus

~/881 7RL2012267 A. Glace lates

ZRL2012120 A. duplocingulatus

ZRL2013348 A. duplocingulatus

1.0/100

CA637 A. campestris

0.2

Figure 1. Phylogeny of Agaricus sect. Brunneopicti generated from MrBayes analysis of ITS

sequences rooted with A. campestris. New sequences are set in bold font. Bayesian posterior

probability (PP) values >0.9, and parsimony bootstrap support (BS) values >50% are given at the

nodes.

Agaricus species new for China... 875

Results

Phylogenetics analyses

The dataset comprised 45 sequences representing 17 species with A. campestris

as outgroup (Zhao et al. 2011). The final alignment comprised a total of 663

characters, of which 517 were constant, 39 parsimony-uninformative, and

107 parsimony-informative. The phylogenetic trees generated by MP and

Bayesian methods exhibited very similar topologies, with the Bayesian tree

shown in Fic.1. Collections ZRL2013266 (from China) and ZRL133 (from

Thailand) clustered in the A. brunneosquamulosus clade with strong support

(1/96); and Chinese collections ZRL2013328, ZRL2012267, ZRL2012120, and

ZRL2013348 grouped with NTT34 (from Thailand; Zhao et al. 2011) in the

A. duplocingulatus clade with strong support (1/99).

Taxonomy

FIGURE 2. Agaricus brunneosquamulosus (ZRL2013266):

a. Pileipellis; b. Basidiospores; c. Basidia; d. Cheilocystidia.

Agaricus brunneosquamulosus L.J. Chen, R.L. Zhao, K D. Hyde & Callac,

Phytotaxa 192(3): 154 (2015). Fics 2, 4A,B

Macroscopical characters: PILEUs 26 mm in diam, parabolic, convex, margin

decurved; surface dry, with appressed triangular fibrillose squamules, light

brown to brown against white background; LAMELLAE free, crowded, pink to

brown, 3 mm broad, with 3 series of lamellulae. StrPE 60 x 8-16 mm (at base),

thickening towards the base, with long rhizomorphs; surface white, covered

by fine fibrils; context narrow, hollow. ANNuLUus double (two adhering layers):

upper layer a membranous remnant of the partial veil, rigid, pendant, white,

876 ... He, Chen & Zhao

<21 mm diam, 3 mm thick; bottom layer a fibrillose-membranous remnant of

the universal veil, fragile; white above and with brown fibrils below. CONTEXT

3 mm thick, white; discoloration indistinct when touched, none when cut.

Odor not noted.

Macrochemical reactions: KOH—slightly yellow. Schaffer’s reaction—

negative.

Microscopical characters: BASIDIOSPORES 4.1-5.6 x 2.8-3.7 um, [X = 4.8 +

0.4 x 3.3 + 0.3 um, Q = 1.21-1.65, Q_ = 1.47 + 0.13, n = 20], ellipsoid, smooth,

brown, thick-walled. Bastp1a 4-spored, 18.5-30.6 x 5.9-8.7 um, narrowly

clavate to clavate, hyaline, smooth. CHErLocystip1a 14.6-27.6 x 10.9-25.3 um,

pyriform, clavate to broadly clavate, basally septate or catenulate with 2-3

ellipsoid to subspherical cells in chain, hyaline, smooth. PLEUROCYSTIDIA

absent. PILEIPELLIS a cutis, hyphae 4.3-11.5 um diam, cylindrical, light brown,

smooth, sometimes slightly constricted at the septa.

Hasir: Solitary in forest.

SPECIMENS EXAMINED: CHINA, YUNNAN PRovINcE, Teng Chong County, Laifeng

Mountain, 18 July 2013, Zhou Junliang, ZRL2013266 (HMAS, SWFC; GenBank

KR812340). THAILAND, CHIANG Mal! PROVINCE, Mae Taeng District, Ban Pha Deng

Village, Mushroom Research Centre, 15 May 2008, Zhao Ruilin, ZRL133 (HMAS;

GenBank KR812337).

¢ _10um d Sum

FIGURE 3. Agaricus duplocingulatus (ZRL2013328):

a. Basidiospore; b. Basidia; c. Pileipellis; d. Cheilocystidia.

Agaricus duplocingulatus Heinem., Bull. Jard. Bot. Natl. Belg. 50: 32 (1980). Fras 3,

4C-H.

Macroscopical characters: PiLEus 27-87 mm diam, convex, margin

decurved when young, becoming straight when mature; surface dry, covered

Agaricus species new for China... 877

5mm & 10mm ‘

10mm 4a K 10mm 20mm

“Wy

Figure 4. A. brunneosquamulosus (a, b: ZRL2013266). Habit.

A. duplocingulatus (c, d: ZRL2012120; e, f: ZRL2012267; g, h: ZRL2013328). Habit.

with reddish brown appressed fibrillose squamules on a white background;

squamules congregated on the disc and concentrically arranged elsewhere;

fibrillose or mud-cracked [areolate?]; surface becoming dull red when wet.

LAMELLAE free, crowded, lamellulae in 5 tiers, 1-6 mm broad, pink when

young, brown when mature. STIPE 30-43 x 4-9 mm, cylindrical with enlarged

(4-23 mm diam) base, with rhizomorphs, hollow; surface silky; white, turning

reddish brown when bruised or cut. ANNULUs double, two separate layers: upper

layer wider (47 mm diam), persistent, membranous, white, smooth above and

fibrillose to floccose below; lower layer, narrower (12 mm diam), bracelet-like,

fugacious, movable, white and floccose above, smooth with a brown margin

below. CONTEXT 4 mm broad, firm; discoloration variable: distinctly yellow

when touched and red when cut or no discoloration when touched or cut. Odor

almond-like.

Macrochemical reactions: KOH reaction yellow. Schaffer’s reaction negative.

Microscopical characters: BASIDIOSPORES 4.5-6.5 x 3.6-4.5 um [X =

5.7 + 0.3 x 4.0 + 0.2 um, Q = 1.30-1.7, Q. = 1.40 + 0.10, n = 20], ellipsoid,

smooth, brown, thick-walled. Basip1a 4-spored, 14.5-19.3 x 4.9-7.5 um,

878 ... He, Chen & Zhao

clavate, hyaline, smooth. CHEILOCysTIDIA 14.2-27.3 x 8.1-19.6 um, globular

to pyriform, hyaline, smooth. PLEUROCysTIpDIA absent. PILEIPELLIS a cutis,

hyphae 2.3-7.0 um diam, cylindrical, light brown, smooth, slightly constricted

at the septa.

Hasir: Solitary in forest.

SPECIMENS EXAMINED: CHINA, YUNNAN PROVINCE, Ying Jiang County, Tong Biguan

National Natural Reserve, 19 July 2013, Yu Qinghua, ZRL2013328 (HMAS, SWFC;

GenBank KR812341); 20 July 2013, Yu Qinghua, ZRL2013348 (HMAS, SWFC; GenBank

KR812342); Cang Yuan county, Nan Gunhe National Natural Reserve, 7 July 2012, Zhao

Ruilin, ZRL2012120 (HMAS, SWFC; GenBank KR812338); An Kang Village, 11 July

2012, Philippe Callac, ZRL2012267 (HMAS, SWFC; GenBank KR812339).

Discussion

Agaricus brunneosquamulosus, originally described from Thailand,

is characterized by small sporocarps, tiny ferruginous brown appressed

triangular squamules on the pileus surface, and a complex annulus with

woolly scales and cortinate fibrils on its lower surface (Chen et al. 2015). The

Chinese material generally agrees with original description, except for the

uncertain annulus morphology of annulus and occasional presence of short

catenulate cheilocystidia. These annular and cheilocystidial differences may be

due to the immaturity of the Chinese collection. The ITS sequence alignment

does indeed show a five nucleotide difference between the Thai and Chinese

collections; however, all the differences are transition mutations (T/C or A/G),

polymorphisms commonly observed in intraspecific variation (Zhang et al.

2009, Yadav 2014). In the absence of significant morphological differences

between the Thai and Chinese material, we for the time recognize the Chinese

material as A. brunneosquamulosus. Collection of more young to mature

materials in near future will undoubtedly refine our determination.

Agaricus duplocingulatus, first described from Singapore and recently

reported from Thailand, is characterized by brown ferruginous appressed

squamules on the pileus surface, a double annulus with the lower one movable,

an ochraceous to reddish discoloration when cut or bruised, and catenulate

cheilocystidia (Heinemann 1980, Chen et al. 2015). The four Chinese

collections, which generally match the species circumscription, are more

variable in sporocarp size (pileus 27-87 mm diam). Comparison of the ITS

sequences reveals nucleotides that differ at four polymorphic positions in

more than 16 positions, also by Chen et al. (2015). This degree of intraspecific

variability is rarely observed in Agaricus, and multi-gene analysis may be

helpful in determining whether A. duplocingulatus represents a complex of

species. Presently, we adopt the identification by Chen et al. (2015).

Agaricus species new for China... 879

Acknowledgements

This work was supported by grants from the National Natural Science Foundation

of China to RLZ (Project IDs 31470152 and 31360014) and the Opening Foundation

of State Key Laboratory of Mycology, Microbiology of Institute, Chinese Academy of

Sciences. The authors wish to thank Kanad Das (Cryptogamic Unit, Botanical Survey of

India, Howrah) and Samantha C. Karunarathna (World Agroforestry Centre (ICRAF),

Kunming Institute of Botany, China) for presubmission review.

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megalosporus: a new species in section Minores. Cryptogamie, Mycologie 33: 145-155.

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Kerrigan RW, Callac P, Guinberteau J, Challen MP, Parra LA. 2005. Agaricus section Xanthodermatei:

a phylogenetic reconstruction with commentary on taxa. Mycologia 97: 1292-1315.

http://dx.doi.org/10.3852/mycologia.97.6.1292

Kerrigan RW, Callac P, Parra LA. 2008. New and rare taxa in Agaricus section Bivelares

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MY COTAXON

October-December 2016— Volume 131, pp. 881-887

http://dx.doi.org/10.5248/131.881

Entoloma ochreoprunuloides from Italy, with notes

on its geographical distribution and allied species

FRANCESCO DOVANA!, ALFREDO VIZZINI' , FABRIZIO BOCCARDO?,

MARCO MUCCIARELLI: & MARCO CLERICUZIO?

‘Department of Life Sciences and Systems Biology, University of Torino,

Viale P.A. Mattioli 25, 10125 Torino, Italy

’Via Filippo Bettini 14/11, I-16162 Genova, Italy

° Dipartimento di Scienze dell Ambiente e della Vita (DISIT), Universita del Piemonte Orientale, Via

T: Michel 11, 15121 Alessandria, Italy

* CORRESPONDENCE TO: alfredo. vizzini@unito.it

ABSTRACT—An Italian collection of E. ochreoprunuloides |= E. prunuloides var. obscurum] is

described. The specimen was identified by means of morphology, and by the analysis of its

nrITS sequence. The European distribution of the species is also discussed. The sequence from

a single Italian specimen of E. luteobasis suggests that E. luteobasis and E. ochreoprunuloides

may be conspecific.

Key worps—Basidiomycota, Agaricomycetes, tricholomatoid clade, taxonomy

Introduction

Entoloma (Fr. ex Rabenh.) P. Kumm. is a large genus of Agaricomycetes, with

worldwide distribution; more than 1500 taxa have been described (Morgado et

al. 2013). Until a few years ago, the systematics of Entoloma was based only on

morphological data (Noordeloos 1981, 1992, 2004), but recent studies based

on DNA sequences have started to appear in the literature (e.g., Co-David

et al. 2009; Baroni et al. 2011; Kokkonen 2015). The consequence of these

investigations is that the infrageneric subdivision is continuously evolving,

but this process is just at the beginning. For example, Noordeloos (1981, 1992,

2004) treats Entoloma as a single genus subdivided into several subgenera, while

Largent (1994) recognizes Entoloma sensu stricto and many segregate genera,

882 ... Dovana & al.

most corresponding to the subgenera used by Noordeloos. Recently Baroni et

al. (2011) proposed Entocybe T.J. Baroni et al. as a new genus that includes a few

select species from Entoloma subg. Entoloma and a few other anomalous species

previously placed in Rhodocybe sect. Rhodophana (Kihner) Singer. These taxa

produce bumpy spores reminiscent of those found in Rhodocybe and abundant

clamp connections that are not typical of Rhodocybe. Entocybe was erected for

a distinct clade in Entoloma subg. Entoloma based on both morphological and

molecular data.

At the species level, even some well-known species appear far more complex

when investigated at the molecular level. For example, Morgado et al. (2013)

focused on the subgenus Entoloma using a multigene approach (mtSSU, nrLSU,

rpb2, nrITS) to find that E. bloxamii (Berk. & Broome) Sacc., E. prunuloides

(Fr.) Quél., and E. sinuatum (Bull.) P. Kumm. each represent species complexes

containing different taxa when considered from a worldwide perspective.

Kokkonen (2015), who examined nrITS, nrLSU and rpb2 sequences of several

Entoloma taxa mainly in E. sect. Rhodopolia (Fr.) Noordel. (actually elevated at

subgeneric level), also proposed several novel taxa and combinations.

The present contribution concerns the E. prunuloides complex in E. sect.

Entoloma, a section characterised by small, somewhat isodiametric and

obscurely angular spores and pileipellis hyphae arranged as a simple cutis or

ixocutis. From a phylogenetic point of view, the species of this section form a

clade that appears basal to the bulk of the species in the Entoloma sensu lato

clade. Morgado et al. (2013) demonstrated that at least three different species

have been covered under the name “E. prunuloides”—(1) E. pseudoprunuloides

Morgado & Noordel., for a Canadian collection; (2) typical E. prunuloides,

probably with an exclusively European distribution; and (3) E. ochreoprunuloides

[= E. prunuloides var. obscurum], from France, Germany, and United Kingdom.

We here describe the finding of an Italian collection of E. ochreoprunuloides,

whose nrITS sequence was examined and confirmed as conspecific with the

holotype collection.

Materials & methods

Morphology

Fresh basidiomata were photographed in the field using a Nikon D80 digital camera.

Microscopic features were examined using a Nikon Eclipse E200 light microscope and

observations were made on mounts in Congo red, followed by 5% NH, solution.

DNA extraction, PCR amplification, and sequencing

DNA was extracted from two herbarium specimens (E. ochreoprunuloides TO 3327;

E. luteobasis WU 17842) with NaOH (Osmundson et al. 2013); 2 mg of dry sample

were homogenized in 250 ul of soda at 0.5M with a pestle. After 5 minutes to allow for

Entoloma ochreoprunuloides in Italy ... 883

sedimentation, 5 ul of the extract were removed and diluted in 195 ul of 100mM Tris-

HC] at pH 8.0, and 1 ul of the dilution was used as template DNA. The nrITS region was

amplified with primers ITS1F (Gardes & Bruns 1993) and ITS4 (White et al. 1990). PCR

was performed in 25 ul reaction volumes following Gardes & Bruns (1993).

PCR products were purified and sequenced by IGA Technology Services (Udine,

Italy).

Results

The ITS sequence of our Italian collection (TO 3327; GenBank KU984720)

shared 99% (582/583) of nucleotides with the German holotype of

E. ochreoprunuloides (L [E. Arnolds 01-142]; GenBank KC710092) and 100%

homology with two independent sequences from an Italian collection labelled

as E. luteobasis (WU 17842; GenBank KU984721 [this paper], LN850613

[Kokkonen 2015]).

Taxonomy

Entoloma ochreoprunuloides Morgado & Noordel., Persoonia 31: 171. 2013.

Figs 1A, C-F

= Entoloma prunuloides var. obscurum Arnolds & Noordel., Fungi Europaei

vol. 5a: 838. 2005 [non Entoloma obscurum Hesler 1967].

SELECTED ICONOGRAPHY: Noordeloos (2004; Pl. 1b, p. 1166), Morgado et al. (2013; Fig.

g 1-2, p. 168)

Description of the Tuscan collection (TO 3327)

Priteus 4-6.5 cm broad, convex, or even conico-convex when young, then

more applanate but with a consistently broad (sometimes acute) central umbo;

surface fibrillose (even strongly so), greasy; colour sepia brown to ochre, darker

in the center, lighter and more brown-yellow at the periphery; hygrophanous;

margin irregular, fimbriate, strongly involute, striate. LAMELLAE emarginate to

almost adnate, crowded, at first whitish or light grey, then pink. StrpE 5-9 x

0.5-1.4 cm, cylindrical, base ending in a distinctly tapered tip; longitudinally

fibrillose, concolourous with the pileus, whitish towards base. CONTEXT rather

thin in the pileus, thicker near the umbo, whitish; sMELL intensely mealy

(farinaceous), TASTE mealy.

SpoRES 6.0-7.6 x 5.4-7.1 um, av. 6.8 x 6.4 wm, Q = 1.0-1.17(-1.26), Q, = 1.08,

somewhat isodiametric, 4-5(-6)-angled, angles blunt under the light

microscope. Basip1A 27-40 x 7-11 um, 4-spored, clavate. HYMENOPHORAL

TRAMA made up of cylindrical to fusoid, parallel hyphae, 35-60 x 4-8 um.

PILEIPELLIS three-layered, with the outermost layer (suprapellis) an ixocutis

of very narrow (1.5-2.5 um diam) hyphae dispersed in a gelatinous matrix,

the middle layer (mediopellis) a cutis of parallel cylindrical hyphae (4-6 um

884 ... Dovana & al.

diam), and the bottom layer (subpellis) of broad, inflated hyphae measuring

25-50 x 15-25 um. Pigments pale, intracellular, diffuse. STIPITIPELLIS a cutis

of narrow (3-5 um diam) cylindrical hyphae. CAuLOcysTIDIA absent. CLAMP-

CONNECTIONS frequent in all tissues.

HasitaT densely gregarious to sub-cespitose in a broad-leaved wood

composed mainly of Quercus cerris L. (Turkey oak; Fagaceae) and Carpinus

betulus L. (hornbeam; Betulaceae).

MATERIAL STUDIED: Entoloma_ ochreoprunuloides: ITALY, Tuscany, Grosseto,

Scansano, Monte Auto, under Quercus cerris and Carpinus betulus, 6.11.2013, legit M.

Clericuzio (TO 3327; GenBank KU984720).

ADDITIONAL MATERIAL STUDIED: Entoloma luteobasis: ITALY, APULIA, Foggia, Vieste,

Val del Tesaro, under Q. cerris, 16.11.1997, legit A. Hausknecht & F. Reinwald (WU

17842; GenBank KU984721)

Discussion

The Italian collection of E. ochreoprunuloides was made by one of us (MC)

in Tuscany (Italy) in a mixed hardwood forest. The micro-morphologic

data revealed small, rather rounded isodiametric spores averaging ca. 7 um;

pileipellis hyphae arranged in an ixocutis; abundant clamp-connections; and

broad inflated hyphae in the subpellis and hymenophoral trama. All these

data supported it as a species of Entoloma sect. Entoloma (sensu Noordeloos

1981, 1992, 2004). From a macro-morphological point of view, the basidiomes

recalled the E. prunuloides group but differed from those of a typical

E. prunuloides not only in pileus colour—sepia ochraceous rather than whitish—

but also for their slender aspect (quite unlike the stout and fleshy basidiomes of

E. prunuloides; Noordeloos 1992, 2004). This collection was therefore assigned

to E. prunuloides var. obscurum, a variety described in 2004 and later raised to

species rank and renamed as E. ochreoprunuloides (Morgado et al. 2013).

The morphologically closest species to E. ochreoprunuloides is E. luteobasis

Ebert & E. Ludw. (described from Germany and Italy), which differs mainly

by its slightly to distinctly yellow or ochraceous stipe base (Ebert et al. 1992,

Noordeloos & Hausknecht 1998, Noordeloos 2004). Because the holotype of E.

luteobasis is missing from the Leiden herbarium (L; Nicolien Sol, pers. comm.),

we turned to an Italian collection of E. luteobasis (WU 17842) described in

Noordeloos & Hausknecht (1998) and Noordeloos (2004). Our ITS sequence

analyses revealed that this collection is conspecific with E. ochreoprunuloides.

This suggests that E. luteobasis (proposed in 1992) might be considered an

earlier name for E. ochreoprunuloides (published in 2013). However, additional

collections and sequences of both taxa and further phylogenetic analyses will

be required to test this potential synonymy.

Entoloma ochreoprunuloides in Italy ... 885

Fig. 1. Entoloma ochreoprunuloides (TO 3327). A. Habit. B. Map of E ochreoprunuloides

distribution. C. Pileipellis. D. Spores: E. Basidia. E. Spores (in water). Scale bars: A = 1 cm;

C-F = 10 um.

From a morphological point of view, delimiting species in the E. prunuloides-

bloxamii group is no easy task. Morgado et al. (2013) published a box plot

of spore dimensions for all the species of the group that showed

E. ochreoprunuloides spores as among the smallest in the whole section.

However, our Tuscan collection falls within the limits of spore size and shape

for the species (5.9-7.1 x 5.7-7.2 um, Q = 1.0-1.16, Q., = 1.04; Morgado et

al. 2013). Our data also agree well with the WU 17842 (E. luteobasis) figure

and spore dimensions in Noordeloos & Hausknecht (1998; 6.6-7.6 x 6.2-7.5

um, av. 7.0 x 6.6 um, Q. = 1.05). Spore dimensions can be used to distinguish

E. ochreoprunuloides from E. prunuloides (with a spore average ca. 1 um larger;

Morgado et al. 2013). Typically, E. prunuloides tends to be more robust and

to have pale (whitish to light ochraceous) tinges on the pileus (Noordeloos

1992, 2004), but it should be stressed that pileus colours are rather variable

and should be used only in connection with other characters. In contrast,

our collection showed sepia brown ochraceous colours, which are probably

the most typical for the species; Noordeloos & Hausknecht (1998) reported

a brown (from chocolate brown through orange brown) pileus with a greyish

886 ... Dovana & al.

margin. Morgado et al. (2013) also described a new form (E. ochreoprunuloides

f. hyacinthinum) with violet or pink tinges on both pileus and stipe. DNA

sequences from two collections of this form, however, show an almost complete

identity with the typical form (Morgado et al. 2013).

With its dark pileus colors and small spores, the Canadian species

E. pseudoprunuloides is morphologically closer to E. ochreoprunuloides than to

E. prunuloides. However, the phylogenetic analysis by Morgado et al. (2013)

placed it as sister to E. prunuloides.

Our report widens the distribution of E. ochreoprunuloides to include Italy,

in addition to Germany (NordRhein-Westfalen, Ibbenbturen, holotype), UK,

and France (Corsica). A map with all of the localities is presented in Fic. 1B.

Acknowledgments

The authors are grateful to Anton Hausknecht (Maissau, Austria) for giving us a

collection of Entoloma luteobasis and to Nicolien Sol (Naturalis Biodiversity Center,

Leiden, The Netherlands) for providing information on the holotype collection of

E. luteobasis. Thanks are due also to Gabriel Moreno (Univ. Alcala de Henares, Madrid,

Spain) and Tim Baroni (State University of New York - College at Cortland, New York)

for reviewing the manuscript and providing useful suggestions.

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Entolomataceae (Agaricomycetes, Basidiomycota) based on morphological and molecular

evidence. North American Fungi 6(12): 1-19. http://dx.doi.org/10.2509/naf2011.006.012

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

Ebert H, Ludwig E, Rodig TH 1992. Neue oder seltene Arten aus der Gattung Entoloma. Zeitschrift

fiir Mykologie 58(2): 185-196.

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

application to the identification of mycorrhizae and rusts. Molecular Ecology 2(2): 113-118.

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

Kokkonen K. 2015. A survey of boreal Entoloma with emphasis on the subgenus Rhodopolia.

Mycological Progress 14: 116. [52 p.]. http://dx.doi.org/10.1007/s11557-015-1135-y

Largent DL. 1994. Entolomatoid fungi of the western United States and Alaska. Mad River Press

Inc., Eureka, California. 516 p.

Morgado LN, Noordeloos ME, Lamoureux Y, Geml J. 2013. Multi-gene phylogenetic

analyses reveal species limits, phylogenetic patterns, and evolutionary histories of key

morphological traits in Entoloma (Agaricales, Basidiomycota). Persoonia 31: 159-178.

http://dx.doi.org/10.3767/003158513x673521

Noordeloos ME. 1981. Introduction to the taxonomy of the genus Entoloma sensu lato (Agaricales).

Persoonia 11: 121-151.

Noordeloos ME. 1992. Fungi Europaei vol. 5: Entoloma s.l. Ed. Candusso, Saronno, Italy. 760 p.

Noordeloos ME. 2004. Fungi Europaei, vol. 5A: Entoloma s.l., supplement. Ed. Candusso, Alassio,

Italy. 617 p.

Entoloma ochreoprunuloides in Italy ... 887

Noordeloos ME, Hausknecht A. 1998. Rezente R6tlingsfunde aus Osterreich und Italien

Osterreichische Zeitschrift fiir Pilzkunde 7: 227-261.

Osmundson TW, Eyre CA, Hayden KM, Dhillon J, Garbelotto MM. 2013. Back to basics: an

evaluation of NaOH and alternative rapid DNA extraction protocols for DNA barcoding,

genotyping, and disease diagnostics from fungal and oomycete samples. Molecular Ecology

Resources 13(1): 66-74. http://dx.doi.org/10.1111/1755-0998.12031

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

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

methods and applications. San Diego, Academic Press.

http://dx.doi.org/10.1002/em.2850160211

MY COTAXON

October-December 2016—Volume 131, pp. 889-896

http://dx.doi.org/10.5248/131.889

Phaeocollybia pakistanica sp. nov.,

the first representative of the genus from Pakistan

JUNAID KHAN’, HASSAN SHER’ & ABDUL NASIR KHALID?

‘Center for Plant Sciences and Biodiversity, University of Swat, Pakistan

*Department of Botany, University of the Punjab,

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

“ CORRESPONDENCE TO: Junaid. botany@gmail.com

ABSTRACT—A new species of Phaeocollybia, P. pakistanica, is described that represents

the first record of the genus from Pakistan. Diagnostic characters include purplish red

to brownish red pilei, lilaceous gills, a monopodial radicating cartilaginous stipe, small

ellipsoidal verruculose brown spores, and thin-walled subcapitate cheilocystidia. ITS-nrDNA

sequence analyses support P pakistanica as an independent new species.

KEY worps—Agaricomycetes, Hymenogastraceae, coniferous forest, pseudorhiza, Swat

district

Introduction

Phaeocollybia R. Heim is a genus of agaricoid fungi, currently accepted

in Hymenogastraceae (Matheny et al. 2006). The genus is easy to recognize

in the field due to the presence of the cartilaginous stipe and deeply rooting

pseudorhiza, moist to viscid umbonate pilei, and brown spores. Microscopically

the genus is characterized by gelatinous tissues, roughened brown spores

with an apical callus, the presence of cheilocystidia, and tibiiform diverticula

(Norvell & Exeter 2008). The genus is widely distributed in forested temperate

regions worldwide and currently comprises about 95 described species

(http://www.indexfungorum.org/).

Species later placed in Phaeocollybia were first included by Fries (1838) in

the heterogeneous assemblage of brown-spored agarics within the [unranked;

section] Gymnoti under [unranked; subgenus] Naucoria. Kammer (1871) and

Quélet (1872) independently raised Naucoria to generic level. Heim (1931)

890 ... Khan, Sher & Khalid

established Phaeocollybia with P. lugubris (Fr.) R. Heim as type to accommodate

Naucoria species characterized by yellow brown spores, viscid caps, and

cartilaginous radicating stipes.

The genus is widely distributed throughout forests in Great Britain, Europe,

Asia, Australasia, and South, Central, and North America (Norvell 1998a).

Nowhere common, the genus can be considered quite rare for Pakistan, and

there is no previous Phaeocollybia record for the country (Ahmad et al. 1997).

In this paper we report the first occurrence of Phaeocollybia for Pakistan and

describe a new species based on morphological and molecular analyses.

The specimens were collected from moist temperate forests of Malam Jabba,

Khyber Pakhtunkhwa, Pakistan. Malam Jabba valley, an important biodiversity

hotspot for Pakistan that hosts a variety of important medicinal and

aromatic plants (Sher & Al-Yemeni 2011), lies in northern Pakistan between

the Himalayas and the Hindu Kush foothills. The region (35°20’-35°45’N

72°12’-73°32’E) lies 52 km from Saidu Sharif, the capital of Swat District in

the Malakand division of Khyber Pakhtunkhwa, bordering Shangla District to

the northeast and District Buner to the southwest. Altitudes range from 990

m (valley entrance) to 2880 m (the highest peak of Shagar Sar; Ali et al. 2011).

The valley benefits from varied agro-climatic conditions and geographic areas

that facilitate a large number of important plants (Ali & Qasier 1986). The lush

green coniferous forests of the Malam Jabba are also home to large numbers of

important herbs, and the forest floors rich in humus and moist environments

favor the growth of many macrofungi.

Materials & methods

Collection, morphological and anatomical study of basidiomata

Basidiomes were collected according to Lodge et al. (2004) slightly modified by

excavation to trace their pseudorhizal origins (Norvell 1998b). Fresh specimens were

photographed and important ephemeral characters and habitat data were recorded in

the field. Surface features were examined using a hand lens. The collected specimens

were covered in blotting paper prior to transport to the laboratory where they were

dried at 40-50°C using a fan heater.

Macroscopic descriptions are based on the fresh field collections. Colors were

coded according to Munsell (1975). Certain morphological and developmental terms

(e.g., tibiiform diverticula, vertical-monopodia, pseudorhizae) follow Norvell (1998a,b).

The dried specimens have been deposited in Swat University herbarium (SWAT) and

Herbarium, University of the Punjab, Pakistan (LAH).

For microscopic examination, tissues were rehydrated in distilled water and mounted

in 5% KOH, with Congo red as a stain. Anatomical features were measured using

calibrated Piximétre software connected to a BOECO BM 120 compound microscope

through a Byomic MVV 3000 microscopic camera and visualized on a computer screen.

Twenty basidiospores, basidia, and cystidia were measured; Q = the length/width ratio

Phaeocollybia pakistanica sp. nov. (Pakistan) ... 891

JN102520 Phaeocollybia gregaria

GQ165650 Phaeocollybia gregaria

GQ165653 Phaeocollybia gregaria

EU846286 Phaeocollybia fallax

EU846281 Phaeocollybia fallax

JN102535 Phaeocollybia olivacea

96 | JN102530 Phaeocollybia olivacea

711 GQ165679 Phaeocollybia olivacea

JN102495 Phaeocollybia ammiratit

99'GQ165629 Phaeocollybia ammirati

JN102546 Phaeocollybia redheadii

N102545 Phaeocoliybia redheadit

JN 102541 Phaeocollybia redheadii

77; GQ165682 Phaeocollybia oregonensis

99 |! GQ165685 Phacocollybia oregonensis

GQ165684 Phaeocollybia oregonensis

KJ450910 Phaeocollybia benzokaufimanii

KJ450907 Phaeocollybia benzokaufimanti

99|KJ450917 Phaeocollybia benzokaufimani

KJ450916 Phaeocollybia benzokaufimann

JN 102499 Phaeocollybia attenuata

JN102500 Phaeocollybia attenuata

100 | GQ165634 Phaeocollybia attenuata

JN 102497 Phaeocollybia attenuata

JN102515 Phaeocollybia fallax

ai JN102512 Phaeocollybia fallax

400 | KF219601 Phaeocollybia phaeogaleroides

KF219600 Phaeocollybia phaeogaleroides

100 - @ KY007615 Phaeocollybia pakistanica

@ KY007616 Phaeocollybia pakistanica

96 | JN 102548 Phaeocollybia sipei

EU644706 Phaeocollybia sipei

EU644705 Phaeocollybia sipei

KF219568 Phaeocollybia dissiliens

EU669354 Phaeocollybia dissiliens

63! KF219569 Phaeocollybia dissiliens

AJ585496 Galerina marginata

0.02

Fic. 1. Maximum Likelihood phylogeny of Phaeocollybia spp. Cluster support percentage is

shown next to the branches. The 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 representing number

of substitutions per site. The analysis involved 34 nucleotide sequences. There were a total of 422

positions in the final dataset.

892 ... Khan, Sher & Khalid

of a single spore; Qe = average length/width ratio of all spores; Me = average L x W of

all spores measured.

DNA extraction, amplification, sequencing, and molecular phylogenetic analysis

About 50 mg of dried gills were ground into fine powder using liquid nitrogen, and

the DNA was extracted according to Porebski et al. (1997). The universal primer pair

ITS1F and ITS 4 (White et al. 1990, Gardes & Bruns 1993) was used to amplify the rDNA

ITS (ITS1+5.8s+ITS2) region in 50 uL reaction volumes following Gardes & Bruns

(1993). The PCR amplified product was sequenced by Beijing Genomic Institute (Hong

Kong). BLAST analysis was performed using the National Center for Biotechnology

Information (USA) database, and closely matching sequences were downloaded for

further phylogenetic analysis. Sequences were aligned using MEGA 6 software (Tamura

et al. 2013). Galerina marginata (Batsch) Kiihner was selected as outgroup. Sequences

are deposited in GenBank.

Results

Phylogenetic analysis FIG. 1

Amplification of the entire ITS region using the ITSI and ITS4 primers

generated a 690bp sequence. The initial BLAST comparison of the Pakistan

sequences showed 85% identity and 96% query cover with Phaeocollybia

dissiliens A.H. Sm. & Trappe (KF219569, KF219568) and 84% identity and

96% query cover with P sipei A.H. Sm. (JN102548, EU644705, EU644706).

The MEGA6 maximum likelihood analysis clustered our Pakistan sequences

with the P. dissiliens and P. sipei sequences in a separate clade with a strong

bootstrap value of 81%, thereby supporting P pakistanica phylogenetically as

an independent species.

Taxonomy

Phaeocollybia pakistanica J. Khan, Sher & Khalid, sp. nov. Figs 2, 3

MycoBAnk MB 818193

Differs from Phaeocollybia sipei by possession of clamp connections, and from both P

dissiliens and P. sipei by its lilaceous cap and gill color.

Type: Pakistan, Khyber Pakhtunkhwa Province, Swat District, Malam Jabba, 2500 m

a.s.l, small groups on forest soil near Abies pindrow (Royle ex D. Don) Royle and Pinus

wallichiana A.B. Jacks., 14 Sep. 2015, Junaid Khan MJ1560 (Holotype, SWAT15-1560;

GenBank KY007615)

Erymo oey: referring to Pakistan, the country where the species was first collected.

Piteus 70-90 mm diam., conical to cuspidate when young, becoming convex

and papillate at maturity, margin initially straight, later uplifted to revolute and

undulating; edge and outer margin striate to rugulose almost two-thirds toward

the central disc; viscid to glutinous when wet, shiny when dry; hygrophanous

with a darker edge, color initially purplish red (LORP6), dulling to brownish red

(10R7/4) and finally brownish (10R6/2) on exposure. CONTEXT concolorous

with or slightly paler than pileus, thin, <3 mm thick at the disc and 1 mm at the

Phaeocollybia pakistanica sp. nov. (Pakistan) ... 893

aa oh

cay

Fic. 2. Phaeocollybia pakistanica (holotype, SWAT15-1560). A. basidiomata in situ; B. detail

of shiny pileus and violet lamellae; C. purplish-red stipe and lamellae; D. excavated basidioma

showing purplish stipe apex. Scale bars = 10 mm.

outer edge, moist to dry, soft; odor fruity to mildly mushroomy; taste mild, not

distinctive. LAMELLAE adnexed to slightly sinuate, close, color purplish with

a reddish tone (7.5 RP7) at least when young before turning brown (10R4/8),

edges eroded, more or less straight, crisped or curled toward the stipe in some

specimens; lamellulae frequent, with crisped terminal ends, L+ll/cm <25.

StTrPE including pseudorhiza overall <160 mm, aerial portion <100 mm long,

apex 5 mm diam.; central, terete, cylindrical or slightly widening towards

the base, even slightly bulbous in some specimens; concolorous with the cap

or slightly paler, with a paler purplish tinted (7.5 R8/2) upper portion and

darker (7.5R6/8) lower portion without any purplish tints, glabrous or slightly

fibrous when young, not bruising, hollow, cartilaginous, cortex 1-1.5 mm

thick, lined with fibrils. PsEUDORHIZA <60 mm, (approximately 1/3 of overall

stipe+pseudorhiza length), vertical monopodial, tapering with a blunt origin,

more or less concolorous with the aerial stipe, but slightly paler orange in upper

part and slightly darker below.

BASIDIOSPORES (6—)6.4—7.1(-7.2) x (3.4-)3.6-4.5 um, Q = (1.3) 1.5-1.8

(1.9), Me = 6.7 x 4.1 um, Qe = 1.6, ellipsoid to elongate in side view, ovoid in

face view, finely verruculose in oil immersion, apiculate, brownish amber in

KOH. Basrp1A <30 um long, narrowly clavate to cylindrical, hyaline, 4-spored,

894 ... Khan, Sher & Khalid

Fic. 3. Phaeocollybia pakistanica (holotype, SWAT15-1560). A. basidiospores; B. basidiole and

basidium; C. subcapitate cheilocystidia; D. hymenium showing immature and sterigmate basidia;

E. pileipellis with narrow, branched and anastomosing hyphae of the suprapellis (top) overlying

shorter and wider unbranched subpellis hyphae (middle) and wider thick-walled tramal hyphae

(bottom); FE. pseudorhiza with rhizopellis layer shown above thick-walled tramal cells. Scale bars:

A = 4.5 um; B = 12 um; C = 20.5 um; D = 10 um; E = 21 um; F= 35 um.

sterigmata <3 um long. CHEILOCYSTIDIA <40 um long, sub-capitate, numerous,

thin-walled. PrrerPeLiis bilaminate, suprapellis a <23 um-thick layer with

narrow (<2-3 um diam) branched, gelatinized, pale brownish to hyaline

hyphae with occasional clamp connections at the septa overlying a slightly

darker (KOH) subpellis composed of somewhat inflated thick-walled cells

(<10 um diam and lacking clamp connections) interspersed with <10 um diam,

non-septate, golden brown, branched oleifers. PILEUS TRAMA cells shorter,

<25 um diam., thick-walled. StrprTIPELLis cells <100 x 10 um, cylindrical, thick-

walled. PSEUDORHIZAL PELLIS cells narrow, <5 um diam, elongate, without any

clamps. STIPE & PSEUDORHIZA TRAMAS Sarcodimitic, with thinner branching

flexuous cells intermixed among ‘vessel elements’ that are thick-walled, <250

x 30 um, more or less fusoid, brown to golden in KOH. CLAMP CONNECTIONS

very rare; observed occasionally in pileipellis. TrsFORM DIVERTICULA present

on pseudorhizal pellis.

ADDITIONAL SPECIMENS EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA PROVINCE,

SwaT District, Malam Jabba, 2500 m asl, in small groups under Abies pindrow, 10 Aug

Phaeocollybia pakistanica sp. nov. (Pakistan) ... 895

2014, Junaid Khan MJ65 (LAH35009); 31 Jul, 2016, Junaid Khan MJ1660 (SWAT16-

0021)

Discussion

Molecular analysis clusters the present Pakistani collection with P. dissiliens

and P. sipei, but P pakistanica is both morphologically and molecularly well

separated from those two species. Phaeocollybia dissiliens (Smith & Trappe

1972) and P. sipei (Smith 1957), both originally described from the North

American Pacific coast, are distinguished by their bright orange to brownish

orange coloration and lack the lilaceous gill colors characterizing P. pakistanica.

The vertical monopodial pseudorhiza of P. pakistanica lacks the thread-like to

racemose pseudorhiza of P dissiliens and P. sipei noted by Norvell & Exeter

(2008). The finely verruculose or punctate roughened ellipsoidal basidiospores

are also morphologically similar to the two North American species. The

rarity of clamp connections in the Pakistani collections (with a few found

only in the suprapellis) further distinguishes P pakistanica from P. dissiliens

(with clamp connections relatively frequent in the pileus suprapellis and at the

cheilocystidial and basidial bases) and P. sipei (which lacks clamp connections

throughout).

Phaeocollybia spoliata E. Horak, described from India, slightly resembles

P. pakistanica in its reddish coloration (although orange-brown more than

purplish) and striate cap margin but is easily distinguished by its larger (9.5-10

x 5-5.5 um) more heavily ornamented almond-shaped spores (Horak 1974).

Phaeocollybia rancida E. Horak, also described from high coniferous forests

in India and characterized by lilaceous violet lamellae, capitate cheilocystidia,

and ellipsoid basidiospores, is easily separated by its ochraceous coloration,

glutinous pilei, slightly smaller (5-6 x 3-3.5 um) basidiospores, and absence of

clamp connections (Horak 1974).

Acknowledgements

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

financial assistance under NRPU Project. Sincere thanks to Dr. Najam-ul-Sahar Afshan,

Assistant Professor (Centre for Undergraduate Studies, University of the Punjab, Lahore,

Pakistan) and Dr. Lorelei Norvell (Pacific Northwest Mycology Service, Portland

Oregon U.S.A.) for presubmission reviews.

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Washington, Seattle, Wash. 391 p.

Norvell LL. 1998b. Observations on development, morphology and biology in Phaeocollybia.

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Norvell LL, Exeter RL. 2008. Phaeocollybia of Pacific Northwest North America. U.S. Dept. of the

Interior, Bureau of Land Management, Salem District. USDI BLM/OR/WA/GI-08/100-1792.

ISBN-13: 978-0-9791310-1-1; ISBN-10: 0-9791310-1-4. 228 pp.

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plants containing high polysaccharide and polyphenol components. Plant Molecular Biology

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

MYCOTAXON

October-December 2016—Volume 131, pp. 897-906

http://dx.doi.org/10.5248/131.897

Mucor indicus isolated from the semiarid region of Brazil:

a first record for South America

Car os A.F. DE SouzA’, DioGo X. LIMA’, RAFAEL J.V. DE OLIVEIRA’,

LucIANA M.S. GURGEL”, ANDRE L.C.M. DE A. SANTIAGO’

‘Post-graduate course in Fungal Biology, Universidade Federal de Pernambuco,

Av. Prof. Nelson Chaves, s/n, 50670-420, Recife, PE, Brazil

?Instituto Agronédmico de Pernambuco (IPA),

Av. General San Martin, 1371 Bongi, Recife, PE, Brazil

* CORRESPONDENCE TO: carlos_fragosol1@hotmail.com

ABSTRACT—A specimen of Mucor indicus was isolated from a dung sample collected in the

city of Sertania in the Brazilian state of Pernambuco and its identity confirmed through LSU

rDNA sequence analysis. The taxon is described and illustrated for the first time from South

America. An identification key is provided for Mucor species from the northeast Brazilian

semiarid region (Caatinga).

Keyworps—lower fungi, phylogenetic analysis, Mucoromycotina, Zygomycota

Introduction

Mucor Fresen. (Mucoraceae) comprises species characterized by the

formation of non-apophysate sporangia, producing simple or branched

sporangiophores that emerge directly from the substrate. Few species have

rhizoids and stolons are not produced (Benny 2014). This genus has a worldwide

distribution and most of the species described are saprobes commonly isolated

from a wide variety of substrates such as soil, stored grains, fruits, rotting

vegetables, fleshy agarics, and herbivore dung (Viriato & Trufem 1985, Alves

et al. 2002, Schoenlein-Crusius et al. 2006, Jacobs & Botha 2008, Santiago &

Souza-Motta 2008, Santiago et al. 2013).

Although over 300 species have been cited in the literature (Jacobs & Botha

2008), the exact number of authentic taxa is unknown. According to Gherbawy

et al. (2010), it is possible that the number of authentic species could range

898 ... Souza & al.

between 50 and 75. Schipper (1973, 1975, 1976, 1978) described 39 species,

four varieties, and 11 forms of Mucor. Subsequently, 17 additional species have

been proposed (Mehrotra & Mehrotra 1978, Mirza et al. 1979, Subrahmanyam

1983, Chen & Zheng 1986, Schipper 1989, Schipper & Samson 1994, Watanabe

1994, Zalar et al. 1997, Pei 2000, Alves et al. 2002, Jacobs & Botha 2008, Alvarez

et al. 2011, Hermet et al. 2012, Madden et al. 2012).

Previous studies have treated Mucor as polyphyletic (O'Donnell et al. 2001,

Kwasna et al. 2006, Jacobs & Botha 2008, Budziszewska & Piatkowska 2010,

Alvarez et al. 2011). Based on the phylogeny of ITS and LSU rDNA regions

of several mucoralean species, Walther et al. (2013) observed that some

Mucor species with curved sporangiophores represent a natural grouping with

Backusella Hesselt. & J.J. Ellis, and transferred nine Mucor species to Backusella.

The aim of the present study was to describe and illustrate M. indicus

from dung samples collected in the city of Sertania, Pernambuco, Brazil. An

identification key for Mucor species from the semiarid region of Brazil is also

provided.

Material & methods

Isolation and morphological & physiological identification of M. indicus

Samples of goat (Capra hircus Linnaeus) dung were collected at the Agronomic

Institute of Pernambuco stations, located in the city of Sertania (8°03’38”S 37°13’32”W)

in the state of Pernambuco, 326 km away from Recife, Brazil. The mean annual

temperature is 25°C, with rainfall of 635 mm per year. The area has a hyperxerophilic

Caatinga vegetation. The samples were placed in clean plastic bags for transportation to

the laboratory and then incubated in triplicate in moist chambers for 10 days. The plates

were left on a bench at room temperature (28 + 2°C) exposed to alternating light and

dark periods. Fragments of mycelium were removed directly from Petri dishes under

a Leica EZ4 stereomicroscope and transferred to Petri dishes with malt extract agar

(MEA) (Benny 2008). The specimen was identified through observation of macroscopic

(color, appearance, and diameter of the colony) and microscopic (e.g., shape and size of

sporangiophores, columellae, sporangia, and sporangiospores) characteristics based on

the descriptions of Schipper (1978). The thermotolerance of M. indicus was confirmed

by growing it at 40°C for 7 days. The colony colors were designated according to Maerz

& Paul (1950).

Molecular analysis

Cultures grown in test tubes containing malt extract were incubated at 28°C for

6 days to generate fungal biomass. The material was transferred to 2 mL microtubes

with screw caps. Subsequently, 0.5 g acid-washed glass beads of two different diameters

(150-212 um and 425-600 um, 1:1; Sigma, USA) were added to each tube. The material

was crushed by stirring at high speed in a FastPrep homogenizer. The genomic DNA

extraction procedure was conducted as described by Goes-Neto et al. (2005). The

mycelium was washed with chloroform: isoamyl alcohol (24:1) and then homogenized

0.1

Mucor indicus, new for South America (Brazil) ... 899

soo Cireinella angarensis JN206551

‘00 C. chinensis JN206549

o.zef~ Mucor fuscus JN206442

°°L M. fuscus JN206443

M. exponens JN206441

M. laxorrhizus JN206444

1,00

100 ogg M. mucedo HM849687

°L__ WV. piriformis HM849681

ee — ™. flavus JN206469

One M. luteus HM849685

|e BSS M. japonicus JN206446

i M. endophyticus JN206448

Og. M. hiemalis f. hiemalis HM849683

1 M. irregularis JX976213

1.00 | M. irregularis JN206450

0.63. 100

: M. odoratus JN206495

1.00 M. inaequisporus JN206501

083 M. amphibiorum HM849688

M. falcatus JN206509

0.99

res 00. M. variosporus JN206508

oor | 1 M. indicus HM849690

- is we M. indicus KU646991

s.o07/ M. genevensis JN206435

°"L M. genevensis JN206436

— soo ™. circinelloides JN206430

100L__ WV. circinelloides JN206413

991, M. plumbeus HM849677

4.00.” |; M. racemosus HM849676

0.99. M. racemosus JN206433

Fig. 1. Phylogenetic tree of Mucor species constructed using partial LSU rDNA sequences. Circinella

angarensis and C. chinensis were used as outgroup. Sequences are labeled with their GenBank

accession numbers. Branch support values are from Bayesian inference (above) and maximum

likelihood (below) analyses. The sequence obtained in this study is in boldface.

900 ... Souza & al.

in 2% cetyltrimethylammonium bromide (CTAB) buffer. The DNA was precipitated

in chilled isopropanol, washed with 70% ethanol, and resuspended in 50 uL ultrapure

water.

The primer pairs LR1/LSU2 were used to amplify a fragment of the large subunit

(LSU) nuclear ribosomal DNA (rDNA) (van Tuinen et al. 1998, Santiago et al. 2014).

The polymerase chain reactions were carried out as described by Oliveira et al. (2014).

The newly obtained sequence was deposited in the National Center for Biotechnology

Information GenBank database (accession number KU646991).

Phylogenetic reconstructions were obtained by analyzing the partial LSU rDNA

sequences. The fungal sequences were aligned with ClustalX (Larkin et al. 2007)

and edited with BioEdit (Hall 1999). Prior to the phylogenetic analysis, the optimal

nucleotide substitution model was estimated using Topali 2.5 (Milne et al. 2004).

Bayesian inference (two runs over 10° generations with a burn-in of 2500) analysis

was performed with MrBayes 3.1.2 (Ronquist & Huelsenbeck 2003), launched from

Topali 2.5.

Results

Phylogenetic analysis

The Blastn analysis of the LSU sequences showed a 99% similarity between

our Brazilian material (URM7222) and M. indicus (CBS 226.29; GenBank

HM849690). Phylogenetic analysis of the sequence datasets (Fic. 1) showed

that URM7222 formed a well-supported clade (BI 1.00/ML 98%) with the

sequence of M. indicus (HM849690).

Taxonomy

Mucor indicus Lendn., Bull. Soc. Bot. Genéve 21: 258 (1930). Fic. 2

Co.ony initially white and becoming deep yellowish (MP 11C1 - Amber

White) with yellowish reverse (MP 11J6), reaching 9.4 cm in diam. and 9 mm in

height after 4 days in MEA at 28°C. SPORANGIOPHORES erect (rarely circinate),

repeatedly sympodially branched, with long branches, (4—)5-12.5(-14.5) um

diam., hyaline to yellowish, smooth-walled. SpPoranGra globose to subglobose,

(22.5-)32.5-45(-50) um diam., brownish, with diffluent wall. CoLUMELLAE

globose (27.5—)30-47.5(-50) um, subglobose (25.5-)27.5-28.5 x 42.5-45(-47.5),

or applanate (18-)20-21.25 x 27.5-30(-32.5) diam., grayish or yellowish brown,

smooth-walled; CoLLARS small and infrequent. SPORANGIOSPORES hyaline,

smooth-walled, often ellipsoid (3.75-)4-6.25 x 3-6.5(-7.5) um and subglobose

4-6(-7.5) um in diam. CHLAMYDOSPORES present. ZYGOSPORANGIA not

observed. Growth reasonably good at 40°C (7.6 cm in 168 hours) in MEA with

rare sporangiophore production and poor sporulation.

HasiratT: Recorded on goat dung in the semiarid region of Pernambuco,

northeastern Brazil.

Mucor indicus, new for South America (Brazil) ... 901

A B

50 pm | 50 pm

od ER

De’ E

)

»,

» 32

)

»)

D 2

s =)

Dy 3

3) ? »

xy, AY yy

5) >

; }

ua yp ») ; ma

“a eee $e)

gi 2

" =

> ?

2 2 dy 4 =

50 7m 25 pm

Fig. 2. Mucor indicus (URM 7222): A. Sporangiophore simple with sporangia; B. Sporangiophore

simple with columella; C. Sporangiophore branch with columella; D. Chlamydospores;

E. Sporangiospores.

SPECIMEN EXAMINED: BRAZIL, PERNAMBUCO, Sertania: Instituto Agrondédmico de

Pernambuco (IPA), 8°03’38”S 37°13’32”W, from goat dung, 5.X1.2014, leg. C.A.F de

Souza (URM 7222).

GEOGRAPHIC DISTRIBUTION: India (Lendner 1930, Abe et al. 2004), Poland

(Szwedek-Trzaska & Glowacka 2011), Spain (Luna et al. 1986, Garcia-Pantaleén

et al. 1992, Herrero et al. 1996), USA (Oliver et al. 1996, Sobel 2001, Alvarez et

902 ... Souza & al.

al. 2009), and Vietnam (Lee & Fujio 1999). Our Brazilian collection represents

the first report from South America.

Discussion

The morphological characteristics of M. indicus reported here show a

close similarity with the description by Schipper (1978), although there are

differences in the diameter of sporangiospores and columellae. Sporangiospore

sizes reported in the literature (5.4-5.7 x 4.4 um; Schipper 1978) are smaller

than our maximum measurements, whereas columellae were larger (<56 x 53

um; Schipper 1978) than observed in our isolate. Mucor indicus strains may

exhibit morphological similarities to M. circinelloides Tiegh. (Schipper 1978).

However, M. indicus lacks the abundant short branches reported for M.

circinelloides and its predominantly globose columellae differ from the obovoid

type found in M. circinelloides. Moreover, the thermotolerance inherent to

M. indicus is not found in M. circinelloides (Schipper 1978). Walther et al. (2013)

have reported that although some M. indicus cultures morphologically resemble

M. circinelloides, there are clear genetic differences between these two species.

The LSU phylogenetic tree (Fic. 1) suggests that M. indicus and M. vario-

sporus Schipper are genetically closely related. Morphologically, however,

M. indicus colonies are lower than those of M. variosporus, which can reach

20 mm in height (Schipper 1978). Differences in sporangiophore diameters

have also been observed, with M. variosporus producing larger (14(-23) um)

sporangiophores. Finally, M. variosporus sporangia are yellow and larger (<150

um diam.) than those of M. indicus, which are brownish and smaller.

This manuscript reports the first occurrence of M. indicus in South America,

thereby expanding our knowledge of mucoralean distribution. In the semiarid

region of Brazil (Caatinga), other Mucor species have been isolated, such as

M. circinelloides Tiegh. f. circinelloides, M. hiemalis Wehmer, M. luteus Linnem.

ex Wrzosek., M. prayagensis B.S. Mehrotra & Nand ex Schipper, M. racemosus

Fresen., and M. subtilissimus Oudem. (Santiago 2016). During a recent

survey of Mucorales from other Brazilian semiarid regions, M. circinelloides

Tiegh. f. griseocyanus (Hagem) Schipper, M. circinelloides Tiegh. f. janssenii

(Lendn.) Schipper, M. circinelloides Tiegh. f. lusitanicus (Bruderl.) Schipper,

M. racemosus Fresen. f. racemosus, and M. variosporus Schipper were also

isolated (unpublished data).

Identification key for Mucor taxa isolated from

the semiarid region of Brazil

Li Sporangiospares devular in forin1-andi size’ sare ls sh Se sar liv. wipe dy < gts Sepeors Senate a)

1. Sporangiospores variable in size and shape: globose, ovoid, cylindrical, and

PUSHOUIA tc cot em Ncee phrase v attasee oatets Gseheced autect eae tMaN Seas Ne M. variosporus

Mucor indicus, new for South America (Brazil) ... 903

2. Sporangiophores unbranched or slightly sympodially branched ................ 3

2. Sporangiophores repeatedly branched se scwsse ee a sen aee enae o ale eed Bes 6

3. Columellae globose to subglobose or globose to applanate;

sporangiospores <15 um long with or without a granule ................... 4

3. Columellae spherical or ellipsoidal with a truncated base;

SpotansiosporeseSS corel Ovi IONE. eh. masts. Badpens Patel te Bake, wv Boden n Mody oth Boks. fe)

. Sporangiophores unbranched; columellae subglobose to applanate;

sporangiospores ellipsoidal or flattened on one side ........... M. prayagensis

. Sporangiophores sparsely branched; columellae globose to subglobose

(rarely ellipsoidal); sporangiospores ellipsoidal with a granule at

CCR CHGAER Praline pete test ncaa h ieee Tater aigeeh ral eet M. subtilissimus

5. Columellae ellipsoidal with a truncate base; sporangiospores ellipsoid,

sometimes flattened on one side, 2.5-10 x 2-7.5 um .............. M. hiemalis

5. Columellae globose;

sporangiospores long-elliptical and fusiform 2.5-8.1 x 1-5 um....... M. luteus

Gz -ADSenCe ero wAliat AOCE 7 ee Mer teed et tats oleh at Nate A Nady oh ee Gata B a iNath i

GC SOGESHOW1 lh: At AOS 5. eenteetn Mn arden Ba at en peers Babel cer Sah es Sansaharh aesere M. indicus

7. Sporangiophores sympodially branched; chlamydospores present or not,

when present not abundant and never formed in sporangiophores or

COMIC Ae ar ee ater acter tte ge Sige Ph alk gr See rae SM Todi peak geal ual yor STALEY ra 8

7. Sporangiophores sympodially and monopodially branched;

chlamydospores abundant, some formed in sporangiophores and

CONIA ae AT ak Ns oe ot ee ht Me A RP RAO M. racemosus f. racemosus

8. Colonies initially white, then becoming gray;

sporangiophores <10 um in diam.; sporangia brownish-black ............... 9

8. Colonies initially yellow than turning brownish;

sporangiophores <17 um in diam.; sporangia gray-brownish............... 10

o-spotansiospores ellipsoid MAS. OS on wl M. circinelloides f. griseocyanus

9. Sporangiospores globose to slightly subglobose ........ M. circinelloides f. janssenii

10. Columellae obovoid;

sporangiospores ellipsoid .................... M. circinelloides f. circinelloides

10. Columellae globose;

sporangiospores ellipsoid, some irregular in shape . circinelloides f. lusitanicus

Acknowledgments

The authors thank the Fundacao de Amparo a Ciéncia e Tecnologia do

Estado de Pernambuco (FACEPE-APQ 0842-2.12/14), the Conselho Nacional de

Desenvolvimento Cientifico e Tecnolédgico (CNPq - 458391/2014-0), the Programa de

Pesquisa em Biodiversidade do Semiarido (MCT/CNPQ/PPBio - 457498/2012-9). We

also thank Dr. Matias Cafaro and Dr. José Ivanildo de Souza for the critical revision of

the manuscript.

904 ... Souza & al.

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MY COTAXON

October-December 2016—Volume 131, pp. 907-911

http://dx.doi.org/10.5248/131.907

Blodgettia saprophytica sp. nov. and Uberispora tropicalis,

new records from southern China

CHUN-LING YANG’, KAI ZHANG’, JIN-YE WANG’, JI- WEN X14’, YING-RuI Ma’,

JIAN-MEI GAO’, X1U-GUO ZHANG’ & ZHUANG LI’*

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

?Department of Landscaping, Shandong Yingcai University, Jinan, 250104, China

*CORRESPONDENCE TO: sdau613@163.com; zhxg@sdau.edu.cn; liz552@126.com

ABSTRACT—A new species, Blodgettia saprophytica, is described and illustrated from

specimens collected on dead branches in Guangxi Province, China. Uberispora tropicalis is

newly recorded from China.

Key worps—hyphomycetes, taxonomy

Introduction

Blodgettia Harv. (Harvey 1858), comprising algal and fungal elements, has

commonly been considered an illegitimate name under the former ICBN Art.

70 (Stafleu et al. 1972) so that Blodgettia E.P. Wright (Wright 1881) has been

used for the fungal element. However, Seifert et al. (2011), who interpreted

Wright's treatment as emending Blodgettia Harv. by selecting the fungal element,

as now required by the current International Code of Nomenclature (McNeill

et al. 2012), regarded Blodgettia bornetii E.P. Wright as a later synonym of

B. confervoides. Two other species, B. indica Subram. and B. aquatica Udaiyan,

have been included in the genus (Subramanian 1954, Udaiyan 1992).

Here we propose an additional Blodgettia species on dead branches from

southern China. We also report Uberispora tropicalis on dead branches as a new

record from China.

Material & methods

Samples were processed, examined, and photographed following the methods

described in Xia et al. (2014). Adobe Photoshop 7.0 was used to process individual

908 ... Yang & al.

Fic. 1. Blodgettia saprophytica (holotype, HSAUP H7855). A. Colonies on natural substratum.

B. Conidia and hyphae. C. Conidia covered by a mucilaginous tunica. D. Conidia.

Blodgettia saprophytica sp. nov. (China) ... 909

images into photomicrographic plates, with backgrounds modified for esthetic

reasons. In view of our failure to obtain cultures of this species, no molecular data are

presented here. The specimens are deposited in the Herbarium of Department of Plant

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

the Mycological Herbarium, Institute of Microbiology, Chinese Academy of Sciences,

Beijing, China (HMAS).

Blodgettia saprophytica C.L. Yang, Z. Li & X.G. Zhang, sp. nov. Fig. 1

MycoBAnk MB 819496

Differs from Blodgettia aquatica and B. indica by its larger, hyaline or subhyaline conidia

with more numerous septa and their occasional coverage by a mucilaginous tunica.

Type: China, Guangxi Province, Nanning, Longhushan, on dead stems of unidentified

broadleaf tree, 15 Nov. 2015, C.L. Yang (Holotype, HSAUP H7855; isotype, HMAS

245638).

ErymMo oey: In reference to the saprophytic habit on dead wood.

CoLonliEs on the natural substratum effuse, inosculating, hyaline or subhyaline,

twinkling. Mycelium superficial and immersed, composed of very long and

yellowish, mostly unbranched, aseptate, smooth hyphae. Conrp1a solitary,

broadly fusiform, straight or slightly curved, moniliform, sometimes covered

by a mucilaginous tunica, forming strings of eight cells, 7-septate, distinctly

constricted at the septa, with the 2 middle cells greatly enlarged, and guttulate

in every cell, globose or ellipsoidal, smooth, hyaline or subhyaline, the two

apical cells attenuated and sometimes triangular, with a rounded tip, 52.5-64

STE S17 tm,

ComMENTs—Among the known species, Blodgettia saprophytica slightly

resembles B. aquatica and B. indica in conidial shape. However, the conidia

of B. aquatica (32-52 x 15-25 um, 3-5-septate; Udaiyan 1992) and B. indica

(20-50 x 7.5-17.5 um, 3-5(-6)-septate; Subramanian 1954) are smaller and

have fewer septa than those of B. saprophytica. In addition, the conidia of

B. aquatica and B. indica are not covered by a mucilaginous tunica.

Uberispora tropicalis Bhat & W.B. Kendr., Mycotaxon 49: 73, 1993. FIG. 2

COLONIES on natural substrate effuse, brown, velvety. CONIDIOPHORES

mononematous, erect, straight or slightly flexuous, rarely branched, pale

brown, 3-7-septate, 40-120 x 2.5-4.8 um. CONIDIOGENOUS CELLS integrated,

terminal, monoblastic, cylindrical, almost colorless, 8-18 x 0.85-2.6 um.

Conip1a solitary, dry, septate, 17-22 um diam, with a central thick-walled,

dark brown, smooth, angular cell, 14-18 um diam, and 3 lateral, almost

colorless, conical cells with obtuse apices and truncate bases 7-14 x 8.5-12 um,

with a conico-truncate basal cell 1.5-4.5 x 1.3-2.6 um, often carrying the upper

27 eeF

i a

“ee

Fic. 2. Uberispora tropicalis (HSAUP H9783). A. Conidiophores, conidiogenous cells,

and conidia. B. Conidia. C. Conidiophore and conidiogenous cell.

Blodgettia saprophytica sp. nov. (China) ... 911

portion of the conidiogenous cell as a basal frill, seceding rhexolytically, with

seceding conidia sometimes remaining attached to the side of the conidiophore

by a wall remnant.

SPECIMEN EXAMINED: CHINA, GUANGXI PROVINCE: subtropical forest of Lengshui, on

dead stems of unidentified broadleaf tree, 16 Nov. 2015, J.M. Gao (HSAUP H9783).

ComMENtTS—Uberispora tropicalis is reported for the first time from China.

The Chinese specimen closely matches the original description by Bhat &

Kendrick (1993), except that the Chinese specimen has a curved and much

narrower conidiogenous cell (0.85-2.6 um vs 2.5-3.5 um). However, in our

opinion they are basically the same species.

Acknowledgments

The authors express gratitude to Dr. Rafael F Castafieda-Ruiz and Dr. De-Wei Li 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, 31493010, 31493011, 31200013) and the Ministry of Science and

Technology of the People’s Republic of China (Nos. 2006FY 120100).

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Andaman Islands (India). Mycotaxon 49: 19-90.

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to Knowledge 10(2). 142 p.

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S, Marhold K, Prado J, Prud’homme van Reine WF, Smith GF, Wiersema JH, Turland NJ. 2012.

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426 p.

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33: 36-42.

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Proceedings of the Royal Irish Academy 28: 21-26.

Xia JW, Ma LG, Castafieda-Ruiz RE, Zhang XG. 2014. A new species of Sporidesmiopsis and three

new records of other dematiaceous hyphomycetes from southern China. Nova Hedwigia

98(1-2): 103-111. http://dx.doi.org/10.1127/0029-5035/2013/0145

MY COTAXON

October-December 2016—Volume 131, pp. 913-923

http://dx.doi.org/10.5248/131.913

New record of Beauveria pseudobassiana from Morocco

ABDESSAMAD IMOULAN”” , YI L1®3, WEN-JING WANG’,

ABDELLATIF EL MEZIANE? & YI-JIAN YAO”

'State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences,

Beijing 100101, China

? Cadi-Ayyad University, Faculty of Science and Techniques, Marrakesh 40000, Morocco

° College of Plant Protection, Fujian Agriculture and Forest University,

Fuzhou, 350002, China

* CORRESPONDENCE TO: imoulan@gmail.com; yaoyj@im.ac.cn

ABSTRACT—Two species of Beauveria were identified from isolates obtained from endemic

Argania spinosa forests in Morocco, using both morphological characteristics and molecular

data. Although isolates exhibited similar reproductive structures, ITS-rDNA_ based

phylogenetic analysis grouped the Moroccan isolates into two clades with known sequences

of either B. bassiana or B. pseudobassiana. Morphological comparison of the colonies

distinguished the two groups of isolates, in full agreement with the ITS phylogenetic analysis.

Beauveria pseudobassiana is recorded for the first time in Morocco.

KEY worDs—Ascomycetes, entomopathogenic fungus, taxonomy

Introduction

Beauveria Vuill. (Cordycipitaceae, Hypocreales) is a genus of cosmopolitan

entomopathogenic fungi found in various types of habitats and ecosystems

(Meyling & Eilenberg 2007). Its species are known to infect a wide range of

insects from many different orders in natural and agricultural environments

(Roy et al. 2010). Preparations from B. bassiana have been developed and

commercialized as biological control agents (Zimmermann 2007). Other

Beauveria species hold great potential for the discovery of mycoinsecticidal

effect (Thomas & Read 2007) and the production of different bioactive and

pathogenetic compounds (Ames & Walsh 2010). Traditionally Beauveria

species have been identified principally by conidial morphology. However,

914 ... Imoulan & al.

the extensive overlap in morphological characters complicates and limits their

usefulness for species identification. Furthermore, the discovery of cryptic

species using molecular markers (Rehner & Buckley 2005, Ghikas et al. 2010,

Rehner et al. 2011, Meyling et al. 2012) makes species identification a challenge

within the genus (Fisher et al. 2011). Accordingly, accurate identification,

including phylogenetic analyses, has been significantly improved using

molecular methods (Rehner et al. 2011).

Through ITS and EF1-alpha sequence analyses, Rehner & Buckley (2005)

demonstrated that the morphospecies B. bassiana was not monophyletic

but comprised two morphologically indistinguishable clades that have been

recently recognized as separate species (Rehner et al. 2011, Meyling et al.

2012). Recently, robust molecular phylogenies based on multi-loci sequences

have resolved 12 well-supported terminal lineages within Beauveria (Rehner

et al. 2011). Similarly, recent sequence analyses have resulted in the recent

description of four new species (Zhang et al. 2012, Chen et al. 2013, Agrawal et

al. 2014, Robéne-Soustrade et al. 2015).

In a previous study of entomopathogenic fungi in forests of endemic Argania

spinosa (L.) Skeels (Sapotaceae) in Morocco, Beauveria and Metarhizium

species were found but only a few isolates were identified as B. bassiana using

molecular analysis of the internal transcribed spacer regions of ribosomal DNA

(ITS-rDNA) sequences (Imoulan et al. 2011). The present study aims to adapt

the phylogenetic species recognition concept (Rehner et al. 2011) by identifying

the strains obtained from the Moroccan forests based on both morphological

and molecular analyses.

Materials & methods

Fungal isolates, growth conditions, and morphological observation

Beauveria isolates were recovered from soil samples collected from Argania spinosa

forests in Morocco using a Galleria mellonella baiting method and maintained on Potato

Dextrose Agar (PDA) slants at 4°C in complete darkness (Imoulan et al. 2011). The

designation of isolates and their geographical origin are given in TaBLE 1. To establish

monosporic cultures, conidial suspensions of 1 x 10° conidia ml' were prepared from

fungal cultures grown on PDA for 2 weeks and plated on PDA plates. The single colony

propagated from single conidia was transferred into a new PDA dish and incubated at

25°C. Dried agar culture vouchers were deposited in Herbarium Mycologium, Chinese

Academy of Sciences, Beijing, China (HMAS).

Microscopic measurements of conidia were taken from slide-cultures produced by

inoculating a small amount of mycelium on a block of the appropriate nutrient agar

overlaid by a cover slip. Images were acquired using an AxioCam MRc digital camera

on a Zeiss AxioScope microscope. Microscopic measurements were performed with

Beauveria pseudobassiana new to Morocco ... 915

TABLE 1. Beauveria and Cordyceps strains and ITS sequences used in phylogenetic

analysis. GenBank numbers in bold are newly generated sequences.

SPECIES

B. pseudobassiana

B. bassiana

B. australis

B. brongniartii

B. asiatica

B. sungii

B. malawiensis

B. vermiconia

B. caledonica

B. amorpha

B. rudraprayagi

B. varroae

B. sinensis

B. lii

B. kipukae

C. militaris

STRAIN

2706

2629

2634

2640

2649

2650

2653

2659

2660

2661

2687

2689

2645

ARSEF1855

ARSEF3405 (T)

ARSEF6229

2721

2718

ARSEF1040

ARSEE751

ARSEF1564 (T)

ARSEF4622

ARSEF4598 (T)

ARSEF617 (T)

ARSEE7058

ARSEE7517

ARSEF4850 (T)

ARSEF4384

ARSEF 1685 (T)

ARSEE7279

ARSEE7760

BCC17613

ARSEF2922 (T)

ARSEF2251

AESEF2567 (T)

ARSEEF2641 (T)

ARSEF4149

MTCC8017 (T)

ARSEF8257 (T)

ARSEF8259

RCEF3903 (T)

RCEF5500 (T)

ARSEEF7032 (T)

ARSEF5050

LOCALITY

Morocco

Canada

USA

China

Morocco

Japan

Vietnam

Italy

Australia

France

USA

Japan

Korea

China

Korea

Malawi

Australia

Chile

Brazil

Scotland

Brazil

Australia

India

France

China

USA

Host/SUBSTRATE

Soil

Coleoptera: Scolytidae

Lepidoptera: Tortricidae

Coleoptera: Scolytidae

Soil

Lepidoptera: Bombycidae

Coleoptera: Chrysomelidae

Lepidoptera: Arctiidae

Orthoptera: Acridiidae

Soil

Coleoptera: Scarabaeidae

Hymenoptera: Formicidae

Coleoptera: Scarabaeidae

Coleoptera: Cerambycidae

Coleoptera: Scarabaeidae

Coleoptera: Cerambycidae

Soil

Coleoptera

Soil

Hymenoptera: Formicidae

Coleoptera: Scarabaeidae

Silkworm

Acari: Varroidae

Lepidoptera: Geometridae

Coleoptera: Coccinellidae

Homoptera: Delphacidae

Lepidoptera

GENBANK NO.

KU364339

KU364340

KU364341

KU364342

KU364343

KU364344

KU364345

KU364346

KU364347

KU364348

KU364349

KU364350

KU364351

HQ880796

AY532022

HQ880799

KU364352

KU364353

AY531972

AY532045

HQ880761

HQ880788

HQ880789

HQ880776

HQ880773

HQ880767

AY531936

AY532026

AY531990

HQ880813

DQ376247

HQ880824

AY532012

AY532003

AY532006

AY532008

HQ880804

JQ266173

HQ880800

HQ880801

HQ270152

JN689372

HQ880803

HQ880829

916 ... Imoulan & al.

AxioVision Rel. 4.6 software (Zeiss, Welwyn Garden City, UK). Length : width ratios

are given as Q, and mean values are indicated by L™ (length), W™ (width), and Q™ (Q).

Genomic DNA extraction, PCR and sequencing

Isolates recovered from single conidia were grown as mycelia for 2 weeks in 250-ml

flasks containing 100ml of potato dextrose broth. Cultures were then shaken at 150 rpm

on a rotary shaker at 25°C for 7d in darkness. The mycelial samples were pelleted from

liquid cultures by centrifugation for 10 min at 3500 rpm (Eppendorf AG Centrifuge,

Hamburg), subsequently washed twice with sterile double distilled water, and then

lyophilized and stored at -80°C.

For each isolate, 400—500mg of lyophilized mycelium was ground in a sterile mortar

using a pestle. Total genomic DNA was extracted following the modified CTAB method

described by Yao et al. (1999). The mixtures were incubated for 2 hours at 65°C, then

extracted twice with 24 : 1 chloroform : isoamy] alcohol, and centrifuged at 10,000 rpm

at 4°C for 15 min. The supernatant was transferred to a clean tube and mixed with 1/10

volume of sodium acetate (3 M, pH 5.2). Total genomic DNA was precipitated by adding

2/3 (v/v) of cold isopropanol and chilled overnight at -20°C. DNA was recovered by

centrifugation at 10,000 rpm, 4°C for 10 min and washed twice with 70% alcohol, dried

at room temperature and resuspended in TE buffer (10Mm Tris-HCl, pH 8.0; lmM

EDTA, pH 8.0). The extracted DNA was stored at -20°C until use.

Genomic DNA was used as template for PCR amplification of ITS region using

universal primers ITS5/ITS4 (White et al. 1990). The PCR reactions were performed

in a final volume of 50 ul containing 25ul 2x Taq PCR Master Mix (Tiangen Biotech

Co., Ltd, China), 0.5 ul of each primer (10 uM), 1 ul of genomic DNA and 23 ul of

RNase-Free water. The PCR reactions were performed in a GeneAmp PCR System 9700

thermocycler (Applied Biosystems) as follows: 94°C for 5 min, followed by 35 cycles of

94°C for 30 s, 53°C for 30 s, 72°C for 45 s and final extension step of 72°C for 8 min. PCR

products were checked by electrophoresis on an agarose gel (1%) at 100 V in 0.5x TAE

buffer (40 mM Tris-Acetic acid, pH 8.0, 1 mM EDTA), and sequenced with the same

primer pairs. The sequencing was performed at Beijing Genomics Institute (Beijing,

China) by using an Applied Biosystems 3730 Analyzer capillary sequencer (Foster

City, CA, USA). All sequences generated in this study (KU364339-KU364353) were

submitted to GenBank (TABLE 1).

Phylogenetic and data analyses

Only two (of 100) B. bassiana strains sequenced for ITS from Morocco were used

as representatives, while all Moroccan strains identified as B. pseudobassiana were

included in the analyses. Also included were 29 representative Beauveria sequences

and one sequence of Cordyceps militaris (ARSEF 5050; as outgroup) retrieved from

GenBank (TABLE 1). The sequences were aligned using ClustalW Multiple alignment

tool (Thompson et al. 1997) and edited manually to avoid some obvious misalignment

using BioEdit ver. 7.2.5 (Hall 1999).

The sequence data set was analyzed for maximum parsimony (MP) and Bayesian

inference (BI). MP analyses were conducted with PAUP* 4.0b10 (Swofford 2003) and

Beauveria pseudobassiana new to Morocco ... 917

executed with 1000 replicates of heuristic search option of random sequence additions

with branch swapping algorithm by tree bisection reconnection (TBR). Alignment gaps

were treated as missing data, and all characters were unordered and equally weighted.

Branch support was estimated by bootstrap analysis with 1,000 replicates (Felsenstein

1985) executing the same search strategy as described above. Bayesian inference (BI)

analyses were carried out in MrBayes 3.1.2 (Ronquist & Huelsenbeck 2003) using

a general time-reversible model and gamma-distributed rate variation to account

for rates of variation across sites, with a proportion of invariant sites. The remaining

parameters were default values. One tree was saved every 1,000 generations from a total

of 5,000,000 Markov chain Monte Carlo (MCMC) generations; the first 25% of the trees

were discarded as burn-in. Clades with bootstrap values for MP =50% and Bayesian

posterior probability =>70% are labeled above the nodes in the phylogenetic tree.

Results

Morphological observation

Microscopically Beauveria isolates from Morocco appeared typical to those

described elsewhere (e.g., Humber 1997, Rehner et al. 2005). The colony

characteristics were: white or pale-yellow mycelium closely (or not) appressed

to agar surface, becoming farinaceous during sporulation and the reverse side

uncoloured or yellowish white (Fics 1, 2). Conidiophores appeared like dense

spherical clusters of subglobose to flask-shaped conidiogenous cells extending

upward with an elongating sympodial denticulate rachis (zigzag appearance)

giving rise to sessile, hyaline, holoblastic smooth conidia (Fics 3, 4). Two

isolate groups were morphologically distinguished based on the colony colour

as shown by aerial hyphae during conidiation: off-white and loosely floccose vs.

yellowish-brown and densely farinaceous (Fics 1, 2).

Phylogenetic analyses

The amplification of ITS-rDNA region produced a uniform fragment size of

560bp. Using Blast search, the sequences exhibited high sequence homology to

those named B. bassiana or B. pseudobassiana in GenBank.

The ITS dataset comprised 571 characters after the exclusion of ambiguous

positions from both ends. There were 488 constant, 37 parsimony-

uninformative, and 46 parsimony-informative characters. The heuristic search

yielded six most-parsimonious trees with tree length (L) = 139, consistency

index (CI) = 0.719, homoplasy index (HI) = 0.281, retention index (RI) = 0.807.

MP and BI phylogenetic analyses produced trees with similar topologies

that resolved most Beauveria lineages in separate terminal branches (Fic. 9).

Independently of the algorithm used for phylogenetic analyses, B. brongniartii

and B. australis were not well distinguished as unique clusters based on

ITS-rDNA sequences.

918 ... Imoulan & al.

Moroccan strains of Beauveria were grouped into two individual terminal

clades corresponding to B. bassiana and B. pseudobassiana with MP bootstrap

support 276% and BI posterior probabilities >99% (Fic. 9). These two groups

corresponded well to their colony colours, off-white in the B. bassiana clade

and yellowish-brown in the B. pseudobassiana clade.

Taxonomy

Imoulan et al. (2011) previously reported Beauveria bassiana from Morocco.

Here we describe B. pseudobassiana as a new record for Morocco.

Beauveria pseudobassiana S.A. Rehner & Humber, Mycologia 103: 1068

(2011) Fics 2-8

Colonies growing fairly well on PDA at 25°C in the dark, reaching a diameter

of 28-37 mm after 7 days incubation, and attaining a diameter of 36—42mm

in 10 days; velutinous to cottony with an overgrowth of aerial hyphae up to

the agar surface, white first and changing to yellowish-brown or pale yellow;

becoming less powdery in older cultures and rarely with a dense farinaceous

surface layer. Reverse side yellow with white margin. Vegetative hyphae septate,

branched, hyaline, smooth-walled, 1-3 um wide. Conidiogenous cells solitary

but usually consisting of dense lateral clusters, base subspherical to flask-shaped

(sometimes ampulliform), 2.0—-9.0 x 1.5-3 um, apex with an indeterminate

denticulate rachis, produced laterally on aerial hyphae or from subtending

cells. Conidia globose to subglobose, 2—4 x 1-3.5 um, Q = 1-2.5 (L™ = 2.6 um,

W” = 2.1 um, Q™ = 1.4), rarely ellipsoid; hyaline, walls thin and smooth;

produced from conidiogenous cells and occasionally directly from hyphal tips

or laterally from hyphae.

MATERIAL EXAMINED: MOROCCO, Ovypa; from soil under Argania spinosa tree, 23

March 2009, coll. A. Imoulan, strain numbers 2629, 2634, 2640, 2645, 2649, 2650, 2653,

2659, 2660, 2661 (HMAS 254118-254127, dried agar cultures); Tamanar, 28 March

2009, coll. A. Imoulan, strain numbers 2687, 2689 (HMAS 254128-254129, dried agar

cultures); Amskroud, 29 March 2009, coll. A. Imoulan, strain number 2706 (HMAS

254131, dried agar culture).

REMARKS: Beauveria pseudobassiana from Morocco differed from B. bassiana

by its colony surface, which is yellowish-brown to pale yellow (Fic. 2)

becoming less powdery when old, and by its occasional production of ellipsoid

conidia (Fic. 7). The colony surface of B. bassiana is usually white to off-white

Fics 1-8. Beauveria bassiana (strain 2718): 1. colony and aerial hyphae. Beauveria pseudobassiana

(strain 2629): 2. colony and aerial hyphae; 3-8. conidiophores, conidiogenous cells, and conidia —

note conidiogenous cell produced from subtending cells (6), conidia forming from a hyphal tip (7),

and conidia produced directly from mycelium (8). Scale bars: 1, 2 = 10 mm; 3-8 = 5 um.

Beauveria pseudobassiana new to Morocco ... 919

920 ... Imoulan & al.

(Fic. 1) becoming powdery when old, owing to the production of a large

number of globose to subglobose conidia.

Discussion

The study of mycology in Morocco is less developed with few works being

published on Moroccan fungi, particularly on entomopathogenic fungi

(Imoulan et al. 2011, Imoulan & El Meziane 2013). The new species record

for Morocco reported here demonstrates that B. pseudobassiana occurs in

A. spinosa forests along with other entomopathogenic fungi such as B. bassiana,

Metarhizium anisopliae, and Paecilomyces lilacinus identified previously by

Imoulan et al. (2011).

As shown in the most recent taxonomic revision based on the phenotypic

criteria combined with molecular evidence (Rehner & Buckley 2005, Rehner

et al. 2011), B. pseudobassiana and B. bassiana comprise a morphospecies

group, morphologically indistinguishable yet phylogenetically only distantly

related. Interestingly, our morphological observation nonetheless revealed that

characters of both colony and conidial spores were sufficient to discriminate

the two species. As these morphological distinctions have not been mentioned

elsewhere, it is unclear whether our experience represents an unusual case or

if it is possible to find additional characters to distinguish Beauveria species

morphologically. It would be very useful if such characters could support

DNA-based phylogenies in Beauveria.

In the current study, ITS-rDNA-based phylogenetic analyses succeed in

distinguishing B. bassiana and B. pseudobassiana among the species analyzed.

Because Beauveria includes cryptic species that cannot be resolved solely

by ITS analyses, multilocus-based phylogenies are becoming increasingly

important for accurate assignment of strains to specific clades. Molecular

phylogenies involving multiple genes of translation elongation factor-1a (TEF),

RNA polymerase II largest subunit (RPB1), RNA polymerase II second largest

subunit (RPB2), and the Bloc nuclear intergenic have become well-established

for determining unique terminal lineages for all Beauveria species (Rehner

et al. 2011, Zhang et al. 2012, Chen et al. 2013, Agrawal et al. 2014, Robéne-

Soustrade et al. 2015). However, our ITS sequence analyses performed well in

distinguishing 12 of the 15 species included, with B. australis and B. brongniartii

grouping in a single unresolved clade, and B. rudraprayagi (MTCC8017)

grouping with B. pseudobassiana (Fic. 9). Although the additional genes are

required in resolving closed related species, ITS sequence is still powerful in

identifying most of the known species of Beauveria as shown in Fie. 9.

Beauveria pseudobassiana new to Morocco ... 921

2718

2533

2721

7659 ARSEF 1020 B. bassiana

7795

ARSEF 1564

ARSEF 751

ARSEF 1567

B. caledonica

1OO'100 ARSEF 2251

ARSEF 2922 __» B. vermiconia

soo. | ARSEF 2641 Bp omorgha

ARSEF 4149

43 Moroccan

$5100 strains é ete

55:73 ARSEF 1855 o PAGER Y Ore Rt

ARSEF

ARSEF 3.405 ,

MTCC 3017 __, B. rudrapravagi

52:59 | ARSEF 1685 B. sungii

723 ARSEF 7279

6599 RCEF 3903 —__» B. sinensis

toorco | ANSEF 775 B. malawiensis

5oC 17613

ARSEF 7032 ——-+» B. kipukae

135 ARSEF £334 Basiarica

ARSEF £69)

RSEF .

vax | ARSEF 4553] australis

oat ARSEF 4822

ARSEF 617

ARSEF 7053 B. brongniatii

ARSEF 7517

age | ARSEF 8257

ARSEF 8259

B. varroas

RCEF 5500 ——-» B. li

ARSEFSOSO Cordvceps militaris

— icange

Fic. 9. Phylogenetic tree of Beauveria based on Maximum parsimony and Bayesian inference of the

ITS-rDNA sequences. Bootstrap values >50% and posterior probabilities >70% are labeled above

branches and separated by /. Terminal clades are labeled according to ARSEF accession numbers

of individual isolates reported in Rehner et al. (2011) except RCEF5500 and RCEF3903 (Zhang et

al. 2012, Chen et al. 2013).

922 ... Imoulan & al.

Acknowledgments

The authors are very thankful to Dr Nicolai V. Meyling and Pavel Hyrsl for providing

Galleria mellonella used in the isolation of Beauveria spp. from soil. We are grateful to

Drs Paul Kirk and Bo Huang for reviewing the manuscript. This work was financially

supported by a grant to AI from the World Academy of Sciences and Chinese Academy

of Sciences.

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MY COTAXON

October-December 2016— Volume 131, pp. 925-937

http://dx.doi.org/10.5248/131.925

New records of Graphis from Cameroon,

with a key to African species of Graphis

SANTOSH JOSHI’, D.K. UPRETI', ANDREW ENOW EGBE? & JAE-SEOUN HuR?*

'Lichenology Laboratory, CSIR-National Botanical Research Institute,

Rana Pratap Marg, Lucknow (UP)-226001, India

? Life Sciences Laboratories, Faculty of Science, University of Buea,

PO. Box 63, Buea, Cameroon

*Korean Lichen Research Institute, Sunchon National University, Suncheon-540 950, Korea

* CORRESPONDENCE TO: jshur1@sunchon.ac.kr

ABSTRACT—New records of Graphis species are reported from Cameroon, West Africa:

G. ajarekarii, G. alboglaucescens, G. brahmanensis, G. daintreensis, G. exalbata, G. gloriosensis,

G. gonimica, G. handelii, G. immersella, G. novopalmicola, G. pseudoaquilonia, and

G. supracola. The material was collected in the tropical rain forests of Mount Cameroon. The

diagnostic characters of the species are briefly discussed and illustrated. An artificial key is

provided to facilitate identification of Graphis species known from the African Palaeotropics.

Key worps—corticolous, crustose lichens, taxonomy, Graphidaceae, Ostropales

Introduction

Mount Cameroon (ca. 4°N 9°E) is the highest mountain (4095 m) in West

Africa, being located near the Gulf of Guinea in the Southwest Region of

Cameroon (Fonge et al. 2005, Proctor et al. 2007). The Mount Cameroon region

has a diversity of complex ecosystems due to frequent disturbances, (especially

volcanic activities) that have made it possible to study different successional

stages in a relatively confined area (Focho et al. 2010). The western rain forests

of Africa, despite having luxuriant forests with high organismic diversity,

are little explored in terms of lichen diversity (including the Graphidaceae)

compared to the Eastern Palaeotropics in Asia.

In the course of an ongoing research project, crustose lichens, particularly

graphidoid representatives, were collected from rainforests at different

926 ... Joshi & al.

elevations at Mount Cameroon. The thelotremoid lichens in the Graphidaceae

have been exhaustively studied in the region (Frisch et al. 2006); however, no

detailed account on graphidoid taxa from this region is available and only

scattered reports from the African Palaeotropics exist in the literature for this

group (Miller 1890, Staiger 2002, Liicking et al. 2009).

Here we report twelve new species representing the genus Graphis Adans.

collected in the Mount Cameroon area: G. ajarekarii, G. alboglaucescens,

G. brahmanensis, G. daintreensis, G. exalbata, G. gloriosensis, G. gonimica,

G. handelii, G. immersella, G. novopalmicola, G. pseudoaquilonia, and

G. supracola. We also present an artificial key to the species of Graphis known

from the African Palaeotropics.

Materials & methods

During January 2015 a field excursion organized by Prof. Andrew Enow Egbe,

University of Buea, targeted one area in the Mount Cameroon region, Federal

Republic of Cameroon. Lichen specimens are deposited in the lichen herbarium of

the Korean Lichen Research Institute, Suncheon, South Korea (KoLRIJ), and duplicates

are preserved in the Faculty of Science in the University of Buea, Cameroon. Samples

were studied in CSIR-National Botanical Research Institute, Lucknow, India. The

material was examined for morpho-anatomical and chemical features using a MSZ-

TR dissecting microscope and a Leica DM 500 compound microscope. The protocol

for color spot reaction tests and thin-layer chromatography (TLC) was derived from

Orange et al. (2010). Thin-layer chromatography was performed in solvent systems A

[toluene (180): 1, 4-dioxane (45): acetic acid (5)] and C [toluene (170): acetic acid (30)].

Lugol's solution (I) was used to check amyloidity of the ascospores. Illustrations were

prepared using Corel Draw (vers. 12).

Taxonomy: new records

Graphis ajarekarii Patw. & C.R. Kulk., Norw. J. Bot. 26: 45, 1979. Dirk

Thallus epiperidermal, slightly uneven, dull to + glossy, <250 um thick;

prothallus dark brown; lirellae immersed to erumpent, strongly branched,

1-2 cm long, with a lateral to apically thin complete thalline margin; disc

concealed; labia entire, epruinose; proper exciple laterally carbonized becoming

completely carbonized in old lirellae, 50-70 um thick; epihymenium brownish

and granular, 15-20 um high; hymenium hyaline, clear, 90-100 um high;

subhymenium 20-30 um high; asci 8-spored, 100-130 x 20-25 um; ascospores

fusiform, transversely septate, 8-10-locular, amyloid, 28-48 x 8-11 um.

CHEMISTRY—Norstictic and stictic acids detected with TLC.

DISTRIBUTION & ECOLOGY—Found growing in association with other

graphidoid taxa on rough barked trees at higher altitudes.

Graphis spp. new to Cameroon ... 927

SPECIMEN EXAMINED: CAMEROON. SOUTHWEST PROVINCE: Mount Cameroon,

4°09’01”N 9°17'02”E, on bark, 25 February 2015, J-S. Hur CR150020 (KoLRI).

REMARKS—Graphis filiformis Adaw. & Makhija, which otherwise resembles

G. ajarekarii in producing norstictic acid and small ascospores, lacks stictic

acid. Graphis ajarekarii was previously known from the Eastern Palaeotropics

(Licking et al. 2009).

Graphis alboglaucescens Adaw. & Makhija, Mycotaxon 99: 312, 2007. PL. 1B

Thallus epiperidermal, ecorticate, farinose, greenish-white to glaucous

green; lirellae immersed, elongate (1-2 cm long) and irregularly branched with

lateral thalline margin; disc slightly exposed, white pruinose (scripta-morph);

labia entire; proper exciple apically carbonized, 30-40 um thick; epihymenium

brownish, granular, 10-15 um high; hymenium hyaline and clear, 90-100 um

high; subhymenium indistinct; asci 8-spored; ascospores fusiform, transversely

septate, 5-10-locular, amyloid, 24-26 x 4-6 um.

CHEMISTRY—Norstictic acid detected with TLC.

DISTRIBUTION & ECOLOGY—Found in small patches on hard and rough

barked trees in evergreen forests at higher altitudes.

SPECIMEN EXAMINED: CAMEROON. SOUTHWEST PROVINCE: Mount Cameroon,

4°07'26”N 9°11'17’E, alt. ca. 1200 m on bark, 26 February 2015, J-S. Hur CR150067

(KoLRI).

REMARKS—In its anatomy, G. alboglaucescens resembles G. glaucescens Fée,

which is distinguished by its lack of secondary metabolites and occasionally

striate labia. Graphis alboglaucescens was originally described as producing

lirellae covered up to the top by a thalline margin (Adawadkar & Makhija

2007), a character Licking et al. (2009) later defined as a lateral thalline margin.

The species exhibits a pantropical distribution (Licking et al. 2009).

Graphis brahmanensis Aptroot, Lichenologist 41: 434, 2009. PRTC

Thallus epiperidermal, thin, pale-brown, fawn to off-white, 80-100 um,

evanescent, indistinctly corticate reflecting bark; algal layer continuous, 28-30

um thick; medulla mostly endoperidermal; lirellae prominent, 1-1.5 cm long,

sparsely and irregularly branched with basal thalline margin (striatula-morph);

labia striate (3-4-striations), epruinose; proper exciple laterally carbonized,

50-100 um thick; epihymenium brownish, 10-15 um high; hyaline and clear;

hymenium 90-100 um high; subhymenium 10-15 um high; asci 8-spored;

ascospores fusiform, transversely septate, 5-8-locular, amyloid, 25-30 x 6-8

um.

CHEMISTRY—Stictic acid detected with TLC.

928 ... Joshi & al.

DISTRIBUTION & ECOLOGY—Found growing in association with other

corticolous lichen taxa at low altitudes on smooth barked trees of tropical rain

forests.

SPECIMEN EXAMINED: CAMEROON. SOUTHWEST PROVINCE: Mount Cameroon,

4°05’41”N 9°22’24’E, alt. ca. 100 m, on bark, 26 February 2015, J-S. Hur CR150072

(KoLRI).

REMARKS—In most characters, G. brahmanensis resembles G. vittata Mull.

Arg., which differs in producing lirellae with a lateral thalline margin. The

otherwise similar G. duplicata Ach. lacks secondary compounds.

Graphis brahmanensis was previously known from the Neotropics,

(particularly in the southeastern USA) and Eastern Palaeotropics (Lticking et

al. 2009).

Graphis daintreensis (A.W. Archer) A.W. Archer, Telopea 11: 72, 2005. PL. iD

Thallus epiperidermal, silver-gray to greenish gray or whitish-gray, off-

white, glossy, uneven, 200-250 um thick, corticate; cortex continuous, 20-30

uum thick; algal layer 40-140 um thick, embedded with large and small crystals;

indistinct to endoperidermal medulla; indistinct to off white prothallus; lirellae

elongate (1-2 cm long), densely and irregularly branched, prominent with basal

thalline margin (hossei-morph); disc concealed; labia entire and epruinose;

proper exciple laterally carbonized, 40-60 um thick; epihymenium brownish,

crystalline, 15-20 um high; hymenium hyaline and clear, 130-150 um high;

subhymenium indistinct; asci broadly clavate, 1-spored, 110-120 x 25-30 um;

ascospores muriform, multi-celled, amyloid, 60-90 x 20-25 um.

CHEMISTRY—No chemicals detected with TLC.

DISTRIBUTION & ECOLOGY—In Cameroon, this taxon was collected from

the rainforests at very low altitudes, and was found growing in large patches on

rough barked trees.

SPECIMEN EXAMINED: CAMEROON. SOUTHWEST PROVINCE: Mount Cameroon,

4°04’09”N 9°22’36’E, alt. ca. 30 m, on bark, 26 February 2015, J-S. Hur CR150081

(KoLRI).

REMARKS—Graphis nanodes Vain., which resembles G. daintreensis in most

taxonomic characters, differs in its lirellae with lateral thalline margins

(lineola-morph) and smaller ascospores (<50 um long).

Graphis daintreensis was previously known only from the Eastern

Palaeotropics (Licking et al. 2009).

Graphis exalbata Nyl., Lich. Ins. Guin.: 28, 1889. PL. 1E

Thallus epiperidermal, whitish, smooth to slightly uneven, glossy, indistinctly

verruculose, continuous, ecorticate, 100-200 um, and embedded with crystals;

Graphis spp. new to Cameroon ... 929

algal layer <90 um thick, embedded with crystals; medulla endoperidermal;

prothallus indistinctly whitish; lirellae + immersed to erumpent, elongate

(1-2 cm long), radially branched with lateral thalline margin; disc concealed;

labia indistinctly striate, white pruinose (glaucescens-morph); proper exciple

apically carbonized, 30-60 um thick; epihymenium grayish crystalline, 5-10

um high; hymenium hyaline and clear, 90-100 um high; subhymenium 15-20

um high; asci 8-spored, 90-100 x 10-15 um; ascospores transversely septate,

8-10-locular, amyloid, 30-38 x 8-9 um.

CHEMISTRY—Norstictic acid detected with TLC.

DISTRIBUTION & ECOLOGY—Found growing in large patches on thin and

smooth barked trees in evergreen forests.

SPECIMEN EXAMINED: CAMEROON. SOUTHWEST PROVINCE: Mount Cameroon,

4°09’01”N 9°17'02”E, on bark, 25 February 2015, J-S. Hur CR150024 (KoLRI).

REMARKS—Graphis eburnea Adaw. & Makhija, which is morphologically

and chemically close to G. exalbata, is distinguished by its epruinose labia

(nigrosina-morph).

Liicking et al. (2009) previously reported G. exalbata from the Palaeotropics.

Graphis gloriosensis A.W. Archer & Elix, Telopea 11: 454, 2007 PL. 1F

Thallus epiperidermal, thin, pale-brown, fawn to off-white, indistinctly

corticate 50-100 tum thick, reflecting bark; algal layer continuous,

28-30 um thick; medulla mostly endoperidermal; lirellae prominent,

sparsely and irregularly branched, 1-1.5 cm long with lateral thalline margin

(marginata-morph); labia entire, epruinose; proper exciple completely

carbonized, 50-100 um thick; epihymenium brown, granular,10-15 um high;

hymenium hyaline and densely inspersed (with oil droplets), 90-100 um

high; subhymenium 10-15 um high; asci 8-spored; ascospores fusiform,

transversely septate, 10-15-locular, amyloid, 40-70 x 10-15 um.

CHEMISTRY—Stictic and hypostictic acids detected with TLC.

DISTRIBUTION & ECOLOGY—Collected from tree bark in evergreen forests

at higher altitudes.

SPECIMEN EXAMINED: CAMEROON. SOUTHWEST PROVINCE: Mount Cameroon,

4°07’40”N 9°11'54”E, alt. 1150 m, on bark, 26 February 2015, J-S. Hur CR150060

(KoLRI).

ReMARKS—In thallus color and texture and in chemistry G. gloriosensis

superficially resembles G. brahmanensis, which differs in its striate labia,

laterally carbonized proper exciple, and clear hymenium.

Liicking et al. (2009) reported G. gloriosensis from the Eastern Paleotropics.

930 ... Joshi & al.

Graphis gonimica Zahlbr., Ann. Mycol. 30: 431, 1932. PL. 1G

Thallus epiperidermal, whitish-gray, silver-gray, white or dark-gray,

glossy, <200 um thick; cortex 30-40 um thick, continuous; algal layer

45-60 um thick; medulla white, crystalline, 60-100 um thick; prothallus dark

brown; lirellae immersed, short (0.5-1 cm long) and scarcely furcated with

lateral thalline margin (lineola- or deserpens-morph); disc concealed; labia

entire and epruinose; proper exciple + completely carbonized, 60-150 um

thick; epihymenium brownish, granular, 10-15 um high; hymenium densely

inspersed (with oil droplets), 140-160 um high; subhymenium indistinct; asci

8-spored; ascospores fusiform, transversely septate, 6-12-locular, amyloid,

30-50 x 7-9 um.

CHEMISTRY—Norstictic acid detected with TLC.

DISTRIBUTION & ECOLOGY—Found growing in small patches associated

with Glyphis cicatricosa Ach. and Arthonia sp. on uneven and thick barked trees

in evergreen forests.

SPECIMEN EXAMINED: CAMEROON. SOUTHWEST PROVINCE: Mount Cameroon,

4°09’01”N 9°17'02”E, on bark, 25 February 2015, J-S. Hur CR150035, CR150036

(KoLRI).

REMARKS— The morphologically similar G. cincta (Pers.) Aptroot differs in its

laterally carbonized proper exciple.

Liicking et al. (2009) previously reported G. gonimica from the Paleotropics.

Part of our material sometimes produces stictic acid and atranorin (in

traces) as additional compounds, but we refrain from proposing a taxon new to

science until further collection and evaluation.

Graphis handelii Zahlbr., Symb. Sinic. 3: 44, 1930. PL. 1H

Thallus epiperidermal, off-white, grayish-green to gray, 100-150 um

thick; cortex continuous, 20-30 um thick; algal layer continuous, 30-40 um

thick; medulla white crystalline, epi- to endoperidermal, 50-80 um thick;

lirellae erumpent to prominent, short (<5 mm long), simple to furcate; disc

exposed and epruinose, brown; labia entire and epruinose; proper exciple

laterally carbonized 20-50 um thick; epihymenium brownish, 10-15 um

high; hymenium hyaline and inspersed (with oil droplets), 90-100 um high;

subhymenium 20-30 um high; asci 8-spored, 70-90 x 10-15 um; ascospores

fusiform, transversely septate, 5-9-locular, amyloid, 28-32 x 5-7 um.

CHEMISTRY—Norstictic acid detected with TLC.

DISTRIBUTION & ECOLOGY—Collected from smooth barked trees in

evergreen forests.

Graphis spp. new to Cameroon... 931

SPECIMENS EXAMINED: CAMEROON. SOUTHWEST PROVINCE: Mount Cameroon,

4°09’01”N 9°17'02”E, on bark, 25 February 2015, J-S. Hur CR150028, CR150038

(KoLRI).

REMARKS—Graphis crebra Vain. is similar but is distinguished by its white-

pruinose disc.

Liicking et al. (2009) report a pantropical distribution for G. handelii.

Graphis immersella Mill. Arg., Bull. Herb. Boissier 3: 319, 1895. Pai

Thallus epiperidermal, off white, greenish-gray, <150 pm_ thick;

cortex continuous, 30-50 um thick; algal layer 30-40 um thick; medulla

endoperidermal; prothallus blackish; lirellae erumpent, short (<1 cm long) and

sparsely branched (lineola-morph) with lateral thalline margin; disc concealed;

labia entire and epruinose; exciple laterally carbonized, 20-60 um thick proper;

epihymenium dark brown, crystalline, 10-15 um high; hymenium hyaline

and clear, 100-150 um high; subhymenium indistinct; asci broadly clavate,

8-spored, 90-100 x 10-15 um; ascospores transversely septate, 8-11-locular,

amyloid, 35-55 x 8-10 um.

CHEMISTRY—Stictic acid detected with TLC.

DISTRIBUTION & ECOLOGY—Collected from rather smooth barked trees in

evergreen forests.

SPECIMEN EXAMINED: CAMEROON. SOUTHWEST PROVINCE: Mount Cameroon,

4°07’41”N 9°12’20”E, on bark, 26 February 2015, J-S. Hur CR150057 (KoLRI).

REMARKS—Graphis pinicola Zahlbr. is morphologically similar to G. immersella

but lacks lichen compounds.

Liicking et al. (2009) report a palaeotropical distribution for G. immersella.

Graphis novopalmicola A.W. Archer & Licking, Lichenologist 41: 439, 2009. PL. 1J

Thallus epiperidermal, pale-gray to yellowish-gray, dull, loosely corticate,

150-200 um thick, continuous in large patches; cortex 20-30 um thick

embedded with crystals; algal layer continuous, 40-50 um thick; medulla

mostly endoperidermal and crystalline, 100-150 um thick; prothallus whitish;

lirellae erumpent to prominent, simple to furcate, short to elongate (1-1.5 cm

long) with a thick lateral thalline margin (subserpentina-morph); concealed

disc; entire and epruinose labia; proper exciple completely carbonized, <50 um

thick; epihymenium brownish crystalline, 10-15 um high; hymenium densely

inspersed (with oil droplets) 150-170 um high; subhymenium indistinct to 20

um high; asci broadly clavate, 1-spored; ascospores large, muriform, multi-

celled, amyloid, 90-110 x 20-30 um.

CHEMISTRY—Norstictic acid detected with TLC.

932 ... Joshi & al.

DISTRIBUTION & ECOLOGY—Collected from smooth barked trees in

evergreen forests near foothills.

SPECIMENS EXAMINED: CAMEROON. SOUTHWEST PROVINCE: Mount Cameroon,

4°04’09”N 9°22’36’E, alt. ca. 30 m, on bark, 26 February 2015, J-S. Hur CR150079,

CR150080 (KoLRI).

REMARKS—Graphis leprographa Nyl., which resembles G. novopalmicola in

having 1-2-spored asci and similar chemistry, differs in its very short and

unbranched lirellae (dussii-morph).

Liicking et al. (2009) report a pantropical distribution for G. novopalmicola.

Graphis pseudoaquilonia Licking, Lichenologist 44: 393, 2012. PL. ak

Thallus epiperidermal, cartilaginous, grayish-green, dark-gray to gray,

continuous, glossy, corticate, spreading in large wide patches; lirellae prominent,

elongate (1-2 cm long), sinuous, irregularly branched with complete thalline

margin; disc concealed; labia striate (archeri-morph); proper exciple completely

carbonized, 90-100 um thick; epihymenium brownish, granular, 10-15 um

high; hymenium hyaline and clear, 100-150 um high; subhymenium 20-30

um high; asci 2-6-spored; ascospores fusiform, terminally muriform, amyloid

[(10-15-septate in the middle x 1-2 longitudinal septa per cell in the terminal

segments)], 50-80 x 9-15 um.

CHEMISTRY—Norstictic acid detected with TLC.

DISTRIBUTION & ECOLOGY—Found growing in large patches on uneven and

thick barked trees in evergreen forests.

SPECIMENS EXAMINED: CAMEROON. SOUTHWEST PROVINCE: Mount Cameroon,

4°09’01”N 9°17'02”E, on bark, 25 February 2015, J-S. Hur CR150011, CR150025

(KoLRI).

REMARKS—Graphis norvestitoides Sutjaritt., which is otherwise similar to

G. pseudoaquilonia in all characters, is distinguished by its strongly inspersed

hymenium (Licking et al. 2012).

Graphis pseudoaquilonia has previously been reported from the Neotropics

and Eastern Palaeotropics (Licking et al. 2009, Liicking et al. 2012).

Graphis supracola A.W. Archer, Aust. Syst. Bot. 14(2): 267, 2001. PLah

Thallus whitish-gray to gray, greenish-gray, loosely corticate, <100 um thick,

smooth to slightly verruculose, spread in patches; cortex discontinuous <15 um

thick, overlying a discontinuous layer of crystals; algal layer 40-60 um thick;

medulla mostly endoperidermal; prothallus dark brown; lirellae immersed,

simple to furcate, 1-2 cm long with lateral thalline margin; disc concealed to

sometimes slightly open; labia entire and white pruinose (caesiella-morph);

Graphis spp. new to Cameroon ... 933

proper exciple laterally carbonized, 30-50 um thick; epihymenium brownish,

5-10 um high; hymenium hyaline and clear, 50-100 um high; subhymenium

35-45 um high; asci 8-spored; ascospores fusiform, transversely septate,

6-10-locular, amyloid, 25-32 x 7-10 um.

CHEMISTRY—Protocetraric acid detected with TLC.

DISTRIBUTION & ECOLOGy—Found growing on tree barks in evergreen

forests at low altitudes.

SPECIMENS EXAMINED: CAMEROON. SOUTHWEST PROVINCE: Mount Cameroon,

4°09’01”N 9°17'02”E, on bark, 25 February 2015, J-S. Hur CR150023 (KoLRI);

4°05’41”N 9°22’24’E, alt. ca. 100 m, on bark, 26 February 2015, J-S. Hur 150069

(KoLRI).

REMARKS— This taxon can be confused with Graphis distincta Makhija &

Adaw., which differs in additionally producing stictic and constictic acids.

Licking et al. (2009) report a pantropical distribution for G. supracola.

Key to Graphis species recorded from the African Palaeotropics

Taxonomic key characters of Graphis species not included in our collections derived

primarily from Licking et al. (2009) and Broeck et al. (2014)

—_

. Lirellae orange-pigmented, prominent to sessile, short to elongate,

sparsely to irregularly branched; ascospores terminally muriform,

70-120 x 10-15 um; no lichen substances present .......... G. subchrysocarpa

Poflite Wace Gli cheno diy on pias o apie ae «gine x eos Sonata eames bnypt nae tegheer es See pee et Z

2*labia.entire: hymeniunr cleator inspersed ic iiis< ns ee ape inne ee nee ean eee Sees 8

Ds rabia-sthiate alin tn eH TMC leak fic. Fea aw. c- Fire oncedie 4 acces nna dse te ancedAt nen tens 2, EA eee 32

5..-broper-exciple completely carbonizede ...5 ta%s.0 teats teeth) tetatd beats waists allt t 4

3.. Proper-exciple apically-to laterally carbonized ims: Lites bbivinblysnbees noes) 16

ASELVTMCHIETIT INS DELSEU! sk MR PT NPT talk, ciadll rk ah tk cath ear alt tat eect tat acl rk ld Berk 5

AS ELYVINISTIMAN COAT Soc nce wala a Bielo < Miteye SM Bite o 6 Ato EA Ree cE to Ste wh ie eM wt ahd 8

5. Ascospores muriform, 90-110 x 20-30 um;

Horstictie dcid. present flo Bl Fl eles hipster tgs See bw deeb pres G. novopalmicola

5. ASCOSPOLES TANS VETSE]Y SEP TALS Hace 6 He cwiies« WBcwieno Mcwijare Memes Mbeya ewice 2 was sewing 6

6. Lichen substances absent; lirellae erumpent

with complete thalline margin (subserpentina-morph);

ascospores 18-20-locular, 100-120 x 14-18um .................. G. superans

Gp Licherssubs tame es, presente <. tetas cates neem atten seeteeen aes haters Pee aoe eben s 7

7. Stictic acid present; ascospores 10-15-locular, 50-90 x 10-15 um .... G. gloriosensis

7. Norstictic acid present; ascospores 6-12-locular, 30-50 x 7-9 um ...... G. gonimica

St LACM SMMSLIDStALICES IPOS TIL gauss sx seed cs BSA a see sro ugl deh road! dem Bev dat gree detox ee BAL em De 9

S, MIGHenest DSUAR Ce ssa DSOIN ao ne ag.c <ptgs ager tnghece eagtan «Auten wap egeeeen tenet gl 11

934 ... Joshi & al.

9. Constictic and stictic acids present; lirellae prominent,

+ simple with lateral to almost complete thalline margins;

ascospores muriform, 60-110 um long ................. G. vandenboomiana

9. Stictic acid present; ascospores transversely septate .............. 0. eee ee eee 10

10. Lirellae with apically thick complete thalline margin (subserpentina-morph);

ASCOSPOTES 5 20 =) Onptt OTS Io esl, ce give nna gl seth mgtee Gun alnunse-ciaheit & G. schroederi

10. Lirellae with thick lateral thalline margin (marginata-morph);

ascOspores45 120) pitt LONG. 5 5s 426i abe s bsg wih sus! dab sha nese G. rustica

11. Ascospores muriform, 20-30 um long; lirellae erumpent, short to sparsely

branched with basal thalline margin (subregularis-morph) ......... G. comma

ll. Ascosporestransversely septate, S50,liny long Fics 08 haath s Sets haath g be lig watts 12

i> sLirellaewith-complete thallinie margin gr gacs.8 facets bests Best s Seales Sed oats: 13

12. Lirellae with basal to lateral thalline margin .......................000 0008. is)

13. Unknown lichen substances present; ascospores 3-septate,

} AN Ee 0 00 1G) 9 Cac A AD RO G. aptrootiana

13. No lichen substances present; ascospores 3- or more septate ..............-.. 14

14. Lirellae short and sparsely branched (negrosina-morph) ............ G. negrosina

14. Lirellae elongate, irregularly branched (farinulenta-morph) ............ G. sitiana

15. Lirellae short, stellate with basal thalline margin (geraensis-morph) G. dracaenae

15. Lirellae elongate, irregularly branched with

thick lateral thalline margin (marginata-morph) ................. G. oxyclada

16. Proper exciple apically carbonized; hymenium clear ...................0005. MZ

16. Proper exciple laterally carbonized; hymenium clear or inspersed............. 18

17. Lirellae immersed to erumpent, short, sparsely or stellately branched

(lineola- or coarctata-morph); ascospores muriform,

25-45 x 13-20 um; lichen substances absent .................. G. pergracilis

17. Lirellae immersed, ascospores transversely septate,

24-26 x 4-6 um; norstictic acid present................06. G. alboglaucescens

PSS y PEN FUL ATIS CES CU cca FE a ccsaly hy seas Senate st pe net tt once ge reese peace aah 19

1S Elymenitina clears: satin. foe eh Sty eh Stk ee hae cee pees eign cee tine e ins 22

TORAscospores-miUritOrMt, 6.9 Sk ee EGS ee Leo Ri Eh bey kee 20

[osAscospores transverse h- septate st 48.51 At a Aha er tha ia tok anit ear berk ea os 21

20. Norstictic acid present; lirellae immersed to erumpent, elongate &

irregularly branched with thick lateral to complete thalline margin

(subserpentina-morph); ascospores 50-110 x 15-30 um........... G. insulana

20. Lichen substances absent; lirellae erumpent, short to elongate,

sparsely to irregularly branched with lateral thalline margin

(lineola- or deserpens-morph); ascospores 35-50 x 8-15 uum ....... G. subvelata

Graphis spp. new to Cameroon ... 935

PLATE 1. Graphis species from Cameroon. A. G. ajarekarii; B. G. alboglaucescens; C. G. brahmanensis;

D. G. daintreensis; E. G. exalbata; EF. G. gloriosensis; G. G. gonimica; H. G. handelii; I. G. immersella;

J. G. novopalmicola; K. G. pseudoaquilonia; L. G. supracola. Scales: A, B, D, EF, G, J, L = 10 mm,

C)E, Hy liK = 0-3-1,

21. Disc exposed, epruinose (handelii-morph); norstictic acid present;

ascospores 5-9-locular, 28-32 5-7 UM ... 2. eee eee eee eee G. handelii

21. Disc concealed, labia epruinose (lineola-morph); no lichen substances;

ascospores 6-10-locular, 20-40 x 6-9 UM «2... eee eee eee ee G. lineola

227 ASC OSPOVES MIE TOT out psbg%s nt cSeda ent f atoms te ete tate bentia tate wt-n Tah) cathy cole sade cele ey ae 73

PDS ASCOSPOLESMTAlISVOTSELY,SE PLAate = Wiig. Na Non Ma Nau ctiay nts aes ty sa oprah oectre afl cra ae. 2S

23. Norstictic acid present; lirellae erumpent, elongate & irregularly

branched with lateral thalline margin (deserpens-morph);

ascospores. 20=35-< 8= 1A inh fox, clue en relat im al als deeds tones) decd G. renschiana

23. Lichen substances absent; ascospores more than 50 um long ................. 24

936 ... Joshi & al.

24.

25,

26.

27s

2H.

28.

29.

29;

30.

aU:

32:

32,

ao:

a3:

34.

a3:

35.

36.

OY,

37.

Lirellae prominent with basal thalline margin (hossei-morph) ..... G. daintreensis

Lirellae erumpent with lateral to apically thick complete thalline margin

(SUDSEMPENING-THORDIY) 23 6 9th a Mell yk all on ease Te G. subhiascens

Lichen substances absent; ascospores <45 um long .......... eee eee eee ee 26

Lichen substances present; ascospores <60 um long........... 0.0... eee eee 27

Thallus partly ecorticate; lirellae thin, flexuous with gently sloping in thalline

margin; labia white pruinose (caesiella-morph) ................... G. furcata

Thallus corticate; lirellae thick; straight or curved with abruptly sloping thalline

margin; labia epruinose (lineola- or deserpens-morph) ............ G. pinicola

Norstictic or stictic or protocetraric acids present ................- eee ee eee 28

Stictic acid additionally with atranorin or norstictic acid present ............. S.

Norsitietic acid, présént:... oo 82.56 55448 deny Sex gh Bey phone pee ee ye ee eee ams 29

Morsticticacidalsenie hit Pht Real Mall Rea Si el Pet eataal eal ae teed 30

Lirellae erumpent, ascospores <45 um long ............. 00. e eee eee eee G. librata

Lirellae immersed to erumpent, short & sparsely branched (Jineola-morph);

ascospores 45-60 um long ........... cece eee eee eee ee G. erythrocardia

Stictic acid present; labia epruinose (lineola-morph) .............. G. immersella

Protocetraric acid present; labia white pruinose (caesiella-morph) ... G. supracola

. Stictic acid & traces of atranorin present;

ascospores 4-locular, 12-17 um long ...................000- G. tetralocularis

. Stictic acid & norstictic acid present;

ascospores 8-10-locular, 35-55 um long ...................004. G. ajarekarii

Propercexciple:completelycakDOniZed ore < Semis « Phowng in Phemed a fete a, fe neport Werner a elects 33

Proper exciple apically to laterally carbonized; ascospores transversely septate .. 34

Ascospores muriform, 30-65 x 10-15 ym; lirellae erumpent, elongate & irregularly

branched with lateral thalline margin; disc concealed; labia epruinose

(tenella-morph); no lichen substances present ................. G. plurispora

Ascospores terminally muriform, 50-80 x 9-15 um;

NOPSLICtCACIC Present Stl alr ead See were ep eee G. pseudoaquilonia

Propercexcipleapically carbonized. Sc s «Pee se Mecwites< Smcmne,e Ssruijess Necense< Stee oir 35

Propeniexcipleélaterallys carbonized’. ..29 ba's28 gash-2 bast 8 heat g be sth Beales watts 36

Norstictic acid present; ascospores 30-38 um long.................. G. exalbata

Lichen substances absent; ascospores 25-35 um long ............ G. glaucescens

Stictic acid present; ascospores 5-8-locular, 25-30 x 6-8 um .... G. brahmanensis

rchiemrsubstances-absenit 6 Aviat cutters ws othe a MaMa tS te et te a area 37

Lirellae erumpent, short & sparsely branched with lateral thalline margin

(tenella-morph); ascospores 15-45 xX 6-9 UM ....... eee eee eee eee G. tenella

Lirellae prominent, elongate & irregularly branched, lacking or with basal thalline

margin (striatula-morph); ascospores 30-65 um x 7-12 um ....... G. striatula

Graphis spp. new to Cameroon ... 937

Acknowledgments

This work was supported by a grant from the National Research Foundation of Korea

(NRF-2014K1A3A1A09063058) and the Korea National Research Resource Center

Program (NRF-2014M3A9B8002115). Santosh Joshi is grateful to the Director, CSIR-

NBRI, Lucknow, India for providing laboratory facilities. Authors are grateful to Dr.

Robert Liicking (Botanical Garden and Botanical Museum, Berlin) and Dr. Ze-Feng Jia

(College of Life Sciences, Liaocheng University, China) for their valuable comments on

the manuscript.

Literature cited

Adawadkar BA, Makhija UV. 2007. New species and new records of Graphis from India with

partially carbonized exciples and transseptate ascospores. Mycotaxon 99: 303-326.

Broeck DV, Liicking R, Ertz D. 2014. Three new species of Graphidaceae from tropical Africa.

Phytotaxa 189(1): 325-330. http://dx.doi.org/10.11646/phytotaxa.189.1.23

Focho DA, Fonge BA, Fongod AGN, Essomo SE. 2010. A study of the distribution and diversity

of the family Orchidaceae on some selected lava flows of mount Cameroon. African Journal of

Environmental Science and Technology 4(5): 263-273.

Fonge BA, Yinda GS, Focho DA, Fongod AGN, Bussmann RW. 2005. Vegetation and soil status on

an 80 year old lava flow of Mt Cameroon, West Africa. Lyonia 8(1): 17-39.

Frisch A, Kalb K, Grube M. 2006. Contribution towards a new systematic of the lichen family

Thelotremataceae. Bibliotheca Lichenologica 92: 556 p.

Liicking R. 2009. The taxonomy of the genus Graphis sensu Staiger (Ascomycota: Ostropales:

Graphidaceae). Lichenologist 41: 319-362. http://dx.doi-org/10.1017/S0024282909008305

Licking R, Sutjaritturakan J, Kalb K. 2012. Validation of three species names and description of

a new species in the genus Graphis (Ascomycota: Ostropales: Graphidaceae). Lichenologist 44:

391-394. http://dx.doi.org/10.1017/S0024282911000855

Mangold A, Elix JA, Lumbsch HT. 2009. Thelotremataceae. Flora of Australia 57: 195-420.

Miiller J. 1890. Lichenes Africae tropico-orientalis. Flora 74: 334-347.

Orange A, James PW, White FJ. 2010. Microchemical Methods for the Identification of Lichens.

British Lichen Society.

Proctor J, Edwards ID, Payton RW, Nagy L. 2007. Zonation of forest vegetation and soils of Mount

Cameroon, West Africa. Plant Ecol. 192: 251-269. http://dx.doi.org/10.1007/s11258-007-9326-5

Rivas Plata E, Licking R, Sipman HJM, Mangold A, Kalb K, Lumbsch HT. 2010. A world-wide

key to the thelotremoid Graphidaceae, excluding the Ocellularia-Myriotrema-Stegobolous clade.

Lichenologist 42: 139-185. http://dx.doi.org/10.1017/S0024282909990491

Staiger B. 2002. Die Flechtenfamilie Graphidaceae. Studien in Richtung einer natiirlicheren

Gliederung. Bibliotheca Lichenologica 85. 526 p.

MY COTAXON

October-December 2016—Volume 131, pp. 939-945

http://dx.doi.org/10.5248/131.939

Revision of some Peziza collections in herb. TAAM (Tartu)

GIANFRANCO MEDARDI”*, ANGELA LANTIERI? & GABRIELE CACIALLI?

'Via Giuseppe Mazzini 21, I-25086 Rezzato (Brescia), Italy

?Via Novaluce 38, I-95030 Tremestieri Etneo (Catania), Italy

°Via Goito 25, I-57127 Livorno, Italy

* CORRESPONDENCE TO: Gianfranco.medardi@gmail.com

ABSTRACT—Forty samples representing the genus Peziza kept in the Mycological Herbarium,

Tartu, Estonia (TAAM) were studied. Collection data and taxonomic comments are presented.

KEY worps—Ascomycota, Pezizales, Pezizaceae

Introduction

This work, part of a series of papers concerning the revision of specimens

assigned to the genus Peziza Dill. ex Fr. kept in various herbaria, is intended

to confirm their importance for the studies on mycological diversity and

taxonomy.

Forty samples, initially labelled under various names and located in the

Mycological Herbarium, Institute of Agricultural and Environmental Sciences,

Estonian University of Life Sciences, Tartu, Estonia (TAAM) (founded in 1950

at the Institute of Biology of the Estonian SSR Academy of Sciences; Parmasto

2011), were studied. Our examinations revealed that not all specimens were

originally correctly determined; various specimens were shown to correspond

to different species, while others could not be definitely identified. Some of

these samples have also been used to carry out molecular analyses, the results

of which have been published in previous papers (Medardi et al. 2012, Medardi

et al. 2014).

Materials & methods

Morphological and microscopical examinations were carried out on Dried

specimens were rehydrated in water or KOH 5% and mounted in water with lactic blue

9AO ... Medardi, Lantieri & Cacialli

to highlight spore ornamentation and/or with Melzer’s reagent to observe the iodine

reaction of the asci. The tissues were examined microscopically with an Optika BK

1301 optical microscope with 40x or 100x (oil immersion) objectives; dimensions were

determined from 50 mature spores per collection.

Results

Peziza alcis Harmaja, Karstenia 26(2): 44. 1986.

MATERIAL EXAMINED: ESTONIA: JOGEVAMAA Co, Kursi, Puurmani Commune,

Altnurga, Forestry sq. 95, on moose dung, 8.10.1997, leg. et det. M. Opik (TAAM

171114, as P. fimeti); RAGAVERE CoMM., Uljaste, Laaéne-Viru Co., on ground and on

dung, in moist Betula—Picea forest, s.d., leg. K. Kalamees, det. A. Raitviir (TAAM 71260,

as P. fimeti). FINLAND: LAPLAND, Kevo, National Park, on moose dung, 28.08.1981,

leg. K. Kalamees, det. A. Raitviir (TAAM 122042, as P fimeti).

Notes: These materials, labeled in the herbarium as P. fimeti, have been

redetermined as P alcis. The spores were smooth and averaged 15-17(-18)

x (7-)7.5—9 um; the excipulum comprised only one layer (textura globulosa

or globulosa-angularis), differentiable into one subhymenium, one medullar

excipulum and one ectal excipulum by the cell sizes consistently wider towards

the outside of the apothecium. Molecular analysis of TAAM 171114 also

confirmed identification of P alcis (Medardi et al. 2012).

TAAM 122042 was judged not to be completely mature, based on the

numerous spores still contained in the asci. Sporormiella australis (Speg.) S.I.

Ahmed & Cain was also noted on the dung.

Peziza arvernensis Roze & Boud., Bull. Soc. Bot. Fr. 26 (Suppl.): LXXVI. 1879.

MATERIAL EXAMINED: DENMARK: ZEALAND, Grib Skov, on ground among stones,

14.08.1965, leg. P. Milan Petersen, det. H. Dissing (TAAM 187941); ESTONIA: Tartu

Co., along the road toward Tallinn, in a Populus forest, 13.05.2007, leg. L. Tedersoo, det.

K. Hansen (TAAM 192338).

Notes: The spores measured (16-)17-20 x (8-)9-10.5 um and were

ornamented with manifest punctiform warts. A section showed the typical

tissues of P arvernensis: subhymenium of textura globulosa-angularis, the

medullar excipulum comprising three layers (the upper and lower layers a

textura globulosa and the middle a textura intricata), and an ectal excipulum of

textura intricata. The habitat of this species is rather variable; that reported for

these two collections is within specifications.

Peziza atrospora Fuckel, Fungi Rhenani Exsic.: no. 1224. 1864.

MATERIAL EXAMINED: ESTONIA: Tartu Co., Nodo Comm., Peedu, on ground,

26.07.2000, leg. et det. B. Kullman (TAAM 179346, as P. pseudovesiculosa Donadini).

TAAM Peziza collections revised ... 941

Notes: TAAM 179346 comprises two specimens, both originally identified

as representing Peziza pseudovesiculosa. Our redetermination of the species

to P. atrospora is supported by spores measuring 17-18 x 7—9 um, most with

1-2 oil drops and with elongated warts often connected in short crests <1 um

high. Other characters include a subhymenium of textura intricata, a medullar

excipulum of textura globulosa-angularis with cells 60-80(-110) um diam.,

and an ectal excipulum of textura globulosa-angularis.

Peziza cinatica Pfister, Mycotaxon 8(1): 189. 1979.

MATERIAL EXAMINED: ESTONIA: Harju Co., Viimsi Comm., Prangli Island, near

Idaotsa Farm, on clay soil of a well in Pinus forest, 25.08.1960, leg. P. Poldmaa, (TAAM

028962, TAAM 028963, as PR domiciliana Cooke).

Notes: We redetermined this material, bearing the herbarium label

P. domiciliana, to P. cinatica. The ascospores, which measured 11—13(-14)

x (5-)6—-7 um and contained 2 oil droplets, were ornamented with fine

punctiform warts <1 um. The medullar excipulum is a textura intricata with

brownish hyphae (ca. 5 um diam.) often perpendicular to the hymenium, and

the ectal excipulum is a textura globulosa, with some rounded (20-50 um

diam.) and elongated (20—40 x 15—25 um) cells intermixed.

Peziza cinatica is not widely distributed but does not appear to be restricted

to one habitat type. Previously reported substrates include ash (Pfister 1979)

and burn residue (Dougoud 2001), but the above material (TAAM 028962,

TAAM 028963) was collected on clay soil.

Peziza fimeti (Fuckel) E.C. Hansen, Vidensk. Meddel. Naturhist. Foren. Kjobenhavn

59: 267. 1877 [“1876”].

MATERIAL EXAMINED: RUSSIA: Kamcuatka, 47 km from Krapivnaya, on dung of

Ursus ursus, 6.08.1978, leg. B. Kullman (TAAM 187899, TAAM 187900); KRASNODAR,

Umpyr, Caucasus Nature Reserve, on dung, 10.08.1976, leg. M. Pallo, det. A. Raitviir

(TAAM 064318); Malaya Laba, Caucasus Nature Reserve, on dung, 12.08.1976, leg. M.

Pallo, det. A. Raitviir (TAAM 064369); Kurix ISLANDS, Kunashir Island, Lagunnoye, on

excrements, 12.08.1970, leg. B. Kullman (TAAM 061513); RESPUBLIKA TyvVA, Erzin, on

dung, 18.07.1972, leg. B. Kullman (TAAM 065761). TAJIKISTAN. Ramit State Nature

Reserve, Mount Hissar, on dung, 12.04.1977, leg. et det. A. Raitviir (TAAM 064569);

HOVALING, on bovine excrement, 22. 04.1985, leg. B. Kullman, det.. A. Raitviir (TAAM

115898). UZBEKISTAN: Dshisaki, Turkestan Chain, Kulsai, Zaamini Nature Reserve,

in Juniperus forest, 24.05.1980, leg. K. Kalamees, det. A. Raitviir (TAAM 199165).

Notes: Ascospores measuring (19-)20.5—22(—22.5) x (10-)11—12 um and

flesh composed of globose or globose-angulated cells differentiated into a

subhymenium, a medullar excipulum (with some filamentous hyphae visible),

and an ectal excipulum confirm Peziza fimeti.

942 ... Medardi, Lantieri & Cacialli

Our molecular analyses (Medardi et al. 2012) of TAAM 187900 and TAAM

064318 also supported Peziza fimeti. Sporormiella australis was also found in

TAAM 064318. TAAM 115898 is immature.

Noteworthy is the fact that the apothecia TAAM 187899 and TAAM 187900

were found (dung of Ursus), a previously unreported substrate.

Peziza howsei Roze & Boud., Bull. Soc. Bot. Fr. 26 (Suppl.): LXXV. 1879.

MATERIAL EXAMINED: ESTONIA: Hiru Co., Hiiesaare Light House, on ground,

18/09/2001, leg L. Vaher, det. B. Kullman ([TAAM 179773, as Pachyella celtica (Boud.)

Haffner); Tartu Co., Vonnu Comm., Jarvselja Forest [sq n. 226 (4)], on ground,

27.09.2001, leg. et det. B. Kullman (TAAM 179788).

Notes: Both samples were characterized by a subhymenium of textura

intricata (hyphae 5-6 um diam.), a 2-layered medullar excipulum (upper a

textura globulosa-angularis with cells <25 um diam.; lower a textura + intricata

toward the upper layer and tending to globulosa-angularis with cells similar to

those of the upper layer), and an ectal excipulum of textura globulosa with cells

10-25 um diam. Ascospores were 17.5—19.5(-22.5) x 8.5—9.5 um, with fine

punctiform warts (0.5-1 um diam., <0.5 um high), more or less isolated but

occasionally joined to form short crests.

Peziza howsei is morphologically similar to P emileia Cooke, but despite

several overlapping characters, their different ITS sequences support them as

two separate species (Medardi et al. 2014).

Peziza lividula W. Phillips, in Cooke, Mycographia: 161. 1877.

MATERIAL EXAMINED: ESTONIA: PARNU Co., Koonga Comm., between Mihkli and

Koonga, in Picea forest, 16.08.1960, leg. et det. A. Raitviir (TAAM 040951, as P. howsei);

SAARE Co., Limanda Comm., Koimia, on ground, 24.08.1960, leg. et det. A. Raitviir

(TAAM 041035, as P. howsei); H11ru Co., Kardia Comm., next to Kardia, near the road to

Kaina, in Corylus forest, 4.09.1960, leg. et det. A. Raitviir (TAAM 041235, as P. howsei);

LAANE Co., Hanila Comm., Puhtulaid islet, near Virtsu, in a forest of mixed deciduous

trees, 6.09.1960, leg. et det. A. Raitviir (TAAM 041243, as P. howsei); TARTU Co., Vonnu

Comm., Jarvselja forest [sq n. 226 (4)], on ground, 27.09.2001, leg. et det. B. Kullman

(TAA 179791, as P. howsei). RUSSIA: IRKUTSKAYA OBLAST, Ust-Kutskiy, Erlenga, along

a path in a forest, 27.08.1967, leg. M.A. Bondarceva, det. A. Raitviir (TAAM 199080, as

P. howsei).

Notes: In this group all samples were at first labelled P howsei. We

redetermined them to P lividula based on the following characters: ascospores

15-19 x 8—-10(-10.5) um, each with 2 oil droplets and punctiform warts +

wide (1—1.5 um diam.), at times more elongate and closed near the extremities

where they resemble low skulls; a subhymenium of textura prismatica or

prismatica-angularis.

TAAM Peziza collections revised ... 943

Peziza perparva Harmaja, Karstenia 26(2): 47. 1986.

MATERIAL EXAMINED: RUSSIA: SVERDLOVSKAYA OBLAST, Kytlym, on unidentified

dung, 20.07.1973, leg. A. Raitviir (TAAM 062952, as P fimeti).

Notes: We redetermined TAAM 062952 (labeled as P. fimeti) to P perparva.

The ascospores measured (11-)12—13 x 6—7 um and were ornamented with

a weak +open reticulum; the tissues were arranged differently than those

found in P fimeti: subhymenium of textura intricata mixed with a few globose

cells, medullar excipulum of textura globulosa or globulosa-angularis, and

ectal excipulum with a structure similar to that of the medullar layer but with

smaller cells. Also diagnostic of P perparva are the very small apothecia, only

1-3 mm diam.

TAAM 062952, which represents just the second collection of P perparva

in Europe (the type collected in Finland and published by Harmaja 1986),

expands the range and habitat of this remarkable fungus. Harmaja (1986) stated

that P perparva fruits “in moist place on a forest path used by elks, growing in

dead plant litter apparently earlier impregnated by elk urine. . ” The Russian

specimens appear to grow instead on rodent dung (showing that the fungus is

not restricted to elk) at a lower latitude (Lantieri et al. 2011).

Sporormiella australis was also identified on some of the dung associated

with the Russian collection.

Peziza varia (Hedw.) Alb. & Schwein. Consp. Fung. Lusat.: 311. 1805.

MATERIAL EXAMINED: ESTONIA: Tartu Co., Tartu Comm., 57 Voru Street, along the

border of a woody floor in a cottage, 12.09.1962, leg. et det. A. Raitviir (TAAM 043232,

as P. domiciliana); near a cement base, 19.12.1977, leg. et det. B. Kullman (TAAM

199053, as P. domiciliana); 27-632 Narva Street, on a plastered block of a construction,

20.06.1994, leg. V. Kastanje, det. A. Jakobson (TAAM 199058, as P domiciliana);

VILJANDI Co., Kolga-Jaani Comm., Potaste, Vaibla Forest [sq n. 29], Pedja Natural

Reserve, on ground, 11.09.1997, leg. et det. B. Kullman (TAAM 157677, 2 collections,

as P. arvernensis). RUSSIA: KAMCHATKA, 47 km from Krapivnaya, on dung of Ursus

ursus, 6.08.1978, leg. B. Kullman (TAAM 116381, as P. fimeti); YAMALO-NENETSKIY

AVTONOMNYY OKRUG, Ovgort, on dung of Ursus ursus and ground around, 28.08.1976,

leg. Murdvee (TAAM 110066, as P. fimeti).

Notes: We redetermined the original identifications of TAAM 116381 and

TAAM 110066 (P fimeti); TAAM 157677 (P. arvernensis, both specimens); and

TAAM 043232, TAAM 199053, and TAAM 199058 (P. domiciliana) to Peziza

varia. We detected a faint superficial roughness on the spores, which measured

(14.5-)15—-16 x (7-)7.5—8.5(—9) um (smaller than those of P fimeti at 20—22.5

x 10-12 um), and the medullar excipulum showed a median, more or less thin

layer of textura intricata, typical of P. varia.

944 ... Medardi, Lantieri & Cacialli

Our study of TAAM 116381 and TAAM 110066 revealed that P. varia is

also able to grow on dung, solving the long-standing question regarding two

conflicting interpretations given in P. fimeti literature (Medardi et al. 2012).

The excipular structure of P varia overlaps with that of P arvernensis and

P. domiciliana, which differ from P. varia in their larger spores with more or

less evident punctiform warts (P arvernensis, 16—19(-20) x 9-10(-11) um;

P. domiciliana, 15—17(-20) x 7—9(-10) um, with occasional warts). The builder's

rubble habitat of TAAM 043232, TAAM 199053, and TAAM 199058 belongs

to the large assortment of substrata on which P. varia is able to fruit (Medardi

et al. 2012).

Peziza cf. ampliata

MATERIAL EXAMINED: RUSSIA: CHELYABINSKAYA OBLAST, Miass, on unidentified

dung, 16.08.1973, leg. I. Parmasto (TAAM 062922, as P. fimeti).

Notes: TAAM 062922, bearing the herbarium label of P. fimeti, was

redetermined as P cf. ampliata. This specimen was previously studied by

Hansen et al. (2002). Our molecular analyses group it with P ampliata Pers.

(Medardi et al. 2012), although its macro- and microscopic features correspond

to those of P fimeti: ascospores measuring 18—20.5(—21) x (8.5-)9—11(-11.5)

um and a medullar excipulum comprising large, globose-angulated cells mixed

with filamentous hyphae.

Peziza cf. fimeti

MATERIAL EXAMINED: ESTONIA: JOGEva Co., Puurmani Comm., Alam-Pedja Nature

Reserve, on beaver dung, 21.08.1997, leg. A. Kollom, det. A. Raitviir, (TAAM 128087,

as P fimeti).

Notes: This material, labeled as P. fimeti, is the “Peziza sp. h” analyzed by

Hansen et al. (2002), who stated it is phylogenetically different from P. alcis,

despite its similar microscopic characters: smooth ascospores 15.5—17 x (7-)

8—9(—9.5) um and an excipulum consisting of practically only one tissue of

(textura globulosa or globulosa-angularis), with three layers differing only in

the cell dimensions.

Peziza sp.

MATERIAL EXAMINED: ESTONIA: PARNU Co., Koonga Comm., Mihkli Oaks, on ground

in oak forest, 16.08.1960, leg. et det. A. Raitviir (TAAM 040944, as P howsei); SAARE

Co., Part of “N” Reserve, Viidumae Natural Reserve, in Corylus forest, 26.08.1960, leg.

et det. A. Raitviir (TAAM 041077, as P. howsei); LAANE Co., Vormsi Comm., Huitbergi,

on ground near fir roots, 22.10.2010, leg. et det. B. Kullman (TAAM 190485, as Pachyella

celtica). RUSSIA: REsPpUBLIKA Tyva, Kyzyl, Mazaloek, on ground, 4.08.1972, leg. A.

Raitviir, det. B. Kullman (TAAM 065930, as Pachyella celtica); KRASNODARSKIY KRAY,

TAAM Peziza collections revised ... 945

Guzeripl, on ground, 25.05.1975, Leg. et det. A. Raitviir (TAAM 063473, as P. howsei);

KaMCHATKA, 47 km from Krapivnaya, on dung of Ursus ursus, 6.08.1978, leg. B.

Kullman (TAAM 187898, as P fimeti).

Notes: Our examinations of these samples did not provide sufficient data for

a sure identification, mainly due to the scarcity or lack of needed characters.

Acknowledgments

The authors thank Prof. G. Consiglio and Dr. C. Losi for critically reviewing the

manuscript. We want to express a particular gratitude to the late Dr. E. Parmasto, keeper

of TAAM (Tartu, Estonia), for his kindness in putting at our disposal the samples and

for permission to conduct molecular analysis on some of them, which enabled us to

publish our work.

Literature cited

Dougoud R. 2001. Clé des Discomycétes carbonicoles. Documents Mycologiques 30(120): 15-29.

Hansen K, Lessoe T, Pfister DH. 2002. Phylogenetic diversity in the core group of Peziza

inferred from ITS sequences and morphology. Mycological Research 106(8): 879-902.

http://dx.doi.org/10.1017/S0953756202006287.

Harmaja H. 1986. Studies on the Pezizales. Karstenia 26(2): 41-48.

Lantieri A, Cacialli G, Medardi G. 2011. Peziza perparva, seconda segnalazione europea, individuata

dallo studio di una raccolta russa. Rivista di Micologia 54(4): 307-313.

Medardi G, Lantieri A, Pfister DH, LoBuglio K, Cacialli G. 2012. Clarification of

Peziza fimeti with notes on P. varia collections on dung. Mycotaxon 121: 465-476.

http://dx.doi.org/10.5248/121.465.

Medardi G, LoBuglio K, Pfister DH, Lantieri A. 2014. Morphological and molecular study

of Peziza emileia and P. howsei, two distinct taxa. Mycological Progress 13: 1227-1234.

http://dx.doi.org/10.1007/s11557-014-1013-z

Parmasto E. 2011. Fungal herbarium EAA in Tartu (Estonia). Folia Cryptogamica Estonica 48:

69-72.

Pfister DH. 1979. Type studies in the genus Peziza. V. Species described by Rehm. Mycotaxon 8(1):

187-192.

MYCOTAXON

October-December 2016— Volume 131, pp. 947-959

http://dx.doi.org/10.5248/131.947

BOOK REVIEWS AND NOTICES’

LORELEI L. NORVELL

Pacific Northwest Mycology Service, Portland OR 97229-1309 USA

ABSTRACT—Books reviewed include: GENERAL—The Outer Spores: Mushrooms of

Haida Gwaii (Kroeger & al. 2012); LICHENS & LICHENIZED FuNGI—Rare Lichens of

Oregon (Exeter, Glade & Loring 2016); Lichens of Uttar Pradesh (Nayaka & Upreti

2013); AscomycoTa—Plant pathogenic and endophytic Botryosphaeriales known from

culture (eds: Phillips & al. 2013); A polyphasic taxonomy of Daldinia (Xylariaceae)

(Stadler & al. 2014); Hypocrealean lineages of industrial and phytopathological

importance (eds: Lombard, Groenewald & Crous 2015).

GENERAL

The Outer Spores: Mushrooms of Haida Gwaii. i

By Paul Kroeger, Bryce Kendrick, Oluna

Ceska & Christine Roberts. 2012. Mycologue

Publications, Sidney-by-the-Sea BC [www.mycolog. Wahab pes

com] in cooperation with the Haida Gwaii Museum THE OUTER SPORES “=,

(Skidegate BC), Canada. 189 p., ~300 color photos MUSHROOMS

and drawings, paperback. ISBN—978-0-9692237-

3-3 Price (excl. postage): $20 (Canadian) Email:

bryce@mycolog.com.

of oluna ceska

HAIDA GWAII christine roberts

I have been happily immersed in the pages of this gem since last week, which I

found temporarily forgotten among the ‘books I must read soon” shelf. When

labors of love are as entertainingly written as this one, the reader definitely

benefits, and THE OUTER Sporss will charm and edify a great many other

mycologists and nature-lovers. The 15 x 23 cm volume covers the mushrooms

* Book reviews or books for consideration for coverage in this column should be sent to the

Editor-in-Chief <editor@mycotaxon.com> 6720 NW Skyline, Portland OR 97229 USA.

948 ... MycoTAxon 131(4)

as well as the history of Haida Gwaii (the post-2010 name for the Queen

Charlotte Islands), “a wild and unspoiled corner of the world and the home of

the Haida, a people with a deep and rich culture.” This overview of 635 species

identified from >2900 mushroom collections gathered during a 5-year survey

constitutes the ‘first ... guide dedicated to the fungi of Haida Gwaii.”

The obligatory first chapter “about mushrooms” (here defined as “fungal

fruiting bodies large enough to see’) packs an impressive amount of

information in ten pages and bears the imprimatur of the indomitable Bryce

Kendrick (author of THE FirTH KINGDOM) by answering What are fungi? and

What are Mushrooms? before moving on to ascomycete and basidiomycete

basics, biological strategies, and a delightful drawing from Oluna’s field

notebook. Next follows a brief description of the 150 islands “that comprise

a sort of miniature continent” accompanied by a map showing collection sites

on the Queen Charlotte Windward Mountains, Skidegate Plateau, and Queen

Charlotte Lowlands. A brief cultural history of the indigenous peoples follows;

although the Haida did not traditionally consume mushrooms for food, they

did venerate Tree-Fungus Man and used bracket fungi for tinder, medicines,

and pigments.

Edible mushroom coverage begins with the inevitable safety cautions; here

it appears that getting lost in the archipelago is more worrisome than the

possibility of death by mushroom: the local forest district and search & rescue

crews (motivated by the “significant cumulative cost of numerous searches”)

now find it more cost effective to distribute free survival kits (contained within

a 1-litre water bottle) to pickers. With much of the area closed to mushroom

picking, the authors devote five pages to where, when, and how to collect and

eat/or preserve the mushroom quarry, closing with two duplicate (somewhat

oddly, given the index on pp. 133-139) lists of the region’s 28 choice edibles

with references to later comments and/or photos, the first alphabetized by Latin

name and the second by common name. Coverage of the profitable commercial

mushroom harvest (primarily of golden, rainbow, blue, and winter chanterelles)

precedes a discussion of current and future forest management practices.

Species accounts of 14 edible and 22 “notable” mushrooms, accompanied

by generally excellent photos and distribution maps (plus one shot of a

dominant herbivore cluster of millipedes munching an Ganoderma tsugae in

the ‘edible’ mushroom chapter) precede observations on some little known

roles and interactions of fungi, such as the truffle-tree-animal triumvirate, sand

ecosystems, and nitrogen-seeking Hebeloma, Laccaria, and Onygena ‘corpse

finders’. Appropriately enough, next on the menu are toxins, symptoms, and

good to somewhat blurry portraits of and maps for the region’s 15 poisonous

Book Reviews ... 949

mushroom species. Found here is Inocybe calamistrata (a mushroom Id never

thought of consuming, but its blue pigmented ‘foot’ might attract those vainly

hoping for a hallucinogenic high). Also portrayed is a mushroom labeled

as Clitocybe (now the type species of Ampulloclitocybe) clavipes looking far

too slender, small, white, and suspiciously far more like Clitocybe fragrans,

another known poisonous mushroom. This is one book that stresses the deadly

poisonous Amanita franchetii—an otherwise beautiful mushroom common

throughout Haida Gwaii.

Moving from the deadly poisonous to the strictly hallucinogenic, the

next chapter focuses on two “main candidates” for “visionary mushrooms

traditionally used as ritual or spiritual intoxicants in Haida Gwaii:” the liberty

cap (Psilocybe semilanceata, probably introduced by immigrant farmers) and

the fly agaric (Amanita muscaria, an abundant endemic). Six pages are devoted

to the modern natural and legal history surrounding these “magic” mushrooms.

The atlas comprises 139 photos (of variable quality) covering 105 species,

including a salal leaf infected by Valdensia heterodoxa (which escaped the

clutches of the final checklist, although not the index). As this volume is

not intended to serve as an identification guide, these photos merely whet

the appetite for further information, and the atlas concludes with a list of

recommended guides and references.

The authors next summarize the scope of their prodigious five-year inventory

efforts: “Over the five years, we made eight field trips to Haida Gwaii of ten to

fourteen days. In all, 113 areas were visited ... and we made and preserved 2906

collections representing 635 species and documented 812 species of fungi (not

including lichens) from all available sources... We collected on nineteen islands

ranging in size of 0.5 hectares to the over 648,000 hectares of Graham Island.

The mushrooms ... remained largely unknown until 2003, when we began our

survey.’ The general bibliography precedes six wonderful photos showing the

authors (and Oluna’s photographer and botanist husband Adolf) in the field.

The final index and acknowledgments follow a 23-page checklist of the 635

species identified thus far from the surveys. Having participated in several long-

term fungal surveys (see Norvell & Exeter 2003), I understand all too well how

much work was involved in identifying the collections after all the fieldwork was

completed. To that end, I do wish the authors had provided a bit more detail on

which references they used to identify their finds. In his 2013 book review, Steve

Trudell suggested that in the absence of the usual identification caveats (e.g., cf.,

aff., sensu lato) the identifications should be “taken with a grain of mycological

salt” He recently amended this (Trudell 2017) by adding that although the

authors had originally included such identification disclaimers, the publisher

950 ... MycoTaxon 131(4)

unfortunately removed them from the published checklist. Misidentifications

or misapplications are to be expected, although the authors’ strengths in certain

genera (e.g., dark-spored saprobes for Paul, Inocybe for Oluna, and Russula for

Christine) lend more credibility to the checklist. All of us who have conducted

similar studies know full well that the real work of identification will continue

years after the fieldwork is complete and will applaud the authors for having

the courage to publish their results (however preliminary they might be) so

promptly and in so engaging a format.

For the most part there are relatively few typographical or spelling errors—

the few I noted were ‘Beauvaria’ for Beauveria, ‘Scerotinia’ for Sclerotinia,

and ‘prescottii’ for prescotii - and the nomenclature is more or less up-to-

date for 2012. I counted three synonym doublets: Bondarzewia mesenterica +

B. montana, Cystolepiota sistrata + Lepiota seminuda, and Galerina autumnalis

+ G. venenata (the last two synonyms of G. marginata). Oddly, while

‘Lichenomphalia is shown on p. 9, only Omphalina ericetorum (accepted as

L. umbellifera after a 25-year nomenclatural battle of epic proportions)

appears on the list. Other nomenclatural holdovers include Boletus (instead

of Chalciporus) piperatus, Clitocybe (instead of Arrhenia) hohensis, Coprinus

(instead of Coprinopsis) atramentarius, lagopus & phaeosporus, Gomphus

(instead of Turbinellus) kauffmanii, Mycena (instead of Roridomyces) rorida,

Omphalina viridis (instead of Arrhenia chlorocyanea), Paxillus (instead of

Tapinella) panuoides, Phlogiotis (instead of Guepinia) tremelloides, and Rozites

(instead of Cortinarius) caperata. Fortunately, as all names can easily be updated

using Index Fungorum, these discrepancies are hardly serious.

All in all, I find THe OUTER SpoREs: MUSHROOMS OF THE Harpa Gwall

eminently readable, enchanting, and educational and recommend it as an

excellent inspiration and guide for other surveys of hitherto untrammeled

wildernesses.

Kendrick, B. 2000. The Fifth Kingdom, 3" edn. Hackett Publishing, 400 p. + CD

ROM. ISBN 978-1-58510-022-4

Norvell LL, Exeter RL. 2004. Ectomycorrhizal epigeous basidiomycete diversity

in Oregon's coastal montane Pseudotsuga menziesii forests - preliminary

observations. IN Fungi in Forest Ecosystems: Diversity, Ecology, and Systematics,

Cathy Cripps (ed.). Memoirs of the New York Botanical Garden 89: 159-189.

Trudell S. 2017. Book Review: The Outer Spores: Mushrooms of Haida Gwaii. THE

MyYcopuiLeE May-June 53(3): 14-15. 2017 reissue accessed on 8 January 2017:

http://www.namyco.org/the_outer_spores_mushrooms_of.php

Book Reviews ... 951

LICHENS & LICHENIZED FUNGI

Rare Lichens of Oregon. By Ronald Exeter, ,

Charity Glade, Scot Loring. 2016.

Government Printing Office, Salem (Northwest

Oregon) District, Bureau of Land Management,

Oregon, USA. 190 p., ~290 color photos &

drawings, hard print + computer disc. ISBN— eggs

133.2978-0-9791310-6-6. 10:: 0-979-1310-6-5; = - ook

$30 (incl. postage, order by phone 1-503-375- Rare lechone

5646). Low resolution web version online:

https://www.researchgate.net/publication/309032897

of Oregon

Ronald L. Ex - Charity Glade - Scot Lorin

RARE LICHENS OF OREGON is the fourth lavishly illustrated and impressive

identification guide featuring rare, threatened, or endangered species

shepherded through the government printing office maze by the first author.

Ron, a botanist and colleague who adopted Ramaria as a genus needing

closer scrutiny during our six-year field studies of ectomycorrhizal fungi,

first approached the USDI-Bureau of Land Management Salem District

about printing a photographic regional monograph covering a good many

“species of interest’ cited by the Northwest Forest Plan. RAMARIA OF THE

PACIFIC NORTHWESTERN UNITED STATES (Exeter & al. 2006), which combined

descriptions derived from research papers and personal collections with

photographs taken by the original authorities and in the field, proved an

immediate success. It was followed two years later by the equally well-received

PNW Phaeocollybia monograph (Norvell & Exeter 2008) and then eight years

later by the outstanding RARE BRYOPHYTES OF OREGON (Exeter & al. 2016).

This valuable addition to Pacific Northwest lichen species literature

thoroughly covers 78 special status lichen species cited in the most recent RARE,

THREATENED AND ENDANGERED SPECIES OF OREGON (ORBIC 2016) plus

Leptogium compactum (Stone et al. 2016), a not-yet-listed recently described

species currently regarded as rare in Oregon. The 8%” x 11” print copy with

sturdy paper cover and glossy pages is an excellent size for displaying the many

detailed full color photos and maps.

Acknowledgments, a color bar key to sections, and table of contents

precede a brief 2-page introduction, color-coded map of Oregon's counties

and eco-regions, and a 3-page table summarizing in which eco-regions each

species has been found. The authors refer readers to MACROLICHENS OF THE

PaciFic NorTHWEST (McCune & Geiser 2009) and LICHENS OF NORTH

952 ... MyCOTAXON 131(4)

AMERICA (Brodo & al. 2001) for detailed lichen structure and biology, keys,

and glossaries, which are not included in this volume. The nomenclature has

been checked with IndexFungorum (www.indexfungorum.org), and special

note is made of nomenclatural changes for four species recently updated in

the ORBIC list: Circinaria (= Aspicilia) rogeri, Gyalolechia (= Caloplaca)

stantonii, Pseudocyphellaria hawaiiensis (= P. perpetua), and Usnea subgracilis

(= U. schadenbergiana).

The species descriptions are divided into two sections: 58 macrolichens

and 21 microlichens, with the species in each section listed alphabetically,

particularly handy given the lack of an index. Left-hand text pages headed by

the scientific name face right-hand illustration pages with (1)2-6 illustrations

(superbly sharp photos, most taken by coauthor Scot Loring, are occasionally

accompanied by lovely and equally detailed pencil habit drawings). Recent

synonyms, common names, a field summary, and diagnostic characters precede

a brief description of the thallus, apothecia or ascomata, chemistry, ecology,

distribution, and comparison with similar species. Each treatment concludes

with a list of references, flagging those with color plates. Not surprisingly in a

book devoted to rare species, there are ten macrolichen and five microlichen

species that are illustrated in color here for the first time.

The volume concludes with seven pages of maps showing distribution by

county, a list of abbreviations and symbols, two pages of micro-images of

the photobionts Dictyochloropsis, Stichocoocus, and Trentephohlia, a 9-page

bibliography, a map of North America, and the CD-disc in a sleeve on a back

cover illustrated with six (unfortunately not identified) lovely lichen photos.

The inclusion of the algal partner recalled this mycologist’s early days of trying

in vain to match one lovely ‘fungus’ (strangely always fruiting on a powdery

grayish green ‘substrate’) with the few Hymenoscyphus photos in Breitenbach &

Kranzlin (1984). (My gratification at eventually discovering the lichenological

nature of Icmadophila ericetorum only slightly offset my embarrassment at not

recognizing its ubiquitous algal symbiont in the first place.)

The only drawback to this lovely and welcome addition to Oregon's BLM

publishing pantheon is the retirement of its first author as Salem botanist

shortly after publication. We wish our man in Salem well, but who now will

serve as mycological guardian angel at the Northwest Oregon District Office?

Breitenbach J, Kranzlin F. 1984. FUNGI OF SWITZERLAND 1. ASCOMYCETES. Lucerne:

Edition Mykologia. 310 p.

Brodo IM, Sharnoff SD, Sharnoff S. 2001. LICHENS oF NORTH AMERICA. Yale

University Press, New Haven. 795 p.

Book Reviews ... 953

Exeter RL, Norvell L, Cazares E. 2006. RAMARIA OF THE PACIFIC NORTHWESTERN UNITED

StaTEs. USDI BLM/OR/WA/PT-06/050-1792, Salem, Oregon 157p. Lo-res web version:

https://www.researchgate.net/publication/255719422

Exeter RL, Harpel J, Wagner D. 2016. RaRE BRYOPHYTES OF OREGON. USDI-BLM Salem

District. ISBN—13: 978-0-29791310-4-2. 379 p. Lo-res web version: https://www.

researchgate.net/publication/305807271

McCune B, Geiser L. 2009. MACROLICHENS OF THE PACIFIC NORTHWEST, 2™ edn. Oregon State

University Press, Corvallis. 448 p.

Norvell LL, Exeter RL. 2009 (2008”). PHAEOCOLLYBIA OF PACIFIC NORTHWEST NORTH

America. USDI-BLM/OR/WA/GI-08/100-1792, Salem, Oregon 228 p. Lo-res web version:

https://www.researchgate.net/publication/307601665

ORBIC (Oregon Biodiversity Information Center). 2016. Rare, Threatened and Endangered

Species of Oregon. Institute for Natural Resources, Portland State University, Portland,

Oregon. 130 p.

Stone DF, Hinds JW, Anderson FL, Lendemer JC. 2016. A revision of the Leptogium saturninum

group in North America. THE LICHENOLOGIST 48(5): 387-421. https://doi.org/10.1017/

$00242829 16000323

Lek

Lichens of Uttar Pradesh. By Sanjeeva Nayaka & ff

D.K. Upreti. 2014 (“2013”). U.P. State Biodiversity

Board (printed by Shivam Arts), Lucknow, UP India.

177 p., ~405 color illustrations. PDF available online: UTTAR PRADESH

http://www.upsbdb.org/pdf/2014/09/Books/2-

Lichens_of_Uttar_Pradesh.pdf. Dever”

Uttar Pradesh State Biodiversity Board

LICHENS OF UTTAR PRADESH, which arrived by post some time ago, has been

buried on my desk patiently awaiting rediscovery. Prepared by two expert

lichenologists, this handy-sized (~14 x 22 cm) volume endeavors to expand

the lichen coverage of a diminished Uttar Pradesh after the 1999 partition of

Uttarakhand state, where until then most Uttar Pradesh collections had been

collected. This 2014 publication is based on “critical observation of more than

2250 lichen specimens in NBRI and based on field studies in 35 (out of 75)

districts in the state.” Nayaka & Upreti, who treat 135 taxa representing 46

genera in 25 families, contributed 41 new records, thereby adding 61 species

(11 new to India) to earlier lists, and excluded 15 cited in Nayaka & Upreti

(2011) based on erroneous identification or doubtful occurrence.

954 ... MycoTAXON 131(4)

Two forwards and an author preface precede a brief but engagingly written

introduction that touches on the symbiotic nature of lichens (“at a given time

[a] lichen can have members from two to three kingdoms altogether: Fungi,

Protista, and Monera sensu Cavalier Smith 2002)” as well as outlines their

household uses, medicinal potential, and importance in perfumery and dyes

and as air pollution indicators. I was intrigued to learn that lichens form an

integral ingredient in Garam Masala (India’s famed powdered spice) and that

several different species (35 identified in one spice mixture) may be used in a

single spice. Lichens are believed to have curative powers for everything from

headaches to leprosy and Uttar Pradeshis use smoke produced by burning

Thamnola vermicularis to kill germs in milk storage containers.

A brief overview of the geography of the state (home to almost 200 million

people) covers the climate, topography, and vegetation. There follow pages on

the status of lichen diversity in India, an outline of lichen study in Uttar Pradesh,

a note on the current state of knowledge on lichen diversity in the state, a list

of the now excluded previously reported 16 lichen taxa, and a conspectus of

the 135 taxa covered taxonomically. The introductory pages conclude with

methodological notes, a map of Uttar Pradesh districts, and photos of six

different lichen habitats.

A 5-page key to the lichen genera of Uttar Pradesh introduces the 139-

page taxonomic section, organized alphabetically by genus. Generic names

(with authorities and family) are accompanied by formal generic descriptions,

numbers of species reported both worldwide and in India, and important

literature references. Where needed, a species key precedes individual species

treatments, each of which presents a full morphological description, chemistry

analysis, and ecological notes accompanied by 3-4 illustrations with photos

depicting the lichen macromorphology and/or important anatomical characters

plus a district-linked distribution map. Although the photos are small and have

a somewhat soft focus, they are definitely adequate for identification purposes,

making the book quite useful in the field. References, a welcome 5-page

glossary, and a taxonomic index to families and species organized by genus

conclude the volume.

Nomenclaturally, the authors have definitely done their homework and

have updated names in accordance with Ertz (2009) Liicking (2009), Liicking

et al. (2009), Mangold et al. (2009), Sharma et al. (2012), and Staiger (2012); a

quick comparison of their names with those cited in IndexFungorum confirms

that the nomenclature is current. The care taken to exclude misdetermined (or

misreported) species from their 2011 list suggests that their taxonomy is equally

reliable. Unfortunately this care does not extent to proof-reading, as there

Book Reviews ... 955

are a great many typographical or grammatical errors sprinkled throughout

the text—a firm ‘fooling’ (for ‘footing’ in the first forward), Anisomeridium

‘calicicolum’ (p. 22, for calicolum,’ which should, however, be correctly

transcribed in Latin as calicola), and “Moussurie (p. 166, for “Missouri’)

Botanical Gardens are but a few—but fortunately these errors generally do not

obscure the meaning and do not detract significantly from the utility of the

book.

Given the size of the state, it is certain that additional lichen species will be

found. The authors are to be commended for providing such a useful resource

for continuing lichen research in Uttar Pradesh.

Ertz D. 2009. REVISION OF THE CORTICOLOUS OPEGRAPHA SPECIES FROM THE

PALAEOTROPICS. Bibl. Lichenol. 102. J. Cramer, Berlin & Stuttgart.

Licking R. 2009. The taxonomy of the genus Graphis sensu Staiger (Ascomycota;

Ostropales; Graphidaceae). LICHENOLOGIST 41(4): 319-362.

Licking R, Archer AW, Aptroot A. 2009. A world-wide key to the genus Graphis

(Ascomycota; Ostropales; Graphidaceae). LICHENOLOGIST 41(4): 363-452.

Mangold A, Elix JA, Lumbsch HT. 2009. Thelotremataceae. 195-420 in FLORA OF

AUSTRALIA VOLUME 57. LICHENS 5 (PM McCarthy, ed.). ABRS and CSIRO

Publishing, Canberra and Melbourne.

Nayaka S, Upreti DK. 2011. An inventory of lichens in Uttar Pradesh through

bibliographic compilation. 24-35 in NATIONAL CONFERENCE ON FOREST

BIODIVERSITY: EARTH’S LIVING TREASURE. Uttar Pradesh State Biodiversity

Board, Lucknow.

Sharma B, Khadlikar P, Makhija U.2012. New species and new combinations in the

lichen genera Fissurina and Hemithecium from India. LICHENOLOGIST 44: 339-

362.

Staiger B. 2012. Die FLECHTENFAMILIE GRAPHIDACEAE. STUDIEN IN RICHTUNG

EINER NATURLICHEN GLIEDERUNG. Biblioth. Lichenol. 85. J. Cramer, Berlin &

Stuttgart.

ASCOMYCOTA

In perusing my bookshelves, I was startled to discover several excellent texts

that MycoTtaxon—except for an occasional brief announcement—has pretty

much ignored. Because these invaluable SrupiEs In MycoLocy volumes

(published by the Centraalbureau voor Schimmelcultures) merit much closer

scrutiny, I have taken the liberty of briefly summarizing on the next pages the

contents of three past issues, all open access and thus immediately accessible

online through ScienceDirect.

All papers are accompanied by excellent phylogenetic analyses, superb

photographic plates (most tending toward the spectacular), and scientifically

rigorous taxonomic descriptions.

956 ... MycoTAXxoN 131(4)

4} Plant pathogenic and endophytic

Botryosphaeriales known from culture.

Alan J.L. Phillips, Bernard Slippers, Johannes

Z. Groenewald & Pedro Crous, eds. 2013.

STUDIES IN MycoLoGy 76 (September 2013).

PDFs online:

http://www.sciencedirect.com/science/

journal/01660616/76

This issue, dedicated to Dr. Robert A. Shoemaker, a Canadian plant pathologist

well known for his extensive studies on Dothideomycetes, presents three

papers. “A phylogenetic re-evaluation of Phyllosticta” by S Wikee, L Lombard,

C Nakashima, K Motohashi, E Chukeatirote, RCheewangkoon, EKH McKenzie,

KD Hyde & PW Crous (pp. 1-29) redefines Phyllosticta (noting “in moving to

a unit nomenclature for fungi...Phyllosticta was chosen over Guignardia) and

resurrects the older family name Phyllostictaceae. A multigene DNA dataset

used to investigate 129 Phyllosticta isolates (and representing ~170 species

names) showed that many were synonyms of P. capitalensis and supported

12 new species and two new combinations.

“Phylogenetic lineages in the Botryosphaeriales: a systematic and evolutionary

framework” by B Slippers, E Boissin, AJL Phillips, JZ Groenewald, L Lombard,

MJ Wingfield, A Postma, T Burgess & PW Crous (pp. 31-49) covers the

phylogenetic evaluation of botryosphaerialean genera known from culture

and recognizes six families: the previously named Botryosphaeriaceae,

Phyllostictaceae, and Planistromellaceae and the newly proposed

Aplosporellaceae, Melanopsaceae, and Saccaharataceae.

“The Botryosphaeriaceae: genera and species known from culture” by

AJL Phillips, A Alves, J Abdollahzadeh, B Slippers, MJ Wingfield, JZ Groenewald

& PW Crous (pp. 51-167) focuses on the phylogenetic recognition of 17 genera

and 110 species presently known only from culture. Keys to the genera and

species are provided, and one new species (Neofusicoccum batangarum) and 12

new combinations (in Botryosphaeria, Cophinforma, Dothiorella, Lasiodiplodia,

Neoscytalidium, and Sphaeropsis) are proposed.

Book Reviews ... 957

A polyphasic taxonomy of Daldinia

(Xylariaceae). By Marc Stadler. Thomas

Leessge, Jacques Fournier, Cony Decock,

Beata Schmieschek, Hans-Volker Tichy &

Derek PerSoh. 2014. Srupigs 1s MycoLocy 77

(March 2014): 1-143. PDF online:

http://www.sciencedirect.com/science/

journal/01660616/77

For someone only dimly aware that there were more species in Daldinia than

D. concentrica, this thorough exposition of 47 taxa and their morphology,

biogeography, chorology, ecology, and secondary metabolites was most welcome.

The genus, divided into 5 major groups based on morphological and chemo-

taxonomic characters and supported by molecular analysis, demonstrates the

additional taxonomic value that fungal secondary metabolite profiles can provide

beyond the species rank. The genus as amended incorporates many new

synonymies, excludes several previously published names, and admits 6 new

species and two new combinations to the genus. The rDNA-based phylotree

separates Daldinia from Annulohypoxylon and Hypoxylon while nesting

representatives of small predominantly tropical genera within its clade.

Hypocrealean lineages of industrial and

phytopathological importance. By Lorenzo

Lombard, Johannes Z. Groenewald & Pedro

Crous, eds. 2015. Srupirs In MycoLocy

80 (March 2015). PDFs online: http://www.

sciencedirect.com/science/journal/01660616/80

Dedicated to Amy Rossman, an American

mycologist “well known to mycologists and

plant pathologists around the world for her

scientific passion for ... hypocrealean fungi”

958 ... MycoTAxon 131(4)

[not to mention her engagingly cheerful and encouraging personality], these

five papers focus on the taxonomy and revision of the generic and species

concepts in the Hypocreaceae and Nectriaceae (Hypocreales).

“Biodiversity of Trichoderma (Hypocreaceae) in southern Europe and

Macaronesia” by WM Jaklitsch & H Voglmayr (pp. 1-87) recognizes 90

Trichoderma species (the 17 new to science dazzlingly illustrated by excellent

photos) supported by TEF1-, RPB2-, and acLi-based sequence analyses from

>650 identified specimens.

“Diversity and potential impact of Calonectria species in Eucalyptus

plantations in Brazil” by RF Alfenas, L Lombard, OL Pereira, AC Alfenas, and

PW Crous (pp. 88-130) identifies 20 Calonectria species new to science plus

C. pseudopteridis (nom. nov. for Cylindrocladium macrosporum) based on

sequence analyses of the 6-tubulin, cMp, TEF1a, and HIs3 DNA regions and

reveals the C. pteridis complex as the most common species complex in Brazil's

Eucalyptus plantations.

“Novel taxa in the Fusarium fujikuroi species complex from Pinus spp.’

by DA Herron, MJ Wingfield, BD Wingfield, CA Rodas, S Marincowitz &

ET Steenkamp (pp. 131-150) explores the diversity of Fusarium species

associated with four species of diseased pine in Colombian plantations and

nurseries. 6-tubulin and TEF1a sequence analyses of 57 isolates sampled from

plants displaying stem cankers, branch dieback, and seedling root/collar rot

identified more than ten Fusarium species, of which five are named as new.

Four of the new species displayed a level of pathogenicity on Pinus patula

comparable to that of the pitch canker pathogen F. circinatum.

“New species, hyper-diversity and potential importance of Calonectria spp.

from Eucalyptus in South China” by L Lombard, SF Chen, X Mou, CD Zhou,

PW Crous & MJ Wingfield (pp. 151-188) summarizes surveys of Eucalyptus

displaying symptoms of leaf blight in Guangdong, Guangxi, and Hainan

provinces that produced a large number of Calonectria isolates. Of the 21 isolates

identified using the Consolidated Species Concept employing morphological

characters and DNA sequence comparisons of the B-tubulin, cmp, TEF1a,

and HIS3 gene regions, 18 represented new taxa (12 in the Sphaero-Naviculate

Group; 6 in the Prolate Group). Southeast Asia appears to represent a centre of

biodiversity for the Sphaero-Naviculate Group, which could be an important

constraint to eucalypt forestry in China.

“Generic concepts in Nectriaceae” by L Lombard, NA van der Merwe,

JZ Groenewald & PW Crous (pp. 189-245). Nectriaceous species are unified by

phenotypic characters such as uniloculate ascomata that are yellow or orange-

Book Reviews ... 959

red to purple and exhibit phialidic asexual morphs. The family’s generic concepts

have been poorly defined due to the unavailability of DNA sequence data

for many genera. A multi-gene phylogenetic analysis of known nectriaceous

genera using partial 28S LSU sequences and the ITS1+5.8S+ITS2, AcL1, RPB1,

RPB2, ACT, TUB2, CMDA, HIS3, & TEF1 regions from available type and authentic

strains support six new genera (Aquanectria, Bisifusarium, Coccinonectria,

Paracremonium, Rectifusarium, Xenoacremonium) that accommodate species

previously classified based solely on morphology. Additionally several

generic names are proposed for synonymy based on the abolishment of dual

nomenclature, and a new family (Tilachilidiaceae) is introduced for two genera

previously accommodated in the Nectriaceae.

MY COTAXON

October-Dece, ber 2016— Volume 131, p. 961

http://dx.doi.org/10.5248/131.961

NOMENCLATURAL NOVELTIES AND TYPIFICATIONS

PROPOSED IN MYCOTAXON 131(4)

Abieticola Hyang B. Lee [MB 811702], p. 755

Abieticola koreana Hyang B. Lee [MB 811703], p. 755

Alternaria parvicaespitosa Gannibal & D.P. Lawr. [MB 816666], p. 784

Apiosordaria hamata B. Wu, K.D. Hyde, Jing Z. Sun & Xing Z. Liu

[MB 812280], p. 852

Arthromoniliphora S.S. Silva, Gusmao & R.F. Castaneda

[MB 813427], p. 822

Arthromoniliphora araucariae S.S. Silva, Gusmao & R.E Castaneda

[MB 813428], p. 822

Blodgettia saprophytica C.L. Yang, Z. Li & X.G. Zhang [MB 819496], p. 909

Discopycnothyrium Hongsanan & K.D. Hyde [IF 551024], p. 862

Discopycnothyrium palmae Hongsanan & K.D. Hyde [IF 551023], p. 863

Entalbostroma J.D. Rogers & P.R. Johnst. [MB 817225], p. 766

Entalbostroma erumpens J.D. Rogers & P.R. Johnst. [MB 817226], p. 768

Nawawia oviformis J. Peng & Z.F. Yu [MB 815592], p. 737

Pezicula chiangraiensis Ekanayaka & K.D. Hyde, [IF 551789], p. 744

Phaeocollybia pakistanica J. Khan, Sher & Khalid [MB 818193], p. 892

Podosporiopsis Jian Ma, X.G. Zhang & R.F. Castafieda [IF 551862], p. 774

Podosporiopsis obclavata Jian Ma, X.G. Zhang & R.F. Castaneda

[IF 551864], p. 775

Podosporiopsis sinensis Jian Ma, X.G. Zhang & R.E Castafieda

[IF 551863], p. 774

Podosporium bacilliforme J.Y. Wang, Yu M. Cai & X.G. Zhang

[MB 819468], p. 842

Strigula sinoaustralis S.H. Jiang, X.L. Wei & J.C. Wei [FN 570267], p. 797

Thelenella haradae J. Halda, Xin Y. Wang, J.A. Ryu & Hur [MB 819398], p. 806

Zasmidium oleae Y.L. Guo, X.W. Xie & B.J. Li [FN 570159], p. 791

MY COTAXON

October-December 2016—Volume 131, pp. 963-1000

http://dx.doi.org/10.5248/131.963

Richard Paul Korf (1925-2016): A Celebration

LORELEI L. NORVELL, EDITOR

Mycotaxon Editor-in-Chief, 6720 NW Skyline Boulevard, Portland OR 97229 USA

ITHACA JOURNAL, DECEMBER 9, 2016—“Richard Paul Korf, age 91, died August 20, 2016, at

home in Ithaca, NY, leaving behind his wife, Kumi, with whom he was enchanted for over

50 years, and his adored children Noni, Mia, Ian, and Mario, and grandchildren Maia Vidal,

Zak Korf, Zephyr Gettelman, Mason Korf, and Matéa LeBeau. Dick’s academic life began at

Cornell University in 1941 at the age of 16, and he received his Ph.D. in Mycology from the

Department of Plant Pathology in 1950. He taught one year in the Botany Department of

Glasgow University (Scotland) before returning to his alma mater where he continued in his

teaching and research until his retirement as an Emeritus Professor in 1992. Dick was elected

President of the Mycological Society of America, received two Fulbright awards (Japan and

Belgium) and an NSF Senior Postdoctoral Fellowship (Japan). He was offered but declined

a Guggenheim Fellowship, and has received several honorary awards for his teaching and

research.”

ABSTRACT— With this section, MycoTaxon celebrates the life of its mentor and co-founder,

Richard Korf, who died after a brief illness at home in 2016 surrounded by his family and

faithful companion, Meena. Materials from other publications and web posts accompany

reminiscences by Dick’s colleagues, students, authors, and family.

PREFACE: Eliciting contributions from friends and colleagues after any death is difficult,

but arranging them into a coherent whole in a scholarly research journal established

by the ‘honoree’ himself has proved doubly so. Those who knew Prof. Korf as ‘Dick’

submitted fond memories that amuse, inspire, and make readers wish they had known

him better. His many contributions were so varied that we have chosen to cite only a few.

Dick would undoubtedly be gratified that this collection of reminiscences is published

more or less as submitted and forgive us for allowing his full accomplishments to be

enumerated elsewhere; he would, however, heartily disapprove of my shameful use of

material previously published or blogged elsewhere.

A complete scholarly obituary should include all publications written, offices held,

organizations joined, and end with one sentence dispensing of family relationships.

However, Korf’s papers and books are listed on www.MycoTaxon.com/pubsRPK.html

and the Richard P. Korf Wikipedia entry cites three genera and 16 species named after

964 ... Norvell (editor)

him and outlines his formidable curriculum vitae, and so we opt instead to celebrate

his life with an eclectic mixture of photos and fond reminiscences. Mourning the

passing of our self-professed curmudgeon and in the spirit (if not the words) of

Shakespeare’s Marc Anthony, we all have come here to praise him.

K.E. Loeffler; courtesy of the CUP image collection

Fic. 1—Dick at his microscope in his Cornell office in 1985

Mycotaxon remembers

Not surprisingly, I first became acquainted with Dick through the colorful

MycoTaxon volumes on the Oregon Mycological Society library shelves.

Informed by 1979 OMS Foray guest mycologist Harry Thiers that we needed

a species checklist, I had begun compiling a master list of Pacific Northwest

macrofungi from journals, books, and reprints. The tedious page-by-page

scrutiny of MycoTAXON prompted me to write to its Editor seeking some sort

of index. By return mail a manila envelope arrived from a Dr. Richard P. Korf

filled with a bundle of typed sheets covering the first 8 volumes accompanied

by request for a quick return of this, his only copy, as he soon intended to

publish a cumulative index (i.e., Korf & Gruff 1985). Researchers today—with

immediate access to INDEX FUNGORUM, MycoBank, and the Internet—cannot

appreciate the true value of what Dick so generously shared with a complete

stranger whose only credential was an interest in fungi. It did, however, net him

an appreciative individual subscriber.

R.P. Korf (1925-2016): A celebration ... 965

I met Professor Korf in person in 1990 as a first-year graduate student.

My supervisor, Joe Ammirati, dispatched me to escort our esteemed visitor

across the University of Washington campus to the Suzzallo Library. Praising

the MycoTaxon submission procedure, I thanked him again for his 1980

generosity; he in turn proudly spoke to MycoTaxon’s success (despite dire

predictions to the contrary) as a journal prepared by authors mentored by

their chosen peer reviewers. We agreed that open peer review was peculiarly

suited to taxonomy where the ultimate peer is the scientist who must test post-

publication hypotheses and that delays inherent in journal-controlled blind

reviews were unnecessary. (Needless to say, three years later my first research

papers were published in Mycotaxon. Dick’s acceptance letters, generally

concluding with a kindly “Thank you for your fine contribution” were the

source of much preening on my part.)

The 1991 Mycological Society of America meeting (my first) in San Antonio

underscored the extent of Dr. Korf’s prodigious mycological contributions. We

chatted briefly before the awards ceremony where he received the Distinguished

Mycologist award from a society that he had served as Councilor (1958-1960,

1965-1968), Secretary-Treasurer [and MSA Newsletter editor] (1969-1970),

Vice-President, and President (1970-1971). Not confined to one continent, Dick

Fics 2 & 3—Fathers of Mycology:

Fia. 2. Elias Magnus Fries (1794-1878), sometime after his 80" birthday. Fic.3. Richard ‘Elias Fries’

Korf (1925-2016), aged 69 in full Friesian regalia for a performance as Elias Fries at Cornell.

Noni Korf

966 ... Norvell (editor)

Chandler Photographers; Courtesy Amy Rossman

ag Mics eee

Fic. 4—Dick Korf chatting with ‘Case’ Bas at the

First International Mycological Congress at Exeter, UK, in 1971.

Amy Rossman

Fic. 5—Back in Eliasian fieldgear, Dick reprises his performance at Oslo’s

seventh International Mycological Congress in 2002 and gives a big hug to Karen Hansen.

R.P. Korf (1925-2016): A celebration ... 967

had also served on the International Botanical Congress (IBC) Nomenclature

Sections Committee for Fungi & Lichens and was 1973-1977 chair of the

International Mycological Congress's Standing Nomenclature Committee. An

international force to be reckoned with, Dick received the Ainsworth Medal,

the International Mycological Association's highest award, in 2010.

Back in 1994, I waltzed up to his 200-year-old Friesian persona at IMCV

in Vancouver and, in an ill-advised attempt at humor, ‘complimented’ him

with, “you really don't look as OLD as you are!” Immediately drawing himself

up, he glared haughtily at me. I’m still not certain whether he didn't ‘get’ it

or merely remained steadfastly in character. Whatever his true feelings,

I promptly wilted and scurried away, abashed.

Despite his self-proclaimed status as

curmudgeon, Dick (whom in 1998 I still

addressed somewhat timorously as “Dr.

Korf’) offered a sympathetic pair of eyes

and amusing advice during my years as

INocuLuM Editor and MSA Secretary.

On the heels of my second MSA

newsletter, I received a cordial note

with the news that Heinz Clémencon Frc. 6—The infamous “oh no, another erratum”

cartoon shared by Dick

was associated with the University of

Lausanne (not Lucerne) accompanied by a small cartoon to soften his proof

that I was not perfect. This goaded me to establish an ‘Embarrassing omissions,

additions, and corrections’ section, printed far too often to suit either of us.

He followed the cartoon with later gems: [August 1998] “Nothing new to add,

but on page 11 I learned that in 1988 Scot Rogers became a Department of

Botany at the University of Washington. Pll bet he was surprized [sic]!” and

[October 1998] “Dr. Richard Hanlin’s proper first name is “Dick (as in Hanlin),

not Robert (as in Heinlein).” My pleasure at his correctly kenning the source of

my temporary confusion re Dick Hanlin did not, however, lessen my chagrin at

misplacing the name of so amiable an MSA former president and friend.

‘Dr. Korf’s phone call in 2003 asking me to serve as next MyCOTAXON

Editor-in-Chief left me flabbergasted. Thoroughly tired of deadlines, errata,

and other editorial nightmares, I had placed editorships behind me. Despite

the small honorarium, however, Dick’s offer was too professionally tempting

to refuse. It would be fun, I mused, to cobble together something for a change

without having to write anything (ignoring former Editor-in-Chief Jean Boise

Cargill’s dry warning that Don Pfister’s estimate of one day per week for her

editorial work fell short of the mark). I mused wrongly, of course, but accepted

Dick's offer and prepared by ordering my last few back MycoTaxon volumes.

968 ... Norvell (editor)

MycotTaxon was Dick’s mycological

love child. In his farewell to co-

founder Grégoire Hennebert, who

retired from our masthead after 32

years as French Language Editor

(including 17 years as Book Review

Editor), Dick proudly recapitulated

the founding and evolution of their

io}

fad

Bit

journal even as he worried over

the economic feasibility of printed

taxonomic publications (Korf 2007).

From its 1973 inception to 2016's

thwarted free open access, thejournal

remained a source of joy and worry

for its Cornell co-founder. After

| ‘retiring’ from active duty as 1974-

Fic. 7—Grégoire Hennebert and Dick Korf 1991 Managing & English Language

plot out their new journal, Mycoraxon, at the Editor, Dick used the title Editor-in-

Université catholique de Louvain (Belgium) in (Chief for his appointed successors—

ye Jean Boise-Cargill (1991-1998),

Pavel Lizon (1998-2003), and me (2004-present)—who ostensibly handled

the day-to-day production side. Dick nonetheless held onto the reins as chief

Journal Preparation wrangler (1991-2004), Business & Subscriptions Manager

(1991-2006), and Mycotaxon Ltd. Treasurer (1999-2015).

A surprisingly patient Dick projected two volumes of two 250-page issues

for 2004 before returning in 2005 to four 500+-page volumes. That first year we

imposed a cohesive MycoTaxon style upon the previous assortment of differing

font sizes and styles and moved from print-ready pages and reviews mailed in by

authors to computer-prepared files submitted by email. Although prone to fretting

about overworking his all-too-willing new E-i-C, Dick was mightily pleased that

we managed the changeover by the end of 2004, although we both despaired of

making my overly complicated ‘Instructions to Authors’ easily understood by

foreign authors and word-processing neophytes.

Dick granted his E-i-C complete freedom but did wistfully ask that we retain

his journal's title and size. He needn't have worried. The name MycotTaxon is

sacrosanct, and although a book format may appear outdated for hard copy, its

smaller page size permits readers to scan a complete page on-screen without having

to scroll up and down moving from column to column. (We did have a bit of a

‘discussion’ over my 2013 desire to shift the titles and sub-titles left from page center;

but after checking other journal formats, he somewhat grumpily acquiesced.)

R.P. Korf (1925-2016): A celebration ... 969

Nontheless, when his new E-i-C changed the unwieldy subtitle—ANn

international journal for research on taxonomy & nomenclature of fungi,

including lichens—to “THE international journal of fungal taxonomy and

nomenclature, Dick was ‘thrilled’ that Grégoire'’s and his journal now rightfully

merited the word “THE. He fretted that lichenologists might feel excluded until

I pointed out that ‘fungal’ democratically covered symbionts, myxomycetes,

and other fellow travelers.

Dick definitely encouraged my nabbing Shaun Pennycook as Nomenclature

Editor in 2005 when I realized that adding nomenclatural research to author

and reviewer correspondence, manuscript revision, and converting separate

text and illustration files into PDFs was too much for one person. Shaun's

volunteering to take over manuscript accession proved an unexpected boon

and soon neither of us could envision the journal without him.

Dick was passionate in his desire that MyCoTAXON survive without page-

charges through subscriptions alone. [Whenever I asked whether MycoTaxon

showed a profit, Dick would respond, “Occasionally, and then change the

subject by telling me not to concern myself with financial matters.] It had

long been obvious that rising postal rates would soon preclude our mailing

heavy print volumes to subscribers. However, after he paid out of pocket what

I considered an enormous sum to keep the journal solvent for one year, even

Dick realized that our financial model had to change. Aware that the next

International Code would permit publication of names online (see Norvell

2011), Dick urged the editorial advisory board and owners to move to online

publication in 2011. They agreed.

Unfortunately, although less expensive than hard copy, online publication

is not free. Webmaster and daughter Noni found an acceptable online host

(Ingenta), but as subscriber fees still could not cover journal costs, Dick and

Bookkeeper Hannes Maddens agreed to institute page charges. A highly

distressed Dick, who had hoped to offer a completely open-access journal,

discovered that obligatory open access fees were too high for many long-time

authors. Dick was bitterly unhappy that a completely open access MyCOTAXON

was economically unfeasible in 2011.

Naturally two strong-willed editorial types did disagree on occasion, and

there were definitely times when I felt the founder’s axe poised dangerously

close to my neck—usually when a quarterly deadline passed without a

publication. MycoTaxon was founded on a prompt turn-around. Our moves

to editorially prepared copy and rigorous scientific and nomenclatural editorial

review produced countless delays. Predictably submissions lagged, due also to

our newly instituted page charges, competition from an increasing number of

online mycological journals, and in 2012 a “perfect storm” of editorial illnesses.

970 ... Norvell (editor)

Our 500-page target proved increasingly unattainable; Dick felt that anything

under 300 pages did not merit the term ‘volume. We eventually resolved this

unhappy situation by changing ‘volume’ to refer to the annual publication of

four quarterly issues delivered more or less on time. [It is sadly ironic that this

paper has delayed publication of the final issue of volume 131.]

Fortunately such collisions were few, and I always looked forward to our

weekly chats, generally launched with neutral weather and health reports

before moving on to thornier problems, such as the discovery that for seven

volumes Else Vellinga had been identified as “BooL REviEw EpIToR” on the

masthead, and no one had noticed.

Dick also dispatched frequent pep-talks. To banish my doldrums over

author errors, he wrote, “We started with the concept that errors were the fault

of authors, not ours as editors/publishers. Any paper that requires editorial

correction by you (as opposed to corrections suggested by reviewers) should

be rejected outright until authors (or their reviewers) make their corrections.

If the title is incorrectly punctuated or misleading or non-informative, I'd

reject the paper outright till they get it right ... Your health is more important

than always trying to be the best friend of folks who don't deserve your pains!

Curmudgeonly, and simultaneously compassionately, Dick.”

When after too many years of stomach-crunching deadline madness, he

buttressed my decision (to correct only PDFs with editorial errors but not

author errors suddenly discovered by the authors after conversion) with,

“Dear over-worked E-i-C, Good idea. I think all author errors should appear in

the errata; we will change only editorial errors in PDFs before publication. Their

submission is what we print... Anything that will relieve your stress and Shaun's

is an improvement. I also highly applaud a much stricter rejection option.

Authors and reviewers both need to be made aware of their shortcomings.

Fondly, Dick.”

In 2011, amused by his body’s commitment to fungi after the successful

removal of a nasal melanoma, Dick wrote, “One bright yellow “mushroom”

almost as big as a golf ball (but flatter) remains, holding the skin graft from my

neck area in place while the blood vessels grow up into it and my nose becomes

grafted correctly. Mushroom probably has to be there a week or so. Think [ll

stay close to home for the next few days. Dick”

Ever the optimist, Dick always looked forward, as witness his response to

his co-founder’s condolences on our move to online publication. Grégoire had

written, “I do not know who chose the cover color [black] of the last printed issue

of MycoTaAxon after 36 years of printed life. For me it expresses adequately my

feeling receiving the last physical volume of your child. ...Now after its physical

R.P. Korf (1925-2016): A celebration ... 971

death, its soul will continue a virtual life, only easily accessible and usable to

illuminated minds.” Dick responded, “The color choice is always Lorelei’s. Her

feelings were like yours about the death of the printed journal. I did not agree.

I would have preferred some verdant GREEN or STARBURST to celebrate the

journal being reborn in a less environmentally destructive format.”

Richard Paul Korf was golden. Not to mention immortal, as noted by his

former student and friend, Wen- Ying Zhuang: “Dick left us, but his contribution

to mycology will stay forever.”

Unfortunately, that doesn’t mean we don't miss him—fiercely.

—LORELEI NORVELL

MycoTaxon Editor-in-Chief, Portland, OR

Fic. 8 (LEFT)—Meredith Blackwell, H. Peter Kahn, and Don Pfister with Dick during a 1994

Cornell Hoot at the Ringwood Preserve, Cornell Botanic Gardens. Fic. 9 (RIGHT)—Former MSA

presidents Robert W. Lichtwardt (1971-1972) and Richard P. Korf (1970-1971) with President

Orson K. Miller, Jr., and Kumi Korf at the 2000 MSA annual meeting in Burlington, VT.

“I was sad when I heard that Dick passed away. Dick meant a lot to me. He was

like a grandfather, mentor, and close friend. Even though I’m sad that he isn’t

around anymore, | will always have lots of happy memories of all of the great

things he has done.

“IT know I will never forget Dick. As I’m typing this at my desk, there are

reminders of him everywhere I look. ’m typing this on my MacBook, which

Dick got me hooked on after giving me his old Macintosh about 10 years ago.

On top of the stack of mostly MycotTaxon-related mail on my desk, is a bank

statement for one of my first bank accounts that Dick helped me set up. When

I look out of my window at my driveway, I see the forsythia hedge that Dick

helped to pay as a housewarming gift when I bought my house. In the kitchen

is the microwave he bought for my girlfriend and me when we first moved in

together. On the kitchen counter I see bottles of Guinness, one of my favorite

beers that I first tasted during one of the many times Dick took me out to eat.

Hanging on our coat rack is my favorite old fishing hat that Dick gave me about

15 years ago. I still think of him whenever I wear it.

972 ... Norvell (editor)

“These little reminders bring back lots of happy memories about good

conversations and stories during long car rides, fishing and swimming, late

night card games, and the many delicious dinners (like fish head soup) that

Dick prepared. Besides happy memories, all these little reminders of him also

bring back memories of all the good things I have seen him do over the years.

“I'm not going to spend hours writing about all of his kind and charitable acts

towards me and other people. I’m assuming most of his students, colleagues,

and friends must have experienced his kindness themselves. I just want to share

Dick's greatest gift to me.

“Dick’s greatest gift to me was his confidence in me. Dick's trust and

confidence in me has given me so much self-confidence. Dick believed I would

be successful in life, and he was always looking for ways that he could support

me. He always encouraged me to continue my education, but when I wanted

to work as soon as | graduated high school instead, he helped connect me with

people who could give me a job. Maybe he somehow knew I would attend

college the next year. When I went on my first date with my girlfriend, he let

me use his Miata sports car. When I told him I wanted to try living in a cabin

in the woods like Thoreau, he didn’t tell me I was crazy. Instead he sold me a

small piece of land he owned in Dryden. And, what I am most proud of is that

he trusted me to help run the journal MycoTaxon that meant so much to him.

I think Dick’s generosity and kindness towards so many came from the fact that

he believed in the goodness of people the same way he believed in me.

“T like to think that the kindness that Dick showed me lives on in me. Dick

once gave me a gift membership to the American Civil Liberties Union (a

charity I had never heard of), and now I am proud to support this and other

charities that he introduced me to. At work, I enjoy giving students confidence

that may help them get through school, just like Dick did for me.

“Like Dick, I am also trying to help Mycotaxon become an open access

journal, or if that is not possible, I would like go back to allowing taxonomists

to publish manuscripts quickly for free. Dick and I spent lots of time trying to

decide whether to continue to allow authors to publish for free (this was how

Dick ran the journal) or charging authors so that we could make the journal

free to everyone.

“Even though Dick isn’t here now, his memories, his generosity, and his

many kind acts will always be with us.’

— HANnNES MADDENS

Mycotaxon Bookkeeper & Subscriptions Manager, Ithaca NY

R.P. Korf (1925-2016): A celebration ... 973

The Theatre

Anyone acquainted with Dick was fully

aware of his theatricality, but they might not

have known of his real acting chops. Regarding

this extra-mycological passion, friend and

former MycoTAxon E-i-C Pavel noted (Lizon

2016), “Dick was performing as an amateur

actor both on stage and in the recording studio,

and for few years he even served as the Chair of

the Department of Theatre Arts at Cornell?

Dick himself explained, “My acting career

began at an early age in Riverdale Country

School in New York City, eventually being cast in

major roles in three annual outdoor productions

ous 2016

Courtesy of IMA Fun

Fic. 10—Dick’s 1980 actor’s

promotional photo

of Shakespeare's plays. ...The theatre has been my lifelong passion. I performed

during my college years at Cornell University (where I later became a professor)

and I continued, both on stage and in radio dramas. While on my final sabbatical

leave before retirement I took a fling at off-off-Broadway performances of three

plays while in New York City.” (Korf 2006)

Ezra, Cornell's quarterly magazine, revisited Dick's participation in the 1946

production ofa play on the founding of the college (Wilensky 2013): “Richard Korf

‘46, Ph.D. ‘50, Cornell professor emeritus of mycology, played ‘Angus Dangit’ in

‘Once Upon a Hill’ (one of his lines: “‘Humph. Makin’ a school outa Ezry’s farm!’).

Reprinted from Wilensky 2013

FiG.11—The soon-to-be illustrious Professor Korf as a hayseed peeks out at the audience

(extreme stage right, second from bottom) in a 1946 Cornell curtain call.

974 ... Norvell (editor)

(<¢9

It was just a fun thing to be in, he recalls, noting that his dog also got to

be in the show, wearing a Cornell sweater. He also remembers that there was a

spittoon sound effect used off stage, which was ‘used to emphasize some of our

comments.”

Korf at Cornell

Amy Rossman and Wen-

Ying Zhuang (2016) recall Dick's

introduction to his alma mater and

mycology. “Having entered Cornell

University intending to become a

gentleman farmer of Cornish game

hens (as I remember it), he soon

switched to mycology under the

influence of Harry M. Fitzpatrick

(1886-1949), completing his PhD

in 1950. With the untimely death of

his major professor, Dick joined the

faculty of the Department of Plant

Howard H. Lyon; courtesy of the CUP image collection

Pathology at a relatively young age.

He was thus the major professor for

several students who were about his

age or older. He started as Assistant

Fic. 12—Professor Korf at the blackboard Professor in 1951, and eventually

of Plant Science 326 lab in 1972. served as full Professor of Mycology

[at Cornell] from 1961-1992”

In his 90" birthday wishes, Pavel (Lizon 2016) rightly referred to Cornell as

Dick's life-long companion. “Under his guidance, more than 20 PhD students

graduated (Korf 1991), the first three—Robert A. Shoemaker, Robert L. Shaffer,

and Martin A. Rosinski—already in 1955.”

The Cornell Hoot

“When hunting mushrooms, it’s easy to lose students in the woods. ‘That's

why we practice the Cornell Hoot. Learning the Hoot is a highlight of the

first Fall field trip. With students gathered round, I describe the exquisite

art of collecting mushrooms, hand out crisp Cornell apples and maps, and

demonstrate the Cornell Hoot: a rising “Ah-OOOT!” My students shuffle

uncomfortably, but soon they can't help but smile. Now we practice together

Ah-OOT! Ah-OOOOT! Ah-OOOOOT! Even the shy ones can’t resist it. We

do it again in unison, very loudly. A distinctive sound.

R.P. Korf (1925-2016): A celebration ... 975

Courtesy of Pavel Lizon

at the Ringwood Preserve, Cornell Botanic Gardens.

“I've wondered where the Cornell Hoot came from. I learned it from my

predecessor, Dick Korf. Imagine my tingling excitement when I encountered

this passage in the 1903 travel diary of my great-great predecessor at Cornell,

George Atkinson, who was visiting the Botanical Gardens at Kew, in England:

“As time for closing the gates came on I heard musical voices from different

parts of the garden sing “all out, all out.” A custom very old, and now it is such

a perfunctory call that one can scarcely distinguish the words. It often sounds

more like ‘Ah-laio-

“... Then I heard back from Dick Korf. He said ‘Oh, it’s the Cornell Hoot

now, is it?’ And he told me it was ENTIRELY HIS OWN INVENTION. Recent

and local, not at all what I was thinking! It made me laugh out loud. But an

enchanting story still. Here's his tale: ‘It was when I was at Ringwood with one

of my first class field trips, maybe in 1951, and noticed that all students had not

returned. I thought it to be the loudest and most distinctive calls in my vocal

repertoire. I am pretty sure it didn’t come from one of the many plays I did

as an undergraduate and graduate student, learning to project my voice, even

a whisper, to reach the back row in the theatre, which Professor Alexander

Magnus Drummond demanded of us.” — Karuie T. Hopeg, Director

Cornell Plant Pathology Herbarium, Ithaca NY

Fic. 14—Dick chats with MycoTaxon E-i-C Pavel

during the 1994 hoot.

Courtesy of Pavel Lizon

976 ... Norvell (editor)

CUP—Cornell Plant Pathology Herbarium

Four days before my usually scheduled phone call to Dick, I learned of

his sudden demise from Scott LaGreca, who wrote, “We are sad to share that

Richard P. Korf passed away yesterday, at the age of 91. There was a small

reception at Noni’ house last night in Ithaca and I’ve never seen such an up-

beat group, celebrating a persons life. It was heartwarming.” Scott later added,

“At a reception Saturday night in Ithaca, his remarkable family and friends

raised toasts of Guinness (his drink of choice); remembered his generosity,

humor, and wisdom; and sang his favorite songs.’ Below is Scott's formal

contribution to our celebration of Dicks life.

Dick’s many personal and professional connections have made our herbarium

(CUP) what it is today. Dick contributed thousands of specimens from around

the world to our collection. I work every day surrounded by those specimens;

they are part of Dick Korf’s legacy: thousands of specimens, as well as his

notebooks and correspondence. Those specimens have people's names and

places on them, which tell Dick’s entire story.

My very first herbarium specimen (LaGreca no. 1!), also deposited in CUP,

was identified by Dick: Sarcosphaera gigantea (Rehm) Kanouse. I collected it

in 1989 after completing Dick's Field Mycology course. It was a hot summer

day when I brought the specimen to him for identification, and I was shocked

to see him shirtless in his lab: seeing my formal professor in such an informal

situation was strange to me! But I quickly got over my surprise when he

excitedly took the fungi from my collecting bag and started poring over his

identification books, quickly making a slide to examine its spores. Later, he also

showed me how to make the cups “poof” their spores. I'll never forget that day.

Right up until his death, Dick’s collaborations with other scientists around

the world inspired activity and growth for us here at CUP. For example: there

was a mycology student in the Canary Islands (Luis Quijada) who had just

completed his PhD in the systematics and floristics of cup fungi on the Canary

Islands. Dick ran an NSF-funded project on Canary Islands cup fungi for years

(resulting in many publications) and so Luis emailed Dick with some questions,

which led in turn to his requesting multiple loans of Canary Islands specimens

from us, which allowed (a) completion of his dissertation and (b) improvement

of our specimens here at Cornell with Luis’s examinations/annotations.

Another example: Madame Francoise Candoussau, a long-time friend

of Dick’s in France, has just this year decided to give her valuable personal

herbarium (comprising thousands of specimens, mostly from Europe) to

Cornell as a direct result of her friendship and longtime professional association

with Dick Korf.

R.P. Korf (1925-2016): A celebration ... 977

I should also mention that Dr. Korf was innovative in his teaching. At a

time when team-teaching was almost unheard of, he divided the work of his

Introductory Mycology class, inviting specialists of different groups of fungi to

teach for different weeks. Dick himself taught the first half of the course, while

other, ‘guest’ teachers took over the last half of the course. This allowed for a

more thorough mycology education for his grateful students. I know this sort

of thing is commonplace now, but at the time (25 years ago—1991) this was a

rare thing.

After I left Cornell and came back to work here with a PhD in lichenology, I

joked with Dick that hed inadvertently turned me into a lichenologist because

he always dismissed any lichen specimens that students (including me) brought

him. “Lichen!” is all he would say, tossing the specimen aside. I found it

fascinating that such a great mycologist had such a poor opinion of (and lack

of interest in) lichenized fungi, and this spurred me on to learn more. So in an

indirect way, I credit Dick for inspiring my career as a lichenologist.

—ScoTT LAGRECA, Curator

Cornell Plant Pathology Herbarium, Ithaca

—"

Fic. 15—Professor Emeritus Richard P. Korf,

CUP Director Kathie Hodge, and Curator

Robert Dirig pose with some specimens from

Cornell’s Fungi of China collection in 2009.

From China to CUP and back again

“Shu Chun Teng traveled halfway around the world on a scholarship to

study mycology at Cornell University in 1923. He left five years later with a

knowledge of fungi unequaled in China, then spent the next decade traveling

on horseback gathering up molds, lichens, yeasts, rusts and morels in the

forests, fields and marshes of his homeland. ...During the Japanese invasion

in 1937, Teng arranged for his best specimens to be removed from a national

botany institute he directed in Nanking to save them from destruction. During

World War II, they were smuggled by ox cart to Indochina and then by sea to

the United States, and 2,278 of the specimen packets ended up at Teng’s alma

mater.

“At Cornell’s initiative, the university is dividing up and sharing its Fungi of

China Collection with the Academy of Sciences in Beijing to help advance the

978 ... Norvell (editor)

<s oo (Sp

Fic. 16—Dick at the microscope in 1988 at the

Key Laboratory for Systematics & Microbiology in Beijing.

exploration of fungal species. ...In a repatriation ceremony Monday, Cornell

President David Skorton presented a high-level Chinese delegation with a rare

mushroom called Lentinus tigrinus, reafirming the university's desire to share a

collection he said it ‘has held in safekeeping for the global scientific community

since 1940’ ..Some 1,700 specimens will be delivered to China in the fall,

including 57 considered irreplaceable. Cornell will retain fungi that can't be

divided, but make them available to scholars.

“At the start of the devastating Cultural Revolution in the 1960s, Teng was

tagged as a ‘counterrevolutionary academic authority. Discharged from his

lab, he was subjected to daily beatings and mental persecution that ruined his

health and career, according to the science academy. He died in 1970 at age

67. Years later, his family managed to recover Teng’s confiscated manuscripts.

His daughter, Rosaline Deng, worked with a Cornell professor of mycology,

Richard Korf, to complete and publish THE FUNGI oF CHINA in 1996 (Teng

1996).

“Transferring the fungi ‘is our decision, said the 83-year-old Korf, who

taught at Cornell until 1998. “The whole reason really is that it makes scientific

sense, especially with the warming of U.S.-China ties over the last 30 years. ... It’s

clear we are cooperating in ways we never knew we could:

Wen-yen Zhuang; Courtesy of IMA Fungus 2016

R.P. Korf (1925-2016): A celebration ... 979

“Another impulse ... is I had a brilliant student, Wen-Ying Zhuang, come

to work with me from China in the 1980s and get her Ph.D. She became a

major figure in the Chinese mycological field. She certainly pressed me on how

important it would be for Chinese scientists to be able to easily access those

specimens.” —BeEn Dossin, Associated Press (Dobbin 2009)

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Fic. 17—Seeing the sights on the way home

from the 1986 MSA Annual Meeting in Gainesville, Florida.

Students and Fellow Travelers

“Dick obviously enjoyed his students, believed in them, and wanted them

to be successful. As such he encouraged them to attend scientific meetings

doing whatever he could to ensure that they managed to get there. This

included towing his own pop-up camper staying with his students in a

campground near the meeting site.” —Rossman & Zhuang (2016)

“One of the world’s most notable mycologists of all times died on August 20,

after ninety-one years of a well-lived life.

“Dick Korf, Professor at the Plant Pathology Department at Cornell

University, was a leading figure in the study of discomycetes (Leotiomycetes and

Pezizomycetes) and a mentor to many in this field. Not only did he convey his

love and appreciation of fungi but also taught us the importance of rigorously

studying the morphology of fungi and their role in nature and the environment.

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980 ... Norvell (editor)

This came with hours of hours of observations of each specimen under the

microscope, methodically recording morphological and anatomical features

and chemical reactions. He always brought an inquisitive mind, teaching us to

formulate questions, to doubt dogma and try to answer questions with rigor.

At the same time he taught us the intricacies of nomenclature, again with rigor.

“The first issue of MycoTaxon, a journal founded by him, dedicated to

the taxonomy and nomenclature of fungi, came out in 1974. It is today in its

volume 131 and has been a leading journal for descriptive mycology.

“Dick was an advocate for social justice, cared for others, and helped many

in need.

“Today is a sad day for his family, to whom he was completely devoted. It

is a sad day for us, his students, and for the many that he mentored, advised,

and helped through his well-lived life as a caring person and as an eminent

mycologist.” — TERESA ITURRIAGA

MSc (1983), PhD student (1990) Cornell, under R.P. Korf

Fic. 18 (LeEFT)—Dick, Linda Kohn (student & future 1999-2000 MSA president), Trond

Schumacher (future 2000-2006 IMA president), and Japanese colleague in 1983 at the Sanshi Imai

Memorial Discomycete Foray after IMC3 in Nikko, Japan. Fic. 19 (RIGHT)—Kumi and Dick at

Satoshi and Harumi Inoue'’s wedding at Cornell’s Stadler Hotel on September 25, 1995

“When I entered Department of Plant Pathology of Cornell University as a

graduate student in 1990, Dick was one of a few professors I met during my

orientation days. I had graduated from Tohoku University in Sendai in 1981

with Agrimony background and didn’t know much about Mycology. Our

conversation was not so much at that time yet.

“I started my M.S. degree with Dr. Jim Aist and selected Mycology as my

major. One for that decision was that I did not want to take a course “SADISTICS”

(statistics).

“I ended up taking almost of all the courses offered by Dick during my M.S.

degree period. One of the toughest courses that I have ever taken is Advanced

Mycology (PL PA 739) during the 1992 fall semester. The TA for the class was

Julie Carroll and my classmates were Linda Hanson, Ker-Chun Kuo, and Kathie

Satoshi Inoue

R.P. Korf (1925-2016): A celebration ... 981

Hodge (who is currently teaching Mycology at Cornell). Dick gave us a very

strange project and we struggled to collect and identify ten (10) uncommon

species of hyphomycetes.

“I obtained my M.S. degree and entered my Ph.D. program in 1993. In

1995, just after I passed my A-exam, I married Harumi. We moved to Dick and

Kumi Korf’s apartment and lived there for two years until I obtained my Ph.D.

and returned to Japan. Our son, Amane, was born there in 1996. We spent a

very happy time with Dick’s family and have beautiful memories of them. I

remember it as if it were just yesterday.

“With my deepest sympathies on the death of Dick.”

SATOSHI INOUE, PhD (1997), Cornell under R.P. Korf

Director of Forage Seed Department, Kaneko Seeds Co. Ltd., Japan

“When I was a senior at Colgate, I was assigned the task of “refreshing” cultures

of Rhizopus nigricans to restore their ability to form zygospores. Knowing

nothing about a process to accomplish this, I wrote to Korf at Cornell, who

kindly sent me two new cultures and a method to demonstrate zygospore

development.

“And so it was that I applied to Cornell and entered in Fall 1956. My

intention was to perform taxonomy on jelly fungi, but I suffered a self-inflicted

wound when I skipped numerous labs in Plant Path 1 and earned a C in the

course, which violated Cornell’s dictum of grades of Bs or better for continued

enrollment. I was informed that while I might eke out a master’s degree, I surely

would not be allowed to continue thereafter.

“Thus it was that I enrolled at Columbia University under Lindsay Olive.

And the rest is history. In one of my rare subsequent meetings with Dick Korf,

he referred to me as “the one that got away.”

“Although Dick was not high on the marquee in his last years, his name

will surface for generations as the co-founder of MycotTaxon, discomycete

taxonomist, and outstanding personality in the mycological community.”

—Ron PETERSEN. Professor Emeritus & MSA President (1993-1994)

Ecology & Evolutionary Biology, University of Tennessee

“Dick Korf was instrumental in my existence as a mycologist. In 1970 I was a

floundering second year graduate student in mycology with no real idea of a

research project or direction. Dick wrote to my major professor, Bill Denison

(a student of Dick’s) at Oregon State University, inviting him on a collecting

trip in Puerto Rico and Dominica. Bill couldn't go, but I jumped at the chance.

That trip was magic, and I met my lifelong friend Don Pfister. During the day

we groveled in the litter finding tiny discomycetes as well as heaps of delightful

yellow and red perithecia and black, twig-like xylarias. In the evenings we

crowded around the microscope attempting to identify them at least to genus.

982 ... Norvell (editor)

Wen-yen Zhuang;, courtsy of IMA Fungus 2010

Fic. 20—Newly installed MSA President Amy Rossman with Richard ‘Elias Fries’ Korf

at IMCV, Vancouver, BC (Canada) in 1994.

Later I learned, at least for the nectria-like fungi, that, no, I wasn't stupid, there

really were no keys to tropical species, and that was the inspiration for research

on these fungi.

“The home of Dick and his wife Kumi was always open to itinerant

mycologists even when their four children were small. Although chaotic during

the day, Dick would rise early in the morning to work on MycoTaxon in those

quiet hours. He was totally supportive of graduate students and postdocs

writing glowing letters of recommendations—perhaps tapping into his creative

writing tendencies! But they worked, as most of us found good jobs in the

always-tough mycological employment marketplace. He shared his time and

resources, always willing to stop his own work to discuss nomenclature issues

or read over a draft manuscript.’ — Amy Y. RossMAN

Oregon State University, Corvallis

Amy Rossman (IMA Fungus 2016)

Fic. 21—Kumi & Dick at Amy Rossman’s home in Beltsville in 2000.

Fic. 22—At Amy’s in 2005: mmmm... roast QUORN!

R.P. Korf (1925-2016): A celebration ... 983

Fungal taxonomy and nomenclature goes international

These messages and reminiscences are organized alphabetically by country.

VALE DICK KORF

Sad news about the passing of Dick Korf

Among his numerous contributions to mycology, Dick Korf was Secretary

of the NCF (then the Committee for Fungi and Lichens) in the 1980s and co-

founder and long-time supporter of MycoTAxon.

Two enduring images are early career Korf at the blackboard (note the peace

sign belt buckle in Fra. 12): and later, channeling Elias Fries.

— Tom W. May, Secretary, IBC Nomenclature Committee for Fungi

Royal Botanic Gardens Victoria, Melbourne, Australia

We regret so much for Dick Korf.

We'll always remember him for his great mycological contributions and

inspiration. — FRANCISCO CALACA

Lab. de Biodiversidade do Cerrado - Fungos Coprofilos

Unidade Universitaria de Ciéncias Exatas e Tecnoldégica, Anapolis, Brazil

Dick’s passing marks the end of an incredible career and great loss for mycology.

I thank him for his friendship and kindly guidance and send my sincerest

condolences to his family and friends. —Scott REDHEAD, Curator, DAOM

Agriculture & Agri-food Canada, Ottawa

Dick began his association with the

Institute of Microbiology (Chinese

Academy of Sciences) in Beijing

and had worked hard to promote

Chinese mycology, _ especially

through publishing major books

on fungi through MycoTaxon

(IMA 2013).

In China, “an 88" birthday is

regarded as particularly special,

and those that reach it are called

“Mi-shou. In honour Dick's

attainment of “Mi-shou” status, the 4

June 2013 issue of MycosysTEMA AS y=

(on whose editorial board Dick ? iv ‘ = te

served since its founding in 1987) RY VF its Me » be a

was dedicated to him (IMA 2013). ALG e | q ‘ ig

The excellent photos of Dick’s 1988 Fic. 23—Dick at the Jinzhong temple in

Yunnan, China (1988)

sus 2016)

jan

984 ... Norvell (editor)

Korf in China in 1998. Fic. 24 (left): Before Tanzhesi temple in Beijing.

Fic. 25 (right): searching lily pads at the Xishuangbanna Tropical Botanical Gardens.

visit to China (Fics 23-25) were captured by Wen-ying, his student and much-

valued colleague and close friend. —WEN- YING ZHUANG,

Key Laboratory of Systematic Mycology & Lichenology

Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

Courtesy of Pavel Lizon

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Fic. 26—The discomycete team at IMC7 outside the Oslo convention center in 2002.

Wen-yen Zhuang ?

R.P. Korf (1925-2016): A celebration ... 985

Fic. 27—Marc Stadler (Germany), José

Guarro (Spain), Teresa Iturriaga (Venezuela),

Dick (USA), and Rafael Castafieda-Ruiz

(Cuba) represent three continents at the

2011 Latin American Mycological Congress

in Costa Rica.

I have received the terrible news from Teresa Iturriaga. Please pass my

condolences to the family of Richard Korf and share this photo (Fic. 27) of Dick

with friends at the 2011 Latin American Mycological congress in Costa Rica.

Un abrazo, Rafael —RaFAEL F. CasTANEDA-RUIZ

Fundamentales en Agricultura Tropical “Alejandro de Humboldt’,

Santiago de Las Vegas, Cuba

It has just come to my attention that Richard Korf, co-founder of MycoTaxon,

has passed away on Aug. 20th. Please accept my sincere condolences for an

outstanding mycologist and a visionary who will undoubtedly be remembered

as one of the all-time greats in mycology. —MICHAEL LOIZIDES

Cyprus Mycological Association, Limassol, Cyprus

Nobody like Dr. Korf, so professional, so enthusiastic of small discomycetes,

but most of all, a good person. We will never forget him.

—Rosario MEDEL ORTIZ, Instituto de Investigaciones Forestales

Universidad Vercruzana, Vera Cruz, Mexico

Dear Dick

I first met you when you visited me when I was working in the University

of Hong Kong in the mid nineties. You were so generous and supportive of my

work at the time and gave me encouragement to continue my efforts

The next time I met you was at the International Mycological Congress in

Norway. You found it so funny that I did not recognize you dressed up as that

‘other’ famous scientist.

Since then you have always been so supportive and provided advice and

encouragement.

I will dearly miss you. —Kevin Davip HyDE

Editor-in-Chief, FUNGAL DIVERSITY

Director, Center of Excellence in Fungal Research

Mae Fah Luang University, Chiang Rai, Thailand

986 ... Norvell (editor)

Was so sad to learn of the passing of Dick. He was hugely influential in

mycology. Truly one of the greats of the 20th century.

—Davip W. MINTER

President, International Society of Fungal Conservation

Egham, Surrey U.K.

Dick was inspirational and I shall make sure that the BMS office knows. I had

a lovely dinner with him in Louvain in the late 1990’s and I certainly valued his

help, advice, and support. — ANTHONY J.S. WHALLEY

School of Biomolecular Sciences

John Moores University, Liverpool England U.K.

I first met Dick at IMC1 at Exeter (UK) in 1971. [had joined the Commonwealth

Mycological Institute two years before, and this was my first exposure to the

international mycological community. I especially remember a discussion on

nomenclature he chaired, and which led to the establishment of a Nomenclature

Secretariat under the auspices of the IMA. He was especially concerned about

the problems due to the later starting point for names of non-lichenized

fungi, including publishing a series of papers he characteristically entitled

‘Later starting point blues;’ the rules were changed in 1981. As Secretary of

the Nomenclature Committee for Fungi, he played a major role in debates at

International, Botanical Congresses, representing mycological interests, where

his acting skills were often put to good effect.

His love of fungi and fieldwork inspired not only his many graduate

students, but also many others throughout the world, especially tropical areas

still so little known for discomycetes. I was pleased to recognize this a little

when introducing Korfiomyces (along with Teresa Iturriaga) for a little disco

from Venezuela that had puzzled him. Perhaps the most important thing

I learned from my contacts with Dick was the importance of always being

fully aware of the historical background of issues and thoroughly researching

the consequences of any contemplated actions. We did not always agree on

controversial matters, but always respected each other’s views.

His foresight in establishing Mycotaxon to address a then developing

log-jam in getting systematic mycology papers in print, with the innovative

requirement for authors to produce their own camera-ready copy, was an

enormous stimulus to the health of the subject. He was a most generous man,

and kindly ‘loaned’ me a set of the printed volumes after I left the International

Mycological Institute at the end of 1997. The neat rows of brightly coloured

volumes, which I repeatedly have to consult, are an incredible legacy.

Dick was always keen to promote mycology selflessly throughout the world,

not least in China where he helped safeguard collections, get several major

R.P. Korf (1925-2016): A celebration ... 987

works published, and was one of a small group ‘foreign’ mycologists involved

in the establishment of MycosyTema prepared by the Chinese Academy of

Sciences, which has now also grown into a major journal in the region.

An inspirational mycologist who touched the lives of numerous mycologists

around the world, many of whom came to occupy significant positions. The

carefully executed works from their ongoing research—and of those they in

turn train and inspire—that have the stamp of Dick’s concern for accuracy and

quality will be a self-perpetuating memorial. I feel privileged to have known

him, and would have loved to have been able to spend more time benefitting

from his vast experience and insights. —Davip L. HawKsworTH

Comparative Plant and Fungal Biology, Royal Botanic Gardens, Kew, U.K. &

Department of Life Sciences, Natural History Museum, London, U.K.

Fic. 28—Dick, wearing his ‘Reunite

Gondwanaland” t-shirt, makes a fine point

at the 2003 New England Mycological

Foray (NEMF) in Deposit, NY. Of the 24

species he identified, five were new to the

27-year old NEMF master list.

Diane Smith; NEMF foray website

My first acquaintance with Dick Korf was as a new member of Mycological

Society of America in the early 1960’s. He and Clark Rogerson literally

WERE the MSA. He had an imperious personage—handsome, self-confident,

theatrical, and well spoken. He seemed initially remote from young mycologists,

but accepted them as colleagues over time. I was flattered when he asked me to

be a founding member of the editorial board of MycoTaxon, a position that I

enjoyed for many years.

Dick was a consummate teacher of mycologists. He took them on field

trips to places that seemed exotic to a small-town man. His former students

are among the leaders of mycology—Jim Kimbrough and Don Pfister among

many others. He was also a teacher at MSA meetings—quizzing students and

professionals on details of their studies and offering advice on improvements.

It was as perhaps nomenclaturist that he was most widely known. He spent

much of his time solving nomenclatorial problems for colleagues worldwide.

He was dismayed with massive changes in the mycological part of CODE OF

BOTANICAL NOMENCLATURE and I suspect that, had he been younger and

988 ... Norvell (editor)

healthier, that he would have used his vast intellect and assertiveness to prevent

the most irrational changes.

Let us praise this great scholar! Mycology is poorer without him and we

shall never see his like again.

— JACK ROGERS,

Professor Emeritus & (1977-1978) MSA President

Washington State University, Pullman, WA U.S.A.

The Lion in Winter

Except for a 1950-1951 tour as

Lecturer at Scotland’s University of

Glasgow, Dick devoted his entire

research life to Cornell. His heart,

however, belonged to his family, and

an impressive one it is. In its praise

of “Hidden Books: the art of Kumi

Korf”” (2011), the National Museum

of Women in the Arts refers to Kumi

as “one of the most original artists in

the United States.” Works by Dick's

wife have been exhibited worldwide

and (of the 25 collections cited)

include the Library of Congress,

Harvard’s Houghton Library, the

New York Public Library, London's

Victoria and Albert Museum and Tate

Library, Hungary's Savaria Muzeum,

and the Cleveland Museum of Art.

He and Kumiare the proud parents of

Mia (an American film and television

actress), Ian (Associate Director of

Bioinformatics at UC Davis), Ian’s

twin Mario (Principal Technical

Writer at Akamai Technologies),

and Noni (Director of Learning

Innovation & Design at University

of Michigan in Ann Arbor). On his

retirement, Dick obviously basked

in the glow of his family’s creative

energies.

“During his stay at the Yokohama

National University (1957-58) as

a Fulbright fellow, Dick met and

Courtesy of Pavel Lizon

Mia Korf; reprinted from Wilensky 2013

Fic. 29 (above). Dick & Kumi at home in Ithaca

with Pavel in 2005. Fic. 30 (below). Dick relaxing

during the summer of 2013.

R.P. Korf (1925-2016): A celebration ... 989

married Kumiko Tachibana. Kumi graduated in architecture and later print-

making at Cornell. She is very active as a visual artist to this day.’ (Lizon 2016)

Courtesy of Pavel Lizon

Fic. 31—1993-1998 MycoTaxon

E-i-C & close friend Lizon chats

with Dick in Ithaca in 2005.

“Exe Island on Big Rideau Lake in Canada was purchased by the Korfs in

1972 (later transferred to Mycotaxon Ltd as Exe Island Biological Station). The

simple cottage and 3-acre island became a favourite holiday destination for

their four children and friends.

From my own experience I must say that it is a great place for swimming

and diving, sailing and waterskiing, fishing, barbecuing, or just having a good

time. Dick organized famous Crazy Eights card tournaments not only on the

island but also during several mycological meetings and everyone who has

participated remembers having a lot of fun.” (Lizon 2016)

: In mid-2007, an audio gem from

SUBS UCU O SEMI NPIBY | Dick arrived in the editorial mailbox:

his 12-disc audio recording of the

epic poem, JOHN BRown’s Bopy.

Next fall’s solo 1500-mile road trip

transformed a welcome gift into

one of my most valued possessions.

Dick’s voice so mesmerized me

during my Montana journey to visit

JOHN BROWN’S BODY my father that I listened to it all over

by Stephen Vincent Benét again on my return to Oregon. This

An unabridged 1929 tour-de-force grips from the first

Pulitzer Prize-winning book-length poem

Narrated by Dick Korf disc and never bores. The text below

Fic. 32—Dick’s tour de force at age 80. notes and a blog by John Brown's

great-great-great-granddaughter.

Cover design by Noni Korf

990 ... Norvell (editor)

“This recording was made in 2006, a last gasp of an 80-year-old lifelong actor,

the culmination of a 20-year dream. It is dedicated to my actress daughter,

Mia Korf, for encouragement, and to my wife, Kumi, for everything.

“I grew up as a ravenous reader, encountering Stephen Vincent Benét’s John

Browns Body at the age of 14, I was captivated by the book, which I read and

reread over the ensuing sixty-some years. It surely helped form me into an anti-

war activist...

“Poetry has a very special place in my heart, and as a youth I began reading

and writing poetry. I agree with Stephen Vincent Benét: poetry begs to be read

aloud. The skilled poet may embed in his poems frequent ‘stage directions’

in the choice of typographic tools (punctuation, the use of parentheses, italic

typeface, paragraphs, long dashes, indentations), and of course changes in

meter or rhyme. Benét’s use of these tools simplified my narration of the poem;

these are treated here as not only readers’ but narrator's guidelines.” (Korf 2006)

“THURSDAY TREASURE - RECORDING OF JOHN BRown’s Bopy” (MeCoy 2009):

“Over the past 30 years, I have been the recipient of some fascinating letters,

emails, FaceBook notifications, and phone calls from people regarding John

Brown. One such FaceBook connection occurred in June of this year and

resulted in today’s featured treasure. Emeritus Professor of Mycology at Cornell

University, Richard P. Korf wrote me the following:

“T assume you are the lady I read about in an article sent to me by my cousin

from her local paper, indicating that you are a great-great-great-granddaughter

of John Brown of Harpers Ferry fame. I have a strong connection, in that I

was very influenced by the story, and particularly the book-length poem called

John Brown's Body by Stephen Vincent Benét, which received the Pulitzer Prize

in 1929. Probably you have read it. Oddly enough it never was made into an

audio book. I finally discovered that there is a copyright issue, and managed to

get permission to do a “not for sale” version (I am now 84, and a lifetime actor)

which I recorded in 2006. I have a few copies left and would be happy to send

you one (it ison 12 CD discs, 13-1/2 hours long!). You can send me your postal

address if so.

“Think about this, Professor Korf was so enthralled with John Browns Body

by Stephen Vincent Benét, that he spent considerable time, effort, and his own

money to manufacture 100 limited editions of a 12 CD set that he cannot sell

due to copyright regulations.

“I immediately responded that I would be honored to receive one of his

recordings. A few days later I received a package in the mail with a professionally

packaged, produced set of CDs. Professor Korf’s voice is a joy to listen to,

and the set is amazing. The front cover of the box features the Stephen Benét

R.P. Korf (1925-2016): A celebration ... 991

postage stamp, while the back is the 1859 photograph of John Brown with his

full beard. Inside each CD is labeled and tucked into its own pocket. ...A truly

impressive presentation. It is a shame that due to copyright legalities, Professor

Korf is not permitted to offer his incredibly moving rendition of this important

work of literature, but I am truly blessed to have received a copy from him. Iam

holding onto this treasure. When I walk past my bookshelves and see the case, I

always think fondly of the Professor whom I have never met, yet he felt inclined

to share his lifelong dream with me?” (MeCoy 2009)

Kumi'’s mystery fungus, found

at home in August 2015. »

Fig. 33 (LEFT): Tiny red

cups delight Teresa & Dick;

Fic. 34 (RIGHT): The sketch

by Kumi with Don Pfister’s

annotations added; Fic. 35

(BOTTOM): Dick supervises

Teresa's note-taking skills.

KuUMI'S MYSTERY FUNGUS—Here are some

photos (Fics 33-37) of my last visit to Dick in |

August 2015 when he described and made me

take very detailed notes on ‘Kumi’ fungus...

while he described in detail the most impressive

manner how this fungus shattered. When I

returned to the Farlow with the specimen, Don

[Pfister] immediately looked at it under the | :

microscope, made drawings of the spores (also

attached), and identified it to Peziza phyllogena,

which Korf himself had worked on.

On that visit he impressed me once more

with his absolutely keen memory and sense of

humor when he told us the story of Schulzer

von Muggenbruck, who described twice as

new (and from the same specimen) Peziza

heterotropha Schulzer 1878 and later as a new

genus and species, Strossmayeria rackii Schulzer

1881 [now listed as S. basitricha (Sacc.) Dennis

on www.indexfungorum.org].

992 ... Norvell (editor)

The holotype of the name of S. rackii, along with most of the Schulzer

Herbarium, was lost, so Dick decided to undertake an expedition to find a

neotype for this fungus and two other type specimens needed to solve some

other taxonomic issues with other groups, which we described in detail (Korf

& al. 1988).

During such expeditions to collect discomycetes, he taught us how to find

these organisms: swimming among leaf litter, staring at little branches, twigs,

leaf blades, leaf petioles, in a place where at first nothing could be seen, and as

he stated “they'll start winking at you”... and so they did!

Each of those collections was carefully removed from its substrate with a

knife—at lunch time he would love to let us know he was opening the only

sardine can with the “dung knife”—and placed in a glassine envelope. Rigorous

notes were taken of textures, shapes, colors and sizes, and then the magic

of examining the microscopic structures started. Looking at the wonders of

ascospores, septa, ornamentation, crystals, colors, and the Melzer’s reaction

took us always late into the night. Sleep was of no concern to Dick when there

was so much fun looking at these amazing structures!

And by the way, we did make 50 collections of Strossmayeria rackii and thus

were able to neotypify this genus. —TERESA ITURRIAGA

Data Curator, Microfungi Digitization Project, University of Illinois

Fic. 36—Teresita and Dick,

August 2015

In a subsequent message, Teresita referred to a Vladimir Nabokov poem that

so enchanted Dick that he pasted it onto the arm of his microscope. It is only

fitting that her fond reminiscence of her friend and former professor conclude

with the stanzas from the Russian author & lepidopterist’s “On Discovering a

Butterfly” that convey the passion of all those longing to reveal Mother Nature's

hidden framework.

R.P. Korf (1925-2016): A celebration ... 993

Smoothly a screw is turned; out of the mist

two ambered hooks symmetrically slope,

or scales like battledores of amethyst

cross the charmed circle of the microscope.

I found it and I named it, being versed

in taxonomic Latin; thus became

godfather to an insect and its first

describer—and I want no other fame.

—Vladimir Nabokov (1943)

Fic. 37—Dick in the heart of his wife, children, and grandchildren

during the annual gathering of the clan (Summer of 2016).

A daughter's farewell

My dad’s passing means that there are many untold stories that can no

longer be told. Many photographs that can no longer be identified. He was

preceded in death by his older brother and his cousin and, at 91, by many his

friends, colleagues, and students. I would have liked to hear more stories about

his childhood, because looking back there are so many narrative threads that

could be followed to their source.

I do know that the publishing of the journal, MycoTaxon, was not his first

adventure in publishing. As a child he had a small letterpress at which he set

type, printing up a local gossip rag, some stories surely fabricated, which he

then sold to his neighbors. It was a novel and entrepreneurial undertaking,

quite unlike MycoTaxon but the connection is there.

994 ... Norvell (editor)

I remember the genesis of the journal, and the way that my father’s eye

would light up as he would describe the concept—a journal which allowed

authors to publish quickly and for free, that used offset printing technology

to shortcut more time intensive and expensive methods of printing, where the

journal wasn‘ responsible for the review process, and where volumes would be

issued when they reached a certain number of pages rather than a certain time

period. I don't know how novel the journal’s “find your own reviewer” peer-

review process was, but he told me he heard many stories of authors who were

able to get peer-reviews from people that they highly respected and to whom

they would not otherwise have dared to communicate.

Then there was all the stuff. We sold special paper that he designed that

had two sets of lines margins laid out in light blue ink so that authors could

type onto this paper, using either an elite or pica typewriter, and then mail in

camera-ready originals. We also sold, and used ourselves, special Letraset dry

transfers for numbering photos and creating somewhat consistent arrows, page

headers, and other typography. We would receive back from the printer rolls

and rolls of acetate which were cut up for reprints, sometimes mailed back to

authors if they requested it for them to make their own. And there were so

many different sizes of envelopes and boxes for shipping the journal out.

When an issue was published, there would be a stack of some 400 or so

brightly colored journals ready to be sent out. We tried many different ways

of printing up labels for these and back then we had to sort them all by zip code

bad taxonomy

©}

can KILL

Fic. 38—Noni designed this logo years ago for one piece of ‘stuff’—the official MycoTaxon t-shirt

worn proudly by journal disciples. With our move to online publication in 2011, Noni modified the

logo for use on our back covers, beginning with Mycoraxon 115. Dick approved!

Kumi Korf; courtesy of Teresa Iturriaga

R-P. Korf (1925-2016): A celebration ... 995

Fic. 39—Noni, Dick, and Teresita share a happy moment in August 2015

and country before dropping them at the post office. We made stuffing the

envelopes into a game, quizzing each other on the zip codes of certain cities, or

the middle names of our subscribers. I knew many, many names and addresses

of mycologists by heart, learned of libraries from all over the world, imagined

the journal traveling there to be opened by someone who must have wondered

why the journals they received were never the same color. It was a game, it was

a chore, it was something we all chipped in on 4 times a year. One time I slipped

a message into the envelope—it was being shipped to Linda Kohn—and I wrote

“help, I'm trapped in a cottage industry!”

Every now and then someone would order a backorder or two, or sometimes

even a whole run, and this was a cause for excitement, and more boxing, and

M-bags, and international postage. I sometimes thought that the post office was

my father’s home away from home. He was meticulous in understanding the

rules and could read the charts and scales as well as any postmaster. He always

tried to stamp the envelopes with the most exotic and fanciful of US postage,

in case the recipient was a stamp collector. He had many difficulties with the

post office, however, because the journal didn't fit into a certain category of a

quarterly publication, so we didn't qualify for 3rd class mail, and ultimately the

crushing cost of postage was one of the reasons we found we had to move to

publishing online.

As I got older I learned to work on the subscriptions management. At

the time this meant gathering checks, stamping them “for deposit only” and

entering the payments on 3x5 inch index cards. Some people were individual

996 ... Norvell (editor)

subscribers, some were institutional, some we invoiced, some had standing

orders, some were organized through companies such as Ebsco. It was all very

detailed and precise, and hand written. Once I started doing the subscriptions

management I became even better at the box stuffing game of “in what city

does mycologist X live?” As this modernized, the job included running credit

card slips through the embosser and mailing back receipts. Eventually there

were uploads via a dial-up modem. And there were also the odd international

money orders, which my father would have to cash at his home away from

home, the post office. So many memories of these little details and all the things

it took to keep the journal running.

At some point my father embraced the computer, bought a Mac Ie, and

began getting fascinated with the computer as both a administrative tool and

one useful for authors. I think we were all very tired of typing on that special

light-blue lined paper, and needing to use whiteout to make corrections.

The early volumes do have the DIY look of a zine, but the computer made

submissions of articles and reviewer notes much easier for the author, but also

added to the work of the editor. There’s no doubt my father loved working at

the computer, teaching himself new software, designing forms and databases

to track subscriptions, and authoring articles. I can see him in my mind's

eye, hunched over the keyboard, using only two fingers to hunt and peck at a

maniacal speed as he corrected a manuscript, or communicated with a former

student.

The printed word was very important to my father. He was a keen critic

of his own writing as well as that of others, helped many people to edit their

manuscript and was always on the lookout for readable prose and the proper use

of the English language. He would admonish writers to “kill your darlings” and

when we were developing the Mycotaxon website, pointed out that I had failed

to italicize a period. Before my father died we had a project to pull together all

of his writings and bind them together. I regret I wasn't able to complete the

project, the reprints were of so many different sizes I couldn't figure out how

to put them together. But together we marveled at the stack of pages. Over 400

different publications! I so wish I could have also added to the stack his first

published articles, the 1935 gossip rag from White Plains, NY.

I miss my dad, and know he would have edited the hell out of this piece.

NONI KORE

11 January 2017

R.P. Korf (1925-2016): A celebration ...

Ode to RPK

Awake, my Muse, and let us chant again,

for Richard has attained four score and ten,

A life well lived, and never could my song

number the landmarks of a life so long,

so rich, so varied, put to so good use,

for like that busy man, Odysseus,

patient, resourceful, skilled in many arts,

our Richard is a man of many parts:

In lusty youth he journeyed to Japan,

and bore away the fair Kumikosan,

whose beauty lives undimmed, though made to bear

sturdy twin sons, and daughters passing fair.

Master of mushroom lore, taxonomist,

shepherd of mushroom hunters, lengthy list,

whose fungal gleanings will survive the ages

in MycoTaxon’s printer-ready pages.

A scientist, a teacher wise and stern,

his life will sometimes take a different turn;

he is no stranger to the sacred rage,

that brings forth sound and fury on the stage;

sailor intrepid, always northward bound,

until one happy day he ran aground,

and entered on the heros life we know,

lord of the wooded isle in Big Rideau.

There let us leave him, Muse, for you grow faint;

a full length portrait genius alone could paint.

Let’s be content to let our rhyming cease,

a gift of love, though not a masterpiece,

a song with but a simple tale to tell:

Richard, we honor you, and love you well.

—Peter Wetherbee

in honor of Dick’s 90" birthday, 2015

997

998 ... Norvell (editor)

Mycotaxon, the fungal taxonomy journal that

Dick co-founded, will publish a tribute issue. + oti x

a oom pede chien celebrating the life of

you would like to contribute a piece. Once Richard P Korf

published, the issue will be available here:

hetp://tinyurl.com/mycotaxon

1925 - 2016

with special thanks for today’s celebration:

Department of Plant Pathology

Department of Music

Cornell Parking

Dana Paul

and all of you that came today.

cover photograph by Jaroslav Klan

9/20/1978

Bugac Puszta, Hungary

8th European Mycological Congress

Donations may be made to the

Richard P. Korf Graduate Student Excellence Fund

http://tinyurl.com/CornellKorfFund

drawing with morel by Bill Benson, 1989

for a Soirée held at LAuberge de Cochon Rouge

Fic. 4o—On December 18, 2016, the Korf family hosted a celebration of Dick's Life at Cornell. The

back of the program above provided the liner notes from Dick's audio book version of John Brown's

Body and his obituary, featuring the 1972 photo of Dick at the Plant Path lab blackboard. Scheduled

speakers included daughters Noni & Mia Korf, students Don Pfister & Kathie Hodge, sons Mario

& Ian Korf, and grand-daughter Maia Korf followed by memories shared at an open mic during

the reception on stage. Not surprisingly, festiities continued well into the evening at Noni’s place.

Acknowledgments

Many have contributed to this celebration, but special editorial thanks are due

Noni Korf, who has borne much during the past year yet agreed to serve as family

liaison and scribe in addition to MycoTAxon web-master while holding down a fulltime

new job. We both thank everyone who shared memories and photos in this memoriam:

Francisco Calaca, Rafael Castafieda, David Hawksworth, Kevin Hyde, Satoshi Inoue,

Teresita Iturriaga, Michael Loizides Hannes Maddens, Scott LaGreca, Pavel Lizon,

Tom May, Rosario Medel, David Miner, Ron Petersen, Scott Redhead, Jack Rogers,

Amy Rossman, Tony Whalley, and Wen-ying Zhuang. We also thank Pedro Crous,

David Hawksworth, Manon van den Hoeven-Verweij, Pavel Lizon, Amy Rossman, and

Wen-ying Zhuang for granting permission to reprint excerpts from IMA Funcus.

The Editor-in-Chief, who assumes sole responsibility for shamelessly purloining

reminiscences and photographs from the Internet, is especially grateful to Kathie Hodge

and Scott LaGreca for providing such entertaining and informative blogs about Cornell.

The photograph of Elias Fries downloaded from the MusHRooM, THE JOURNAL website

(www.mushroomthejournal.com) is elsewhere listed as in being the public domain.

Other attributions are placed next to the photos, presented in the text above, or covered

in the references cited.

Kathie Hodge

R.P. Korf (1925-2016): A celebration ... 999

ite " ray!

Fic. 41. Watkins Glen State Park, NY

Excuse me, while I disappear.

References cited

Dobbin B. 2009 (posted 14 April). An Ivy League school is giving China back its treasured

mushrooms.” Chicagoer News [Accessed 12-29-16]:

http://www.chicoer.com/article/ZZ/20090414/NEWS/904149781

Hodge K. 2013. The Cornell Hoot. Cornell Mushroom Blog: blog.mycology.cornell.edu [accessed

January 16, 2017]

IMA Fungus. 2010. Ainsworth Medal: Richard P Korf. IMA Fungus 1(2): 15-16.

IMA Fungus. 2013. Personalia— Richard P. Korf - Mi-shou. IMA Fungus 4(1): 15.

Iturriaga T. 2016 (posted August 22). Richard Korf and his love for the discomycetes. MiCC

(Microfungi Collections Consortium). www.facebook.com/microfungi.org/ [Accessed online

December 27, 2016]

Korf RP. 1991. An historical perspective: mycology in the Departments of Botany and of Plant

Pathology at Cornell University and the Geneva Agricultural Experiment Station. Mycotaxon

40: 107-128.

Korf RP. 2006. John Brown's Body by Stephen Vincent Benét, narrated by Dick Korf. Mycotaxon

Ltd, Ithaca NY. Available for download at audio.ithaca-ny.com/jbb

Korf RP. 2007. A tribute to Grégoire Laurent Hennebert and Mycotaxon’s 100" volume. Mycotaxon

100: 1-4.

Korf RP, Gruff SC. 1985. Mycotaxon cumulative index for volumes i-xx (1974-1984). Mycotaxon,

Ltd., Ithaca NY. ISBN 0-930845-00-5.

Korf RP, Iturriaga T, Zhuang WY. 1988. Lost and found: a discomycete pilgrimage. Mycotaxon 31:

85-88.

1000 ... Norvell (editor)

Kumi. [accessed January 11, 2017]. Artist’s statement. www.kumikorf.com

Lizon P. 2005. Birthday Greetings: Richard (“Dick”) P. Korf’s 90" birthday. IMA Fungus 6(1): 20.

Mecoy AK. 2009 (November 13). Thursday Treasure - Recording of John Brown’ Body. John

Brown Kin. [Accessed December 12, 2016-January 18, 2017]

http://johnbrownkin.blogspot.com/2009/11/thursday-treasure-recording-of-john.html

Norvell LL. 2011. Fungal Nomenclature. 1. Melbourne approves a new Code. Mycotaxon 116:

481-490. https://doi,org/10.5248/116.481

Obituary. 2016. Korf, Richard Paul. Dec. 9, 2016. Ithaca Journal.

Rossman AY, Zhuang WY. 2016. In Memoriam: Richard Paul Korf (1925-2016): leading specialist

on discomycetes and inspiring mentor. IMA Fungus 7(2): 62-64.

Teng SC. 1996. Fungi of China, Richard P. Korf, ed. Mycotaxon, Ltd., Ithaca NY. xiv + 586 pp. ISBN

0-930845-05-6.

Wilensky J. 2013 (Spring). Once upon a hill: Cornell's story as told by a 1946 student music hall

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