IV ... MycoTAxon 130 (4)

MY COTAXON

VOLUME ONE HUNDRED THIRTY (4) — TABLE OF CONTENTS

COVER SECTION

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RESEARCH ARTICLES

The chestnut pathogen Gnomoniopsis smithogilvyi (Gnomoniaceae,

Diaporthales) and its synonyms

Lucas A. SHUTTLEWORTH, DONALD M. WALKER & DavVID I. GUEST

Distribution of Alternaria species among sections. 2. Section Alternaria

PHILIPP B. GANNIBAL

The lichen genus Kroswia in China = Hua-Jig Liu, JIAN-SEN Hu & Cuao Li

New reports of macromycetes from Mexico Oxtvia RoprRiGuEz,

Maria DE JESUS HERRERA FONSECA & ADRIAN GALVAN CORONA

Two new records of microfungi from the Brazilian Atlantic Forest

MARCELA A. BARBOSA, MARINA A.G. ARAUJO, MAYRA S. OLIVEIRA,

PHELIPE M.O. Costa, ELAINE MALOSSO & RAFAEL FE. CASTANEDA-RUIZ

Pyramidospora quadricellularis sp. nov. on submerged leaves

from Brazil Mayra S. OLIVEIRA, Marina A.G. ARAUJO,

MaArRcELA A. BARBOSA, JESSICA C, SILVA,

ELAINE MALOSSO & RAFAEL F, CASTANEDA-RUIZ

Tretolylea, a new genus from the Brazilian semiarid region

TAIMY CANTILLO-PEREZ, Luis FERNANDO PASCHOLATI GUSMAO &

RAFAEL FE, CASTANEDA-RUIZ

Acaulospora reducta sp. nov. and A. excavata—two glomeromycotan

fungi with pitted spores CAMILLA MLR. PEREIRA,

BRUNO TOMIO GOTO, DANIELLE KARLA ALVES DA SILVA,

ARAESKA CARENNA DE ALMEIDA FERREIRA, FRANCISCO ADRIANO DE SOUZA,

GLADSTONE ALVES DA SILVA, LEONOR C. MAIA & FRITZ OEHL

Lecanicillium uredinophilum sp. nov. associated with rust fungi

from Korea MI-JEONG PaRK, SEUNG-BEOM HONG & HYEON-DONG SHIN

DNA barcoding is an effective tool for differentiating

Pisolithus species from Macedonia KATERINA RUSEVSKA,

MITKO KARADELEV, CHERDCHAI PHOSRI, MARGARITA DUENAS,

M. TERESA TELLERIA, Roy WATLING & Maria P. MARTIN

929

941

951

961

967

O77

953

900

1007

OCTOBER-DECEMBER 2015 ... V

Five new species of Hymenoscyphus (Helotiaceae, Ascomycota)

with notes on the phylogeny of the genus

Huan-D1 ZHENG & WEN-YING ZHUANG 1017

Phyllachora jianfengensis, a new species from China

Na Liu & MENGZHEN Li 1039

A new species and a new combination in Codinaea from Brazil

Mayra S. OLIVEIRA, ELAINE MALOSSO & RAFAEL F CasTaNeEpDA-Ruiz 1045

Conidial fungi on Araucaria angustifolia: Trichoconis foliicola sp. nov.

and two new records from Brazil SILVANA SANTOS Da SILVA,

Luis FERNANDO PascHOLATI GUSMAO & RAFAEL FE. CASTANEDA-Ruiz 1059

Entyloma scandicis, a new smut fungus on Scandix verna

from Mediterranean forests of Israel KyRyLo G. SAVCHENKO,

Lori M. Carris, LisA A. CASTLEBURY, VASYL P. HELUTA,

SOLOMON P. WASSER & EVIATAR NEvo 1061

Myxomycetes of Chihuahua (México) 4.

Central Plains of the Chihuahuan Desert Marcos LizARRAGA,

GABRIEL MORENO, MARTIN ESQUEDA, CYNTHIA SALAZAR-MARQUEZ

& MARTHA L. Coronapo 1073

Three Trichoderma species associated with wood discoloration

in South Korea

SEOKYOON JANG, YEONGSEON JANG, GyU-HYEOK KIM & JAE-JIN Kim 1103

Terriera fici sp. nov. on Ficus vasculosa from Hainan Province,

China YUAN Wu, SHI-JUAN WANG,

YAN-QIONG MENG, YAN-PING TANG & YING-REN Lin 1111

Six Russula records from Turkey

Hasan HisseyIn DoGan & OyKiim Ozturk 1117

Dictyosporium amoenum sp. nov. from Chapada Diamantina,

Brazil CAROLINA RIBEIRO SILVA, Luis FERNANDO PASCHOLATI GUSMAO

& RAFAEL FE CasTANEDA-Ruiz 1125

Synchaetomella aquatica sp. nov. from submerged leaves

from Brazil PaTRiCIA OLIVEIRA FIUZA,

Luis FERNANDO PASCHOLATI GUSMAO & RAFAEL F. CasTANEDA-Ruiz 1135

Tuber petrophilum, a new truffle species from Serbia

MIROLJUB MILENKOVIC, TINE GREBENC,

MrrosLtav MARKOVIC & Boris IvANCEvIC 1141

Pouzarella alissae, a new species from northwestern California,

United States Davip L. LARGENT & SARAH E. BERGEMANN 1153

The discovery of Syncephalis obliqua (Zoopagomycotina, Zoopagales)

in the Neotropics ROGER FAGNER RIBEIRO MELO,

LEONOR Costa MAIA & ANDRE LuIZ CABRAL MONTEIRO DE AZEVEDO SANTIAGO 1165

vi ... MyCOTAXON 130 (4)

Two new records of Agaricus spp. from Ethiopia

R. S1ToTaw, Y. Li, T.-Z. WEI, D. ABATE & Y.-J. Yao 1171

Phoma candelariellae sp. nov., a lichenicolous fungus from Turkey

ZEKIYE KOCAKAYA, MEHMET GOKHAN HALICI & MustaFa Kocakaya 1185

Neoalbatrellus subcaeruleoporus sp. nov. (Scutigeraceae) from

western North America SERGE AUDET & BRIAN S. LUTHER 1191

New Acrocordia and Candelariella records for Turkey

ZEKIYE KOCAKAYA & MEHMET GOKHAN Hatici 1203

BOOK REVIEWS AND NOTICES Ese C. VELLINGA 1209

REGIONAL MYCOBIOTAS NEW TO THE MYCOTAXON WEBSITE 1213

Gasteroid mycobiota (Agaricales, Geastrales and Phallales)

from Espinal forests in Argentina

Maria L. HERNANDEZ CAFFOT, XIMENA A. BROIERO, MARIA E. FERNANDEZ,

LEDA SILVERA Ruiz, ESTEBAN M. CRESPO & EDUARDO R. NOUHRA

A checklist of Hymenochaetaceae from Northeast Brazil

CARLA R. S. DE LIRA, GEORGEA S. NOGUEIRA-MELO,

LEIF RYVARDEN & TATIANA B. GIBERTONI

Corticioid fungi of the western Canary Islands. Chorological additions

ESPERANZA BELTRAN-TEJERA, J. LAURA RODRIGUEZ-ARMAS, M. TERESA TELLERIA,

MARGARITA DUENAS, IRENEIA MELO, ISABEL SALCEDO & J. CARDOSO

The lichenicolous fungi of Burdur province (Turkey)

KENAN YAZICI & JAVIER ETAYO

NOMENCLATURAL NOVELTIES AND TYPIFICATIONS

PROPOSED IN 130(4) 1215

OCTOBER-DECEMBER 2015 ... VII

REVIEWERS — VOLUME ONE HUNDRED THIRTY (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 quarter.

Joe Ammirati

André Aptroot

Nahara Ayala Sanchez

Juliano M. Baltazar

Timothy J. Baroni

Dominik Begerow

Janusz Blaszkowski

Wolfgang von Brackel

Jie Chen

Vagner G. Cortez

Bao-Kai Cui

Jorge Raul Deschamps

Frank M. Dugan

Eduardo Furrazola

J. Ginns

Nils Hallenberg

Jae-Gu Han

Adriana Hladki

Cheng-Lin Hou

Mikael Jeppson

Ze-Feng Jia

Mitko Karadelev

Bryce Kendrick

Kerry Knudsen

Francisco Kuhar

Ivana Kusan

Daniel P. Lawrence

James D. Lawrey

De-Wei Li

Jing Luo

Seonju Marincowitz

Neven Matocéec

Eric H.C. McKenzie

David Minter

Josiane S. Monteiro

Gregory M. Mueller

Karen K Nakasone

Lorelei L. Norvell

Toru Okuda

Shaun R. Pennycook

V. Ramirez-Cruz

Jolanda Roux

Amy Y. Rossman

Leif Ryvarden

Roger Graham Shivas

José Ivanildo de Souza

Gi-Ho Sung

Andrei Tsurykau

Gennadii Urbanavichus

Yusuf Uzun

Else C. Vellinga

Yei-Zeng Wang

Martin Westberg

Anthony J.S. Whalley

Merlin White

Lidia Yakovchenko

Ming Ye

Xiu-Guo Zhang

vul ... MyCOTAXON 130 (4)

ERRATA FROM PREVIOUS VOLUMES

VOLUME 125

p.305, 9 line from bottom [Mattirolia ohiensis] FOR: 154 READ: 155

p.305, 8" line from bottom [Mattirolia platensis] FOR: p. 154 READ: p. 155

p.306, 4" line from bottom [Setosynnema yunnanense] FOR: p. 22 READ: p. 82

VOLUME 126

p.249, lines 5 & 6 [Ambomucor] FOR: seratioinflatus

READ: seratoinflatus

VOLUME 127

p-233, lines 17 & 18 [Helicodochium] FOR: p.5 READ: p. 6

p.233, 5" line from bottom FOR: p. 154 READ: p. 146

VOLUME 130(2)

Validation of the name Matsushimiella paraensis

JOSIANE SANTANA MONTEIRO, Luis FERNANDO PASCHOLATI GUSMAO

& RAFAEL E CASTANEDA-RUIZ

The proposed name “Matsushimiella paraensis J.S. Monteiro, Gusmao & R.E

Castaneda, sp. nov.’ on page 312 of MycoTaxon 130(2) was not validly published

because the cited registration number, “MB 807635’, was incorrect and is assigned

to a separate name. Art. 42.1 of the International Code of Nomenclature (McNeill

& al. 2012) requires that after January 1, 2013, ‘the citation in the protologue of the

identifier issued by a recognized repository for the name (Art. 42.3) is an additional

requirement for valid publication’ We hereby validate the name Matsushimiella

paraensis J.S. Monteiro, Gusmao & R.E Castafieda, sp. nov. with the assigned

number MB 810689, with its description published in MycoTaxon 130: 312. 2015

and its holotype designated as HUEFS 210425. Additional details were published in

the original publication.

OcCTOBER-DECEMBER 2015 ... IX

FROM THE EDITOR-IN-CHIEF

FUNGAL IDENTIFICATION NUMBERS REDUX — The IBC permanent Nomenclature

Committee for Fungi is currently engaged in a spirited discussion as to whether

citing an incorrect fungal identification number after a newly proposed name

SHOULD render that new name invalid. There is no question as to whether the current

INTERNATIONAL CODE OF NOMENCLATURE FOR ALGAE, FUNGI, AND PLANTS in fact

DOES specify that a name published with the wrong number is an invalidly published

name. Article 42.1 requires “the citation in the protologue of the identifier issued,” and

leaves no apparent room to correct mistakes. Therefore, until the Melbourne Code

is amended to regard a typographical slip of the pen producing an incorrect number

as a ‘correctable’ error (as is now permitted for epithets misspelled by authors—most

frequently by those ignorant of the grammatical rules of Latin gender agreement),

FUNGALNAME, INDEXFUNGORUM, and MycoBank identification numbers must be

presented with the new names exactly as they have been issued by the repository.

The ‘new’ fungal identification numbers are important enough that we again ask

all our authors to make certain that the numbers match the names for which they

have been issued. MycoTaxon also now includes them in the list of nomenclatural

novelties and typifications that closes each issue. Correctly, we hope!

MyYCOTAXON 130(4) contains 29 papers (+ links to 4 new regional mycobiotas) by 118

authors representing 24 countries and revised by 57 expert reviewers.

Within its pages are one new genus (Tetrolylea from Brazil) and 21 species new

to science representing Acaulospora, Codinaea, Dictyosporium, Pyramidospora,

Synchaetomella, Tetrolylea, and Trichoconis from Brazil; Entyloma from Israel;

Hymenoscyphus, Kroswia, Phyllachora, and Terriera from China; Lecanicillium from

Korea; Neoalbatrellus from western Canada and the USA; Phoma from Turkey;

Pouzarella from the USA; and Tuber from Serbia.

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

also offer one new combination in Codinaea, Gnomoniopsis smithogilvyi as the correct

name for the chestnut pathogen, a complete list of the species currently accepted

in Alternaria sect. Alternaria, a monographic treatment of Kroswia in Korea, a

multigene phylogeny of Hymenoscyphus, the DNA bar-code assisted identification

of three Pisolithus species in Macedonia, an overview of myxomycetes in Chihuahua

Desert, a pictorial key to Dictyosporium conidia, and a revised key to the American

and European species of Scutiger sensu lato.

With our very best wishes for a joyful and productive 2016,

Lorelei L. Norvell (Editor-in-Chief)

29 December 2015

x ... MyCcoTAXoN 130 (4)

PUBLICATION DATE FOR VOLUME ONE HUNDRED THIRTY (3)

MYCOTAXON for JULY-SEPTEMBER, (I-X + 601-928)

was issued on October 9, 2015

OcCTOBER-DECEMBER 2015 ... XI

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MY COTAXON

http://dx.doi.org/10.5248/130.929

Volume 130, pp. 929-940 October-December 2015

The chestnut pathogen Gnomoniopsis smithogilvyi

(Gnomoniaceae, Diaporthales) and its synonyms

Lucas A. SHUTTLEWORTH ', DONALD M. WALKER”? & DAvID I. GUEST ™

‘Faculty of Agriculture and Environment, University of Sydney,

1 Central Avenue, Australian Technology Park, Eveleigh NSW 2015, Australia

*Department of Natural Sciences, The University of Findlay,

1000 North Main St., Findlay, OH 45840, U.S.A.

* Department of Biology, Tennessee Tech University,

1100 N. Dixie Avenue, Cookeville TN 38505, U.S.A.

* CORRESPONDENCE TO: david.guest@sydney.edu.au

ABSTRACT — Two species, Gnomoniopsis smithogilvyi and G. castaneae, were described

independently in 2012 as causal agents of chestnut (Castanea) rot in Australasia and Europe.

A comparative morphological analysis and five-marker phylogenetic analysis of ITS, TEF1-a,

B-tubulin, MS204, and FG1093 confirm that both names refer to the same species, and an

investigation of their exact publication dates demonstrates that G. smithogilvyi has priority

over G. castaneae.

Key worps — canker, endophyte, Gnomonia, nut rot

Introduction

Species of Gnomoniopsis have a mainly northern hemisphere distribution

and are economically important pathogens of the rosaceous hosts blackberry,

raspberry, and strawberry (Bolay 1971, Monod 1983, Maas 1998, Sogonov et al.

2008, Walker et al. 2010).

Shuttleworth et al. (2012a) described a new species, Gnomoniopsis

smithogilvyi, as the causal agent of chestnut rot, as an endophyte of reproductive

and vegetative tissues, and as a saprobe of European chestnut (Castanea sativa

Mill.) and hybrids of Japanese chestnut and European chestnut (Castanea

crenata Siebold & Zucc. x C. sativa) in southeastern Australia (Shuttleworth

et al. 2012a,b). Shuttleworth et al. (2012a) also found G. smithogilvyi occurring

in New Zealand on C. crenata, C. sativa, and Castanea sp. and associated with

C. sativa in India and Italy.

930 ... Shuttleworth, Walker & Guest

Visentin et al. (2012) described a new species, Gnomoniopsis castaneae [as

“castanea” |, as the causal agent of nut rot, endophyte, and saprobe of C. sativa

in France, Italy, New Zealand, and Switzerland. They referred to the fungus

reported earlier as a Gnomonia sp. (informally named “G. pascoe”) as the main

causal agent of chestnut rot in Australia and New Zealand (Smith & Ogilvy

2008). Sequences from Swiss isolates have been deposited as G. castaneae

(Dennert et al. 2014), and an Italian isolate from necrosis of insect-induced

galls of Castanea has also been identified as G. castaneae (Vinale et al. 2014).

The aim of this paper is to clarify the taxonomic relationship of

G. smithogilvyi and G. castaneae and, if they are conspecific, to determine

which name has priority.

Materials & methods

Morphology

Observation of cultures of G. smithogilvyi CBS 130190 (ex-type) from Australia,

G. castaneae MUT 401 (ex-type) from Italy, and “Gnomonia pascoe” MUT 411 from

New Zealand were made from herbarium material. Herbarium specimens of the sexual

phase were not examined in this study.

Sequencing and phylogenetic analyses

Fragments of four protein coding markers TEF1-a, B-tubulin, MS204, FG1093

were amplified and sequenced for Gnomoniopsis smithogilvyi CBS 130190 (ex-type),

CBS 130188 and CBS 130189, G. castaneae MUT 401 (ex-type) and “Gnomonia pascoe”

MUT 411 using primer pairs TEF1-o: EF1-728F (Carbone & Kohn 1999)/EF1-1567

(Rehner 2001), B-tubulin: T1/T2 (O’Donnell & Cigelnik 1997), MS204: amplification

E1FI/E5R1, sequencing E3F1/E4Rla/E5Rla, FG1093: EIFla/E3Rla (all MS204 and

FG1093 primers; Walker et al. 2012).

Thermocycling conditions were identical to Walker et al. (2010, 2012). In addition,

ITS sequences and reference sequences were downloaded from GenBank (TABLE 1).

Alignments were completed with the MAFFT alignment option (Katoh et al. 2002) of

Geneious® R7 v7.1.2 (Biomatters Ltd., New Zealand) with default settings applied. Six

alignments were completed, one for each of the five individual marker datasets and one

for the combined five-marker dataset.

For the TEF1-a, B-tubulin, MS204, FG1093 and the combined five-marker dataset,

nineteen isolates were selected including reference species of Gnomoniopsis. The ITS

analyses included an addition four isolates from India, six from Italy, two from France,

six from New Zealand, and two from Switzerland. Sequences from studies in India and

Switzerland associated with cankers of C. sativa were also included (Dar & Rai 2013,

2015, Pasche et al. 2015).

Two outgroup isolates were selected based on Walker et al. (2012), specifically

Ophiognomonia setacea CBS 128354 and Plagiostoma sp. CBS 128351. Gnomoniopsis

guttulata was excluded from the five-marker dataset because sequences for four of

the five markers were not available. Although MS204 and FG1093 sequences were not

available for G. racemula, this taxon was included in the five-marker dataset.

931

Synonymy of Gnomoniopsis smithogilvyi ...

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Synonymy of Gnomoniopsis smithogilvyi ... 933

Two criteria were used for phylogenetic species recognition: genealogical

concordance phylogenetic species recognition (GCPSR; Taylor et al. 2000) and the

genealogical sorting index (Gs1; Cummings et al. 2008). A conditional comparison test to

determine GCPSR was completed to identify support for the G. smithogilvyi clade on the

individual marker datasets using maximum parsimony bootstrap support (MPBS) 270%

(Mason-Gamer & Kellogg 1996, Kellogg et al. 1996). The ast is a statistical measure

to determine if predefined groups on a phylogenetic tree have reached reciprocal

monophyly thus sharing exclusive ancestry. It is interpreted on a 0 to 1 continuum

where 0 = lack of genealogical divergence from other groups and 1 = monophyly

(Cummings et al. 2008). All five single marker and the combined marker alignments

were independently tested with Gs1 using 100 randomly selected trees calculated from

the GARLI maximum likelihood bootstrap (MLBS) tree distribution computed with

the GARLI (v2.1) web service hosted at molecularevolution.org (Bazinet et al. 2014,

Zwickl 2006). The Gs analyses were performed with a web service hosted at

molecularevolution.org (GSI v0.92; Cummings et al. 2008) using 10,000 permutations

to determine statistical significance (P-value <0.05). Independent csi values from the

MLBS tree distribution were pooled to calculate an ensemble statistic Gs1,, for each

marker and the combined marker alignment (TABLE 2).

TABLE 2. Phylogenetic species recognition of Gnomoniopsis smithogilvyi

MARKER Sia gsi p-value

(MPBS %) #

ITS 99 1 0.0001

TEF1-a 100 1 0.0001

B-tubulin 100 1 0.0005

MS204 100 1 0.0001

FG1093 100 1 0.0003

Concatenated dataset 100 1 0.0001

Maximum parsimony bootstrap support (MPBS) 270% indicates strong support for the G. smithogilvyi

clade. The gene sorting index (Gs1) is based on a 0 to 1 continuum, 0 = lack of genealogical divergence

from other groups and 1 = monophyly; p-values <0.05 indicate the Gs is statistically significant.

Maximum parsimony (MP), maximum likelihood (ML), and Bayesian analyses were

used to infer phylogenetic trees. MP and ML trees were inferred with MEGA6 (Tamura

et al. 2013). The Bayesian trees were inferred with MRBAYES (Huelsenbeck & Ronquist

2001). The MP analyses used the heuristic search option with 1000 random sequence

addition replicates, the tree-bisection reconnection (TBR) branch-swapping option

with MULTTREES on, and MAXTREES set at 1000. All characters had equal weight.

For the ML and Bayesian analyses, jModeltest 2.1 (Posada 2008) was used to determine

the optimal model that best fit the individual marker and the five-marker datasets.

The resulting model was GTR+1+G for all datasets.

934 ... Shuttleworth, Walker & Guest

Branch support in ML and MP analyses were determined with maximum likelihood

bootstrap (MLBS) and maximum parsimony bootstrap (MPBS) analyses in MEGA6.

The MLBS and MPBS were calculated using 1000 bootstrap replicates, with 10 random

addition replicates per bootstrap replicate in the MPBS analysis (Felsenstein 1985).

A threshold of 270% support was used as a cut-off for strongly supported tree nodes

for both MPBS and MLBS. The Bayesian analyses used the Markov Chain Monte Carlo

(MCMC) method. Identical nucleotide substitution models were used ML and Bayesian

analyses. Four heated chains were run with 5 million generations, a subsampling

frequency of 2000 generations and a burn-in of 500,000 generations (10%).

Results & discussion

Morphology

Measurements of asexual culture characters agreed with the published

descriptions of Shuttleworth et al. (2012a) and Visentin et al. (2012).

However, conidia of all isolates were hyaline and mostly multi-guttulate

(although occasionally uni- and bi-guttulate). The differences in the published

descriptions of the sexual states were the biseriate arrangement of ascospores

in G. smithogilvyi (Shuttleworth et al. 2012a) and the uniseriate or irregularly

multiseriate arrangement in G. castaneae (Visentin et al. 2012).

Morphologically, the sexual states of Gnomoniopsis spp. are characterised by

small black perithecia that are immersed in host tissue, solitary, lacking a stroma

or in groups aggregated in a minimal stroma, with a single central, marginal or

lateral neck (Sogonov et al. 2008). Asci are oval to fusiform with a visible apical

ring and contain eight spores. The ascospores are one-septate, oval to fusiform,

and without appendages (except in G. tormentillae; Barr 1978). The known

asexual states of Gnomoniopsis have subglobose pycnidia with one-celled

hyaline conidia that are oval, oblong, globose or femur-shaped. Gnomoniopsis

smithogilvyi fits morphologically into this description of Gnomoniopsis.

Gnomoniopsis smithogilvyi can be separated morphologically from its sister

species G. paraclavulata Sogonov and G. clavulata (Ellis) Sogonov by having

smaller ascospores (G. smithogilvyi mean length x width = 7 um x 2 Um

[(Shuttleworth et al. 2012a); G. paraclavulata 9.5 um x 3.5 um (Sogonov et al.

2008); G. clavulata 9 um x 4 um (Sogonov et al. 2008)].

Host range

Most Gnomoniopsis species show host specificity to genera in Fagaceae,

Onagraceae, or Rosaceae (Walker et al. 2010). Gnomoniopsis smithogilvyi,

G. paraclavulata, and G. clavulata can be separated by their known host ranges.

All three species are reported on fagaceous hosts, but Gnomoniopsis smithogilvyi

is reported only on Castanea spp., G. paraclavulata is reported only on

Quercus spp., and G. clavulata is reported on Fagus sylvatica and Quercus spp.

(Sogonov et al. 2008).

Synonymy of Gnomoniopsis smithogilvyi ... 935

G. castanea MUT 401 C. sativa Robilante ITALY

G. smithogilvyi CBS 130190 Castanea sp. Mullion Creek NSW AUSTRALIA

G. smithogilvyi CBS 130188 Castanea sp. Bright VIC AUSTRALIA

G. smithogilvyi CBS 130189 Castanea sp. Mullion Creek NSW AUSTRALIA

“Gnomonia pascoe” MUT 411 Castanea sp. NEW ZEALAND

G. paraclavulata CBS 123202

G. clavlata CBS 121255

G. occulta CBS 125678

G. chamaemori CBS 804.79

G. alderdunensis CBS 125680

G. racemula CBS 121469

G. tormentillae CBS 904.79

G. comari CBS 806.79

G. sanguisorbae CBS 858.79

G. fructicola CBS 208.34

G. macounii CBS 121468

G. idaeicola CBS 125676

Gnomoniopsis

smithogilvyi

100/100

78/79

100/99

Plagiostoma sp. CBS 128351 arc

0.05

Ophiognomonia setacea CBS 128354 substitutions/site

Fic. 1. ML phylogeny of combined ITS/TEF1-c/B-tubulin/MS204/FG1093 sequences of G.

smithogilvyi, G. castanea, and “G. pascoe”, with single isolates of Gnomoniopsis reference taxa and

outgroup (ML score = -InL score of 16532.47). Maximum likelihood bootstrap (MLBS), maximum

parsimony bootstrap (MPBS) values 270%, and Bayesian posterior probabilities (PP) 20.95 are

displayed at each branch in the order MLBS/MPBS/PP.

Phylogenetic analyses

The alignment lengths for the six datasets including gaps were ITS: 508 bp,

TEF1-c: 917 bp, B-tubulin: 593 bp, MS204: 774 bp, FG1093: 351 bp, and the

combined five-marker: 3137 bp. The topology of the ML five-marker phylogeny

grouped Gnomoniopsis isolates from Castanea in Australia, Italy and New

Zealand in the same strongly supported lineage (MLBS = 100%; Fic. 1). Of the

3137 total-character MP five-marker dataset, 763 were parsimony informative,

471 parsimony uninformative, and 1903 constant. The MP heuristic search

produced three most parsimonious trees of 2717 steps (CI = 0.687523,

RI = 0.701477, RCI = 0.482281 for all sites, and iCI = 0.601034, iRI = 0.701477,

iRCI = 0.421611 for parsimony informative sites). The topology of the MP five-

marker phylogeny was similar to the ML phylogeny, grouping Gnomoniopsis

isolates from Castanea in Australia, Italy, and New Zealand in the same strongly

supported lineage (MPBS = 100%). The most likely ML five-marker tree had

-InL score 16532.47. The Bayesian distribution of likelihood scores was

-InL 16403.508-16414.583, and the PP of the branch supporting the isolates

from Castanea was 1.00.

The MLITS analysis resulted in a most likely tree with a -InL score of 1866.45.

Twenty-five isolates from Castanea collected in Australia, France, India, Italy,

936 ... Shuttleworth, Walker & Guest

G. smithogilvyi Ge1 C. sativa Geneva SWITZERLAND

G. castanea MUT 810 C. sativa Biasca SWITZERLAND

G. smithogilvyi AU/DBT305 C. sativa INDIA

G. smithogilvyi AU/DBT306 C. sativa INDIA

G. smithogilvyi AU/DBT296 C. sativa INDIA

G. smithogilvyi INDC(BSF5)A C. sativa INDIA

G. castanea TNS C. sativa Trentino ITALY

G. castanea Fl C. sativa Tuscany ITALY

G. castanea LSV908 C. sativa Oise FRANCE Gnomoniopsis

“Gnomonia pascoe” MUT 411 Castanea sp. NEW ZEALAND smithogilvyi

G. castanea TO C. sativa Piedmont ITALY

G. smithogilvyi |CMP:14492 C. sativa NEW ZEALAND

G. smithogilvyi \CMP:14080 C. crenata NEW ZEALAND

Gnomoniopsis sp. CBS 121918 C. crenata NEW ZEALAND

Gnomoniopsis sp. CBS 121917 Castanea sp. NEW ZEALAND

Gnomoniopsis sp. CBS 121477 C. crenata x C. sativa NEW ZEALAND

G. smithogilvyi |CMP:14035 C. sativa NEW ZEALAND

G. smithogilwi CBS 130190 Castanea sp. Mullion Creek NSW AUSTRALIA

G. smithogilvi CBS 130189 Castanea sp. Mullion Creek NSW AUSTRALIA

G. smithogilwi CBS 130188 Castanea sp. Bright VIC AUSTRALIA

G. castanea MUT 401 C. sativa Robilante ITALY

G. castanea MUT 815 C. sativa Sisteron FRANCE

G. paraclavulata CBS 123202

G. clavulata CBS 121255

G. idaeicola CBS 125676

G. macounii CBS 121468

G. occulta CBS 125678

G. chamaemori CBS 804.79

G. guttulata MS 0312

G. alderdunensis CBS 125680

G. racemula CBS 121469

G. tormentillae CBS 904.79

G. comari CBS 806.79

G. fructicola CBS 208.34

G. sanguisorbae CBS 858.79

Plagiostoma sp. CBS 128351

0.05

Ophiognomonia setacea CBS 128354 substitutions/site

Fic. 2. ML phylogeny of ITS sequences of G. smithogilvyi, G. castanea, and “G. pascoe” with single

isolates of Gnomoniopsis reference taxa and outgroup (ML score = -InL score of 1866.45). Country

of collection, and species of Castanea that the isolate was collected from are next to the isolate

number. Maximum likelihood bootstrap (ML), maximum parsimony bootstrap (MP) values 270%,

and Bayesian posterior probabilities (PP) 20.95 are displayed at each branch in the order MLBS/

MPBS/PP.

New Zealand, and Switzerland clustered in the same lineage with 99% MLBS

support (Fic. 2). One isolate from France, LSV908, displayed minor inter-

specific variation with 1 bp different from the other isolates from Castanea.

The MP ITS dataset had 508 total characters: 68 parsimony informative,

56 parsimony uninformative, and 384 constant. The MP heuristic search

produced three most parsimonious trees with length of 228 steps (CI = 0.754386,

RI = 0.87037, RCI = 0.656595 for all sites and iCI = 0.652174, iRI = 0.870370,

Synonymy of Gnomoniopsis smithogilvyi ... 937

iRCI = 0.567633 for parsimony informative sites). The MP ITS phylogeny

grouped all Gnomoniopsis isolates from Castanea in the same strongly supported

clade (MPBS = 99%). The Bayesian distribution of likelihood scores for the ITS

dataset was -InL 1948.241-1998.552 and the PP of the branch supporting the

isolates from Castanea was strongly supported with a value of 1.00.

Phylogenetic species were characterized using genealogic concordance

phylogenetic species recognition (GCPSR) and the genealogical sorting

incidence (Gs1). The conditional comparison test (GCPSR) was satisfied for the

G. smithogilvyi clade with MPBS for ITS = 99%, TEF1-a = 100%, B-tubulin = 100%,

MS204 = 100% and FG1093 = 100%. Independent marker Gast, values for

G. smithogilvyi were all equal to 1 (p <0.0001-0.0005), suggesting that this

species is a distinct evolutionary lineage from other Gnomoniopsis species.

The phylogenies of the ITS and five-marker datasets indicate all the isolates

associated with Castanea represent one species, G. smithogilvyi.

Taxonomy

Gnomoniopsis smithogilvyi L.A. Shuttlew., E.C.Y. Liew & D.I. Guest,

Persoonia 28: 143. 4 June 2012.

= Gnomoniopsis castaneae Tamietti, J. Plant Pathology 94: 416. 21 July 2012, as “castanea.”

SPECIMENS EXAMINED:

MORPHOLOGICAL AND MOLECULAR EXAMINATION: AUSTRALIA, NEw SouTH WALES,

Mullion Creek, “Brittle Jacks’ chestnut orchard, saprobic on dead burrs of Castanea sp.,

Dec. 2009, collected & isolated by L.A. Shuttleworth (holotype CBS H-20623; isotype

RBG 5586; ex-type culture CBS 130190 = RBG 5585); Mullion Creek, endophytic in

living leaf of Castanea sp., Dec 2009, collected & isolated by L.A. Shuttleworth (culture

CBS 130189); VicrortA, Bright, diseased chestnut kernel of Castanea sp., April 2009,

collected & isolated by L.A. Shuttleworth (culture CBS 130188). ITALY, PrepMoNT,

Robilante, diseased fruit of Castanea sativa, 2007, isolated by G. Tamietti & S. Gentile

(culture MUT 401). NEW ZEALAND, diseased fruit of Castanea sativa, 2007, isolated

by H. Smith (culture MUT 411).

MOLECULAR EXAMINATION ONLY: FRANCE, PICARDY, Oise, stem of Castanea sp., 2013,

collected by S. Carole (culture LSV 908); PROVENCE-ALPES-COTE D'AZUR, Sisteron,

diseased Castanea sativa fruit, 2011, isolated by P. Gonthier & S. Gentile (culture MUT

815). INDIA, bark of Castanea sativa, collected & isolated by M.A. Dar & M. Rai

(culture AU/DBT30); bark of Castanea sativa, collected & isolated by M.A. Dar & M.

Rai (culture AU/DBT306); on bark of Castanea sativa, collected & isolated by M.A. Dar

& M. Rai (culture INDC(BSF5)A); bark of Castanea sativa, collected & isolated by M.A.

Dar & M. Rai (culture AU/DBT296). ITALY, TRENTINO-SOUTH TyROL, Val d’Adige,

Trento, fruit with brown rot, October 2011 (culture Gn_TN5); PIEDMONT, Val di Susa,

Turin, October 2011 (culture Gm_TO); Tuscany, Mugello, Florence, October 2011

(culture Gm_FI). SWITZERLAND, Ticino, Biasca, diseased fruit of Castanea sativa,

2011, isolated by M. Jermini (culture MUT 810); GENEVA, branch wood of Castanea

sativa, Aug 2013 (culture Gel, specimen voucher UASWS1319). NEW ZEALAND,

Bay OF PLENTY, Tauranga, nut of Castanea sativa, 1 February 2002, collected by K.

Eade (culture ICMP 14492); WarkaTo, nut of Castanea crenata, 1 April 2000, isolated

938 ... Shuttleworth, Walker & Guest

by K.D.R. Wadia (culture ICMP 14080); from Castanea crenata x C. sativa, isolated by

K.D.R. Wadia (culture CBS 121477); from Castanea sp., collected & isolated by H. Smith

(culture CBS 121917); isolated from Castanea crenata (culture CBS 121918).

ECOLOGY & DISTRIBUTION— Gnomoniopsis smithogilvyi occurs in Australia on

Castanea sativa and hybrids of Castanea crenata x C. sativa (Shuttleworth et al.

2012a,b). In New Zealand it occurs on C. crenata, C. sativa, C. crenata x C. sativa

hybrids, and Castanea sp.; and in France, India, Italy, and Switzerland on

C. sativa (Shuttleworth et al. 2012a, Visentin et al. 2012, Maresi et al. 2013). The

species is reported as a causal agent of chestnut rot/nut rot in Australia, France,

Italy, New Zealand, and Switzerland (Shuttleworth et al. 2012a,b; Visentin et al.

2012), and is reported as the cause of canker of C. sativa in India (Dar & Rai

2013, 2015) and Switzerland (Pasche et al. 2015). The fungus is documented as

an endophyte of reproductive and vegetative tissues of C. sativa in Australia,

Italy, and New Zealand, and of C. crenata x C. sativa hybrids in Australia and

New Zealand (Shuttleworth et al. 2012a,b). It also associated with necrosis of

leaves and galls induced by Dryocosmus kuriphilus (chestnut gall wasp) on

Castanea spp. in Italy (Magro et al. 2010; Vinale et al. 2014).

ComMMENTS— The name Gnomoniopsis smithogilvyi was first published on-

line on 4 June 2012 in Shuttleworth et al. (2012a). The name G. castaneae was

first published on-line on 21 July 2012 in Visentin et al. (2012) according to

L. Rubino, Editor of the Journal of Plant Pathology (pers. comm.). These

on-line publications were both in accordance with Article 29 of the ICN

(McNeill et al. 2012). Thus, G. smithogilvyi has priority over G. castaneae. The

earlier informal name, “Gnomonia pascoe” was never validly published.

Acknowledgments

Thank you to Dr. Amy Rossman (Department of Botany & Plant Pathology, Oregon

State University, U.S.A.) and Prof. Jolanda Roux (Forestry and Agricultural Biotechnology

Institute, University of Pretoria, South Africa) for reviewing the manuscript. Thank

you to Mycotheca Universitatis Taurinensis, University of Turin, Italy, and the Royal

Botanic Garden Sydney, Australia for providing cultures. We gratefully acknowledge the

University of Sydney, Faculty of Agriculture and Environment, Horticulture Innovation

Australia Ltd., Chestnuts Australia Inc., the Tree Pathology Co-operative Programme

(TPCP), Forestry and Agricultural Biotechnology Institute, University of Pretoria, and

the National Research Foundation (NRF) of South Africa for their financial support.

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MY COTAXON

http://dx.doi.org/10.5248/130.941

Volume 130, pp. 941-949 October-December 2015

Distribution of A/ternaria species among sections.

2. Section Alternaria

PHILIPP B. GANNIBAL

Laboratory of Mycology and Phytopathology, All-Russian Institute of Plant Protection,

Shosse Podbelskogo 3, Saint Petersburg, 196608, Russia

CORRESPONDENCE TO: phbgannibal@yandex.ru

ABSTRACT — Among several groups of small-spored Alternaria species, A. sect. Alternaria

(containing the generic type) has been found to be isolated, based on phylogenetic as well as

morphological data. Molecular phylogenetic data confirm inclusion of twenty-one species

in this section besides the type species. Examination of additional material, conforming to

the morphological description for the section but not as yet phylogenetically characterized,

allow confirmation of an additional 37 species. All 59 species are listed, and an emended

description of Alternaria sect. Alternaria is presented.

Key worps — Alternaria alternata, A. herbiphorbicola, A. gomphrenae, Pseudoalternaria

Introduction

The genus Alternaria Nees is a large taxonomic group that comprises

approximately 280 species (Simmons 2007). A series of large-scale works

has attempted to resolve the phylogeny of Alternaria and other alternarioid

hyphomycetes by employing 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). Several well-supported phylogenetic lineages

have been revealed within the alternarioid hyphomycetes, resulting in several

taxonomic novelties. The genus Alternaria was divided into eight taxonomic

sections by Lawrence et al. (2013). Presenting an alternative concept,

Woudenberg et al. (2013) collapsed all alternarioid hyphomycetes into one

genus, Alternaria, with 24 sections, 11 of which referred to Alternaria sensu

stricto.

All works support a monophyletic status of a particular Alternaria group,

one of several groups having members with catenate, small-spored conidia.

942 ... Gannibal

Both research teams (Lawrence et al. 2013; Woudenberg et al. 2013) agreed

that based on both molecular phylogenetic and morphological data, this group

(including the type species A. tenuis [= A. alternata]) should be treated as a

well-defined section. This section, which contains the type of the genus, is an

autonymous section that must repeat the generic name as its epithet, Alternaria

sect. Alternaria, and is not followed by any author citation (McNeill et al 2012:

Art. 22.1). Lawrence et al. (2013) were mistaken in presenting this section

as a sect. nov. with themselves as authors. Woudenberg et al. (2013), who

repeated the erroneous attribution to Lawrence et al., misspelled the epithet as

“Alternata’.

Lawrence et al. (2013) referred thirty-one strains to A. sect. Alternaria.

However, six strains belonged to A. gossypina, A. grisea, A. grossulariae, A. lini,

A. maritima, or A. nelumbii, species denoted by Simmons (2007) as taxa that

are not distinguishable morphologically from other small-spored Alternaria

species, that lack isolates from identified field material, that were considered as

INCERTAE SEDIS, or whose only isolate(s) sporulate inadequately. Another strain

(CBS 1010.26) is labelled with the invalid name “Alternaria iridis’, referring to

Macrosporium iridis Cooke & Ellis, which is a Stemphylium (Simmons 2007:

722). Strains of three other species used by Lawrence et al. (2013) — A. malvae,

A. rhadina, and A. tomato — were not authentic or representative. Strain CBS

595.93 was deposited as “A. rhadina” by the species author, E.G. Simmons, but

he subsequently reported that this strain was mislabelled and did not represent

an authentic A. rhadina (Simmons 2007: 512). Here I regard it as representing

an unnamed Alternaria species and exclude it from my list.

An isolate of A. tomato (CBS 114.35) clustered in a clade referred to as A. sect.

Alternaria (Lawrence et al. 2013). However, the credible species description

in Simmons (2007) suggests that this species resides better in A. sect. Porri.

According to Simmons (2007: 356), no correctly identified A. tomato isolates

are known. Hence, we suggested that A. tomato sensu Simmons belongs in

A. sect. Porri (Gannibal 2015).

Lawrence et al. (2013) placed A. resedae CBS 175.80 in A. sect. Alternaria.

The molecular phylogenetic analyses by Woudenberg et al. (2013) placed the

representative strain of A. resedae Neerg. CBS 115.44 in A. sect. Cheiranthus.

Earlier Simmons (2007) had suggested that this name is a synonym of

A. septorioides (Westernd.) E.G. Simmons in A. sect. Brassicicola. Obviously

CBS 175.80 and CBS 115.44 belong to different species and most likely they

do not both correspond to the description used by Simmons. Hence I do not

recommend using the name A. resedae until a species study has been completed.

Alternaria sect. Alternaria ... 943

Based on ITS1-2 rDNA sequence analysis Hoog & Horré (2002) included

33 strains of 7 species in group alternata. Apart from the well-studied species

A. lini, A. mali and Cylindrocarpon lichenicola were placed in the group. Strain

“C. lichenicola” (current name Fusarium lichenicola) was obviously incorrectly

identified. The status of A. lini has been discussed above. Strain IFO 8984

A. mali was not ex-type. The same strain and the type strain of A. mali have

been studied by Rotondo et al. (2012). The strains did not demonstrate a close

relationship, but both do appear to be members of A. tenuissima/A. alternata

group, which is placed in A. sect. Alternaria.

Woudenberg et al. (2013) added Alternaria daucifolii to A. sect. Alternaria,

confirming 21 species (in addition to the type species) in the section. Also three

putatively representative strains corresponding to the names Clathrospora

heterospora, Comoclathris magna, and Nimbya gomphrenae were found to

be closely related to species in A. sect. Alternaria. Presumably the strains

Clathrospora heterospora (CBS 175.52) and Comoclathris magna (CBS 174.52)

were lost during cultivation and were replaced by A. alternata group isolates

(Woudenberg et al. 2013). A strain of Nimbya gomphrenae (CBS 108.27)

was isolated by Togashi and deposited at CBS in 1927, but Simmons (1989)

maintained that Togashi actually isolated two fungi, one the distinctive large-

spored A. gomphrenae [= N. gomphrenae] and the other a small-spored, chain-

forming species in the A. alternata group. This suggests that the strain used by

J. Woudenberg et al. most likely is a contaminant.

Initially A. sect. Alternaria was estimated to comprise about 60 species

(Gannibal 2012; Woudenberg et al. 2013). The number of species approved by

phylogenetic species recognition approaches may differ from that revealed by

morphological analysis, and it is very likely that a morphological concept would

favor splitting. For instance, phylogenetic analyses were performed with 150

small-spored Alternaria isolates using sequence data from three genomic loci

(Andrew et al. 2009). Strict congruence between morphology and phylogenetic

lineage was not found among isolates morphologically defined as A. alternata

or A. tenuissima. Although 5-9 well-supported clades were evident among

isolates, it was unclear whether these clades should be considered phylogenetic

species or emerging evolutionary lineages.

In other works (Peever et al. 2004, 2005) lineages inferred from phylogenetic

analysis of the combined dataset were in general agreement with described

morphospecies; however, three clades contained more than one morphological

species. The authors agreed to collapse all morphologically diverse small-

spored, citrus-associated isolates of Alternaria into a single phylogenetic

species, A. alternata.

944 ... Gannibal

The main diagnostic morphological feature of many Alternaria sect.

Alternaria species is short conidia (usually no longer than 60 um in culture)

produced in chains (Lawrence et al. 2013; Woudenberg et al. 2013). Sexual

morphs have not yet been discovered. Many strains of most well-studied species

produce toxic metabolites, such as derivatives of dibenzo-a-pyrone (alternariol,

alternariol monomethy] ether, altenuen, etc.), tetramic acid (tenuazonic acid),

and perylene (altertoxin I, II and III) (Andersen et al. 2001, 2002; Lou et al.

2013). Almost all species in this section are saprobes or facultative parasites

of plants with no strong host specialization. Only a few species (or — in an

alternative classification — pathotypes of A. alternata) are known to be strong

but specialized parasites, each able to infect only a few genotypes (cultivars)

of a host plant (Tsuge et al. 2013). Such species become pathogenic due to a

limited group of genes associated with synthesis of host-selective toxins, but

presumably they have all the same adaptations necessary for saprotrophic

growth as seen in closely related species.

It is unlikely that all 280 Alternaria species will be molecularly characterized

in the foreseeable future. Moreover, for several species living isolates are not

known and herbarium material is too old and scanty for successful DNA

extraction. On the other hand, the morphological diversity of Alternaria

is well studied and available in a monograph (Simmons 2007), and classical

comparative morphological approaches can still be utilized to assign all

Alternaria species to sections.

This work continues the series begun with A. sect. Porri (Gannibal 2015).

The aim of the present work is to detect which phylogenetically unexamined

Alternaria species fit the current morphological circumscription of A. sect.

Alternaria and to emend the morphological concept of this section.

Materials & methods

Morphology of almost all Alternaria species with legitimate names was analyzed

with regard to conformity with criteria of Alternaria sect. Alternaria. The morphological

assessment was based on descriptions made by Simmons (2007). As A. sect. Alternaria

is the autonymous section including the genus type, it contains all species that have not

yet been segregated into other sections or designated as INCERTAE SEDIS. In this article I

consider only species that are phylogenetically or morphologically closely related to the

type species and do not treat “omitted” or “obscure” species.

Results & discussion

Evaluation of all available descriptions of Alternaria species allowed for the

addition of 37 species to A. sect. Alternaria, here recognized as comprising the

59 species listed in TABLE 1.

Alternaria sect. Alternaria ...

TABLE 1. List of the 59 Alternaria spp. assigned to A. sect. Alternaria

> > BS

FPP PP Peer PP Se Se Be DB

> p> DR DR DR DD D

alocasiae T.Y. Zhang & M.X. Gao [a, b]

alternata (Fr.) Keissl. [= A. tenuis Nees] (Pryor & Bigelow 2003; Hong et al. 2005;

Lawrence et al. 2013; Woudenberg et al. 2013)

. amorphophalli V.G. Rao [a]

. angustiovoidea E.G. Simmons (Lawrence et al. 2013; Woudenberg et al. 2013)

. arborescens E.G. Simmons (Pryor & Bigelow 2003; Hong et al. 2005;

Lawrence et al. 2013; Woudenberg et al. 2013)

. asclepiadea (Cooke) E.G. Simmons [a]

. betae-kenyensis E.G. Simmons

. brassicinae E.G. Simmons

. broussonetiae T.Y. Zhang, W.Q. Chen & M.X. Gao

. burnsii Uppal, Patel & Kamat (Lawrence et al. 2013; Woudenberg et al. 2013)

. catharanthicola T.Y. Zhang [a, b]

caudata (Cooke & Ellis) E.G. Simmons

cerealis E.G. Simmons & C.F. Hill (Lawrence et al. 2013; Woudenberg et al. 2013)

. citri Ellis & N. Pierce [a]

. citriarbusti E.G. Simmons (Lawrence et al. 2013; Woudenberg et al. 2013)

. citricancri E.G. Simmons

. citrimacularis E.G. Simmons (Lawrence et al. 2013; Woudenberg et al. 2013)

. colombiana E.G. Simmons (Lawrence et al. 2013; Woudenberg et al. 2013)

. daucifolii E.G. Simmons (Woudenberg et al. 2013)

. destruens E.G. Simmons (Pryor & Bigelow 2003; Lawrence et al. 2013;

Woudenberg et al. 2013)

. dumosa E.G. Simmons (Hong et al. 2005; Lawrence et al. 2013;

Woudenberg et al. 2013)

. floridana (Cooke) E.G. Simmons [a]

. franseriae E.G. Simmons [a]

gaisen Nagano ex Hara (Lawrence et al. 2013; Woudenberg et al. 2013)

. godetiae (Neerg.) Neerg.

. graminum (Cooke) P. Joly [a]

herbiphorbicola E.G. Simmons (Lawrence et al. 2013; Woudenberg et al. 2013)

. hesperidearum (E. Pantan.) E.G. Simmons

. hibisci Bouhot ex E.G. Simmons [a]

. interrupta E.G. Simmons

. limoniasperae E.G. Simmons (Hong et al. 2005; Lawrence et al. 2013;

Woudenberg et al. 2013)

. longipes (Ellis & Everh.) E.W. Mason (Pryor & Bigelow 2003; Hong et al. 2005;

Lawrence et al. 2013; Woudenberg et al. 2013)

. mali Roberts (Rotondo et al. 2012)

malvae Roum. & Letendre [a]

obclavoidea E.G. Simmons [a]

palandui Ayyangar

945

946 ... Gannibal

A. pellucida E.G. Simmons

A. perangusta E.G. Simmons (Lawrence et al. 2013; Woudenberg et al. 2013)

A. petalicolor (Sorokin) E.G. Simmons [a]

A. platycodonis Z.Y. Zhang & H. Zhang

A. postmessia E.G. Simmons (Lawrence et al. 2013; Woudenberg et al. 2013)

A. pulvinifungicola E.G. Simmons

A. rhadina E.G. Simmons [a]

. rhizophorae E.G. Simmons

. rugosa McAlpine [a]

. sanguisorbae M.X. Gao & T.Y. Zhang

. senecionicola E.G. Simmons & C.F. Hill

. soliaegyptiaca E.G. Simmons

. spadicea (Thim.) E.G. Simmons [a]

. tangelonis E.G. Simmons (Lawrence et al. 2013; Woudenberg et al. 2013)

. tenuissima (Kunze) Wiltshire (Pryor & Bigelow 2003; Hong et al. 2005;

Lawrence et al. 2013; Woudenberg et al. 2013)

. tinosporae E.G. Simmons

> PF BP Be BB

. tomaticola E.G. Simmons & Chellemi

. toxicogenica E.G. Simmons (Lawrence et al. 2013; Woudenberg et al. 2013)

. tropaeolicola T.Y. Zhang [a, b]

. turkisafria E.G. Simmons (Lawrence et al. 2013; Woudenberg et al. 2013)

A. uredinis (Ellis & Barthol.) E.G. Simmons [a]

A. yaliinficiens R.G. Roberts

A. zhengzhouensis T.Y. Zhang [a, b]

Names with parenthetical annotations = species with phylogenetic data (in the cited references).

Bold font names = species assigned to Alternaria sect. Alternaria previously based on phylogenetic

data (Lawrence et al. 2013 and/or Woudenberg et al. 2013 or Rotondo et al. 2012).

Unannotated names = species with reliable living cultures and herbarium specimens.

Names with square bracketed annotations = species for which type material and/or representative

strains are not available: [a] = no known living isolates; [b] = type specimen is not available on

loan.

> Pp PB BP DB

The most complicated task was differentiating species of A. sect. Alternaria

from species belonging to A. sect. Infectoria and Pseudoalternaria. Typical

representatives of those two groups can easily be identified by a number of

cultural and microscopic morphological features (Lawrence et al. 2014). But

the intermediate morphology of several species or strains tends to blur the

morphological border between these two groups.

Although molecular analyses and morphological observations coincide

for most species, they are discordant in the case of A. herbiphorbicola. In

this species the conidia are short (conidial body is mainly 15-22 um long)

and produce secondary conidiophores that can slightly exceed the length

Alternaria sect. Alternaria ... 947

of the body. The three-dimensional sporulation pattern resulting from such

relatively long secondary conidiophores with several conidiogenous loci is

similar to that of Pseudoalternaria. But in many Pseudoalternaria species

a portion of conidia usually have longer apical secondary conidiophores

(30-80 um). In Pseudoalternaria lateral secondary conidiophores usually have

1-2 conidiogenous loci, while several species of Alternaria sect. Alternaria,

including A. herbiphorbicola, can produce such conidiophores with up to

4 pores.

Despite ambiguities resulting from the lack of authentic living cultures, both

A. malvae and A. rhadina should be placed in A. sect. Alternaria based on the

morphological characters documented by Simmons (2007).

Here I emend (and somewhat expand) the description of Alternaria sect.

Alternaria given previously by Lawrence et al. (2013). Characters are quantified

when appropriate, and examples are provided for species whose quantified

characters either depart from the section norm or conform to it.

Alternaria Nees sect. Alternaria emend. Gannibal

TYPE: Alternaria tenuis Nees [= Alternaria alternata]

On PCA primary conidiophores are straight or curved, simple or branched,

short (15-60 um; A. brassicinae, A. gaisen) or long (<300-435+ um; A. betae-

kenyensis), 3-4 um wide. Conidiophores bear one or several (ranging from

1-2 to 1-5 in different species) conidiogenous loci at the apex. Conidia form

simple or branched chains that are moderately long (2-6 conidia; A. godetiae,

A. toxicogenica) to long (10-20+ conidia; A. daucifolii, A. perangusta). Conidial

ageregates usually present as simple straight to branched chains of conidia

or as loose to tightly packed branching clusters (<125 conidia; A. alternata).

Young terminal conidia often are short ovoid or ellipsoid. Mature conidia are

obclavate, long ellipsoid or ellipsoid, septate, 15-50(-60) x 7-13 um in culture

and somewhat larger in nature (long secondary conidiophores are not included

in calculation). Conidia mainly distoseptate, with numerous transverse septa

(ranging from 3-7 to 5-12 in different species) and 1-2 longitudinal septa in

one or a few of the transverse divisions. In some species fully developed conidia

have 1-3 eusepta. Conidia are slightly constricted near eusepta and sometimes

near some distosepta. Most conidia narrow gradually into a tapered beak or

secondary conidiophore. Apical secondary conidiophores are short, usually

not exceeding 5-10 um (but sometimes longer, in some species <100-110+

um; A. angustiovoidea, A. tinosporae), bearing one to several conidiogenous

loci (ranging from 1-2 to 1-4 in different species). Some conidia produce short

solitary lateral secondary conidiophores. They are simple or with very short

branches, 5-10(20)x3-4 um, with 1-4 conidiogenous loci.

948 ... Gannibal

Acknowledgments

It is my pleasure to acknowledge the attention of Dr. Frank Dugan and Dr. Daniel

Lawrence for their presubmission reviews of this article. This work was supported by the

Russian Science Foundation (project #14-26-00067).

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MY COTAXON

http://dx.doi.org/10.5248/130.951

Volume 130, pp. 951-959 October-December 2015

The lichen genus Kroswia in China

Hua-Jre Liu, JIAN-SEN Hu’ & CHAO LI’

"College of Life Sciences, Hebei University, Baoding, 071002, China

? College of Life Sciences, Shandong Normal University, Jinan, 250014, China

* CORRESPONDENCE TO: liuhuajie@foxmail.com

ABSTRACT —Three species belonging to the lichen genus Kroswia are reported from China:

Kroswia epispora sp. nov., K. crystallifera, and K. gemmascens. The colourless ellipsoid

ascospores with epispore in K. epispora provide new evidence for the close relationship

between Kroswia and Fuscopannaria. Descriptions and comments for the three species and a

key to all Kroswia species are presented.

Key worps —Ascomycota, Pannariaceae, Peltigerales, taxonomy

Introduction

Kroswia P.M. Jorg. is a small, paleotropical genus belonging to Pannariaceae,

Peltigerales, Lecanoromycetidae, Ascomycota. It differs from other genera in the

family by its gelatinous, homoiomerous, and ecorticate thallus and the presence

of terpenoids and fatty acids (Jorgensen 2002, 2003a). The genus contains three

species: Kroswia crystallifera (the type), K. gemmascens, and K. polydactyla

(Jorgensen 2002, 2003a, 2004; Jorgensen & Gjerde 2012).

Only K. gemmascens is known to be fertile, and its hymenium characters

suggest a close relationship with Fuscopannaria P.M. Jorg. (Jorgensen 2007).

This was further confirmed by a recent molecular study suggesting that

K. cystallifera, the type of the genus, should be included in Fuscopannaria

(Magain & Sérusiaux 2014), making the genus Kroswia a later synonym of

Fuscopannaria (Magain & Sérusiaux 2015). However, another recent molecular

phylogeny of Pannariaceae failed to obtain PCR products from Kroswia

samples, and the authors therefore retained Kroswia as a separate genus

(Ekman et al. 2014). Because the placement of the other two Kroswia species in

Fuscopannaria lacks molecular support, we follow here the traditional concept

of Kroswia.

952... Liu, Hu & Li

Two Kroswia species have been reported from China, K. crystallifera

from Taiwan (Jorgensen 2002) and K. gemmascens from mainland China

(Zahlbruckner 1930; Wei 1991; Jorgensen 2002, 2007). Here we describe

Kroswia epispora, and present a key to all Kroswia species.

Materials & methods

Specimens are deposited in Herbarium Mycologicum Academiae Sinicae-Lichenes,

Beijing, China (HMAS-L), the Herbarium of Kunming Institute of Botany, Chinese

Academy of Sciences, Kunming, China (KUN), the Herbarium of Nanjing Normal

University, Nanjing, China (NJNU), and the Herbarium of Shandong Normal University,

Jinan, China (SDNU). Dissecting microscopes (Motic SMZ-140 and Olympus S$Z51)

and light microscopes (Motic B2 and Olympus CX21) were used for the morphological

and anatomical studies. Photographs were taken with BX51 fluorescence microscope.

Color tests and thin-layer chromatography (TLC) were routinely performed as

described by Culberson & Kristinsson (1970), Culberson (1972), Culberson et al. (1977),

Huneck & Yoshimura (1996), and Orange (2010). The K, C, and P reagents were used

for color tests. The solvent system C for TLC was used to develop acetone extraction of

the thallus fragments on a silica gel-coated glass plates. Lugol’s solution was added to the

apothecial sections for Iodine reaction.

Taxonomy

Kroswia epispora H.J. Liu & Chao Li, sp. nov. Pre

MycoBAnk MB 811551

Differs from Kroswia crystallifera by the presence of apothecia and absence of gymnidia.

Type: China. Zhejiang Province, Mt. Jiulongshan, 28°22 N 118°52 E, alt. 1600 m, on

bark, 8/V/1987, Shu-Fan Chen Z0923 (Holotype, NJNU).

EryMo.oGcy: The epithet refers to the epispore of the ascospores.

THALLUS foliose, orbicular to irregular spreading, 4-9 cm in diam., partially

fenestrate, without distinct hypothallus; opus rounded, up to 4-5 mm broad;

UPPER SURFACE Olivaceous brown, glabrous, uneven to irregularly warted,

sparsely wrinkled (warts and part of wrinkles sometimes bearing white powder

crystals), without gymnidia, marginally with white marbling and bearing white

terpenoid crystals; LOWER SURFACE bluish gray near margin, blackened towards

center, smooth to finely wrinkled, with scattered tufts of rhizines. APOTHECIA

PLATE 1. Kroswia epispora (holotype, NJNU Chen, Z0923). A. Thallus with apothecia; B. Upper

surface, showing lobe margins with white terpenoid crystals; C. Upper surface, showing fenestrated

thallus and sparse wrinkles with white powder crystals; D. An apothecium, showing thalline

margin with white powder crystals; E. Lower surface; F. Cross-section of thallus; G. Cross-section

of apothecium, showing the ecorticate and well-developed apothecial margin; H. Ascus and

ascospores, showing the amyloid ring structure of ascus apex, and subglobose ascospores in ascus;

I. Ascospores, showing distinct, smooth epispore. Scale bars: A= 1 cm; B, C = 0.4 mm; D = 1 mm;

E=5 mm; — G= 150 um; H = 20 um; 1 = 10 um.

Kroswia epispora sp. nov. (China) ... 953

954 ... Liu, Hu & Li

common, laminal, mainly adnate upon the center of the thallus, 2-3 mm in

diam.; Disc plane to convex, reddish brown; THALLINE EXCIPLE thick, glabrous,

smooth to warted, crenate at margins, beset with white powder crystals.

THALLUS homoiomerous, 300-400 um thick, <500 um thick when wet;

BOTH CorRTICES lacking, brown at the uppermost 20 um and lowermost 10 um

portions, colourless or somewhat bluish at the rest portions; PHOTOBIONT

Nostoc in long chains, consisting of more than 10 cells of 4-5 um in diam.;

HYPHAE regularly interwoven.

APOTHECIA lecanorine; THALLINE EXCIPLE well-developed, 350-450 um

thick at the margin, without cortex, with an algal layer of long chains of Nostoc

and radiating hyphae; PROPER EXCIPLE poorly-developed or lacking; HYMENIUM

100-140 um high, hemiamyloid (I+ blue, rapidly turning red-brown), with a

thin, pale yellow-brown epithecium; PARAPHYSES simple, 1.5-3 um thick,

slightly thickened at the apices; SUBHYMENIUM well-developed, <100 um high,

of non-oriented, closely compacted hyphae; asci cylindrical-clavate, apically

with an internal amyloid ring structure, 8-spored; ascospores colourless,

subglobose to broadly ellipsoid in asci, ellipsoid when released, tapered at both

ends, with visible, smooth epispore, 25-32 x 10-12 um.

Corticolous.

CHEMIstTRy: All reactions negative (color test); terpenoids and fatty acids

present (TLC).

ADDITIONAL SPECIMENS EXAMINED: CHINA. SICHUAN PROVINCE, Mt. Emeishan, on

bark, alt. 3160 m, 17/VIII/1963, J.D. Zhao & L.W. Xu 07659 (HMAS-L 036844), 7773-

1 (HMAS-L 131145); alt. 3100 m, on bark, 14/XI/1964, J.C. Wei 2252-1 (HMAS-L

131144), 2903-1 (HMAS-L 105161).

REMARKS: The new species is corticolous in montane forests from Sichuan and

Zhejiang Provinces of China. It is characterized by a homoiomerous thallus

without cortices, white marbled lobe margins, ellipsoid colourless ascospores

with an epispore, absence of gymnidia, and presence of terpenoids and fatty

acids.

Kroswia gemmascens is also fertile, but has gymnidia and light brown

ascospores without epispore; its globose, brown-pigmented ascospores are

unique in Pannariaceae and an important character distinguishing Kroswia

from Fuscopannaria (Jorgensen 2007, Ekman et al. 2014). The ellipsoid

colourless ascospores in K. epispora are similar to those in Fuscopannaria,

providing new evidence for the close relationship between the two genera.

Kroswia polydactyla P.M. Jorg. also lacks gymnidia on the thallus, but it

has dactyliform lobes and has no lichen substances detectable by TLC. The

new species may be a fertile counterpart of K. crystallifera. Both species are

morphologically very similar, but K. crystallifera is sterile with abundant

gymnidia.

Kroswia epispora sp. nov. (China) ... 955

Kroswia crystallifera P.M. Jorg., Lichenologist 34(4): 299 (2002). PL. 2 A-C

= Fuscopannaria crystallifera (P.M. Jorg.) Magain & Sérus., Lichenologist 47(1): 39 (2015).

THALLUS foliose, membranaceous, irregular spreading, 3-9 cm in diam.,

partly fenestrate, without distinct hypothallus; Losus flattened, flabellate,

up to 3 mm broad; UPPER SURFACE Olivaceous grey to olivaceous brown, glabrous,

sparsely wrinkled, with sparse to dense, white marbling, marginally beset with

bluish grey gymnidia (sometimes also spreading to upper surface); GYMNIDIA

always covered with white terpenoid crystals; LOWER SURFACE bluish gray near

margin, blackened towards center, distinctly veined, with scattered tufts of

blackish rhizines.

THALLUS homoiomerous, <500 um thick when wet, brown near both sides,

the rest portions colourless; BOTH CORTICES lacking; PHOTOBIONT Nostoc in

chains, small-celled, 4-6 um in diam.; HYPHAE loosely interwoven.

APOTHECIA not seen.

On bark, rarely on moss covered rocks.

CHEMIsTRY: All reactions negative (color test); terpenoids present (TLC).

SPECIMENS EXAMINED: CHINA. ANHUI PROVINCE, Mt. Huangshan, on bark, alt.

1330 m, 09/VIII/1978, Z.G. Qian, 01484a (NJNU); alt. 1000 m, 09/IX/1981, Z.G. Qian

03203 (NJNU). GuizHoU PROVINCE, Mt. Fanjingshan, on shrubs, alt. 2100 m, 04-05/

VII/1988, L.S. Wang 88-187, 88-192, 88-248 (KUN). HuBEI PROVINCE, Shennongjia,

on bark, alt. 2650 m, 20-30/VI/1984, A.T. Liu 8401312, 8401430, 8401510 (NJNU); alt.

1350 m, 09-10/VIII/1984, J.N. Wu 8401821, 8401867 (NJNU). SICHUAN PROVINCE,

Yanyuan County, on bark, alt. 3450 m, 26/VII/1983, L.S. Wang 83-1339 (KUN), alt.

3100 m, 11/VIHI/1983, L.S. Wang 83-1509 (KUN); Luding County, Mt. Gonggashan,

on rotten branches, alt. 2800 m, 01/1X/1996, L.S. Wang 96-16287 (KUN). ZHEJIANG

PROVINCE, Mt. Jiulongshan, on bark, alt. 980-1450 m, 4-8/V/1987, S.F. Chen Z1334,

Z1348, Z0544 (NJNU); 29/IV/1987, S.E. Chen Z0792, Z0796 (NJNU); Mt. Tiantaishan,

on bark, alt. 730 m, 24/VII/1986, Z.G. Qian, 04514 (NJNU).

REMARKS: The species is characterized by a glabrous sparsely wrinkled

and white marbled upper surface, lobe margins with gymnidia, a veined

lower surface, a homoiomerous thallus without cortices, and the presence

of terpenoids. It has been reported from Australia, East Africa, Madagascar,

Reunion, South Asia, and Taiwan of China (Jorgensen 2002, 2003a, b, 2004;

Magain & Sérusiaux 2014). The new materials reported here were collected

on barks in subtropical montane forests and extend the Chinese range from

Taiwan westward to southern Sichuan.

Kroswia crystallifera is the type of the genus (Jorgensen 2002), and has been

suggested to belong to Fuscopannaria (Magain & Sérusiaux 2014, 2015), from

which it differs, however, by the homoiomerous thallus without proper cortices.

It differs from K. gemmascens in the white marbled upper surface (especially

noticeable near the margins), less wrinkled lobes, a fenestrate foliose thallus,

956 ... Liu, Hu & Li

PLATE 2. Kroswia crystallifera (NJNU Liu 8401430). A. Thallus; B. Lobes, showing slightly wrinkled

upper surface and gymnidiate lobe margins; C. Cross-section of thallus. Kroswia gemmascens

(HMAS-L 073208). D. Thallus; E. Lobes, showing wrinkled upper surface and gymnidiate margins;

FE. Cross-section of thallus. Scale bars: A, D = 1 cm; B, E= 0.5 mm; C, F = 100 um.

and the absence of apothecia. Kroswia epispora and K. polydactyla differ by the

absence of gymnidia.

Kroswia epispora sp. nov. (China) ... 957

Wu & Qian (1989) reported Physma gemmascens and P. pergranulatum

(both synonyms of K. gemmascens) from eastern China without citing any

specimens, but most of the specimens in NJNU that they identified by these

names represent K. crystallifera.

Kroswia gemmascens (Ny]l.) P.M. Jorg., Lichenologist 34(4): 302 (2002). PL. 2D-F

= Pannaria gemmascens Nyl., Lich. Japon.: 36 (1890).

= Physma gemmascens (Nyl.) Asahina, Journ. Jap. Bot. 38: 66 (1963).

= Physma pergranulatum Zahlbr., Symb. Sin. 3: 75 (1930).

THALLUS squamulose, <1.5 cm diam., forming rounded to irregular rosettes

<3-7 cm diam., without distinct hypothallus; Lopes rounded to somewhat

elongated, auriform, 1-2 mm wide; UPPER SURFACE light brown to dark brown,

without white marbling, often wrinkled when dry, gelatinous and considerably

swelling when wet; GYMNIDIA marginal, rarely also spreading to upper surface,

granular, bluish-white, often covered with white terpenoid crystals; LOWER

SURFACE marginally light brown to whitish, darker towards the center, partly

with bluish-black rhizohyphae in the center.

THALLUS homoiomerous, <400 um when wet; BOTH CorRTICEs lacking;

PHOTOBIONT Nostoc in chains.

APOTHECIA not seen.

On barks and mosses.

CHEMIstTry: All reactions negative (color test); terpenoids and fatty acids

present (TLC).

SPECIMENS EXAMINED: CHINA. ANHUI PROVINCE, Mt. Huangshan, on bark, alt. 1610

m, 20/VIII/1962, J.D. Zhao & L.W. Xu 5532 (HMAS-L 073208). GuIzHOU PROVINCE,

Kaili City, Mt. Leigongshan, on bark, alt. 1800 m, 23/VIII/2010, D.F. Jiang 20102857

(SDNU); Daozhen County, Dashahe, on bark, alt. 1500 m, 10/VI/1987, X.L. Wu 2960

(HMAS-L 073223). HUNAN PROVINCE, Guidong County, Sidu Town, on mosses, 17/

VIII/2000, M.R. Huang 005 (HMAS-L 036431). JIANGXI PROVINCE, Mt. Lushan, on

mosses, alt. 1150 m, 11/TI/1965, J.C. Wei 3129 (HMAS-L 036429).

REMARKS: The species is characterized by a squamulose non-fenestrate thallus,

auriform lobes, a wrinkled and non-marbled upper surface, marginal gymnidia,

and a homoiomerous non-corticate thallus. It is a fertile species with globose

brown spores (Jorgensen 2007), but our materials are all sterile.

The species has been recorded from Japan and China [Sichuan, Xizang, and

the Jiangxi/Fujian border] (Jorgensen 2002, 2003a, 2007; Zahlbruckner 1930,

as Physma pergranulatum). ‘The new materials reported here were collected on

barks or mosses from montane forests and extend the Chinese range to Anhui,

Guizhou, Hunan, and Jiangxi Provinces.

Wu & Qian (1989) reported this species (without citing any specimens)

from Anhui, Fujian, and Jiangxi (as Physma pergranulatum) and from Zhejiang

958 ... Liu, Hu & Li

and Hubei (as P. gemmascens), but most of the specimens at NJNU that they

identified by these names represent Kroswia crystallifera. Both K. gemmascens

and K. crystallifera are gymnidiate species; for a comparison see the remarks

under K. crystallifera, above.

Key to species of Kroswia

1 GY T Fa PLES CITE ha se iar ine dee Bont en de as Badly oa inde woes Mndey OO dl Ph andy Pe alls « 2

GynifiidiaabSents, bc okies hcg: pete chee y mne eign ee Binns Meine Ae nse ee B

2 Thallus foliose; lobes flabellate; upper surface sparsely wrinkled; apothecia and

ASCOsporestumicnOwier ke bee cee bo Ae nat tee je ey Ree oe K. crystallifera

Thallus squamulose; lobes auriform; upper surface densely wrinkled; ascospores

globose, pale brown, without a distinct epispore .............. K. gemmascens

3 Lobes dactyliform; terpenoids absent; apothecia unknown ..... K. polydactyla

Lobes flabellate; terpenoids present; ascospores ellipsoid, colourless,

WAH AsCISTING TODS POLE’. ne < cmt giz bese | pesmere. CR canes pee ett Peceneres Cleceort: A oe K. epispora

Acknowledgements

This study was supported by the National Natural Science Foundation of China

(31093440, 31493010 & 31493011) and Natural Science Foundation of Hebei Province

(C2014201032). The authors are indebted to Li-Song Wang M.Sc. (Kunming Institute

of Botany, CAS), Prof. Jiang-Chun Wei and Ms. Hong Deng (Institute of Microbiology,

CAS) for sending the specimens on loan. The authors are grateful to Drs. André Aptroot

(ABL Herbarium, The Netherlands) and Dr. Ze-Feng Jia (College of Life Sciences,

Liaocheng University, China) for reading and improving the manuscript, and for acting

as presubmission reviewers.

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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

Culberson CF, Culberson WL, Johnson A. 1977. Second supplement to “Chemical and botanical

guide to lichen products”. American Bryological and Lichenological Society, St Louis. 400 p.

http://dx.doi.org/10.2307/2418579

Ekman S, Wedin M, Lindblom L, Jorgensen PM. 2014. Extended phylogeny and a revised generic

classification of the Pannariaceae (Peltigerales, Ascomycota). Lichenologist 46(5): 627-656.

http://dx.doi.org/10.1017/S002428291400019X

Huneck S, Yoshimura I. 1996. Identification of lichen substances. Springer-Verlag, Berlin. 493 p.

http://dx.doi.org/10.1007/978-3-642-85243-5

Jorgensen PM. 2002. Kroswia, a new genus in the Pannariaceae (lichenized Ascomycetes).

Lichenologist 34(4): 297-303. http://dx.doi.org/10.1006/lich.2002.0401

Jorgensen PM. 2003a. Conspectus familiae Pannariaceae (Ascomycetes lichenosae). Ilicifolia 4:

1-78. (Revised version 2006).

Kroswia epispora sp. nov. (China) ... 959

Jorgensen PM. 2003b. Notes on African Pannariaceae (lichenized ascomycetes). Lichenologist

35(1): 11-20. http://dx.doi.org/10.1006/lich.2002.0424

Jorgensen PM. 2004. Additional lichen records from Australia 53. Kroswia crystallifera P.M. Jorg.

and Pannaria molkenboeri Mont. & Bosch. Australasian Lichenology 54: 25.

Jorgensen PM. 2007. New discoveries in Asian pannariaceous lichens. Lichenologist 39(3):

235-243. http://dx.doi.org/10.1017/S0024282907006858

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Magain N, Sérusiaux E. 2014. Do photobiont switch and cephalodia emancipation act as

evolutionary drivers in the lichen symbiosis? A case study in the Pannariaceae (Peltigerales).

PLOS ONE 9(2): e89876. http://doi.dx.org/10.1371/journal.pone.0089876

Magain N, Sérusiaux E. 2015. The lichen genus Kroswia is a synonym of Fuscopannaria

(Pannariaceae). Lichenologist 47(1): 35-42. http://dx.doi.org/10.1017/S0024282914000553

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edition. British Lichen Society, London. 101 p.

Wei JC. 1991. An enumeration of lichens in China. International Academic Publishers, Beijing.

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Wu JN, Qian ZG. 1989. Lichens. 158-266, in: Xu BS (ed.) Cryptogamic flora of the Yangtze Delta and

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Springer, Vienna. 254 p. http://dx.doi.org/10.1007/978-3-7091-4178-6

MYCOTAXON

http://dx.doi.org/10.5248/130.961

Volume 130, pp. 961-965 October-December 2015

New reports of macromycetes from Mexico

OLIVIA RODRIGUEZ ,

Maria DE JESUS HERRERA FONSECA

& ADRIAN GALVAN CORONA

Departamento de Botanica y Zoologia, Universidad de Guadalajara,

Apartado postal 1-139, Zapopan, Jalisco, 45101 México

* CORRESPONDENCE TO: oliviaro@cucba.udg.mx

ABSTRACT — Two species of macromycetes, Phellodon tomentosus and Pulvinula constellatio,

are reported by first time for the Mexican mycobiota. The studied materials were collected

from the state of Jalisco in pine-oak forest.

Key worps — Ascomycota, Basidiomycota, taxonomy

Introduction

The present work contributes records of two genera poorly known from

Mexico. Pulvinula Boud. (Pyronemataceae, Ascomycota) has not previously been

reported from the Mexican mycobiota (Medel & Guzman 1999, Medel 2007),

and only four species of Phellodon P. Karst. (Bankeraceae, Basidiomycota) have

been reported from Mexico: Phellodon alboniger (Peck) Banker, P. confluens

(Pers.) Pouzar, P melaleucus (Sw. ex-Fr.) P. Karst., and P. niger (Fr.) P. Karst.

(Cifuentes 1999).

Materials & methods

Specimens were collected from a pine—-oak forest in the Mexican state of Jalisco, and

deposited at the herbarium of the Institute of Botany of the University of Guadalajara,

Mexico (IBUG). Macro- and micro-morphological descriptions and drawings were

made of the studied materials. Photographs of fruiting bodies are provided for each

taxon. Microscopic sections were mounted in a 3% KOH aqueous solution and Melzer’s

reagent. Specimens were identified after consulting Cifuentes (1999), Dennis (1970,

1978), Maas-Geesteranus (1975), Hall & Stuntz (1971), and Stalpers (1993).

962 ... Rodriguez, Herrera-Fonseca & Galvan-Corona

Species descriptions

PiaTE 1. Pulvinula constellatio (Rodriguez 2359): a) discoid ascomata, b) asci with globose

ascospores, c) ascal crozier, d) septate paraphyses with curved hyaline tips.

Pulvinula constellatio (Berk. & Broome) Boud., Hist. Classific. Discomyc. Europe:

70. 1907. PLATE 1

APOTHECIA 1-5 mm diam., discoid, reddish orange and lighter orange

pink towards the center, surface glabrous, margin finely wavy. ASCOSPORES

13-15(-16) um, globose uniseriate in the ascus, hyaline with numerous drops.

Asci 216-246 x 13-14 um, cylindrical with bifurcate base formed by a crozier,

hyaline. PARAPHYSES 2 um diam., filiform, slender with curved apex, forked,

septate with numerous yellowish drops.

SPECIMEN STUDIED — MEXICO. JA.isco: Municipality of Zapopan, El Valle de las

Tortugas, La Primavera, 17 Oct 2000, O. Rodriguez 2359 (IBUG).

HaBitTaT — On ground, gregarious, in pine-oak.

COMMENTS — Our material matches the description by Breitenbach & Kranzlin

(1984) in ascospore morphology (13-17 um diam., globose, with one or several

drops) and the bifurcating paraphyses with curved apices. Dennis (1978) also

described 13-15 um diam. ascospores and curved branched paraphyses. Our

specimen, however, had septate paraphyses whereas no septa were noted by

those authors.

New Pulvinula and Phellodon records for Mexico ... 963

According to Dennis (1978), the distinctive forked ascal base of Pulvinula

constellatio is also found in P. cinnabarina (Fuckel) Boud., which differs by its

larger ascospores (<18 um diam.) and asci (<320 um long). Lantieri (2008)

described P. cinnabarina with ascospores of 15-16.5 um diam. and asci of

250 x 17-18 um.

Pulvinula differs from Lamprospora by its more pulvinate ascoma and its slender

and much forked non-clavate paraphyses with curved apices (Dennis 1978).

ihcaes

YT fee ae

PLATE 2. Phellodon tomentosus (Herrera 1260): a) basidiomes, b) subglobose echinulate (spiny)

basidiospores, c) lageniform hyaline cheilocystidia, d) generative hyphae.

Phellodon tomentosus (L.) Banker, Mem. Torrey Bot. Club 12: 171.1906. | PLATE 2

PiLEus 20-35 mm diam., zonate, velutine to tomentose, more conspicuous

towards the center, with the margin white to whitish-yellowish and the disc

964 ... Rodriguez, Herrera-Fonseca & Galvan-Corona

orange-brown to reddish-brown and dark brown. HYMENOPHORE toothed or

with spines, whitish to cream coloured, with gray-pink tones in dried material.

STIPE central to eccentric, surface also zonate and concolorous with the pileus

but obscured in the base by a reddish-brown colour. CONTEXT zonate, reddish-

brown, woody, odor agreeable, nut-like.

BASIDIOSPORES 3-4.5 X 3-4 um, subglobose, echinulate, hyaline.

CHEILOCYSTIDIA 29-37 X 4-6 um, lageniform, thin-walled, the majority

with a long neck and some flexuose, hyaline, abundant, with basal septa and

clamp connections absent. HYPHAL SYSTEM monomitic with generative hyphae

1.5-4 um diameter, light yellowish coffee colour, and without clamp connections.

SPECIMEN STUDIED — MEXICO. Jatisco: Municipality of San Sebastian del Oeste,

Camino Real Alto - La Bufa, 5.5 km del pueblo de San Sebastian del Oeste, 1 Aug 2009,

M. Herrera 1260 (IBUG).

HasitTat — On soil and needle litter, gregarious, in pine-oak.

ComMENTs — Phellodon tomentosus is characterized by circular stipitate

basidiomes that form into small colonies of five or six specimens with fused

pilei, each pileus having its own independent stipe. Although cheilocystidia

have not previously been reported for Phellodon species (Maas-Geesteranus

1975, Pegler et. al. 1997, Stalpers 1993), we observed cheilocystidia over the

entire hymenial surfaces of our material.

The macro- and micro-morphological features agree with those described

by Breitenbach & Kranzlin (1986). Phellodon tomentosus can be confused with

some Hydnellum species based on context and stipe zonation, but Hydnellum

can be differentiated microscopically by its brown spores.

Phellodon tomentosus is a widely distributed but uncommon species; it is

reported as mycorrhizal on conifers (Pegler et al. 1997) and in mixed forests

(Maas-Geesteranus 1975).

Acknowledgements

The authors are grateful to Dra. Nahara Ayala Sanchez and Dra. Virginia Ramirez-

Cruz for their critical review and comments. Thank you also to E. Horak for his valuable

comments and manuscript review. Support from the Universidad de Guadalajara is

gratefully acknowledged.

Literature cited

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Breitenbach J, Kranzlin F. 1986. Fungi of Switzerland, vol. 2. Verlag Mykologia, Lucerna. 411 p.

Cifuentes Blanco J. 1999. Distribucidn y algunos aspectos ecoldgicos de hongos hidnoides

estipitados de México. Universidad Aut6énoma Nacional de México. Facultad de Ciencias. Bases

de datos SNIB-CONABIO proyecto No. H177. México, D.E

Dennis RWG. 1970. Fungus flora of Venezuela and adjacent countries. Cramer, Lehre. 531 p.

New Pulvinula and Phellodon records for Mexico ... 965

Dennis RWG. 1978. British Ascomycetes. Cramer, Vaduz. 585 pp.

Hall D, Stuntz DE. 1971. Pileate Hydnaceae of the Puget Sound area. I. White spored

genera: Auriscalpium, Hericium, Dentinum and Phellodon. Mycologia 63(6): 1099-1128.

http://dx.doi.org/10.2307/3757988

Lantieri A. 2008. Pulvinula johaniis, a new species from Sicily, Italy. Sydowia 60 (2): 247-252.

Maas-Geesteranus RA. 1975. Die terrestrischen Stachelpilze Europas — ‘The terrestrial hydnums

of Europe. Verhandelingen der Koninklijke Nederlandse Akademie van Wetenschappen, Afd.

Natuurkunde, Tweede reeks, 65. 127 p.

Medel R. 2007. Especies de Ascomycetes citados de México IV: 1996-2006. Revista Mexicana de

Micologia 25: 69-76.

Medel R, Guzman G. 1999. Especies de macromicetos citadas de México IX. Ascomycetes, parte III:

1983-1996. Acta Botanica Mexicana 46: 57-72.

Pegler DN, Roberts PJ, Spooner BM. 1997. British chanterelles and tooth-fungi: an account of the

British cantharelloid and stipitate hydnoid fungi. Royal Botanic Gardens, Kew. 114 p.

Stalpers JA. 1993. The aphyllophoraceous Fungi I: keys to the species of the Thelephorales. Studies

in Mycology 35. 168 p.

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

Research 99(12): 1521-1524. http://dx.doi.org/10.1016/S0953-7562(09)80804-6

MY COTAXON

http://dx.doi.org/10.5248/130.967

Volume 130, pp. 967-970 October-December 2015

Two new records of microfungi

from the Brazilian Atlantic Forest

MARCELA A. BARBOSA’, MARINA A.G. ARAUJO', Mayra S. OLIVEIRA’,

PHELIPE M.O. Costa’, ELAINE MALOSSO” & RAFAEL F. CASTANEDA-RUIZ?3

'Programa de Pés-Graduagao em Biologia de Fungos, Universidade Federal de Pernambuco,

Avenida da Engenharia, s/n. Cidade Universitaria, Recife, PE, 50.740-600, Brazil

*Centro de Ciéncias Biologicas, Departamento de Micologia/Laboratério de Micorrizas,

Universidade Federal de Pernambuco, Avenida da Engenharia, s/n. Cidade Universitaria,

Recife, PE, 50.740-600, 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: elaine.malosso@ufpe. br

ABSTRACT— Two microfungi, Linodochium sideroxyli and Leptodiscella africana, are recorded

for the first time from Brazil. The specimens, collected on decaying leaves in natural areas of

the Brazilian Atlantic Forest, are described and illustrated.

KEY worDs— taxonomy, conidial fungi, tropics

Introduction

Linodochium Hohn. (Hohnel 1909, Dyko & Sutton 1979, Li et al. 2013),

typified by L. hyalinum, comprises five species characterized by cupulate,

superficial, separate or confluent, and yellow, orange, or white sporodochial

conidiomata, closely packed branched conidiophores, and _polyblastic,

denticulate, sympodially extended conidiogenous cells. The conidia are solitary,

cylindrical or falcate, septate, and hyaline (Castafieda-Ruiz & Kendrick 1990a,

Li et al. 2013).

The genus Leptodiscella, introduced by Papendorf (1969) and typified by

L. africana, is distinguished by mono- or polyblastic, denticulate, sympodially

extended conidiogenous cells and solitary, cylindrical to oblong, 1-septate,

hyaline conidia with a subpolar, filiform appendage at each cell. Leptodiscella

comprises four species (Madrid et al. 2012).

968 ... Barbosa & al.

During three mycological surveys of conidial fungi occurring on leaf

litter from the Atlantic rainforest, the microfungi Linodochium sideroxyli and

Leptodiscella africana were found and recorded for the first time in Brazil.

Material & methods

Samples of leaf litter were placed in paper and plastic bags, taken to the laboratory,

and treated according to Castafieda-Ruiz (2005). Mounts were prepared in PVL

(polyvinyl alcohol and lactic acid) and measurements were taken at x1000. Micrographs

were obtained with a Nikon microscope (model Eclipse Ni-U) with bright field and

DIC optics. Specimens were deposited in the Herbarium of Universidade Federal de

Pernambuco, Recife, Brazil (URM).

Taxonomy

Linodochium sideroxyli Dulym., P.M. Kirk & Peerally,

Mycotaxon 73: 317. (1999). Fic. 1A-D

CoLonigs on the natural substrate sporodochial, cupulate or pulvinate,

scattered, amphigenous, yellow, 300-550 um diam. Mycelium superficial

and immersed, composed of septate, branched, smooth, hyaline hyphae,

1-3 um diam. Conip1opHorEs distinct, branched, erect or flexuous, strongly

geniculate above, numerous, tightly aggregated, 1-3-septate, smooth, hyaline,

25-50 x 2-4 um. CONIDIOGENOUS CELLS polyblastic, discrete, terminal,

indeterminate, long sympodial elongated, with broad, flat-topped denticles

forming a geniculate rachis, hyaline, 15-30 x 2-4 um. Conidial secession

schizolytic. Conip1a solitary, cylindrical-filiform, slightly inequilateral, basal

cell 1 um wide, apical cell 1.5-2 um diam., hyaline, smooth, 0-1-septate, mostly

1-septate, with very thick and refractive walls at each end, truncated at the base,

rounded at the apex, 33-40 x 1.5-2 um

SPECIMENS EXAMINED: BRAZIL. PERNAMBUCO: Cabo de Santo Agostinho, Refigio

de Vida Silvestre Matas do Sistema Gurjat, 8°13’S 35°03’W, on decaying leaf of an

unidentified plant, 12-XI-2014, coll. M.A. Barbosa (URM 87703); Abreu e Lima, Mata

de Monjope, 7°52’S 34°55’W, on decaying leaf of an unidentified plant, 30-IH-2015, coll.

M.A.G. Araujo (URM 87704).

Note: The Brazilian specimens have shorter conidia than those of the type

material (32-48 um long; Dulymamode at al. 1999) but otherwise fit well the

original description.

Leptodiscella africana (Papendorf) Papendorf,

Trans. Br. Mycol. Soc. 53: 146 (1969). Fic. 1E-H

CoONIDIOMATA on the natural substrate sporodochial, scattered or confluent,

white. Mycelium superficial and immersed, compose of septate, branched,

subhyaline hyphae, 1-1.5 um diam. CONIDIOPHORES distinct, erect or prostrate,

subhyaline, 0-2-septate, <25 um long, sometimes reduced to conidiogenous

Linodochium & Leptodiscella spp. new for Brazil ... 969

Fic 1. Linodochium sideroxyli (URM 87704): A, B. Sporodochia on the natural substratum.

C. Conidiogenous cell and conidium. D. Conidium Leptodiscella africana (URM 86557):

E. Conidiogenous cells and conidia. F-H. Conidia.

970 ... Barbosa & al.

cells. CONIDIOGENOUS cells mono- and polyblastic, terminal and intercalary,

sympodial extended, cylindrical or irregular, subhyaline, smooth, 5-10 x

1-2 um. Conidial secession schizolytic. Conrp1A solitary, cylindrical or

oblong, 1-septate, rounded or slightly obtuse at the ends, sometime somewhat

truncate at the base, hyaline smooth, 12-18 x 2-3 um, with a filiform, subpolar

appendage at each end, 6-8 um long.

SPECIMEN EXAMINED: BRAZIL. PERNAMBUCO: Cabo de Santo Agostinho, Refugio

de Vida Silvestre Matas do Sistema Gurjau, 8°13’S 35°03’W, on decaying leaf of an

unidentified plant, 12-XI-2014, coll. M.A. Barbosa (URM 86557).

Note: The original description of Leptodiscella africana was derived from a

pure culture on potato-carrot-agar (Papendorf 1967, 1969); however, the

conidial sizes in the type material (11-17.5 x 2-3 um) are almost the same as

in the Brazilian specimen, although the subpolar conidial appendages from the

type are slightly longer than from the Brazilian specimen (6.5-13 um).

Acknowledgments

The authors express their sincere gratitude to Dr. Josiane S. Monteiro and Dr. De-Wei

Li for their critical review of the manuscript. The authors are grateful to the ‘Coordenacao

de Aperfeigoamento de Pessoal de Nivel Superior (CAPES)’ for financial support

through project 88881.062172/2014-01 and the ‘Programa Ciéncia sem Fronteiras’

RFCR is grateful to the Cuban Ministry of Agriculture and ‘Programa de Salud Animal

y Vegeta, project P131LH003033 for facilities. Dr. Lorelei Norvell’s editorial review and

Dr. Shaun Pennycook’s nomenclature review are greatly appreciated.

Literature cited

Castaneda-Ruiz RE 2005. Metodologia en el estudio de los hongos anamorfos. 182-183, in: Anais

do V Congresso Latino Americano de Micologia, Brasilia.

Castaneda-Ruiz RF, Kendrick B. 1990a. Conidial fungi from Cuba. I. University of Waterloo,

Biology Series. 32. 53 p.

Castaneda-Ruiz RF, Kendrick B. 1990b. Conidial fungi from Cuba. II. University of Waterloo,

Biology Series. 33. 61 p.

Dulymamode R, Kirk PM, Peerally A. 1999. Fungi from Mauritius: three new hyphomycete species

on endemic plants. Mycotaxon 73: 313-323.

Dyko BJ, Sutton BC. 1979. A revision of Linodochium, Pseudocenangium, Septopatella, and

Siroscyphella. Canadian Journal of Botany 57: 370-385. http://dx.doi.org/10.1139/b79-049

Hohnel F von. 1909. Fragmente zur Mykologie (VIII. Mitteilung, Nr. 354 bis 406). Sitzungsberichte

der Kaiserlichen Akademie der Wissenschaften, Mathematisch-naturwissenschaftliche Klasse,

Abteilung 1, 118: 1157-1246

Li DW, Chen J, Wang Y. 2013. Hyphomycetes: Linodochium sinense sp. nov. and new records from

fallen Sycopsis sinensis leaves in China. Mycotaxon 126: 15-22. http://dx.doi.org/10.5248/126.15

Madrid H, Gené J, Cano J, Guarro J. 2012. A new species of Leptodiscella from Spanish soil.

Mycological Progress 11: 535-541. http://dx.doi.org/10.1007/s11557-011-0768-8

Papendorf MC. 1967. Leptodiscus africanus sp. nov. Transactions of the British Mycological Society

50: 687-690. http://dx.doi.org/10.1016/S0007-1536(67)80102-5

Papendorf MC. 1969. Leptodiscella africana gen. et comb. nov. Transactions of the British

Mycological Society 53: 145-147. http://dx.doi.org/10.1016/S0007-1536(69)80021-5

MY COTAXON

http://dx.doi.org/10.5248/130.971

Volume 130, pp. 971-976 October-December 2015

Pyramidospora quadricellularis sp. nov.

on submerged leaves from Brazil

Mayra S. OLIVEIRA’, MARINA A.G. ARAUJO', MARCELA A. BARBOSA’,

JESSICA C. SILVA?, ELAINE MALOSSO” & RAFAEL F. CASTANEDA-RUIZ?

‘Programa de Pés-Graduagao em Biologia de Fungos, Universidade Federal de Pernambuco,

Avenida da Engenharia, s/n Cidade Universitaria, Recife, PE, 50.740-600, Brazil

*Centro de Ciéncias Biolégicas, Departamento de Micologia/Laboratério de Micorrizas,

Universidade Federal de Pernambuco, Avenida da Engenharia, s/n

Cidade Universitaria, Recife, PE, 50.740-600, 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: elaine.malosso@ufpe.br

ABSTRACT — Pyramidospora quadricellularis, a new species collected on submerged decaying

leaves in Brazil, is described and illustrated. It is characterized by 4-celled, hyaline, smooth

conidia. Illustrations and a comparative table of the described species of Pyramidospora are

provided.

KEY worDs — asexual fungi, systematics, freshwater fungi

Introduction

Pyramidospora, established by Nilsson (1962) and typified by P casuarinae

Sv. Nilsson, comprises eight species (Ingold 1975, Index Fungorum 2015). The

genus is distinguished by conidiophores that are distinct, septate, and hyaline

and monoblastic determinate terminal conidiogenous cells. The conidia are

more or less pyramid-like, stellate to stauroform, and multicellular, composed

of hyaline globose cells that sometimes form a moniliform branched or

irregularly complex conidial body. A fungus collected during a mycological

survey of freshwater fungi associated with submerged leaves in streams at a

Brazilian Atlantic Forest is herein described as new Pyramidospora species and

compared with other described species (TABLE 1).

Materials & methods

During an expedition in April 2015 through “Reftigio de Vida Silvestre Matas do

Sistema Gurjau,’ samples of submerged decaying leaves were placed in sterile glass jars

972 ... Oliveira & al.

with water from the site, taken to the laboratory, and processed according to Ingold

(1975) and Schoenlein-Crusius & Milanez (1989). Mounts were prepared in lactic acid

90%, and measurements made at a magnification of x1000. Photomicrographs were

obtained with a Nikon Eclipse Ni-U microscope with bright field and DIC optics. The

holotype was deposited in the Herbarium of Universidade Federal de Pernambuco,

Recife, Brazil (URM).

f 50 um

Fic. 1. Pyramidospora quadricellularis (holotype, URM 87706): A, B. Conidia. C-E. Conidiophores,

conidiogenous cells, and conidia.

Pyramidospora quadricellularis sp. nov. (Brazil) ... 973

Taxonomy

Pyramidospora quadricellularis M.S. Oliveira, Malosso &

R.F. Castaneda, sp. nov. Fia. 1

MycosBank MB 812756

Differs from all other Pyramidospora spp. by its 4-celled conidia.

Type: Brazil, Pernambuco, Jaboatao dos Guararapes, Refugio de Vida Silvestre Matas

do Sistema Gurjau, 8°13'S 35°3'W, 85 m alt., on submerged decaying leaves of an

unidentified plant in a stream, 13.IV.2015, coll. M.S. Oliveira (Holotype, URM 87706).

EryMo_oey: Latin, quadri- (= four) + -cellularis (= celled).

CoLonigs on the natural substrate effuse. Mycelium mostly superficial.

Hyphae septate, branched, hyaline, 2-2.5 um diam, smooth. CONIDIOPHORES

distinct, erect, straight or decumbent, mostly single, sometimes branched,

cylindrical, abruptly attenuated at the apex, 0-7-septate, smooth, <50 um long,

3-4 um wide. CONIDIOGENOUS CELLS monoblastic, terminal, determinate,

integrated in conidiophores and branches, 5-15 x 2.5-3 um, denticulate with

small (1.5-2 um long) narrow cylindrical teeth. Conrp1a solitary, acrogenous,

square rounded to tetrad-shaped or shamrock leaf-shaped, 13-15 x 15-17 um,

hyaline, smooth, sometimes guttulate, composed of 4 globose cells, hyaline,

4-6 um diam, sometimes with a germ tube.

Note: With 4-celled conidia, P quadricellularis is unique and does not resemble

any of the described Pyramidospora species (TABLE 1; Fics 2, 3).

TABLE 1. Morphological comparison of conidia of Pyramidospora species

SPECIES SHAPE BRANCHES SIZE (um) REFERENCE

P. casuarinae Pyramid-like 6-8 15-20 x 12-18 Nilsson (1962)

P. constricta Stellate to irregular 2-5 23-47 long Singh (1972)

P. densa Stellate 6-8 29-37 wide Alasoadura (1968)

P, fluminea Stellate 3-5 16-23 long Miura & Kudo

(1971)

P. herculiformis H-shaped 4 11-17 long Singh (1976)

P. quadricellularis Tetrad-shaped 2 13-15 x 15-17 This paper

P. ramificata Grape cluster-shaped 6-7 17-29 long Miura & Kudo

or irregular (1971)

P. robusta Stellate or irregular 8-13 42-57 long Moreira &

moniliform cluster Schoenlein-

Crusius (2012)

P. stellata Stellate 4 15-25 long Sinclair & Morgan-

Jones (1979)

974 ... Oliveira & al.

Fic. 2. Conidia of Pyramidospora species, redrawn or adapted from original descriptions:

A. P. casuarinae (Nilsson 1962). B. P. constricta (Singh 1972). C. P. densa (Alasoadura 1968).

D. P. fluminea (Miura & Kudo 1971). E. P. herculiformis (Singh 1976). F. P ramificata (Miura &

Kudo 1971). Scale bars = 10 um.

Pyramidospora quadricellularis sp. nov. (Brazil) ... 975

Fic. 3. Conidia of Pyramidospora species, redrawn or adapted from original descriptions:

G. P. robusta (Moreira & Schoenlein-Crusius 2012). H. P. stellata (Sinclair & Morgan-Jones

1979). Scale bars = 10 um.

Acknowledgments

The authors express their sincere gratitude to Dr. Josiane S. Monteiro and Dr.

De-Wei Li for their critical review of the manuscript. The authors are grateful to the

‘Coordenag¢ao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)’ for financial

support through project 88881.062172/2014-01 and the ‘Programa Ciéncia sem

Fronteiras. RFCR is grateful to the Cuban Ministry of Agriculture and ‘Programa de

Salud Animal y Vegetal, project P131LH003033 for facilities. We acknowledge the

facilities provided by Dr. P.M. Kirk and Drs. V. Robert and A. Decock through the Index

Fungorum and MycoBank websites. Dr. Lorelei Norvell’s editorial review and Dr. Shaun

Pennycook’s nomenclature review are greatly appreciated.

976 ... Oliveira & al.

Literature cited

Alasoadura SO. 1968. Some aquatic hyphomycetes from Nigeria. Transactions of the British

Mycological Society 51: 535-540. http://dx.doi.org/10.1016/S0007-1536(68)80023-3

Index Fungorum. 2015. http://www.indexfungorum.org/names/names.asp. Accession date: 2015-05-29.

Ingold CT. 1975. An illustrated guide of aquatic and water borne hyphomycetes (fungi imperfecti)

with notes on their biology. Freshwater Biological Association, Scientific Publication 30. 96 p.

Miura K, Kudo MY. 1971. Two new species of filamentous fungi from Japan. The Journal of

Japanese Botany 46: 39-46.

Moreira CG, Schoenlein-Crusius IH. 2012. Nova espécie e novos regsitros para o Brasil de

hifomicetos observados em folhedo submerso colectados no Parque Municipal Alfredo Volpi,

Sao Paulo, SP, Brasil. Hoehnea 39: 521-527.

http://dx.doi.org/10.1590/S2236-89062012000400001

Nilsson S. 1962. Some aquatic hyphomycetes from South America. Svensk Botanisk Tidskrift 56:

351-361,

Schoenlein-Crusius IH, Milanez AI. 1989. Sucessao fingica em folhas de Ficus microcarpa L. f.

submerses no Lago Frontal situado no Parque Estadual das Fontes do Ipiranga, Sao Paulo.

Revista de Microbiologia, S4o Paulo 20: 95-101.

Sinclair RC, Morgan-Jones G. 1979. Notes on hyphomycetes. XXXII. Five new aquatic species.

Mycotaxon 9: 469-481.

Singh N. 1972. Pyramidospora constricta sp. nov., a new aquatic hyphomycete. Transactions of the

British Mycological Society 59: 336-339. http://dx.doi.org/10.1016/S0007-1536(72)80027-5

Singh N. 1976. Pyramidospora herculiformis sp. nov., a new aquatic hyphomycete from Sierra

Leone. Transactions of the British Mycological Society 66: 347-350.

http://dx.doi.org/10.1016/S0007-1536(76)80070-8

MY COTAXON

http://dx.doi.org/10.5248/130.977

Volume 130, pp. 977-981 October-December 2015

Tretolylea, anew genus from the Brazilian semiarid region

TAIMY CANTILLO-PEREZ', LUiSs FERNANDO PASCHOLATI GUSMAO"

& RAFAEL F. CASTANEDA-RUIZ?

"Universidade Estadual de Feira de Santana, Departamento de Ciéncias Bioldgicas,

Laboratorio de Micologia, Avenida Transnordestina, s/n,

Novo Horizonte, 44036-900, 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

Asstract — Tretolylea pleiomorpha gen. & sp. nov. is described and illustrated. It

is characterized by inconspicuous prostrate (or decumbent) brown multi-branched

conidiophores, tretic conidiogenous cells, blastocatenate cylindrical to oblong euseptate

brown conidia, and a Phialophora-like synanamorph that arises from the same assimilative

hyphae and conidia.

KEY worpDs — asexual fungi, sooty moulds, systematics, tropical fungi

Introduction

During investigations of microfungi on dead plant material in the semiarid

region of northeast Brazil, an interesting fungus was collected on decaying

wood of an unidentified plant. This fungus, which shows remarkable differences

from all previously described genera (Seifert et al. 2011), is described as a new

genus and species.

Materials & methods

Samples of decaying wood of unidentified woody plants were collected in small

separate areas with typical vegetation of the semiarid region and placed in paper bags.

In the laboratory the samples were placed in Petri dish moist chambers and stored

in a polystyrene box with sterile water plus glycerol at 25°C for 30 days (Castaneda-

Ruiz 2005). Mounts of slides were prepared in PVL (polyvinyl alcohol, lactic acid,

and phenol), and micrographs were obtained with an Olympus microscope BX 51.

The specimen is conserved in the Herbarium, Departamento de Ciéncias Bioldgicas,

Universidade Estadual de Feira de Santana, Brazil (HUEFS).

978 ... Cantillo-Pérez, Gusmao & Castafteda-Ruiz

Taxonomy

Tretolylea Cantillo, R.F. Castafieda & GusmAo, gen. nov.

MycoBAank MB 812409

Differs from Lylea by its tretic conidiogenous cells and euseptate conidia.

TYPE SPECIES: Tretolylea pleiomorpha Cantillo et al.

EryMoLoGy: Greek treto- (tpntoc) meaning pierced through, referring to the tretic

conidiogenous cells, + -lylea referring to the hyphomycete genus Lylea.

CoLonliEs on the natural substratum effuse, hairy, brown. Mycelium superficial,

composed of smooth, septate, brown hyphae. CONIDIOPHORES inconspicuous,

prostrate or decumbent, multi-septate, brown, resembling assimilative hyphae.

CONIDIOGENOUS CELLS terminal, integrated or discrete, determinate and

producing one conidium or extending sympodially and producing several

conidia sequentially; conidial initiation tretic; conidial secession schizolytic.

Conip1A blastocatenate, cylindrical to oblong, phragmosporous, euseptate,

brown, smooth. Synanamorph Phialophora-like arising from the same

assimilative hyphae and conidia.

Tretolylea pleiomorpha Cantillo, R.F. Castafieda & Gusmao, sp. nov. Fics 1, 2

MycoBank MB 812410

Differs from Lylea catenulata by its tretic conidiogenous cells and euseptate conidia.

Type: Brazil, Rio Grande do Norte State: Portalegre, Chapada do Apodi, 6°01’S 37°59’W,

520 m alt., on decaying wood, 3.VII.2014, coll: T. Cantillo (Holotype: HUEFS 211337).

Erymo ocy: Greek, pleio- meaning more than usual, + -morpha, referring to existing

forms of conidium ontogeny.

Cotoniges on the natural substrate effuse, funiculose, brown. Mycelium

superficial, composed of branched, 6-11 um wide, septate, smooth hyphae.

CONIDIOPHORES inconspicuous. CONIDIOGENOUS CELLS discrete, cylindrical,

determinate and producing one conidium or extending sympodially and

producing several conidia sequentially; conidial initiation tretic; conidial

secession schizolytic. Conrp1a blastocatenate, cylindrical to oblong, straight,

somewhat curved or slightly, sinuate, rounded at the ends, brown, smooth,

2- to 11-euseptate, slightly darker at septa, 29-47 x 10-19 um. SYNANAMORPH

Phialophora-like, conidiophores distinct, branched, conidiogenous cells

forming a slightly penicillate cluster, lageniform, discrete, “phialidic” (ie.,

producing conidia sequentially by a replacement wall-building apex system

from a single fertile locus; Minter et al. 1983), sometimes extending by

percurrent enteroblastic proliferation, pale brown, 8-16 x 6-8 um, with a

conspicuous, narrow collarette. Conidia globose, unicellular, subhyaline to

pale brown or pale olivaceous-brown, smooth, 2-3 um diam., accumulating in

brown masses.

Tetrolylea pleiomorpha gen. & sp. nov. (Brazil) ... 979

Fic. 1. Tretolylea pleiomorpha (holotype, HUEFS 211337).

A, B. Conidia. C. Conidiogenous cells and conidia.

D. Conidiogenous cell and conidia of Phialophora-like synanamorph.

980 ... Cantillo-Pérez, Gusmao & Castafieda-Ruiz

Fic. 2. Tretolylea pleiomorpha (holotype, HUEFS 211337). A. Colonies on the natural

substratum. B, C. Conidiogenous cells and conidia. D. Conidiogenous cell and conidia.

E. Conidial development. F, G. Conidiogenous cells.

Notes: Lylea Morgan-Jones superficially resembles Tretolylea but has

holoblastic conidiogenous cells and distoseptate conidia (Morgan-Jones

1975). Heteroconium Petr. (Castafieda-Ruiz et al. 2008, Ma et al. 2012a,b, Ren

et al. 2012), Taeniolella S. Hughes (Watanabe 1992), and Taeniolina M.B. Ellis

(Seifert et al. 2011) are similar to Tretolylea; these genera produce conidia

in unbranched or branched chains, but conidial development is holoblastic.

The sooty moulds Capnophialophora S. Hughes and Capnosporium S. Hughes

(Hughes 1976) also superficially resemble Tretolylea, but neither has tretic

conidial development (Seifert et al. 2011).

This fungus was collected in the semiarid region of Brazil, also known

as Caatinga. Caatinga ecosystems are unique to Brazil and are undoubtedly

threatened, with only about 1% of the area having formal protection

(www.nature.org/ourinitiatives/regions/southamerica/brazil/placesweprotect/caatinga.xml

Tetrolylea pleiomorpha gen. & sp. nov. (Brazil) ... 981

— accessed 31 May 2015). The type locality is a small remaining fragment

of natural or semi-natural vegetation surrounded by farming and other

developments. The discovery of this new genus here (and of others already

collected by the authors but as yet undescribed) suggests that the Caatinga may

be as rich in fungi as in animals and plants, many of which are endemic. As a

newly discovered species about which very little is known, the conservation

status of Tretolylea pleiomorpha is Data Deficient, but if it is found to be an

endemic of the region, it is will probably be categorized as threatened.

Acknowledgments

The authors express their sincere gratitude to Dr David Minter and Dr De-Wei Li for

their critical review of the manuscript. The authors thank Programa de Pdés-Gradua¢ao

em Botanica (PPGBot/ UEFS), Programa de Pesquisa em Biodiversidade do Semiarido

(PPBIO semiarido/ Ministry of Technology and Science) for financial support. TCP

thanks PEC-PG/CAPES (proc. 12636134/2014) for a grant. RFCR and LFPG are grateful

to the “Coordenacao de Aperfeigoamento de Pessoal de Nivel Superior (CAPES)”

for financial support through project 88881.062172/2014-01 and the “Programa

Ciéncia sem Fronteiras’. RFCR is also grateful to the Cuban Ministry of Agriculture

and “Programa de Salud Animal y Vegetal’, project P131LH003033 for facilities. We

acknowledge the facilities provided by Dr P.M. Kirk and Drs V. Robert and A. Decock

through the Index Fungorum and MycoBank websites. Dr Lorelei Norvell’s editorial

review and Dr Shaun Pennycook’s nomenclature review are greatly appreciated.

Literature cited

Castaneda-Ruiz RE 2005. Metodologia en el estudio de los hongos anamorfos. 182-183, in: Anais

do V Congresso Latino Americano de Micologia, Brasilia.

Castaneda-Ruiz RF, Iturriaga T, Heredia G, Minter DW, Gené J, Stadler M, Saikawa M, Silvera-

Simon C. 2008. Notes on Heteroconium and a new species from Venezuela. Mycotaxon. 105:

175-184.

Hughes SJ. 1976. Sooty moulds. Mycologia. 68: 693-820. http://dx.doi.org/10.2307/3758799

Ma J, Ma LG, Zhang YD, Castafieda-Ruiz RK Zhang XG. 2012a. New species or records of

Endophragmiella and Heteroconium from southern China. Cryptogamie Mycologie 33:

127-135. http://dx.doi.org/10.7872/crym.v33.iss2.2012.127

Ma J, Ma LG, Zhang YD, Castafieda-Ruiz RK Zhang XG. 2012b. New species and records

of Heteroconium (anamorphic fungi) from southern China. Mycoscience 53: 466-470.

http://dx.doi.org/10.1007/S10267-012-0190-3

Minter DW, Sutton BC, Brady BL. 1983. What are phialides anyway? Transactions of the British

Mycological Society 81: 109-120.

Morgan-Jones G. 1975. Notes on hyphomycetes. VIII. Lylea, a new genus. Mycotaxon 3: 129-132.

Ren SC, Ma J, Zhang XG. 2012. Two new Heteroconium species and two other forest microfungi

newly recorded from China. Mycotaxon 119: 361-367. http://dx.doi.org/10.5248/119.361

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

Biodiversity Series 9. 997 p. http://dx.doi.org/10.3767/003158511X617435

Watanabe T. 1992. Taeniolella phialosperma sp. nov. from Japan. Mycologia 84: 478-483.

http://dx.doi.org/10.2307/3760204

MY COTAXON

http://dx.doi.org/10.5248/130.983

Volume 130, pp. 983-995 October-December 2015

Acaulospora reducta sp. nov. and A. excavata —

two glomeromycotan fungi with pitted spores from Brazil

CAMILLA M.R. PEREIRA’, BRUNO TOMIO GOTO’, DANIELLE KARLA ALVES DA SILVA?3,

ARAESKA CARENNA DE ALMEIDA FERREIRA’, FRANCISCO ADRIANO DE SOUZA4,

GLADSTONE ALVES DA SILVA!, LEONOR C. MAIA! & FRITZ OEHL*S”

"Departamento de Micologia, CCB, Universidade Federal de Pernambuco,

Av. da Engenharia s/n, Cidade Universitaria, 50740-600, Recife, PE, Brazil

*Departamento de Botanica e Zoologia, CB, Universidade Federal do Rio Grande do Norte,

Campus Universitario, 59072-970, Natal, RN, Brazil

°Universidade Federal do Vale do Sado Francisco, Campus de Ciéncias Agrarias,

Colegiado de Zootecnia, 56300-990, Petrolina, PE, Brazil

‘Embrapa Milho e Sorgo, Sete Lagoas, Minas Gerais, Brazil

*Agroscope, Federal Research Institute for Sustainability Sciences, Plant-Soil-Interactions,

Reckenholzstrasse 191, CH-8046 Ziirich, Switzerland

“CORRESPONDENCE TO: fritz.oehl@gmail.com

ABSTRACT — Acaulospora reducta sp. nov. and A. excavata, both characterized by large

pits on their spore surfaces, were found in the semi-humid Atlantic rainforest and semi-

arid Caatinga biomes of Northeastern Brazil. Phylogenetic analyses of the ITS regions of

the ribosomal gene place the two fungi in two distinct clades within the Acaulosporaceae.

Acaulospora reducta has whitish yellow, dark yellow to light brown spores (135-205 um in

diam.) and irregularly shaped, often edged to sometimes dumbbell-shaped pits (5.5-19 x

3.5-8.6 um). These large pits have roughened irregular surfaces comprising secondary small

pits (c. 0.5 um broad and deep) and fine ridges. Spores of A. excavata are ochre to yellow

orange (sometimes yellow) and have regular circular to subcircular pits (4-20 x 4-16 um)

with smooth pit surfaces. Most recently, A. reducta was also found in the Cerrado biome of

Minas Gerais, suggesting its wide distribution in tropical Brazil.

Key worps — Glomeromycota, Diversisporales, rDNA

Introduction

Recently, the number of new arbuscular mycorrhizal (AM) fungal species

forming acaulosporoid (sensu lato) spores has greatly increased (e.g., Goto

et al. 2008; Palenzuela et al. 2008, 2011; Btaszkowski 2012; Oehl et al. 2015),

984 ... Pereira & al.

especially in the Acaulosporaceae due to concomitant morphological and

molecular spore analyses. Acaulospora species have been increasingly reported

both from colder climates (e.g. Oehl et al. 2011a, 2012; Palenzuela et al. 2013,

2015) and hot tropical areas (e.g., Goto et al. 2010, 2013; Furrazola et al. 2013;

Oehl et al. 2014), with some species found in both cold and hot climates

(e.g., Oehl et al. 2011b).

During surveys of AM fungal diversity in different agricultural and natural

ecosystems in Brazil, two Acaulospora species were found with numerous

conspicuously large depressions on the spore surfaces. Acaulospora excavata,

which had been described from a similar climate zone in Ivory Coast (West

Africa) and which was easily identified by large regular pits covering the spore

surface (Ingleby et al. 1994), had never been subjected to molecular analyses. In

Acaulosporaceae such prominent pits have rarely been found (e.g., for A. foveata

Trappe & Janos and A. bireticulata FM Rothwell & Trappe; Rothwell & Trappe

1979, Janos & Trappe 1982 as summarized in Blaszkowski 2012 and Oehl et al.

2012, 2014). The second fungus, new and described and illustrated herein as

Acaulospora reducta, has similarly large pits that are more irregular and more

complex than A. foveata and A. excavata. The objective of this study was to

analyze the two fungi concomitantly on spore morphology and the ribosomal

ITS region and to compare both with each other and other Acaulospora species.

Material & methods

Study area and sites

Soil samples were collected during the June 201lwet season and subsequent

March 2012 dry season at the Itapirema Experimental Station, Agronomic Institute

of Pernambuco - IPA, located in the municipality of Goiana (7°38’20”S 34°57'10’W;

13 m asl), Pernambuco State, NE Brazil. Acaulospora reducta was found in plantations

of cassava (Manihot esculenta Crantz; Euphorbiaceae), sapodilla (Manilkara zapota (L.)

P. Royen; Sapotaceae), and mahogany (Swietenia macrophylla King; Meliaceae) of the

experimental station, while A. excavata was found only in the sapodilla and mahogany

plantations. The climate in this sampling area is Ams’ (after K6ppen; Kottek et al. 2006),

semi-humid with a tropical monsoon and a dry summer. Mean annual precipitation is

around 2000 mm with a mean annual temperature of 24°C. The soil type is a Ferralsol

(IUSS Working Group WRB, 2014). Pereira et al. (2014) describe the soil chemical

characteristics; the mean soil pH (H,O) was 6.1, organic carbon 7.7 g kg", and available

P 11.0 mg kg".

Acaulospora reducta was additionally found in a coastal ‘restinga vegetation in

Tamandaré (Pernambuco State; 8°46’52”S 35°06’22”W) and Mataraca (Paraiba State;

6°29’S 34°56’W), in a semiarid area with Caatinga vegetation in Petrolina (Pernambuco

State; 9°03’16.68”S 40°18’59.23”W; 389 m asl) and Asst (Rio Grande do Norte State;

5°34’38"S 36°54’30”W; 60 m asl), and in Cerrado Ferralsol soil samples from Sete

Lagoas (Minas Gerais State; 19°27’46”S 44°10’24”W; 738 m asl). The Cerrado soil

was collected in May 2012 at the Embrapa Maize and Sorghum Experimental Station

Acaulospora reducta sp. nov. (Brazil) ... 985

from a subsoil layer and used to prepare substrate for “On Farm’ AM fungal inoculum

production using maize (Zea mays L.) and Urochloa decumbens (Stapf) R.D. Webster as

host plants. Mean soil pH was 5.4 (H,O) with organic carbon 0.29 g kg" and available

P 0.85 mg kg". Lastly, the fungus was also found in Planaltina (near Brasilia) in a no-till

production site grown with soybean (Distrito Federal, 15°36’S 47°42’W; 1014 m asl) and

in a Sorghum production site near the type location in Itapirema (Pernambuco).

Acaulospora excavata was additionally found in a small home garden in Recife

(Pernambuco State; 7°53’06”S and 34°50’11”W) within the rhizosphere of “Palmeira”

(Dypsis lutescens (H. Wendl.) Beentje & J. Dransf.; Arecaceae), a palm known variously

as bamboo palm, golden cane palm, areca palm, yellow palm, or butterfly palm.

Arbuscular mycorrhizal cultures

The native AM fungal communities from the sampling sites in Itapirema were

propagated in trap cultures over three consecutive 4-month cycles using millet (Panicum

miliaceum L.), maize, and sunflower (Helianthus annuus L.) as hosts in 2 L plastic pots

according to Pereira et al. (2014). At the end of each cycle, the plants were allowed to dry

and cut off, with 50 g soil aliquots collected for spore extraction. The soil was reseeded for

the subsequent cycle. The cultures were maintained at the greenhouse of the Mycology

Department, at the Federal University of Pernambuco, Recife, Brazil. However, the two

fungi under study did not sporulate within three consecutive propagation cycles.

Morphological analyses

Spores of the two fungi were extracted from the field soil samples by wet sieving

and sucrose centrifugation according to Sieverding (1991), separated under a

stereomicroscope, and mounted in polyvinyl alcohol-lacto-glycerin (PVLG) and

PVLG + Melzer’s reagent (Brundrett et al. 1994). Slide mountants were dried (3-5 days

at 60°C) to clarify oil drops and observed with a Nikon compound microscope (Eclipse

Ni). The spore terminology follows Oehl et al. (2012), Furrazola et al. (2013), and Goto et al.

(2013) for acaulosporoid glomeromycotan fungi.

Transmission electron microscopy

The samples of the new fungus were dehydrated by immersion in solutions of

increasing concentrations of ethanol (25%, 50%, and 90%) for thirty minutes each

followed by three baths with 100% ethanol. The samples were then immersed in three

30-minute baths in 100% acetone. Dehydrated spores critically point dried in an Emitech

K850, sputter coated with gold in an Emitech K550, and examined under a Zeiss DSM

940 A scanning electron microscope at an accelerating voltage of 5-15 kV. The analyses

were carried out at the Nucleus of Applied Biology microscopy lab at Embrapa Maize

and Sorghum.

Molecular analyses

After isolation from field soil samples, spores from the two fungi were washed

in ultrapure water and sonicated three to four times. The DNA was extracted from

individual spores placed on a slide in a drop (5-10 ul) of ultrapure water, and crushed

with a sterile needle. Crude DNA extract was used as template for a semi-nested PCR

using the primers ITS5+LSUAC (White et al. 1990; 5’-cCATTACGTCAGCATCCTTAGCG-3’)

and ITS1+28G6 (White et al. 1990; 5’-cGGGATTCTCACCCTCTATG-3’) consecutively.

986 ... Pereira & al.

PCR reactions were carried out in a volume of 50 ul, containing 75 mM Tris-HCl pH

8.8, 200 mM (NH,),SO4, 0.01% Tween 20, 2 mM MgCl, 200 uM each dNTPs, 1 uM of

each primer, and 2 units of DreamTaq DNA polymerase (Thermo Scientific, Maryland,

USA); cycling parameters were one cycle of 5 min at 95°C; 40 cycles of 45 s at 94°C,

1 min at 55°C, and 1 min at 72°C; and a final elongation of 7 min at 72°C following the

last cycle. The amplified products (~600) were purified with a GeneJET PCR Purification

Kit (Thermo Scientific, USA) and cloned with a CloneJET PCR Cloning kit (Thermo

Scientific, USA) following the manufacturer's instruction, and then sequenced by the

Central Laboratory of the CCB/UFPE (Recife, Brazil). The phylogeny was inferred

by ITS sequence analyses. These AM fungal sequences were then aligned with other

glomeromycotan sequences from GenBank in ClustalX (Larkin et al. 2007). Sequences

from Claroideoglomus etunicatum (W.N. Becker & Gerd.) C. Walker & A. Schiissler were

used as outgroup. Prior to phylogenetic analysis, the model of nucleotide substitution

was estimated using Topali 2.5 (Milne et al. 2004). Bayesian (two runs over 3 x 10°

generations with a burn in value of 25%) and maximum likelihood (1000 bootstrap)

analyses were performed, respectively, in MrBayes 3.1.2 (Ronquist & Huelsenbeck 2003)

and PhyML (Guindon & Gascuel 2003) launched from Topali 2.5, using the GTR + I

model. Maximum parsimony analysis was performed using PAUP*4b10 (Swofford

2003) with 1000 bootstrap replications.

Taxonomy

Acaulospora reducta Oehl, B.T. Goto & C.M.R. Pereira, sp. nov. FIGs 1-11

MycoBank MB 812332

Differs from Acaulospora excavata by its more irregular pits on the outer spore wall that

have roughened and slightly pitted inner surfaces.

Type: Brazil, Pernambuco State, Goiana, Experimental Station of Itapirema (Instituto

Agronémico de Pernambuco), from a Ferralsol in a cassava plantation, 1.6.2011, coll.

C.M.R. Pereira (Holotype, URM87697; isotypes, URM87698, ZT Myc 55894; GenBank

KM057063-KM057067).

EryMo_oey: reducta (Latin), referring to the small depressions within the large pits on

the outer spore wall.

SPOROCARPS unknown. Acaulosporoid spores form singly in soils laterally on

the tapering hyphal neck of sporiferous saccules.

SPORIFEROUS SACCULES are hyaline to subhyaline to light yellow, rarely

dark yellow, globose to subglobose, 140-210 um in diam. and form terminally

on hyphae. They are generally detached from mature spores. The saccule wall

generally is bi-layered with a rapidly degrading evanescent hyaline to light

yellow outer layer (0.5-1.5 um) and a semi-persistent sub-hyaline to light

yellow to dark yellow inner layer, which is 1.8-2.8(-4.4) um.

SPORES form singly on the saccule necks. They are globose (135-205 um

diam.) to subglobose to oval (145-210 x 130-195 um), whitish yellow to dark

yellow. The spores form three walls: outer wall (OW), middle wall (MW), and

inner wall (IW).

Acaulospora reducta sp. nov. (Brazil) ... 987

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30 : ~ 50ym Mw” ~ ~___ 40 pm

Fics 1-9. Acaulospora reducta. 1. Spore formed laterally on the neck of a sporiferous saccule (sac);

globose saccule terminus already collapsed; 2-3. Crushed spores with three walls: pitted outer

wall (OW), smooth middle wall (MW) and granular inner wall (IW); 4-6. Large, irregular pits

on the outer OW surface in planar view showing a roughened, irregular inner surface structure

resembling small depressions and projections of c. 0.5-1.5 um in diam. and depth; 7. Pits on OW

in cross view; OW with two layers visible (OWL1-2); inner, small pits not clearly visible within the

large depressions; 8-9. MW and IW with multiple layers visible (MWL1-2; IWL1-2).

OUTER WALL consists of three layers, with the outermost layer (OWL1)

evanescent to semi-persistent, 1.5-2.6 um thick, hyaline to subhyaline. In older

spores, the outer wall layer (OWL1) might degrade completely, but usually

some fragments remain on the OWL2 surface. OWL2 is laminated, 6.0-14.5

uum thick, whitish yellow, dark yellow to light brown and has innumerous

irregularly-shaped, often edged to sometimes dumbbell-shaped pits (5.5-19 x

988 ... Pereira & al.

3.5-8.6 tm) that itself have roughened surfaces consisting of small depressions

of approx. 0.5-1.5 um in diam. and 0.5-1.5 um depth) surrounded by projections

of similar dimensions to fine ridges. OWL3 is thin (0.7-1.2 um), yellowish and

tightly attached to OWL2. None of the OW layers stains in Melzer’s reagent.

MIDDLE WALL is bi-layered, hyaline, semi-flexible to flexible and in total

2.0-3.5 um thick. MWLI is 1.0-1.7 um thick, and generally tightly adherent to

MWL2Z, which is 1.0-1.8 um. None of the MW layers stains in Melzer’s reagent.

INNER WALL is three-layered, hyaline, and in total 3.6-5.8(-9.2) um thick.

The outer layer IWL1 is 0.6-1.3 um, with a granular (‘beaded’) structure, and

tightly adherent to IWL2, which is 2.5-3.5 um thick and sometimes, as being

‘amorphous, expands up to 6.9 um in lactic acid based mountants. IWL3 tightly

adheres to IWL2, and is, as only 0.5-1.0 um thick, usually difficult to detect.

IWL2 stains dark red purple in Melzer’s reagent, but this stain may disappear

completely within months in the mountants.

GERMINATION was not observed.

ARBUSCULAR MYCORRHIZA FORMATION so far unknown.

ADDITIONAL SPECIMENS EXAMINED: BRAZIL, PERNAMBUCO STATE, Goiana,

Experimental Station of Itapirema, from a sapodilla plantation, 1.6.2011, coll. C.M.R.

Pereira (URM87699); from a mahogany plantation, 1.6.2011, coll. C.M.R. Pereira

(URM87700); from a Sorghum bicolor production site, coll. D.M.A. Assis, 18.8.2015

(URM88218); Tamandaré, from a ‘restinga vegetation (1.6.2013, coll. D.K.A.

Silva (URM88213); Petrolina, from a natural Caatinga, 1.6.2014, coll. D.K.A. Silva

(URM88214); ParafBaA STATE, Mataraca, from a ‘restinga; 1.6.2012, coll. D.K.A.

Silva (URM88215); Rio GRANDE DO NorTE STATE, Assu, from a natural Caatinga,

15.9.2013, coll. A.S. Medeiros (URM88216). MINAS GERAIS STATE, Sete Lagoas, from a

natural Cerrado forest savanna, 1.5.2012, coll. RA. Souza (URM88217, ZT Myc 55273);

DIsTRITO FEDERAL, Planaltina, from a no-till production site with soybean, 1.3.2014,

coll. J.S. Pontes (URM88219).

PHYLOGENETIC ANALYSES — rDNA sequence analysis of the ITS region inferred

the phylogeny of Acaulosporaceae, placing A. reducta in a monophyletic clade

with high levels of support near A. scrobiculata and A. minuta and supporting

A. reducta as a new species within Acaulospora (Fic. 12). In BLASTn analysis,

the species most closely related to A. reducta was A. scrobiculata (FR692353)

with 97% identity; no environmental ITS sequences deposited in GenBank

corresponded to A. reducta.

DISTRIBUTION — Acaulospora reducta has been recorded from the semi-

humid Atlantic rainforest, the semi-arid Caatinga, and the Cerrado savanna

biomes of tropical Brazil. In the Atlantic Rainforest biome, the fungus was

isolated from agricultural systems in Itapirema (Goiana, Pernambuco State)

and coastal sand dunes with typical ‘restinga’ vegetation in Tamandaré

(Pernambuco State) and Mataraca (Paraiba State). In the Caatinga biome, it

was found in regeneration areas from the National Forest Flona de Acu in Asst

Acaulospora reducta sp. nov. (Brazil) ... 989

Figs 10-11. Acaulospora reducta (transmission electron microscopy). 10. Uncrushed spore with

innumerous large pits on the spore surface and multiple small depressions and projections on

the pit surfaces; 11. Spore segment showing detail view of the multiple small depressions and

projections within the large pits.

(Rio Grande do Norte State) and in a preserved Caatinga area in Petrolina

(Pernambuco State). Finally, in the Cerrado biome it was found in a forest

savanna of Sete Lagoas (Minas Gerais State). It was also found in mycorrhizal

inoculum production cultures of non-sterile soils, derived from this Cerrado

area and cultivated with maize and Urochloa decumbens. The fungus co-

occurred at the type location with A. excavata, among many other AMF species

(see Pereira et al. 2014).

Acaulospora excavata Ingleby & C. Walker, Mycotaxon 50: 100.1994 Fras 13-16

MycoBAank MB 361679

Spores form laterally on the necks of sporiferous saccules, which are

110-190 x 100-165 um. Spores are globose to subglobose to oval (115-195 x

100-180 um), pale ochraceous to pale yellow to yellow orange to sometimes

bright yellow. The spores form three walls: triple-layered outer wall (OWL1-3),

bi-layered middle wall (MWL1-2), and triple-layered inner wall (IWL1-3).

OWL2 with circular to elliptical pits (3.9-20 x 3.5-16 um in diam.; 2.2-6.3

um in depth). IWL1 with ‘beaded, granular structure; IWL2 staining purple to

dark purple in Melzer’s reagent.

SPECIMENS EXAMINED — COTE D’°IVOIRE, southwest of Tissale, Mopri Forest

Reserve, under Terminalia ivorensis, 30 Oct 1990, C. Walker 1674 (OSC 83345, isotype).

BRAZIL, PERNAMBUCO STATE, Goiana, Experimental Station of Itapirema (Instituto

Agronémico de Pernambuco), from a Ferralsol in a sapodilla plantation, 1.6.2011,

coll.: C.M.R. Pereira (URM87701), and from a mahogany plantation, 1.6.2011, coll.:

C.M.R. Pereira (ZT Myc 55895); PERNAMBUCO STATE, Recife, in the rhizosphere of

990 ... Pereira & al.

Claroideoglomus etunicatum AJ239125

C. etunicatum AF004683

0.1

Kuklospora colombiana FR750063

= K. colombiana AJ239117

54 Acaulospora koskei KP191475

64 A. koskei KP191474

A. koskei KP191476

A. viridis HG421738

A. viridis HG421737

A. viridis HG421736

77 99 | A. entreriana FR750172

69 earl A. entreriana FR750169

0.83 ‘i A. entreriana FR750171

6 tr A. colossica AF133764

62. Lf A. colossica AF133770

0.92 A. colossica AF133772

.... } A. laevis AJ242499

7a A. laevis FM876786

0.85 A. laevis FM876780

A. lacunosa AJ891112

A. lacunosa AJ981111

81 A. lacunosa AJ891113

82 A. foveata LN736025

en 0.90 A. foveata LN736026

wo | 52 A. foveata LN736024

0.51} 58 A. alpina AJ891101

0.59 A. alpina AJ891103

A. alpina AJ891106

A. tortuosa HF567933

A. tortuosa HF567937

- A. tortuosa HF567934

50 Acaulospora sp. FN825906

0.57 Acaulospora sp. FN825907

82) ' Acaulospora sp. FN825904

oan A. pustulata HF 567941

; A. pustulata HF567940

wan A. pustulata HF567939

= . scrobiculata FR692349

0.84 A. scrobiculata FR692351

‘ A. scrobiculata FR692350

A. minuta FR821674

A. minuta FR869690

A. minuta FR821672

A. reducta KM057065

89 A. reducta KM057064

86 A. reducta KM057066

A. reducta KM057067

9 A. reducta KM057063

1.00 A. kentinensis FN547522

90 A. kentinensis FM876828

6 A. kentinensis FN547520

1.00] G A. spinosissima HG422732

: A. spinosissima HG422734

88 A. spinosissima HG422733

67 73 A. excavata KM057069

4.00 A. excavata KM057068

1.00 . A. excavata Km057076

A. excavata KM057075

A. excavata KM057074

A. morrowiae AJ242500

94 A. mellea AJ239116

96 A. morrowiae AM905249

tfooy— A. delicata JF439203

A. delicata JF439093

A. mellea JF439090

A. mellea JF439092

56 69 A. cavernata FM876789

74) A. cavernata FR692348

1.00 0.98]' A. cavernata FR692347

‘ A. punctata FR846383

A. punctata FR846384

A. punctata FR846385

A. denticulata AJ239115

A. paulinae AJ891114

60 A. paulinae AJ891116

62 A. paulinae AJ891115

1.00 A. sieverdingii AM076376

: A. sieverdingli FM876793

A. sieverdingii AM076378

80 A. nivalis HE603643

88 A. nivalis HE603644

4.00 A. nivalis HE603642

67 A. baetica LN810994

59 | A. baetica LN811000

06g J Sut A. baetica LN810998

. 77 |_| A. ignota KP191470

1.00 A. ignota KP191472

A. ignota KP191471

Acaulospora reducta sp. nov. (Brazil) ... 991

Dypsis lutescens, 1.7.2012, coll: I.P. de Monte Junior (URM87702, GenBank

KM057068-KM057069; KM057074-KM057076).

Notes — Our morphological analyses revealed no differences between the

type specimens of A. excavata isolated from Mopri Forest Reserve in Ivory

coast (West Africa) and our specimens isolated from Goiana and Recife, in

Northeastern Brazil (Fics 13-16). Our study is the first to present molecular

phylogenetic data for A. excavata (Fic. 12).

PHYLOGENETIC ANALYSES — The ITS sequence analysis grouped A. excavata

with A. spinosissima and A. kentinensis. In BLASTn analysis, the sequences

closest to A. excavata were from A. spinosissima (HG422733-HG422734)

with 94% identity. No environmental ITS sequences deposited in GenBank

corresponded with A. excavata.

Discussion

Acaulospora reducta and A. excavata represent two species with characteristic

large pits on the outer spore wall that easily distinguish them from all other

species in the Acaulosporaceae. While A. excavata has somewhat regularly

sized pits (concerning both diameter and depth) and smooth inner pit surfaces,

A. reducta has more irregular and often edged pits with roughened surfaces

consisting of small depressions and projections inside the large pits, so that the

pits are doubly ornamented, a diagnostic character within the Glomeromycota.

There are only a few other Acaulospora species that have pits close to as large

as those found in A. reducta and A. excavata, such as A. bireticulata, A. foveata,

and A. verna (Rothwell & Trappe 1979, Janos & Trappe 1982, Blaszkowski

2012). Acaulospora foveata has pitted spores similar to A. excavata, but its

spores are reddish brown to brownish black and generally larger than those of

A. reducta and A. excavata (Janos & Trappe 1982, Ingleby et al. 1994), and the

pits are generally more irregular in A. foveata than in A. excavata (Trejo et al.

2015). Acaulospora bireticulata has light brown to brown spores with a double

reticulum on the outer OW surface and regular and conspicuous spines within

the large pits (Rothwell & Trappe 1979, Walker & Trappe 1981). Acaulospora

verna also has rather large pits (3.2-7.8 um if circular to subcircular, or 3.2-4.2 x

9.8-16.2 um if ellipsoidal to oblong), but its spores are clearly smaller (60-95 um)

than those of A. reducta and the pit surfaces are smooth (Blaszkowski 2012).

Fic. 12. Phylogenetic tree of the Acaulosporaceae obtained by analyses from ITS1, 5.8 rDNA, and

ITS2 sequences of different Acaulospora spp. Sequences are labeled with their database accession

numbers. Support values (from top) are from maximum parsimony (MP), maximum likelihood

(ML), and Bayesian analyses. Sequences obtained in this study are in boldface. Only support values

of at least 50% are shown. Thick branches represent clades with more than 95% of support in all

analyses. The tree was rooted by Claroideoglomus etunicatum. (Consistency Index = 0.50; Retention

Index = 0.86).

992 ... Pereira & al.

eV ®

Figs 13-16. Acaulospora excavata. Crushed spores with three walls: pitted outer wall (OW),

smooth middle wall (MW) and granular inner wall (IW); large pits on the outer OW surface,

with smooth surfaces within the pits. 13-14. OSC 83345, Céte dIvoire; 15. URM87701, Brazil;

16. URM87702, Brazil.

Other species (e.g., A. cavernata, Blaszkowski 1989; A. elegans, Gerdemann &

Trappe 1974) have significantly smaller pits than A. reducta and A. excavata.

Phylogenetically, A. reducta and A. excavata form two distinct clades within

Acaulosporaceae. Interestingly, they do not group closely together (Fic. 12) nor

with A. foveata, which was recently analyzed from its type location in Veracruz,

Mexico (Trejo et al. 2015). It would nonetheless be worthwhile to analyze

also A. excavata from its type location in Ivory Coast, West Africa (Ingleby

et al. 1994), from a climatic zone comparable to semi-humid tropical South

America. Its distinctive morphology (Figs. 13-16), however, does support our

species identification of the A. excavata isolates from Pernambuco (Brazil).

Acaulospora reducta is known from three biomes in NE and SE Brazil,

the semi-humid Atlantic rainforest, the semi-arid Caatinga and the Cerrado

savanna biomes. ‘These findings suggest that A. reducta is widely distributed

in tropical Brazil. The morphology of A. excavata, originally described from

tropical West Africa, supports its occurrence in tropical South America. In the

Acaulospora reducta sp. nov. (Brazil) ... 993

present study, molecular phylogenetic data of this fungus were shown for the

first time.

Acknowledgements

The authors especially acknowledge Prof. Janusz Blaszkowski (West Pomeranian

University of Technology, Szczecin, Poland) and Dr. Eduardo Furrazola (Instituto de

Ecologia e Sistematica, IES-CITMA, de la Habana, Cuba) for reviewing the manuscript

and making helpful comments and suggestions. We also appreciate the valuable

corrections and suggestions by Shaun Pennycook, Nomenclatural Editor, and Lorelei

Norvell, Editor-in-Chief. This work was supported by: Protax, INCT-Herbario Virtual

da Flora e dos Fungos, and Sisbiota, all from the Conselho Nacional de Desenvolvimento

Cientifico e Tecnolédgico (CNPq) that provided research grants to LC Maia, BT Goto,

and GA Silva, a scholarship to CMR Pereira, and fellowships to DKA Silva as ‘postdoc’

and to F Oehl as ‘visiting professor: We also thank Marcio Geraldo Martineli from

Embrapa Maize and Sorghum for technical assistance with the SEM analysis and Inacio

Pascoal de Monte Junior (UFPE in Recife) for providing spores of A. excavata.

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MY COTAXON

http://dx.doi.org/10.5248/130.997

Volume 130, pp. 997-1005 October-December 2015

Lecanicillium uredinophilum sp. nov.

associated with rust fungi from Korea

MI-JEONG PARK’, SEUNG-BEOM HONG? & HYEON-DONG SHIN?

‘Horticultural and Herbal Crop Environment Division, National Institute of Horticultural

and Herbal Science, Rural Development Administration, Wanju 55365, Korea

*Korean Agricultural Culture Collection, National Academy of Agricultural Science,

Rural Development Administration, Wanju 55365, Korea

*Division of Environmental Science and Ecological Engineering,

College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Korea

* CORRESPONDENCE TO: hdshin@korea.ac.kr

ABSTRACT — A novel species of Lecanicillium found on rust fungi is described. Lecanicillium

uredinophilum sp. nov. is closely related to L. longisporum but differs morphologically in

having longer phialides. Multiple gene sequence analysis supports its taxonomic position in

the genus Lecanicillium.

KEY worps — Cordycipitaceae, fungicolous species, hyperparasitism, Verticillium-like

hyphomycete

Introduction

The genus Lecanicillium, typified by L. lecanii, was established to

accommodate entomogenous or fungicolous species that had been previously

assigned to Verticillium sect. Prostrata (Gams & Zare 2001). Subsequently, Zare

& Gams (2001) described 14 Lecanicillium species based on a combination of

morphological characteristics and analysis of the internal transcribed spacer

(ITS) rDNA sequence. Subsequently, nine additional taxa have been placed

in Lecanicillium (Kope & Leal 2006, Zare & Gams 2008, Sukarno et al. 2009,

Kaifuchi et al. 2013). Phylogenetically, the genus Lecanicillium is placed in

Cordycipitaceae Kreisel ex G.H. Sung et al. (Sung et al. 2007). The genus is

characterized by elongated conidia and simple phialides that are held in whorls

or singly, arising from prostrate aerial hyphae. Sung et al. (2007) demonstrated

that the genus Lecanicillium is not monophyletic but paraphyletic, a fact that

was inferred from multigene phylogenetic analysis using the nuclear ribosomal

998 ... Park, Hong & Shin

small and large subunits (SSU, LSU), translation elongation factor 1-a (TEF1),

and the largest and second largest subunits of RNA polymerase II (RPB1,

RPB2). More recently, Kouvelis et al. (2008) showed that phylogenetic analysis,

using the three mitochondrial genes and the ITS region combined, placed some

taxonomically uncertain isolates of Verticillium species into Lecanicillium.

During extensive surveys on the occurrence of hyperparasitic fungi attacking

rusts in Korea, three isolates of a Lecanicillium-like fungus were obtained

from the rust sori. In this study, these fungi are characterized based on their

in vivo and in vitro morphology. Furthermore, the phylogenetic positions of

the isolates have been investigated by sequence analysis of the combined genes

SSU, LSU, TEF1, RPB1, and RPB2. As a result, these isolates are identified as

representing a new species of Lecanicillium.

Materials & methods

Isolates

Three collections of rust infected with Lecanicillium were collected from several

localities of Korea during 2008-2009. Single spore isolates from fresh materials were

successfully obtained and grown on PDA at room temperature for 4 weeks in the dark.

The isolates were deposited in the Korean Agricultural Culture Collection (KACC)

of the National Academy of Agricultural Science, Wanju, Korea. Voucher specimens

are housed in the Korea University herbarium, Seoul, Korea (KUS). For this study,

ex-type strains and reference isolates of Lecanicillium species were gathered from the

Centraalbureau voor Schimmelcultures (CBS, the Netherlands) and analyzed together

with the Korean isolates. Detailed information on all the isolates used is shown in

TABLE 1.

Morphological study

Fungal structures from air-dried specimens were mounted in 3% KOH solution,

whereas those from monoconidial isolates were mounted in sterilized water. They

were measured at magnifications of 400x and 1000x, using an eyepiece micrometer

and a model BX51 microscope (Olympus, Tokyo, Japan). Microscopic images were

photographed using an Axio imager microscope (Carl Zeiss, G6ttingen, Germany).

Molecular phylogenetic analysis

DNA EXTRACTION, PCR AMPLIFICATION, AND SEQUENCING — For genomic DNA

extraction, mycelial mats and conidia of the Lecanicillium isolates were harvested by

scraping the surface of colonies that had been grown on PDA plates for one month.

Total genomic DNA was prepared according to the method of Lee & Taylor (1990). PCR

amplification was performed for the SSU rDNA using primers NS1 and NS4 (White

et al. 1990), and for the LSU rDNA using primers LROR and LR7 (Vilgalys & Hester

1990). The PCRs for these two regions of rDNA were conducted as follows: an initial

denaturation for 5 min at 95°C, followed by 35 cycles of denaturation for 1 min at 95°C,

annealing for 1 min at 58°C, extension for 2 min at 72°C, and a final extension for 10

min at 72°C. A fragment of the TEF1 gene was amplified using the primer pair EF1-983F

999

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Lecanicillium uredinophilum sp. nov. (Korea) ... 1001

and EF1-2218R (Rehner 2001). The PCR cycling conditions were as follows: an initial

denaturation for 2 min at 96°C, followed by 35 cycles of denaturation for 30 s at 94°C,

annealing for 30 s at 60°C, extension for 30 s at 72°C, and a final extension for 7 min at

72°C. A region of the RPB1 gene was amplified with the primer pair CRPB1 and RPB1-

Cr (Castlebury et al. 2004). The PCR cycling conditions used were as those described by

Castlebury et al. (2004). A portion of the RPB2 gene was amplified with the primer pair

fRPB2-5F and fRPB2-7cR (Liu et al. 1999). The PCR cycling conditions were as follows:

an initial denaturation for 5 min at 94°C, followed by 35 cycles of denaturation for 1 min

at 94°C, annealing for 10 s at 55°C, extension for 2 min at 72°C, and a final extension

for 10 min at 72°C. The PCR amplicons were purified using MultiScreen HTSTM PCR

filter plates (Millipore, Carrigtwohill, Cork, Ireland). Purified amplicons were directly

sequenced on an ABI Prism™ 377 DNA sequencer (Applied Biosystems, Foster City,

CA, USA) with the same primer pairs used for the PCR amplification.

SEQUENCE ALIGNMENT AND PHYLOGENETIC ANALYSIS — All resulting sequences

were edited using DNAStar Lasergene Seqman™ and EditSeq™ software version

7.1.0 (DNAStar, Madison, WI, USA). Reference gene sequences were retrieved from

GenBank. The RPB1 and PRB2 gene sequences were unobtainable for a few isolates and

thus were treated as missing data in the phylogenetic analysis, and the areas of ambiguity

in the alignments were excluded. Multiple sequence alignments were performed using

MAFFT version 6.864 (Katoh et al. 2005) with the G-INS-i strategy. The alignments

were manually adjusted, if needed. A phylogenetic tree was reconstructed from

the Bayesian inference analysis. The model of DNA substitution was estimated with

jModelTest version 0.1.1 (Posada 2008). The Bayesian inference analysis was performed

using MrBayes version 3.0b4 (Ronquist & Huelsenbeck 2003). Four incrementally

heated simultaneous Markov chains were run for one-million generations, with a

tree saved at every 100th generation. The first 1000 trees generated from this analysis

were discarded. A majority rule consensus tree showing all compatible partitions was

obtained with the posterior probability (PP) for each group. Only PP values =>50% were

indicated in the resulting tree. Branch lengths were computed as the mean values over

the trees sampled. The phylogenetic trees were viewed with TreeViewX version 0.5.0

(Page 1996). A sequence of Simplicillium lanosoniveum (J.F.H. Beyma) Zare & W. Gams

(CBS 101267) served as outgroup for the multigene phylogenetic analysis.

Results

Molecular phylogenetic analysis

The resulting alignment included the sequences of the five genes from 27

Lecanicillium isolates and one outgroup isolate. The alignment consisted of

3999 characters (including alignment gaps) that comprised 952 characters of

SSU, 759 of LSU, 871 of TEF1, 522 of RPB1, and 895 of RPB2. A majority rule

consensus tree (Fic. 2) was generated from the Bayesian analysis, using the

GTR + I + I model selected by jModelTest. The three Korean isolates found

on uredinia of the rust fungi Pucciniastrum agrimoniae and Coleosporium sp.

clustered into a separate clade. The three isolates (KACC 47756, KACC 44066,

and KACC 44082), forming a distinct group (PP = 1.00), were closely related

1002 ... Park, Hong & Shin

with three Lecanicillium species, L. muscarium (Petch) Zare & W. Gams,

L. pissodis Kope & I. Leal, and L. attenuatum Zare & W. Gams, within a strongly

supported cluster (PP = 1.00).

Taxonomy

Lecanicillium uredinophilum M.,). Park, S.B. Hong & H.D. Shin, sp. nov. FIG. 1

MycoBAank MB 814832

Differs from Lecanicillium longisporum by its longer phialides.

Type: Korea, Gangwon Province, Hongcheon, Seongdong-ri, 37°44’41”N 127°52’01’E,

on uredinia of Pucciniastrum agrimoniae (Dietel) Tranzschel growing on Agrimonia

pilosa Ledeb. (Rosaceae), 26 October 2008, collected and isolated by M.J. Park and H.D.

Shin (Holotype, KUS-F23890; ex-type culture, KACC 44082; GenBank KM283758,

KM283782, KM283806, KM283828, KM283848).

ErymMo.ocy: The epithet refers to the uredinicolous habit of this fungus.

In vivo: Colonies on rust sori, loose or floccose, white. Mycelium partly

immersed in rust sori but mostly superficial. Phialides rather long, 15-50 x 1-3

um, hyaline, tapering towards the apex, arising from prostrate hyphae, solitary

or up to 3-4 per node; secondarily produced phialides present. Conidia 3-6 x

1.5-3 um, hyaline, cylindric to oblong or narrowly ellipsoid, aseptate.

In vitro: Colonies attaining 25-30 mm diameter in 10 d on PDA, white to

cream colored, velvety to cottony; margin entire; reverse cream colored.

Phialides 20-60 x 1-2.5(-3) um, produced singly or in whorls of up to 3-4(-5)

on prostrate hyphae. Conidia 3-9 x 1.8-3 um, cylindric, oblong, or ellipsoid,

mostly aseptate, rarely uniseptate.

ADDITIONAL SPECIMENS EXAMINED:

On uredinia of Coleosporium sp. growing on Aster sp. (Asteraceae): KOREA, GANGWON

PROVINCE, Pyeongchang, Korea Botanic Garden, 37°42’24”N 128°36'49”E, 9 October

2008, collected and isolated by M.J. Park and H.D. Shin (KUS-F23782; culture, KACC

44066).

On uredosori of Pucciniastrum agrimoniae growing on Agrimonia pilosa: KOREA,

NORTH CHUNGCHEONG PROVINCE, Boeun, Okhwa recreational forest, 36°35’52”N

127°41'48”E, 2 August 2009, collected and isolated by M.J. Park (KUS-F24383; culture,

KACC 47756).

Discussion

Three Korean fungal isolates found as rust hyperparasites were shown to

represent a new species, Lecanicillium uredinophilum. The morphology of

Fic. 1. Lecanicillium uredinophilum. A: Colonies overgrowing uredinia of Pucciniastrum agrimoniae

on Agrimonia pilosa. B: Non-hyperparasitized (left) and hyperparasitized (right) uredinia.

C: Rust propagules densely intertwined with hyphae of L. uredinophilum. D-G, O: Verticillate

phialides. H: Branched phialide. I-N: Solitary phialides. P: Conidia. Scale bars: C = 100 um,

D, F = 50 um, E, G-P = 20 um.

Lecanicillium uredinophilum sp. nov. (Korea) ... 1003

1004 ... Park, Hong & Shin

L. uredinophilum resembles that of L. longisporum (Petch) Zare & W. Gams,

which differs by its shorter phialides (20-40 um; Zare & Gams 2001). Based

on their ITS rDNA (not presented in this study) and multigene phylogenies,

the three L. uredinophilum isolates form a well-supported distinct clade that

is distant from other Lecanicillium taxa. Morphological and phylogenetic

analyses show that the isolates represent a previously unknown species, here

described as L. uredinophilum.

Although the main hosts of Lecanicillium species are known to be insects

and mushrooms (Zare & Gams 2001), the present investigation indicates that

rust fungi are another major ecological substrate for Lecanicillium species. The

discovery of Lecanicillium species associated with rust sori suggests that more

species of Lecanicillium and Verticillium-like fungi remain to be discovered as

hyperparasites of rusts worldwide.

Acknowledgments

The authors are grateful to the pre-submission reviewers Eric HC McKenzie and

Gi-Ho Sung for their critical comments on the manuscript. This work was supported

by a grant from the Regional Subgenebank Support Program of Rural Development

Administration, Republic of Korea.

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RNA polymerase II subunit. Molecular Biology and Evolution 16: 1799-1808.

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1253-1256. http://dx.doi.org/10.1093/molbev/msn083

Lecanicillium uredinophilum sp. nov. (Korea) ... 1005

Lecanicillium uredinophilum KACC 47756

1.00 |'L. uredinophilum KACC 44066

L. uredinophilum KACC 44082

oggr L. muscarium CBS 143.62

1.00|'"- 1. pissodis CBS 118231

1.00

any L. attenuatum CBS 402.78

1.00 7 L. lecanii CBS 101247

Oe L. lecanii CBS 102067

1.00 100 L. longisporum CBS 102072

L. longisporum CBS 126.27

1.007 L. dimorphum CBS 345.37

1.00 1.00 L. dimorphum CBS 363.86

abe L. aphanocladii CBS 797.84

L. saksenae CBS 532.81

1.00 L. fusisporum CBS 162.70

L. fusisporum CBS 164.70

1.00 r L. flavidum CBS 300.70D

1.00 L. flavidum CBS 342.80

L. fungicola var. fungicola CBS 992.69

L. fungicola var. aleophilum CBS 357.80

0.72 L. tenuipes CBS 309.85

L. psalliotae CBS 367.86

1.00 0.60 L. antillanum CBS 350.85

L. aranearum CBS 726.73A

1.00 Lecanicillium sp. CBS 639.85

L. acerosum CBS 418.81

L. wallacei CBS 101237

0.50 1.00

Simplicillium lanosoniveum CBS 101267

0.05

Fic. 2. Phylogenetic relationship among taxa belonging to the genus Lecanicillium based on

Bayesian analysis of the combined SSU, LSU, TEF1, RPB1, and RPB2 sequences. Isolates obtained

from this study are in bold. The numbers at the nodes indicate the Bayesian inference posterior

probability (BI PP) values. Only PP values 250% are shown. The number of nucleotide changes

between taxa is represented by branch length and the scale bar indicates the number of nucleotide

substitutions per site.

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http://www.aftol.org/pdfs/EF1primer.pdf

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models. Bioinformatics 19: 1572-1574. http://dx.doi.org/10.1093/bioinformatics/btg180

Sukarno N, Kurihara Y, Ilyas M, Mangunwardoyo W, Yuniarti E, Sjamsuridzal W, Park JY,

Saraswati R, Inaba S, Widyastuti Y, Ando K, Harayama S. 2009. Lecanicillium and Verticillium

species from Indonesia and Japan including three new species. Mycoscience 50: 369-379.

http://dx.doi.org/10.1007/s10267-009-0493-1

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classification of Cordyceps and the clavicipitaceous fungi. Studies in Mycology 57: 5-59.

http://dx.doi.org/10.3114/sim.2007.57.01

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ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: 4238-4246.

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

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

methods and applications. Academic Press, San Diego, USA.

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Simplicillium gen. nov. Nova Hedwigia 73: 1-50.

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the genus Lecanicillium. Mycological Research 112: 811-824.

http://dx.doi.org/10.1016/j.mycres.2008.01.019

MY COTAXON

http://dx.doi.org/10.5248/130.1007

Volume 130, pp. 1007-1016 October-December 2015

DNA barcoding is an effective tool

for differentiating Pisolithus species from Macedonia

KATERINA RUSEVSKA’, MITKO KARADELEV', CHERDCHAI PHOSRI’,

MARGARITA DUENAS?, M. TERESA TELLERIA?, ROY WATLING‘ &

Maria P. MARTIN?

' Ss. Cyril and Methodius University, Faculty of Natural Science and Mathematics,

Institute of Biology, Arhimedova 5, Skopje, 1000, Macedonia

? Biology Programme, Faculty of Science, Nakhon Phanom University,

167 Naratchakouy Sub-District, Muang District, Nakhon Phanom, 48000, Thailand

° Departamento de Micologia, Real Jardin Botdnico, RJB-CSIC,

Plaza de Murillo 2, 28014, Madrid, Spain

‘Caledonian Mycological Enterprises,

Crelah, 26 Blinkbonny Avenue, Edinburgh, EH4 3HU, Scotland

* CORRESPONDENCE TO: kruseska@pmf.ukim.mk

ABSTRACT — With the aim of clarifying the number of Pisolithus species in Macedonia,

molecular analysis was carried out on seven specimens collected in oak forests in Macedonia

during 1987 and 2005. New sequences were compared with previously published sequences

included in public databases. Three Pisolithus species are identified in Macedonia: P. arhizus,

P. capsulifer, and P. tinctorius.

KEY worDs — gasteromycetes, Boletales, ITS nrDNA, phylogeny

Introduction

Pisolithus Alb. & Schwein. is a genus widely distributed across temperate to

tropical regions and forming ectomycorrhiza with many different woody plants

(Marx 1977, Chambers & Cairney 1999). Phylogenetic studies conducted

particularly in the last decade have pointed to the presence of several species in

this genus (Anderson et al. 1998, 2001; Martin et al. 1998, 2002; Gomes et al.

2000; Diez et al. 2001) that was for a long time considered monotypic (Coker &

Couch 1928, Cunningham 1942, Pilat 1958). Recently several new species have

been recognized, such as P orientalis Watling et al., associated with Pinus kesiya

(Phosri et al. 2012); the new combination P capsulifer (Phosri et al. 2012);

P. calongei M.P. Martin et al., mycorrhizal with Cistus (Martin et al. 2013);

1008 ... Rusevska & al.

P. croceorrhizus P. Leonard & McMullan-Fisher associated with Myrtaceae

(Leonard et al. 2013); a close relative of Pisolithus sp. 10 Martin et al. (2002);

and Pisolithus sp. 12/13 (Leonard et al. 2013). These new analytical methods

have confirmed previous hypotheses based on macroscopic and microscopic

variations proposed by Bronchart et al. (1975), Calonge & Demoulin (1975),

and Demoulin & Dring (1975). The only species recognized in Macedonia based

on morphological features is Pisolithus arhizus (Karadelev et al. 2008), which is

included in the Macedonian Red List of Fungi (Karadelev & Rusevska 2013). In

order to determine the precise number of Pisolithus species in Macedonia, we

decided to analyze the internal transcribed spacer region of nuclear ribosomal

DNA (ITS nrDNA) obtained from several different specimens collected in

Macedonia.

Materials & methods

Morphological studies

Dried samples used in this study are deposited in the Macedonian Collection of

Fungi, Mycological Laboratory, Faculty of Natural Sciences and Mathematics, Sts. Cyril

and Methodius University, Skopje, Macedonia (MCF). Morphological identification of

the specimens has been made according to Jiilich (1984), Pegler et al. (1995), Calonge

(1998), Krieglsteiner (2000), Sarasini (2005), Knudsen & Vesterholt (2012). Microscopic

measurements and photographs were made from tissue sections mounted in 5% KOH

and examined at magnifications up to 1000x with an LW Scientific microscope. SEM

was carried out after coating dried glebal samples in gold with Balzers SCD 004 sputter

coater with a Hitachi S-3000N SEM. 30 spores were measured from each collection.

The species distribution map was generated in ArcGIS 10.1 based on Digital Elevation

Model (ASTERGDEM) (http://asterweb.jpl.nasa.gov/gdem.asp, 09. 2012).

Molecular analyses

DNA extraction, amplification, and sequencing of the ITS regions were made

from glebal tissue of dry field collected basidiomes using the protocols described in

Phosri et al. (2009). The ITS region, including the 5.88 rDNA the ribosomal RNA gene

cluster and small flanking parts of SSU and LSU genes, was amplified by PCR with

ITS1F and ITS4 primer pair (White et al. 1990, Gardes & Bruns 1993) according to

the cycling protocol in Martin & Winka (2000). Five pl aliquots of PCR products were

verified by gel electrophoresis in 2% Pronadisa D-1 Agarose (Lab. Conda, Spain). The

amplified products were purified with QIAquick gel PCR purification kit (QAGen,

Valencia, California) and sequenced by Macrogen, Inc. (South Korea). The new

Pisolithus sequences were aligned with 99 sequences obtained from Phosri et al. (2012)

and analyzed using a heuristic search in PAUP 4.0b10 (Swofford, 2003) and under a

Bayesian approach (Huelsenbeck et al. 2000, Larget & Simon 1999) assuming a HKY+G

Fic. 1. Phylogenetic tree (one of 100 most parsimonious trees) obtained with a parsimony analysis

under heuristic search of ITS nrDNA sequences of Pisolithus specimens (indicated with the MCF

herbarium number) and sequences from Phosri et al. (2012), with sequences of Suillus luteus and

Scleroderma citrinum as outgroup.

Pisolithus in Macedonia ... 1009

GU373495 Suillus luteus

FM213344 Scleroderma citrinum

AF415227 Shorea macropera, Malaysia ji A

100 AF415226 Shorea macropera, Malaysia I P_ aurantloscabrosus (P. species)

AY756113 Vateria indica, India =_—-: P. indicus (P. species 13)

100, AFO03915 Afzelia sp., Kenya | Pp jest

AF228653 Afzelia sp., Kenya = Species

FR748119 Shorea sp., Thailand

AB099921 Shorea roxburgii, Thailand

Al | 400 {/K 48099920 Shorea roxburgii, Thailand P. abditus (P. species 12)

FR748120 Thailand

AB099922 Dipterocarpus alatus, Thailand

AF003914 Eucalyptus camaldunensis, Kenya

AF004733 Eucalyptus sp., New South Wales

AF374719 Eucalyptus calophylla, Australia 5

99 AF004734 Eucalyptus sp., New South Wales P. marmoratus (P. species 2)

AF440866 Eucalyptus plantation, China

AF374665 Eucalyptus patens, Australia

AF228641 Cistus ladanifer, Spain

83 FR748140-FR748142 (3 clones) Cistus sp., Spain

100 [| AF228642 Cistus ladanifer, Spain P. calongei (P. species 3)

AF228643 Cistus ladanifer, Spain

AF228644 Cistus ladanifer, Spain q

AF374624 Acacia sp., Australia == P. species 10

AF347700 Eucalyptus sp., Brazil

AF004735 Eucalyptus sp., New South Wales

AF142991 Eucalyptus sp., Brazil

AF228657 Eucalyptus sp., Morocco

AF 140547 Eucalyptus sp., Brazil P. microcarpus (P. species 9)

AF374704 Eucalyptus dunni, Brazil

56 66 U62666 Eucalyptus citriodora, Brazil

AF374661 Eucalyptus camaldunensis, Australia

AF374708 Eucalyptus sp./Acacia sp., Australia

AF004732 Eucalyptus sp., New South Wales

AF270786 Eucalyptus sp., Australia

AF874622 Acacia holosericea, Senegal

FR748122 Eucalyptus camaldunensis, Thailand

FR748123 Eucalyptus camaldunensis, Thailand

AF228656 Eucalyptus sp., Spain

87 AF228655 Eucalyptus sp., Morocco

AF228654 Eucalyptus sp., Morocco

AB099918 Thailand

FR748121 Eucalyptus camaldunensis, Thailand

B ABO099909 Eucalyptus camaldunensis, Thailand

99 AF416589 Eucalyptus tereticornis, India P. albus complex

AF440868 Eucalyptus sp., China (P. species 7)

AF374675 Eucalyptus sp., Queensland

AF004736 unknown potential host, New South Wales

AF004737unknown potential host, New South Wales

FR748124 Eucalyptus camaldunensis, Thailand

AJ629887 Thailand

FR748125 Eucalyptus sp., Hawaii, USA

FR748127 Melaleuca sp., Thailand

AB099911 Thailand

AF374638 Acacia sp./Mangium sp., Malaysia

FR748126 Eucalyptus sp., Australia

AF374646 Eucalyptus globulus, Australia == P. species 10

AF440867 Eucalyptus plantation, China) === P. microcarpus (P. species 9)

AF003916 Pinus caribea, Kenya

AF228645 Quercus ilex/Q. coccifera, Spain

AF228646 Quercus ilex/Cistus ladanifer, Spain

FR748138 Pinus sp., Spain

AF143234 Unknown potentital host, France

58 |! AF143233 Pinus sp., USA

OS5MCF5506

FR748139 Quercus sp., USA

AF228647 Pinus caribea, Kenya

93 AF374625 Pinus kesiya, Thailand

AF374679 Eucalyptus sp., China

AF374711 Pinus sp./Eucalyptus sp., China

AB099919 Pisolithus sp., Thailand

FR748149 Pinus kesiya, Thailand

54 FR748147Pinus kesiya, Thailand 7 A :

FR748146 Pinus kesiya, Thailand P. orientalis (P. species 5)

AB099845 Pisolithus sp., Thailand

FR748145 Pinus kesiya, Thailand

FR748148 Pinus kesiya, Thailand

FR748143 Pinus kesiya, Thailand

FR748144 Pinus kesiya, Thailand

FM213365 Pinus sp., Spain

A ll AF228648 Quercus ilex/Q. coccifera, Spain

FR748128 (clone a),FR748131 (clone d) Will Wood, Sardinia, Italy

02MCF9560

O5MCF5324

87MCF7741 . a

ret ve anione c) Will Wood, Sardinia, Italy P. arhizus (P. species 4)

FR748129 (clone b) Will Wood, Sardinia, Italy

AF228649 Quercus ilex, Spain

AF228651 Quercus ilex, Spain

54| || AF228650 Pinus halepensis/Quercus coccifera, Spain

AF228652 Pinus halepensis, Spain

FR748132 (clone f) Will Wood, Sardinia, Italy

FR748133 Pinus sp., Spain

AF374627 Pinus sp. South Africa

AF374629 Pinus pumila/Betula ermanii, Japan

UDB001206 Pinus forest, Sweden

78) L o3Mcr3428

FR748134 unknown potential host, England P. capsulifer (P. species 14)

02MCF3355

FR748135 unknown potential host, England

FR748138 mixed forest, France

FR748136 Pinus sylvestris/Pinus contorta, England

P. tinctorius (P. species 6)

— 5 changes

1010 ... Rusevska & al.

model with MRBAYES 3.0 (Huelsenbeck & Ronquist 2001), as described in Phosri et

al. (2012). The phylogenetic tree was visualized in Treeview (Page 1996) and edited in

Adobe Illustrator CS3. The names of clades and subclades follow Phosri et al. (2009).

Results

Genomic DNA concentration ranged from 1.5 to 15 ng/ul. The ITS region

was successfully amplified and sequenced from 7 collections of dried Pisolithus

basidiomes using ITS1F/ITS4 primer pair. After purification of the amplified

product, the DNA concentration was greater than 20 ng/ul, and sequenced

directly. The seven new sequences obtained in this study have been submitted

to the international database (EMBL).

The phylogenetic tree (Fic. 1) obtained under heuristic search has similar

topology with the 50% majority-rule tree of the Bayesian analysis (data not

shown). New sequences are well grouped in linage AII (Phosri et al. 2012), but

in three main clades, each corresponding to one Pisolithus species.

One sample grouped in the P tinctorius clade. Four Pisolithus records that

were placed in the P. arhizus clade were recorded from three different localities:

Pogana (kermes oak shrubland), Galichica Mountain (Macedonian oak forest),

and Skopska Crna Gora Mountain (oak-hornbeam forest). Two Pisolithus

collections, both originating from Bistra Mountain, were placed in the

P. capsulifer clade.

Pisolithus tinctorius (Pers.) Coker & Couch,

Gasteromycetes East. U.S.: 170. 1928. RIG. 2

BASIDIOMES pyriform with almost ellipsoid head, slightly flattened at the

apex, 55-60 x 40-50 mm and a pseudostipe, 40-50 x 20-25 mm, narrowing

toward the base, <10 mm; PERIDIUM yellowish olivaceous, very thin (<1 mm),

showing PSEUDOPERIDIOLES as black ellipsoid patches.

BASIDIOSPORES globose, 8.4-11.6 tm, in average 9.9 um (including spines);

spines 1.2-2.3 um (average = 1.6 um) long, more or less isolated.

SPECIMEN EXAMINED: MACEDONIA, SkopskA CRNA GORA MOUNTAIN, 800-900

m a.s.l., in meadow close to deciduous forest (Quercus spp., Carpinus orientalis, and

planted Castanea sativa), 9-Oct-2010, leg. K. Rusevska (OSMCF5506, LK024184).

Pisolithus arhizus (Scop.) Rauschert, Z. Pilzk. 25: 51. 1959. FIG.-3

BASIDIOMES with subglobose to ellipsoid head, 30-60 x (40-)55-90 mm

tapering into irregular pseudostipe <95 mm x (20-)25-40(-50) mm, with

narrow base <10 mm diam; PERIDIUM pale ochre to brown black.

BASIDIOSPORES globose, 8.4-11.6 tum, in average 9.9 um (including spines);

spines 1.2-2.3 um (average = 1.2 um) long, isolated but very compact, in parts

forming small pyramids.

Pisolithus in Macedonia... 1011

Fi. 2. Pisolithus tinctorius (OSMCF5506). a. basidioma; b. spores (LM); c. spores (SEM).

Scale bars: a = 1 cm; b= 10 um; c= 1 um.

Fic. 3. Pisolithus arhizus (OSMCF5324). a. basidioma; b. spores (LM); c. spores (SEM).

Scale bars: a= 1 cm; b= 10 um; c = 5 um.

1012 ... Rusevska & al.

SPECIMENS EXAMINED: MACEDONIA, GALICHICA NATIONAL PARK, between

Oteshevo and Kale, 900-1000 m a.s.l., Macedonian oak forest, 8-Oct-1988, leg. M.

Karadelev (02MCF9560, LK024180); Gradishte, at roadsides, 800 m a.s.l., grassy place

in Macedonian oak forest, 10-Nov-2005, leg. K. Rusevska (OS5MCF5324, LK024181);

PoGANa, 300 m a.s.l, meadow in Quercus coccifera forest, 28-Oct-1987, leg. M.

Karadelev (87MCF7741, LK024178); SkopskKA CRNA GORA, above Chucher-Sandevo

vill, 600 m a.s.1., degraded oak forest (Querco-Carpinetum orientalis), 6-Oct-1998, leg.

M. Karadelev (98MCF4331, LK024179).

Fic. 4. Pisolithus capsulifer (03 MCF3428). a. basidioma; b. spores (LM); c. spores (SEM).

Scale bars: a = 1 cm; b = 10 um; c= 5 um.

Pisolithus capsulifer (Sowerby) Watling, Phosri & M.P. Martin,

Mycotaxon 120: 202. 2012. FIG. 4

BASIDIOMEs with globose to subglobose head, 25-35 x 45 mm, pseudostipe

40 mm long, 20-22 mm wide, and incorporating soil particles; PERIDIUM pale

Pisolithus in Macedonia... 1013

ocher to black; PSEUDOPERIDIOLES dark yellow when cut, becoming darker and

finally black on drying.

BASIDIOSPORES globose, 6.8-11.0 tm, in average 9.3 um (including spines);

spines 1.1-2.2 um (average = 1.6 um) long, mainly united in conical structures.

SPECIMENS EXAMINED: MACEDONIA, BisTRA MOUNTAIN, above Tresonche vill., 1100

m a.s.l., open place in deciduous forest, on cinnamon soil, 1100 m a.s.l., 3-Aug-2002,

leg. K. Rusevska (02MCF3355, LK024182); 12-Jul-2003, leg. K. Rusevska (03 MCF3428,

LK024183).

®@ P arhizus © P capsulifer @ P tinctorius

Fic. 5. Distribution map of Pisolithus collections from Macedonia analyzed in this study.

Discussion

The analyzed Pisolithus samples were obtained from four different localities

in Macedonia: Pogana, and mountains Bistra, Galichica, and Skopska Crna

Gora (FIG. 5).

Bistra Mountain is situated in western Macedonia. Pisolithus capsulifer, a

new species for the mycobiota of Macedonia, was found near Tresonche village.

The samples were collected at approximately 1000 m on cinnamon soil in an

open area with common juniper shrubs. The site is close to a xerophytic and

1014 ... Rusevska & al.

thermophilic community of pubescent oak and hop hornbeam as dominant

trees; Acer monspessulanum and Fraxinus ornus are also present. Pisolithus

capsulifer is known from a few sites in three European countries (Sweden,

France, and England) and Japan (Phosri et al. 2012).

Pogana, which lies in southern Macedonia, is one of three P arhizus

localities. The dominant forest species, evergreen shrub kermes oak, forms a

well-developed forest community (Coccifero-Carpinetum orientalis) together

with Quercus pubescens, as well as Phillyrea media, the dominant evergreen

shrub. Several other plant species also present are Pistacia terebinthus, Fraxinus

ornus, Coronilla emeroides, Colutea arborescens, Crataegus heldreichii, Lonicera

etrusca, and Cistus villosus.

Galichica Mountain is situated between two lakes in southwestern

Macedonia. Pisolithus arhizus is known from two mountain sites (one on the

Lake Ohrid side and the other on the Lake Prespa side) between 800 and 1000

m altitude. The collections were made in Macedonian oak forest with Quercus

trojana dominant and Q. cerris, Q. pubescens, Carpinus orientalis, and Fraxinus

ornus also present.

The mountain Skopska Crna Gora lies in northern Macedonia. Two

Pisolithus species were found on different sites in oak-hornbeam forests.

Pisolithus arhizus was collected at 600 m from a degraded forest, while

P. tinctorius was recorded at 800 m in a well-developed association mixed with

planted sweet chestnut trees.

Using ITS barcodes (see Schoch et al. 2012) to analyze fungi has greatly

clarified the Pisolithus complex in Macedonia. Our study confirms the

presence of three Pisolithus species in the country: P arhizus, P. capsulifer, and

P. tinctorius, all growing near oak trees in deciduous forests. Thus far,

P. capsulifer appears specific to a single locality and substrate (terra rossa on

silicate ground). Pisolithus arhizus, as noted, is already included in the National

Red List of Fungi. The two new species for Macedonia, P capsulifer and

P. tinctorius, are also rare and should be protected.

As P. tinctorius and P. arhizus have the same spore size and P. arhizus and

P. capsulifer have a similar peridium colors, it is almost impossible to separate

these species morphologically. In the face of such similar microscopic and

macroscopic characters, we do not propose a key here.

Acknowledgments

This research received support from the SYNTHESYS Project http://www.synthesys.

info/ (financed by European Community Research Infrastructure Action under the FP7

“Capacities” Program). Thanks to Y. Ruiz for SEM technical assistance. The authors are

grateful to for pre-submission comments and suggestions provided by Mikael Jeppson

and Anthony Whalley.

Pisolithus in Macedonia... 1015

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

MY COTAXON

http://dx.doi.org/10.5248/130.1017

Volume 130, pp. 1017-1038 October-December 2015

Five new species of Hymenoscyphus (Helotiaceae, Ascomycota)

with notes on the phylogeny of the genus

HuANn-D1I ZHENG & WEN- YING ZHUANG

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

No. 1 Beichenxi Road, Chaoyang District, Beijing 100101, China

* CORRESPONDENCE TO: zhuangwy@im.ac.cn

ABSTRACT — Five new species of Hymenoscyphus—H. aurantiacus, H. macrodiscus,

H. ginghaiensis, H. tetrasporus, and H. yui—are recognized by morphological characters

and DNA sequence data. Detailed descriptions, illustrations, and discussions concerning

morphologically similar and phylogenetically closely related species are provided for each

species. The phylogeny of 39 Hymenoscyphus species was explored based on the individual

internal transcribed spacer of nrDNA, D1/D2 domain of 28S nrDNA, and partial B-tubulin

gene sequences, as well as the combined dataset. The sampled species clustered as a highly

supported group in the phylogenetic trees with six main clades formed. Correlations between

the morphological and phylogenetic features are discussed.

Key worps — China, Leotiomycetes, taxonomy

Introduction

The genus Hymenoscyphus was established by Gray (1821) and lectotypified

with H. fructigenus (Bull.) Gray (Dennis 1964). It is one of the largest genera

of Helotiaceae and has a cosmopolitan distribution (Kirk et al. 2008). About

155 species were known in the genus according to Kirk et al. (2008) with a few

more species added subsequently (Han & Shin 2008; Queloz et al. 2011; Baral

& Bemmann 2013; Zheng & Zhuang 2013a,c, 2014; Gross & Han 2015; Gross

et al. 2015).

Species of Hymenoscyphus are mainly saprotrophic on plant materials such

as wood, twigs, fruits, leaves and herbaceous stems, producing small, stipitate

to sessile, white- to yellow-colored apothecia and having an ectal excipulum

composed of non-gelatinized textura prismatica to textura angularis parallel or

at a low angle to the receptacle surface and fusoid, ellipsoid, to scutuloid hyaline

ascospores. Morphological characteristics used to identify Hymenoscyphus

1018 ... Zheng & Zhuang

species include apothecial shape, size, and color; excipular structure; the ascal

apical apparatus, basal structure, shape, and size; and shape, size, guttulation,

septation, and presence of cilia of ascospores. Habitat is another useful feature

for species recognition. Sequence data of the internal transcribed spacer

of nuclear ribosomal DNA (ITS) have been introduced to aid identification

(Zhang & Zhuang 2004, Baral et al. 2006, Han & Shin 2008, Johnston & Park

2013, Zheng & Zhuang 2013a), with multilocus phylogenetic approaches

further applied to establish a clear species concept of the H. albidus complex

(Queloz et al. 2011, Zheng & Zhuang 2014, Gross & Han 2015, Gross et al.

2015).

A total of 42 Hymenoscyphus species have been reported from 28 provinces

of China (Teng 1963; Tai 1979; Korf & Zhuang 1985, 1989; Zhuang 1995,

1996, 1999; Wu et al. 1996; Zhuang & Wang 1998; Yu et al. 2000; Zhang &

Zhuang 2002a,b, 2004; Zheng & Zhuang 2011, 2013a,b,c, 2014), of which 12

were originally described from China (Zhuang 1996; Zhang & Zhuang 2002a,b,

2004; Zheng & Zhuang 2013a,c, 2014; Liu & Zhuang 2015). In this work, five

new species are established based on morphological and molecular data.

Materials & methods

Morphological study

Specimens examined were collected from Hubei, Qinghai, Xinjiang, and Yunnan

provinces and deposited in Herbarium Mycologicum, Academiae Sinicae, Beijing,

China (HMAS). Colors of fresh apothecia were recorded in field notes. Dry apothecia

were rehydrated with distilled water and sectioned longitudinally at a thickness of 8-15

um using a Yidi YD-1508A freezing microtome (Jinhua, China). Measurements were

taken from the longitudinal sections and from squash mounts in lactophenol cotton

blue solution using an Olympus BH-2 microscope (Tokyo, Japan). Iodine reactions of

ascus apparatuses were tested in Melzer’s reagent and Lugol’s solution with or without

pretreatment by 3% KOH (Baral 2009). Features of ascus apical ring were recorded

according to Baral (1986, 1987). Photographs were taken using a Canon G5 digital

camera (Tokyo, Japan) connected to a Zeiss Axioskop 2 Plus microscope (G6ttingen,

Germany) for anatomical structure (lactophenol cotton blue as mounting medium

except for otherwise mentioned) and to a Zeiss Stemi 2000C stereomicroscope

(Gottingen, Germany) for gross morphology.

DNA extraction, PCR amplification, and sequencing

DNA was extracted from dry apothecia using the CTAB procedure (White et al.

1990) with some modifications. Primer pairs used for amplification and sequencing

included ITS1/ITS4 or ITS5/ITS4 (White et al. 1990) for ITS, LROR (Moncalvo et al.

1995) and LRS (Vilgalys & Hester 1990) for D1/D2 domain of 28S nrDNA (LSU) and

Bt3-LM/Bt10-LM (Myllys et al. 2001) for partial B-tubulin gene (BTU).

PCR amplifications had a final volume of 25 ul, containing 2.5 ul 10x PCR buffer

(including MgCl), 0.5 ul dNTP (10mM each), 1.25 ul of each primer (10 mM),

Hymenoscyphus spp. nov. (China) ... 1019

TABLE 1. Sequences of Hyaloscypha (Hya.) and Hymenoscyphus (H.) used in the

phylogenetic analyses, with voucher specimen information and GenBank

accession numbers

SPECIES

Hya. aureliella

Hya. fuckelii

H. albidoides H.D. Zheng &

W.Y. Zhuang

H. albidus (Gillet) W. Phillips

H. aurantiacus

H. berggrenii (Cooke & W. Phillips)

Kuntze

H. brevicellulus H.D. Zheng &

W.Y. Zhuang

H. calyculus

H. caudatus

H. crataegi Baral & R. Galan

H. dehlii M.P. Sharma

H. epiphyllus (Pers.) Rehm

ex Kauffman

H. fraxineus (T. Kowalski) Baral et al.

H. fructigenus

H. fucatus (W. Phillips) Baral &

Hengstm.

H. ginkgonis J.G. Han & H.D. Shin

H. globus W.Y. Zhuang &

Yan H. Zhang

H. haasticus P.R. Johnst.

H. himalayensis (K.S. Thind &

H. Singh) KS. Thind & M.P. Sharma

H. hyaloexcipulus

TABLE 1, concluded on p. 1020

SPECIMEN/

STRAIN NO.

M234

M233

HMAS 264140

HMAS 264142

Lav_01

90812.1

HMAS 264143

HMAS 264144

ICMP 19614

ICMP 19615

HMAS 264018

HMAS 264016

HMAS 264145

HMAS 264146

HMAS 82057

HMAS 82060

F156966

HMAS 264160

HMAS 82075

HMAS 82076

HMAS 264174

HMAS 266580

Oth_01

HMAS 75893

CBS650.92

ASM 1061

M159

HMAS 264028

HMAS 264029

KUS-F51352

KUS-F51854

HMAS 82107

ICMP 19598

ICMP 19603

HMAS 188545

HMAS 188542

ITS

EU940228

EU940230

KF188722

KF188723

GU586884

GU586893

KJ472289

KJ472288

KC164645

KC164647

JX977162

JX977155

KJ472290

KJ472291

AY348576

AY348577

DQ431177

KC416307

AY348580

AY348581

KF188724

KF188726

GU586904

JX977144

GU586933

JX977147

JX977148

EU096525

EU219982

AY348593

KC164648

KC164644

KJ472292

JX977145

LSU

EU940152

EU940154

KJ472226

KJ472227

KJ472228

KJ472229

KC164640

KJ472231

KJ472230

KJ472232

KJ472233

KJ472234

KJ472235

KJ472236

KJ472237

KJ472248

KJ472249

JN673046

EU940157

KJ472238

KC164639

BTU

FJ477050

FJ477052

KF597265

KF597267

KJ472259

KJ472260

KJ472262

KJ472261

KJ472263

KJ472264

KJ472265

KJ472266

KJ472267

KJ472268

KF597268

KF597269

FJ477056

KJ472269

1020 ... Zheng & Zhuang

TABLE 1, concluded

SPECIES

H. immutabilis

H. jinggangensis Yan H. Zhang & W.Y.

Zhuang

H. kiko PR. Johnst.

H. lasiopodius (Pat.) Dennis

H. macrodiscus

H. macroguttatus

H. microcaudatus H.D. Zheng &

W.Y. Zhuang

H. microserotinus (W.Y. Zhuang)

W.Y. Zhuang

H. cf. monticola

H. ohakune PR. Johnst.

H. ginghaiensis

H. scutula (Pers.) W. Phillips

H. scutuloides

H. serotinus

H. sinicus

H. subpallescens Dennis

H, subsymmetricus H.D. Zheng &

W.Y. Zhuang

H. tamaricis R. Galan et al.

H. tetrasporus

H. waikaia P.R. Johnst.

A. yui

SPECIMEN/

STRAIN NO.

HMAS 71809

F155012

HMAS 82036

HMAS 188548

HMAS 264156

ICMP 19613

ICMP 19597

HMAS 71820

HMAS 75878

HMAS 264158

HB7034

HMAS 264159

HMAS 264020

HMAS 68520

HMAS 264030

HMAS 264031

HMAS 188562

ICMP 19601

ICMP 19607

HMAS 264175

HMAS 264178

HMAS 264180

HMAS 82092

HMAS 82093

F093261

F156526

HMAS 71818

HMAS 264193

HMAS 264194

HMAS 264022

HMAS 264026

HMAS 264021

br020

F110125

HMAS 266592

PDD 102886

ICMP 19599

HMAS 266594

HMAS 266595

ITS

AY348584

DQ431175

KJ472293

KJ472294

KJ472295

KC164656

KC164661

AY348585

AY348587

KJ472296

DQ431179

KC416306

JX977156

DQ986481

JX977150

JX977151

KJ472301

KC164649

KC164650

KJ472297

KC416308

KC416309

AY348589

AY348590

DQ431168

DQ431178

KJ472298

KJ472299

KJ472300

JX977154

JX977160

JX977153

DQ431167

KJ472302

KC164667

KC164646

KJ472304

KJ472303

LSU

KJ472239

KJ472240

KJ472241

KC 164642

KJ472242

KJ472243

KJ472244

KJ472245

KJ472246

KJ472247

KJ472250

KJ472251

KJ472252

KJ472253

KJ472254

KJ472255

KJ472256

KJ472257

FJ005156

KC164641

KJ472258

BTU

KJ472270

KJ472271

KJ472272

KJ472273

KJ472274

KJ472275

KJ472276

KJ472277

KJ472278

KJ472279

KJ472280

KJ472281

KJ472282

KJ472283

KJ472284

KJ472285

KJ472286

KJ472287

Hymenoscyphus spp. nov. (China) ... 1021

2.5 units of Taq DNA polymerase (HT-biotech Co., Ltd., Beijing, China), and 2-3 ul DNA

template. PCR reactions were carried out in an Applied Biosystems 2720 thermocycler

(Foster City, CA, USA) under the following conditions: an initial denaturation at 94 °C

for 5 min, followed by 35 cycles of denaturation at 94 °C for 30-60 s, annealing at 50-53

°C for 30-60 s, and extension 1 min at 72 °C, and bya final extension at 72 °C for 10 min.

PCR products were purified using the AxyPrep™ 96-well DNA Gel Extraction Kit

(Corning Life Sciences - Axygen Biotechnology Co., Ltd., Hangzhou, China) and

sequenced on an ABI 3730 XL DNA Sequencer (Applied Biosciences, Foster City, CA,

USA) with the same primer pairs used for PCR amplification at Beijing Tianyi Huiyuan

Bioscience and Technology Inc., China.

Sequence assembly, alignment and phylogenetic analyses

Forward and reverse sequences generated in this study were assembled using BioEdit

7.0.5.3 (Hall 1999) or SeqMan (DNASTAR, Lasergene 7.1.0) to obtain a consensus

sequence. Sequences used in the phylogenetic analyses were either generated by this

study or retrieved from GenBank (TABLE 1). Hyaloscypha aureliella (Ny1.) Huhtinen and

Hyaloscypha fuckelii Nannf. were used as outgroup taxa. Sequences were aligned with

either BioEdit 7.0.5.3 or Muscle 3.8.31 (Edgar 2004), manually edited when needed, and

converted to nexus files in ClustalX 1.83 (Thompson et al. 1997).

Phylogenetic analyses were based on individual ITS, LSU and BTU sequences

as well as combined dataset. All characters were treated as unordered and equally

weighted, and gaps were treated as missing data. Prior to combined analyses, partition

homogeneity tests (PHT) were performed in PAUP* 4.0b10 (Swofford 2003) with

100 replicates to confirm the combination of different gene sequences for phylogenetic

analyses. PAUP*4.0b10 for Windows (Swofford 2003) was used to conduct neighbor-

joining (NJ) and maximum parsimony (MP) analyses. NJ analysis was performed using

default settings. MP analysis was done with the heuristic search option using max trees

set to 1000 and auto-increased by 100, TBR branch swapping. Tree scores, including

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

index (RC), and homoplasy index (HI) were calculated for all the trees generated. The

topological confidence of the resulting trees was tested with bootstrap analysis using

1000 replications, each with 10 replicates of random addition of taxa in both NJ and

MP analyses.

Bayesian inference (BI) analysis was performed using MrBayes 3.1.2 (Huelsenbeck

& Ronquist 2001) under the best-fit evolutionary model estimated by MrModeltest

2.3 with the Akaike information criterion (Nylander 2004). Two parallel runs of four

simultaneous chains of Markov Chain Monte Carlo were performed starting from

random trees for 2,000,000 generations with the trees sampled every 100 generations

(resulting in 20,000 total trees). The first quarter of sampled trees were discarded as

the burn-in phase of the analyses, and the remaining trees were used for calculating

posterior probabilities (PP) in the majority rule consensus tree. The trees were viewed

in TreeView 1.6.6 (Page 1996).

Results

ITS, LSU, and BTU sequences of Hymenoscyphus were analyzed separately

as well as concatenated. The P value of PHT is 0.06 for the combined ITS, LSU,

1022 ... Zheng & Zhuang

and BTU matrix, which indicates no significant incongruence among the three

markers. For Bayesian analysis, GIR+1I+G was selected by MrModeltest as the

best-fit model for all the four datasets. The characteristics of each sequence

dataset are summarized in TABLE 2.

TABLE 2. Sequence characteristics of ITS, LSU, BTU, and combined datasets.

PARAMETER ITS LSU BTU aura

DATASET

Number of Hymenoscyphus sequences

(newly generated) 70 (18) 40 (33) 34 (29) 30 (30)

Number of Hymenoscyphus species /

represented by holotype 39/19 26/8 21/9 19/8

(newly generated / new species + known (9 / 5+4) (20 / 2+18) (20 / 2+18) (19 / 2+17)

species)

Total nucleotides 593 844 804 2193

Constant characters 345 720 512 1592

Variable but parsimony uninformative AA 16 %6 87

characters

Parsimony informative characters 204 108 266 514

Tree length (TL) 966 250 950 1622

Consistency index (CI) 0.424 0.620 0.457 0.533

Retention index (RI) 0.788 0.846 0.685 0.740

Rescaled consistency index (RC) 0.334 0.524 0.313 0.394

Homoplasy index (HI) 0.576 0.380 0.543 0.467

Best-fit model GTR+I+G GTR+I+G GTR+I+G GTR+I+G

The NJ, MP, and BI analyses produced similar tree topologies except

for minor differences in the arrangement of a few terminal branches. The

phylograms inferred from NJ analysis of each dataset are shown in Fics 1-3

and statistical supporting values higher than 50% or 0.50 are shown at the

nodes. In the phylogenetic trees inferred from all datasets, the sampled species

of Hymenoscyphus formed a strongly supported group.

The ITS alignment included 70 sequences representing 39 Hymenoscyphus

species with 593 nucleotides. The ITS barcodes recognized all five putative

new taxa that appeared as independent lineages, which give strong support

to the morphological observations. In the ITS tree (Fic. 1), the genus were

divided into six main clades (A, B, C, D, E, and F) with high statistic supports

and a few scattered species. The LSU dataset consisted of 40 samples from 26

species (including two new species) with 844 characters. The LSU tree (Fic.

2A) discriminated the clades A, D, E, and F, while Clade B split into two parts,

one together with Clade A and the other with species of Clade C. The BTU

matrix comprised 34 sequences from 21 species (including two new species)

Hymenoscyphus spp. nov. (China) ... 1023

so0/99/1,.00) 4. serotinus F093261 (DQ431168)

944-0.92 H. serotinus F156526 (DQ431178)

H. yui HMAS

$6700 78 .yui HMAS 26

H, fucatus HMAS 264028 (JX977147)

toorsi0.e8! H. fucatus HMAS 264029 (JX977148)

99/84/0.72) H. Sinicus HMAS 264193

91/:10.7; H. sinicus HMAS 264194

pene sas H. sinicus HMAS 7 a

rodiscus HMA‘

BAL: oosoart.oo} 4. caudatus HMAS 82057 (AY348576)

H. caudatus HMAS 82060 (AY348577)

99/87/09 H. fructigenus HMAS 75893 (JX977144)

H. fructigenus CBS650.92 (GU586933)

H. dehlii HMAS 264160 (KC416307)

roora.9y H. scutuloides HMAS 82092 (AY348589)

93/77/- H. scutuloides HMAS 82093 (AY348590)

H. macroguttatus HMAS 264159 (KC416306)

61/740.75 macroguttatus HB7034 (Di

asporus HMAS 2

haiensis HMAS

S2001.00 H. fraxineus Oth_01 (GU586904)4

99i96/4.00 |? H. fraxineus HMAS 264174 (KF 188724)

H. fraxineus HMAS 266580 (KF188726)

97/980.90; H. albidus Lav_01 (GU586884)

soorgert.00 H. albidus 90812.1 (GU586893)

H. albidoides HMAS 264140 (KF188722)4

9716910.58! 11 albidoides HMAS 264142 (KF188723)

1620.10.97 gar9a1.00f— H. microserotinus HMAS 264030 (JX977150)

847710.90 H. microserotinus HMAS 264031 (JX977151)

H. microserotinus HMAS 68520 (DQ986481)4

so0g9r1.00} H. ginkgonis KUS-F51352 (EU096525)4

H. ginkgonis KUS-F51854 (EU219982) B

1o0/100/1.00} H. brevicellulus HMAS 264016 (JX977155)

i H. brevicellulus HMAS 264018 (JX977162)4

76/65/1.00 93/81/0.87 H. hyaloexcipulus HMAS 188542 (JX977145)4

99/99/1.00 sertos H. subsymmetricus HMAS 264021 (JX977153)4

H. microcaudatus HMAS 264020 (JX977156)4

oov100/1,00) H. subpallescens HMAS 264022 (JX977154)

H. subpallescens HMAS 264026 (JX977160)

53/-f- H. immutabilis HMAS 71809 (AY348584)

S6rE H. immutabilis F155012 (DQ431175) Cc

89/600 H. crataegi F156966 (DQ431177)

H. globus HMAS 82107 (AY348593)4

seregit.00f H. epiphyllus HMAS 82075 (AY348580)

72/594 H. epiphyllus HMAS 82076 (AY348581)

100/86/0.99 H. tamaricis br020 (DQ431167) D

1o0/00/1.00 -- H. lasiopodius HMAS 71820 (AY348585)

H. lasiopodius HMAS 75878 (AY348587)

100/100/1.00 H. scutula HMAS 264178 (KC416308)

69/-1~ H. scutula HMAS 264180 (KC416309)

oaieg1.00] H. berggrenii \CMP 19614 (KC164645)

61/-4- H. berggrenii \CMP 19615 (KC164647)

go940.977- H. haasticus \CMP 19598 (KC164648)4

H. haasticus \CMP 19603 (KC164644)

Tate soonart.00f H. ohakune ICMP 19601 (KC164649)4

H. ohakune ICMP 19607 (KC164650) E

83/-10.90 99/99/1.00 H. kiko ICMP 19613 (KC164656)4

H. kiko ICMP 19597 (KC 164661)

soononio0 fH. waikaia PDD 102886 (KC164667)4

bias H. waikaia ICMP 19599 (KC164646)

H. himalayensis HMAS 188545

/. aurantiacus ns

99/99/1.00 99/99/100 |. aurantiacus HMAS F

100/100/1.00) H. calyculus HMAS 264145

H. calyculus HMAS 264146

si9e0.85) H. jinggangensis HMAS 264156

400/100/400 H. jinggangensis HMAS 820364

H. jinggangensis HMAS 188548

H. cf. monticola HMAS 188562

70/-/-

Hyaloscypha aureliella M234 (EU940228 )

Hya. fuckelii M233 (EU940230)

Fic. 1. Neighbor-joining tree of selected Hymenoscyphus species inferred from ITS sequences.

Bootstrap values of NJ (left) and MP (middle) > 50%, and Bayesian posterior probabilities values

(right) >0.50 are indicated at the nodes. A indicates an ex-holotype sequence.

containing 804 positions. The clades A, B, C, D, and F were well reconstructed

in the tree inferred from BTU with Clade E missing due to unavailability of

sequence data (Fic. 2B). The combined three-marker alignment consisted of

30 samples representing 19 species and 2,193 characters including two new

species. In the tree generated from the combined dataset (Fic. 3), the clades

A, B, C, D, and F were well recognized.

1024 ...

Fic. 2.

Zheng & Zhuang

87/s3/0.991 1. fraxineus HMAS 264174

A gei99i1,00| | H. fraxineus HMAS 266580

H. albidoides HMAS 2641404

si | 1. albidoides HMAS 264142

95/94/1.00) H. sinicus HMAS 264193

96/91/1.00 H. sinicus HMAS 264194

ui HMAS 2665950 A

110.77, soo/i00/1,007-° H. caudatus HMAS 82057

H. caudatus HMAS 82060

H. fructigenus M159 (EU940157)

95/-" H. fructigenus ASM1061 (JN673046)

10@100/0.96 | H. scutuloides HMAS 82092

55/75/1.00 ee H. scutuloides HMAS 82093

H. macroguttatus HMAS 264159

87/-1- H. microserotinus HMAS 264030

H. microserotinus HMAS 264031

"lH. microcaudatus HMAS 2640204

ggi9e/1.00) H. brevicellulus HMAS 264016 B

719/5710.68 H. brevicellulus HMAS 2640184

H.subsymmetricus HMAS 2640214

100/99/1,00 | H, subpallescens HMAS 264022

H. subpallescens HMAS 264026

a H. immutabilis HMAS 71809 | c

eaieaaoe H. globus HMAS 821074

: 99/-/- H. tamaricis F110125 (FJ005156)

99/67/0.86 H. epiphyllus HMAS 82076

90/-0.51 H. epiphyllus HMAS 82075 D

H. lasiopodius HMAS 71820

98/96/1.00! 1) Jasiopodius HMAS 75878

64/-1- H. kiko ICMP 19597 (KC 164642)

450K. H. haasticus \CMP 19603 (KC 164639)

H. berggrenii ICMP 19614 (KC164640) E

H. waikaia |CMP 19599 (KC164641)

99/99/1.00 H. scutula HMAS 264180

too/100/1.00 | H. jinggangensis HMAS 188548

H. jinggangensis HMAS 820364

H. aurantiacus Hi

400/100/1,00 H. aurantiacus HI F

99/930.817- H. calyculus HAMS 264145

H. calyculus HAMS 264146

Hyaloscypha aureliella M234 (EU940152)

Hya. fuckelii M233 (EU940154)

80/90/1.00

72/73/1.00

62/-/-

58) g3788/1.00

100/100/1.007- H. sinicus HMAS 264193

99/69/0.98 H. sinicus H 718184

S/O 38: ui HMAS.266

B H. fructigenus M159 (FJ477056)

H. macroguttatus HMAS 264159

savage 99/98/1.00 H. caudatus HMAS 82057 A

H. caudatus HMAS 82060

100/100/1.00) H. scutuloides HMAS 82092

ata E H. scutuloides HMAS 82093

io0/99/1.00} H. albidoides HMAS 264140 (KF597265)4

100/100/1.00 H. albidoides HMAS 264142 (KF597267)

H100/99/1.00 H. fraxineus HMAS 264174 (KF597268)

H. fraxineus HMAS 266580 (KF597269)

100/100/1.00 7 H. microserotinus HMAS 264030

70rk H. microserotinus HMAS 264031

61/-0./72 H. microcaudatus HMAS 2640204

100/10011.00| H. subpallescens HMAS 264022 B

H. subpallescens HMAS 264026

da toon00r.00) H. brevisporus HMAS 264016

H. brevisporus HMAS 2640184

Wer H. sybsymmetricus HMAS 2640214

H. scutula HMAS 264178

H. globus HMAS 821074 |

H. immutabilis HMAS 71809 Cc

1-10.68

96/96/1.00

~152(0.72 . ;

1. aurantiacus HM.

58/-/- 4. aurantiacus HM. F

400/100/1.00 H. calyculus HMAS 264145

H. calyculus HMAS 264146

77/96/1.00 100/100/1.00 _H. epiphyllus HMAS 82075

H. epiphyllus HMAS 82076 D

7240.71 1o000/1.00 p H. lasiopodius HMAS 71820

H. lasiopodius HMAS 75878

400/100/1.00 i H. jinggangensis HMAS 264156

H. jinggangensis HMAS 820364

Hya. aureliella M234 (FJ477050)

Hyaloscypha fuckelii M233 (FJ477052)

Neighbor-joining trees of selected Hymenoscyphus species inferred from LSU (A)

and BTU (B) sequences. Bootstrap values of NJ (left) and MP (middle) > 50%, and Bayesian

posterior probabilities values (right) >0.50 are indicated at the nodes. A indicates an ex-

holotyp

e€ sequence.

Hymenoscyphus spp. nov. (China) ... 1025

100/99/1.00 H. caudatus HMAS 82057

100/98/1.00 H. caudatus HMAS 82060

100/99/1..00} H. sinicus HMAS 264193

97/90/0.80 fi HMAS 2665!

100/100/1.00] H. scutuloides HMAS 82092

H. scutuloides HMAS 82093 A

100/99/1.00 99/91/0.92

H. macroguttatus HMAS 264159

100/100/1.00 H. albidoides HMAS 2641404

100/100/1.00 H. albidoides HMAS 264142

H. fraxineus HMAS 264174

100/100/1.00! H. fraxineus HMAS 266580

100/100/1.00[— H. microserotinus HMAS 264030

fk H. microserotinus HMAS 264031

1-10.62 H. microcaudatus HMAS 2640204

100/100/1.00] H. subpallescens HMAS 264022

100/96/1.00 H. subpallescens HMAS 264026

100/100/1.00| H. brevicellulus HMAS 264016

ee H. brevicellulus HMAS 2640184

97/86/0.96 H. subsymmetricus HMAS 2640214

100/99/1.00 H. globus HMAS 821074

H. immutabilis HMAS 71809 | C

1oo/100/1.00} H. epiphyllus HMAS 82075

64/56/0.63 100/96/1.00 H. epiphyllus HMAS 82076 D

1oo100/1.00 H. lasiopodius HMAS 71820

H. lasiopodius HMAS 75878

ane |. aurantiacus was 2a |

100/100/1.00 |. aurantiacus HM) F

soo00/1.00 f H. calyculus HMAS 264145

H. calyculus HMAS 264146

H. jinggangensis HMAS 820364

91/88/0.99

99/87/1.00

Hyaloscypha aureliella M234

Hya. fuckelii M233

0.01

Fic. 3. Neighbor-joining tree of selected Hymenoscyphus species inferred from combined ITS, LSU

and BTU sequences. Bootstrap values of NJ (left) and MP (middle) > 50%, and Bayesian posterior

probabilities values (right) >0.50 are indicated at the nodes. A indicates an ex-holotype sequence.

Taxonomy

Fic. 4. Gross morphology of apothecia (A, C, E, G, I: Rehydrated apothecium; B, D, F, H, J:

Dried apothecia on natural substrates). A, B. Hymenoscyphus aurantiacus (HMAS 264143,

holotype); C, D. H. macrodiscus (HMAS 264158, holotype); E, F. H. ginghaiensis (HMAS 264175,

holotype); G, H. H. tetrasporus (HAS 264195, holotype); I, J: H. yui (HMAS 266595, holotype).

Scale bars: A, B, E, F G, H = 0.5 mm; C, D, I, J = 1 mm.

1026 ... Zheng & Zhuang

Fic. 5. IKI reaction of ascus apical ring (mounted in fetar solution without KOH pretreatment).

A. Hymenoscyphus aurantiacus (holotype); B. H. macrodiscus (holotype); C. H. qinghaiensis

(holotype); D. H. tetrasporus (holotype); E. H. yui (holotype). Scale bars = 5 um.

Fic. 6. Ascus bases showing croziers. A. Hymenoscyphus aurantiacus (holotype); B. H. macrodiscus

(holotype); C. H. qinghaiensis (holotype); D. H. tetrasporus (holotype); E: H. yui (holotype).

Scale bars = 5 um.

Hymenoscyphus aurantiacus H.D. Zheng & W.Y. Zhuang,

sp. nov. FIGS 4A,B, 5A, 6A, 7

FUNGAL NAME FN570094

Differs from Hymenoscyphus imberbis by its orange, distinctly stipitate apothecia and

narrower excipular hyphae.

Type: China. Hubei, Wufeng, Houhe, alt. 800 m, on rotten twig and mossy wood of an

unidentified deciduous tree, 12 Nov. 2004, W.Y. Zhuang & C.Y. Liu 5501 (Holotype,

HMAS 264143).

GENE SEQUENCES EX-HOLOTYPE: KJ472289 (ITS), KJ472228 (LSU), KJ472259 (BTU).

Erymo toey: The specific epithet refers to the color of the apothecia.

Apothecia scattered or 2—3 in a cluster, discoid, stipitate, 0.8-3 mm in diam.;

hymenium surface orange, drying pale brownish to pale orange; receptacle

surface paler, drying cream to cream-orange; stipe concolorous with receptacle,

glabrous or nearly so, with white to orange-red mycelium at base, 0.5—-1.5 mm

long. Outer covering layer absent or with loosely interwoven hyphae at stipe

surface, hyphae cylindrical, ends obtuse. Ectal excipulum of textura porrecta,

20-50 um thick, cells hyaline, 10-35 x 2-6 um. Medullary excipulum of two

layers, outer layer of textura porrecta, 20-150 um thick; inner layer of textura

intricata, 30-180 um thick; hyphae hyaline, 2-4 um wide. Subhymenium

not distinguishable. Hymenium 85—95 um thick. Asci arising from croziers,

clavate, with apex broadly subconical to rounded, 8-spored, J+, apical ring

Hymenoscyphus spp. nov. (China) ... 1027

LA e

a te Z

PLEA

—

ri )

—

Fic. 7. Hymenoscyphus aurantiacus (A—C, E, F, H-K: HMAS 264143, holotype; D, G, L, M: HMAS

264144). A. Longitudinal section of apothecium; B. Excipular structure at flank; C, D. Structure

at the middle of stipe; E-G. Ascus with ascospores; H—L. Ascospore; M. 1-septate ascospore.

Scale bars: A = 100 um; B—D = 20 um; E-G = 10 um; H—M = 5 um.

as two blue lines without KOH pretreatment, Hymenoscyphus-type, 65-80 x

4.5-5.5 um. Ascospores clavate to slipper-shaped, upper part slightly enlarged,

0(-1)-septate, hyaline, eguttulate to occasionally with tiny guttules, uniseriate

to biseriate above and uniseriate below, 7.8-11 x 2.8-3.3 um. Paraphyses

filiform, hyaline, 1-2 um wide, equal to or slightly exceeding the asci.

ADDITIONAL SPECIMEN EXAMINED — CHINA. Huse, Wufeng, Houhe, alt. 800 m,

on rotten seeds of an unidentified plant, 12 Nov. 2004, W.Y. Zhuang & C.Y. Liu 5492

(HMAS 264144).

Notrs—In the phylogenetic trees (Figs 1-3), H. aurantiacus clustered with

H. calyculus (Fr.) W. Phillips (Dennis 1956) in the same clade. Hymenoscyphus

1028 ... Zheng & Zhuang

calyculus differs in its yellow apothecia, broader ectal excipular cells (<10-25 x

4-12 um), and cylindrical, biguttulate and much larger ascospores (15—22 x

3-4.5 um) irregularly biseriate in much larger asci (120-125 x 9-10 Um;

Dennis 1956).

Among other Hymenoscyphus species, H. imberbis (Bull.) Dennis

resembles H. aurantiacus in asci and ascospores but differs in having pure

white apothecia, a short and stout stipe, and broader ectal excipular cells

(10-20 x 5-10 um; Dennis 1956). The new species also resembles H. leucopsis

(Berk. & M.A. Curtis) Dennis in stipitate apothecia and ectal excipulum

structure, but H. leucopsis has larger apothecia (4-7 mm diam.), a pallid to

copper-colored hymenium surface, and smaller asci (38-52 x 4-5 um) and

ascospores (4-9 x 1.5-2.5; Dumont 1981).

Thetwo H. aurantiacus specimens share similar macroscopic and microscopic

characteristics as well as identical sequence data. A minor difference observed

is that the stipe surface is glabrous in the holotype (Fic. 7c) but hairy in the

other collection (Fic. 7D). We treat this as infraspecific variation.

Hymenoscyphus macrodiscus H.D. Zheng & W.Y. Zhuang,

sp. nov. FIGS 4C,D, 5B, 6B, 8

FUNGAL NAME FN570095

Differs from Hymenoscyphus sinicus by its parallel outer covering layer, smaller asci, and

equilaterally elliptical-fusoid smaller ascospores uniseriate in the asci.

Type: China. Xinjiang, Guozigou, alt. 1400 m, on rotten wood of an unidentified

deciduous tree, 11 Aug. 2003, W.Y. Zhuang & Y. Nong 4860 (Holotype, HMAS 264158).

GENE SEQUENCE EX-HOLOTYPE: KJ472296 (ITS).

ErymMo.oey: The specific epithet refers to the large-sized apothecia.

Apothecia scattered, discoid, stipitate, 3-8 mm in diam.; hymenium surface

yellow, drying ochraceous; receptacle surface paler, drying pale ochraceous;

stipe slightly rough, not darkening at base, 1-8 mm long. Outer covering layer

of textura intricata, up to 35 um thick, hyphae 2-3 um wide. Ectal excipulum

of textura prismatica, tissues somewhat gelatinized, 8-30 um thick, cells

hyaline, 5-8 um wide. Medullary excipulum of two layers, outer layer of textura

porrecta, 50-110 um thick; inner layer of textura intricata, 50-200 um thick;

hyphae hyaline, 2-3 um wide. Subhymenium not distinguishable. Hymenium

about 150 um thick. Asci arising from croziers, subcylindrical, tapered towards

base, apex subconical to subtruncate, 8-spored, J+, apical ring as two blue

lines without KOH pretreatment, Hymenoscyphus-type, 112-150 x 6—7.5 um.

Ascospores elliptical-fusoid, pointed at both ends, hyaline, eguttulate or rarely

with very tiny oil drops, obliquely uniseriate, 11-14.5 x 3.3-4.4 um. Paraphyses

filiform, hyaline, 2-3 um wide, not exceeding the asci.

Hymenoscyphus spp. nov. (China) ... 1029

eget 854 15

Wn ee i

Fic. 8. Hymenoscyphus macrodiscus (HMAS 264158, holotype). A. Longitudinal section of portion

of apothecium; B. Structure at the middle of stipe; C. Excipular structure at flank; D. Excipular

structure at the middle of stipe; E-H. Ascus with ascospores; I-N. Ascospore. Scale bars:

A, B = 100 um; C, D = 20 um; E-H = 10 wm; IE-N =5 um.

Notes—In the ITS phylogenetic tree (Fic. 1), H. macrodiscus grouped together

with H. sinicus W.Y. Zhuang & Yan H. Zhang, which is similar in apothecial

gross morphology, excipular structure, and habitat but differs in its outer

covering layer composed of parallel hyphae, larger asci (125-175 x 8-12.5

um), and larger ascospores (16-26 x 4.1-6 lum) that are inequilaterally fusoid,

guttulate, and occasionally with one or two septa (Zhang & Zhuang 2002b).

Hymenoscyphus immutabilis (Fuckel) Dennis has similar ascospores, but differs

in having white and smaller apothecia (0.8-1.5 mm) with a short stipe, angular

cells in ectal excipulum, absence of an outer covering layer, shorter and broader

asci (80-105 x 8-10 um), and foliicolous habitat (White 1943).

1030 ... Zheng & Zhuang

Hymenoscyphus ginghaiensis H.D. Zheng & W.Y. Zhuang,

sp. Nov. FIGS 4E,F, 5C, 6C, 9

FUNGAL NAME FN570096

Differs from Hymenoscyphus phyllogenus by its larger apothecia, broader ectal excipular

hyphae, narrower asci arising from croziers, and narrower ascospores.

Type: China. Qinghai, Minhe Xigou, alt. 2600 m, on rotten leaves of Populus sp., 10 Aug.

2004, W.Y. Zhuang & C.Y. Liu 5248 (Holotype, HMAS 264175).

Fic. 9. Hymenoscyphus qinghaiensis (HMAS 264175, holotype). A. Longitudinal section of

apothecium; B. Excipular structure at flank; C. Excipular structure at the middle of stipe;

D, E. Ascus with ascospores; F—H. Ascospore. Scale bars: A = 100 um; B, C = 20 um; D, E = 10 um;

F-H =5 um.

Hymenoscyphus spp. nov. (China) ... 1031

GENE SEQUENCE EX-HOLOTYPE: KJ472297 (ITS).

ErymMo.oey: The specific epithet refers to the type locality of the fungus.

Apothecia scattered, discoid to flat, stipitate, 0.4-1.4 mm in diam.; hymenium

surface cream to very pale yellow, drying pale orange; receptacle surface drying

cream; stipe concolorous with receptacle surface, glabrous or nearly so, not dark

at base, 0.2-0.7 mm long. Outer covering layer of 1-3 hyphal layers, hyphae

about 3 um wide. Ectal excipulum of textura prismatica, 15-60 um thick, cells

hyaline, 8-30 x 5-10 um. Medullary excipulum of two layers, outer layer of

textura porrecta, 10-55 um thick; inner layer of textura intricata, 30-140 um

thick; hyphae hyaline, 2-4 um wide. Subhymenium not distinguishable.

Hymenium 80-85 wm thick. Asci arising from croziers, clavate, apex obtuse to

conical, 8-spored, J+, apical ring as two blue lines without KOH pretreatment,

Hymenoscyphus-type, 68-89 x 6.7-7.8 um. Ascospores cylindric-ellipsoid-

fusoid, posterior end pointed, anterior end partly asymmetrical, flattened on

one side, hyaline, eguttulate or rarely with very small guttules, uniseriate to

biseriate above and uniseriate below, 11-14.5 x 3.3-4 um. Paraphyses filiform,

hyaline, 1.5-2.5 um wide, not exceeding the asci.

ADDITIONAL SPECIMENS EXAMINED — CHINA. QinGuAlI, Minhe Xigou, alt. 2600

m, on rotten leaves of Populus sp., 10 Aug. 2004, W.Y. Zhuang & C.Y. Liu 5241, 5247

(HMAS 264176, 264177).

Notes—Among the known Hymenoscyphus species growing on rotten Populus

leaves having similar asci and ascospores, H. phyllogenus (Rehm) Kuntze is

most similar to H. ginghaiensis but differs in having much smaller apothecia

(0.3-0.5 mm diam.), narrower ectal excipular cells (15-20 x 4-5 um wide),

broader asci (65-75 x 8.5-11 Um) arising from simple septa, and broader

ascospores (11-14 x 3.8-5 um; White 1943; Dennis 1956). The new species

is also similar to H. albopunctus (Peck) Kuntze, which differs obviously in

broader asci (60-70 x 8-10 um) arising from simple septa, ascospores with

2-4 conspicuous oil drops, and growing on leaves of Fagus sp. (White 1943).

In the ITS phylogenetic tree (Fic. 1), H. ginghaiensis stands in a distinct

lineage within Clade A and of uncertain relationship with other species.

Hymenoscyphus tetrasporus H.D. Zheng & W.Y. Zhuang,

sp. Nov. FIGS 4G,H, 5D, 6D, 10

FUNGAL NAME FN570098

Differs from all the known Hymenoscyphus species by its asci containing 4 ascospores

at maturity.

Type: China. Hebei, Wulingshan Lianhuachi, alt. 1800 m, on herbaceous stem of an

unidentified monocot, 26 Aug. 1989, W.Y. Zhuang 490 (Holotype, HMAS 266592).

GENE SEQUENCE EX-HOLOTYPE: KJ472302 (ITS).

ErymMo.oey: The specific epithet refers to the number of ascospores in mature asci.

1032 ... Zheng & Zhuang

Fic. 10. Hymenoscyphus tetrasporus (HMAS 264195, holotype). A. Longitudinal section of

apothecium; B: Excipular structure at flank; C—E: Mature ascus / asci with four ascospores;

F: Immature ascus with four normal ascospores and four degenerated ones; G: Asci at different

developmental stages; H—M: Ascospore. Scale bars: A = 100 um; B = 20 um; C-G = 10 um;

H-M =5 um.

Apothecia scattered, discoid, stipitate, 1-2 mm in diam.; hymenium surface

yellow, drying pale brownish yellow; receptacle surface drying pallid to light

orange; stipe concolorous with receptacle surface, glabrous or nearly so,

0.5-1.2 mm long. Outer covering layer about 15 um thick, hyphae with walls

roughened, about 2 um wide. Ectal excipulum of textura prismatica, glabrous

or with short hyphal protrusions, 20-50 um thick, cells hyaline, 6-15 x

4-8 um. Medullary excipulum of two layers, outer layer of textura porrecta,

30-40 wm thick, inner layer of textura intricata, 50-90 um thick; hyphae

hyaline, 2-3 um wide. Subhymenium not distinguishable. Hymenium about

125 um thick. Asci arising from croziers, subcylindrical to clavate, apex obtuse,

8-spored when young, with 4 mature spores at maturity, J+, apical ring as two

blue lines without KOH pretreatment, Hymenoscyphus-type, 100-115 x 7-9

um. Ascospores elliptical-fusoid with blunt ends, hyaline, having a conspicuous

central dark stained area in cotton blue, with 2-4 large oil drops and several

Hymenoscyphus spp. nov. (China) ... 1033

small ones, uniseriate, (16-)17.8-22 x 4.5-5.5 um. Paraphyses filiform,

2.5-4 um wide, equal to or slightly exceeding the asci.

Notes—The most distinctive character of H. tetrasporus is that four of the

eight ascospores present in young asci abort during development. Therefore,

each mature ascus contains only four ascospores (Fic. 10c-G). This prominent

character easily distinguishes it from its morphologically most similar

species, H. caudatus (P. Karst.) Dennis and H. hyaloexcipulus H.D. Zheng &

W.Y. Zhuang.

Phylogenetically (Fic. 1) this new species grouped together with

H. macroguttatus Baral et al. and H. scutuloides Hengstm., which are similar

in anatomic structure, the asci arising from croziers, and guttulate ascospores.

Hymenoscyphus macroguttatus differs in 8-spored, shorter and wider asci (77-

97 x 9.5-11 um) and shorter (13.5-18 x 4-5.5 um) and scutuloid ascospores

(Zheng & Zhuang 2013b). Hymenoscyphus scutuloides is distinguished

by 8-spored asci and scutuloid ascospores with cilia at one or both ends

(Hengstmengel 1996).

Hymenoscyphus yui H.D. Zheng & W-Y. Zhuang, sp. nov. FIGS 41,J, 5E, 6E, 11

FUNGAL NAME FN570099

Differs from H. macrodiscus by its outer covering layer of parallel hyphae, ectal excipular

hyphae slightly undulate, shorter asci, and inequilaterally elongate-ellipsoid ascospores.

Type: China, Xinjiang, Guozigou, alt. 1800 m, on rotten twig of unidentified deciduous

tree, 11 Aug. 2003, W.Y. Zhuang & Y. Nong 4877 (Holotype, HMAS 266595).

GENE SEQUENCES EX-HOLOTYPE: KJ472303 (ITS), KJ472258 (LSU), KJ472287 (BTU).

ErymMo.ocy: The specific epithet honors the late distinguished Chinese mycologist,

Prof. Y.N. Yu.

Apothecia scattered, discoid, stipitate, 2-4 mm in diam.; hymenium surface

yellow, drying dull orange to brownish orange; receptacle surface paler,

drying cream-orange; stipe concolorous with receptacle surface, glabrous or

nearly so, having whitish mycelium at base, 0.5-8 mm long. Outer covering

layer of 2 or more hyphal layers, hyphae 2-3 um wide. Ectal excipulum of

textura prismatica, 40-70 um thick, hyphae slightly undulated and somewhat

gelatinized, cells hyaline, 10-25 x 3-7 um. Medullary excipulum of two layers,

outer layer of textura porrecta, 20-80 um thick; inner layer of textura intricata,

40-150 um thick; hyphae hyaline, 2-3 Um wide. Subhymenium not

distinguishable. Hymenium 110-125 um thick. Asci arising from croziers,

subcylindrical, apex rounded, 8-spored, J+, apical ring as two blue lines

without KOH pretreatment, Hymenoscyphus-type, 86-115 x 5.5-8.5 Um.

Ascospores elongate ellipsoid, slightly flattened on one side, hyaline, eguttulate

to occasionally with a few tiny oil drops, uniseriate, 11-15.5 x 3.3-5 Um.

Paraphyses filiform, hyaline, 1.5-2.5 um wide, not exceeding the asci.

1034 ... Zheng & Zhuang

Fic. 11. Hymenoscyphus yui (HMAS 266594). A, B. Longitudinal section of portion of apothecium;

C. Excipular structure at flank; D-F Immature ascus; G, H. Ascospores in ascus. Scale bars:

A, B = 100 um; C = 20 um; D—-F = 10 um; G, H = 5 um.

ADDITIONAL SPECIMEN EXAMINED — CHINA. XINJIANG, Yining, Qabqal, alt. 2000 m,

on rotten wood of an unidentified deciduous tree, 13 Aug. 2003, W.Y. Zhuang & Y. Nong

4911 (HMAS 266594).

Notes — In the ITS tree (Fic. 1), H. yui appears to be a sister of H. serotinus

(Pers.) W. Phillips, which is quite different in ectal excipulum structure, and

bigger (18-28 x 3-4 um), guttulate, strongly heteropolar and curved ascospores

biseriate in asci (Dennis 1956; Baral & Bemmann 2013). In the trees inferred

from LSU, BTU, and multilocus data (Fics 2,3), the new species clusters with

H. sinicus in a highly supported subclade. Hymenoscyphus sinicus is similar

in apothecial size and color, excipulum structure, and lignicolous habitat but

differs in larger asci (125-175 x 8-12.5 um) and guttulate ascospores, which

are larger (16-26 x 4.1-6 um; Zhang & Zhuang 2002b).

The apothecial gross morphology and habitat of H. yui are also similar to

H. macrodiscus, which differs by its interwoven hyphae in the outer covering

layer, straight instead of undulate hyphae in ectal excipulum, longer asci (112-

150 x 6-7.5 um), and equilaterally ellipsoid ascospores.

Hymenoscyphus spp. nov. (China) ... 1035

Discussion

In this paper, five new species of Hymenoscyphus are described based on

morphological characters that are congruent with phylogenetic inference. Our

study revealed that combining morphological with molecular data is important

for defining a species. In the case of H. aurantiacus, the stipe surface morphology

differs somewhat between the two collections as mentioned previously, yet

the sequence identity and major morphological similarities support them as

conspecific. The minor stipe surface differences among collections may be

considered infraspecific variations.

The previous phylogenetic studies of Hymenoscyphus were preliminarily

ITS-based using small sample sizes (Zhang & Zhuang 2004, Baral et al. 2006,

Han & Shin 2008, Johnston & Park 2013, Zheng & Zhuang 2013a). Our

analyses have been based on more extensive sampling and multi-gene sequence

data, which provide a more comprehensive understanding of the genus. Our

phylogenetic reconstruction produced six main clades within Hymenoscyphus.

Hymenoscyphus fructigenus, the type species of the genus, is located in Clade

A together with H. albidoides, H. albidus, H. caudatus, H. dehlii, H. fucatus,

H. macroguttatus, H. fraxineus, H. scutuloides, H. serotinus, and H. sinicus in

addition to four new taxa described here (H. macrodiscus, H. qinghaiensis,

H. tetrasporus, H. yui). These fungi represent the core group of Hymenoscyphus.

Generally, species in this clade have relatively large apothecia, rectangular cells

in ectal excipulum, and scutuloid ascospores.

The seven species—H. brevicellulus, H. ginkgonis, H. hyaloexcipulus,

H. microcaudatus, H. microserotinus, H. subpallescens, H. subsymmetricus—

that comprise Clade B possess rectangular ectal excipular cells and scutuloid or

ellipsoid ascospores usually less than 20 um long. Clade C contains H. crataegi,

H. globus, and H. immutabilis, which share the presence of angular, isodiametric

to subglobose cells in the ectal excipulum and fusoid, symmetrical ascospores

less than 15 um long. The three taxa in Clade D share yellowish apothecia and

the presence of croziers at ascus base.

Hymenoscyphus berggrenii, H. haasticus, H. kiko, H. ohakune, and

H. waikaia, species so far known only from New Zealand (Johnston & Park

2013), comprise Clade E. They are similar in that the paraphyses branch on

their upper portions to form an epithecium-like layer across the top of the

asci and by occurring on Nothofagus leaves. ‘The phylogenetic position of these

fungi may be influenced by geographic separation and host restriction.

Hymenoscyphus aurantiacus and H. calyculus, which form Clade F, resemble

each other in their yellow to orange apothecia and J+ asci with croziers at base.

They appear to be similar to taxa in Clade D but are differentiated by their

sequence data.

1036 ... Zheng & Zhuang

Analyses of individual gene sequences showed that ITS and BTU are

efficient in distinguishing the species investigated, while LSU is problematic

in some cases due to its limited interspecific nucleotide divergence. Based on

the information gathered, it is still difficult to fully understand the phylogeny

of Hymenoscyphus. Evolutionary relationships among species and clades need

further exploration by integrated studies. Along with increasing the number of

taxa and features investigated within the genus, a better and more reasonable

phylogeny of Hymenoscyphus will be displayed.

Acknowledgments

The authors would like to express their deep thanks to Dr. Yei-Zeng Wang,

Dr. Jae-Gu Han, Mr. Hans-Otto Baral, and Dr. Brian Spooner for critical review of the

manuscript, valuable suggestions, and corrections; Ms. Xia Song for technical assistance;

and all collectors of specimens for this study. This work was supported by the National

Natural Science Foundation of China (nos. 31093440, 31270073).

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MY COTAXON

http://dx.doi.org/10.5248/130.1039

Volume 130, pp. 1039-1043 October-December 2015

Phyllachora jianfengensis sp. nov. from China

Na Liu* & MENGZHEN LI

College of Biological Engineering, Henan University of Technology,

Lianhua street, High & New Tech Development District, Zhengzhou 450001, China

* CORRESPONDENCE TO: /iuna3456@163.com

ABSTRACT—A new species, Phyllachora jianfengensis, causing tar spots on leaves of

Miscanthus sacchariflorus is reported from Hainan Province. Phyllachora jianfengensis differs

from similar species by its longer ascospores.

Key worps—Phyllachoraceae, Phyllachorales, taxonomy

Introduction

The conserved generic name Phyllachora Nitschke ex Fuckel was published

by Fuckel (1870) with P graminis (type) and 17 other species. The genus was

initially defined as fungi parasitic on leaves or stems of plants and with obvious

stroma. Following modern research (Doidge 1942, Parbery 1967, Cannon 1991,

Pearce & Hyde 1994), the characteristics of Phyllachora are being parasitic and

having paraphyses, unitunicate 8- or 4-spored asci, and hyaline ascospores.

According to Kirk et al. (2008), Phyllachora belongs to Phyllachoraceae,

Phyllachorales, and includes about 944 species worldwide.

An undescribed Phyllachora species was reported on leaves of

Miscanthus sacchariflorus collected from Hainan Province, China, in 2007.

It differs morphologically from other Phyllachora species on three genera of

Andropogoneae subtribe Saccharinae (Imperata, Miscanthus, and Saccharum)

and is described here as Phyllachora jianfengensis.

Materials & methods

We measured the length and width of leaf spots with a ruler and observed the

presence of holes with a magnifying glass. The characters of asci were observed after

they were separated from ascomata with dissecting needle and stained in lactophenol

blue solution. The asci and ascospore were measured using a C7 ocular micrometer

and Nikon E200MV photomicroscope. We prepared slices by using a Leica CM1850UV

1040 ... Liu & Li

freezing microtome. The holotype of the new species is deposited in the Herbarium

Mycologicum Academiae Sinicae, Beijing, China (HMAS).

Taxonomy

Phyllachora jianfengensis Na Liu & M.Z. Li, sp. nov. PLATE 1

FUNGAL NAME FN570152

Differs from Phyllachora ischaemi, P. miscanthi, P. miscanthidii, and P. tengchongensis by

its longer ascospores.

Type: China, Hainan Province, Jianfengling, alt. 900 m, on Miscanthus sacchariflorus

(Maxim.) Hack. (Poaceae), 18 XI 2007, S.H. He, L. Guo & Z. Li 2014 (HMAS 245921,

holotype).

EryMmo ocy: From Jianfengling mountain, where the new species was collected.

Leaf spots immersed in the mesophyll on ellipsoidal glossy black leaf spots

0.6-3 mm in diam, surrounded by pale yellow haloes, visible both sides of

leaves, sparse, 1-4-loculate, subglobose, ostiolate. AscomaTa 202-278 x

180-213 um, immersed in the mesophyll layer of the leaves, subglobose or

ellipsoidal, upper wall up to 25 um thick composed of black material, lateral

wall up to 18 um thick composed of epidermal cells and black material, and

lower wall up to 13 um thick composed of parenchyma cells; asci rising from

the basal and lateral wall of the ascoma. Paraphyses 1-2 um wide, filiform,

aseptate, unbranched, shorter than asci, thin-walled. Ascr 83-125 x 11-26 um,

cylindrical to clavate, apex round or slightly sharp, thin-walled, 8-spored, long

pedunculate or without peduncle, unitunicate. ASCOSPORES 32-37 x 5-9 um,

biseriate or arranged irregularly, long ellipsoidal with slightly sharp apex to

oblong, unicellular, hyaline, smooth. ANAMORPH not seen.

COMMENTS—Species of Phyllachora with ascospores longer than 30 um are

uncommon. Based on the assumption that Phyllachora species are likely to be

host-specific, we compared our collection with Phyllachora species reported

from the related hosts Miscanthus, Imperata, and Saccharum (Chen 2007,

Hodkinson et al. 2002).

Four Phyllachora species have been recorded on Miscanthus: P. ischaemi

Syd. & P. Syd. with ovato-ellipsoidal ascospores (12-15 x 5-6.5 um) and

cylindraceo-clavate asci (45-60 x 12-18 um; Sydow & Sydow 1915),

P. miscanthi Syd. & P. Syd. with broadly ellipsoidal ascospores (18-24 x

12-14 um) and cylindriceo-clavate asci (160-180 x 18-22 um; Sydow &

Sydow 1917), P. miscanthidii Doidge with ellipsoidal to subclavate ascospores

(22-27.5 x 7.5-10 um) and cylindrical to cylindrico-elliptical asci (125 x 17-20

um; Doidge 1942), and P. tengchongensis Na Liu & L. Guo with narrowly

ellipsoid ascospores (15-26 x 5-10 um) and clavate asci (93-159 x 15-23 um;

Phyllachora jianfengensis sp. nov. (China) ... 1041

PLATE 1. Phyllachora jianfengensis (HMAS 245921, holotype).

1. Leafspots; 2. Ascomata; 3, 4; Asci and ascospores.

Liu et al. 2007). These species all differ from P jianfengensis by their much

shorter ascospores; additionally, P ischaemi differs by its shorter asci and

P. miscanthi by its longer asci and wider ascospores.

1042 ... Liu & Li

Two Phyllachora species have been reported on Imperata: P. oxyspora

Starback (= P. imperatae Syd. & P. Syd., = P. antioquensis Chardon, and

= P. sorghastri Chardon; Species Fungorum 2015) with clavulate ascospores

(20-25 x 5-6 um) and cylindrical asci (96-115 x 9-10 um; Starback 1899) and

“P. imperaticola” Sawada (an invalid name, lacking a Latin description) with

ellipsoidal ascospores (17-19 x 9-10 um) and clavate-cylindrical asci (89-110

x 13-18 um; Sawada 1959). Both species differ from P jianfengensis by their

much shorter ascospores as well as their host specialisation; and P oxyspora

additionally differs by its narrower asci.

Two Phyllachora species have been reported on Saccharum: P. sacchari-

spontanei Syd. & P. Syd. with fusiform ascospores (22-27 x 7.5-8.5 um) and

clavate asci (75-100 x 18-22 um; Sydow & Sydow 1913) and P. sacchari Henn.

(= P. sacchari-aegyptiacae Briosi & Cavara, = P. sorghi Hohn., = P. rottboelliae

Syd. et al., and = P. andropogonicola Speg.; Species Fungorum 2015) with

ellipsoid to ovoid ascospores (12-18 x 9-12 um) and clavate asci (80-120 x

15-18 um; Hennings 1902). Both species differ from P jianfengensis by their

much shorter ascospores as well as by their host specialisation, and P. sacchari

additionally differs by its wider ascospores.

Acknowledgments

The authors would like to thank Dr. Roger Shivas (Brisbane, Australia) and Dr. Jing

Luo (Rutgers University, New Brunswick, USA) for serving as pre-submission reviewers;

Drs. Shuanghui He, Lin Guo, and Zhenying Li for collecting the specimen; and

Dr. Jiamei Li (Henan Agricultural University, Zhengzhou, China) for identifying the

host plant; Dr. Shaun Pennycook (Auckland, New Zealand) for nomenclatural review;

and Dr. Lorelei Norvell for selfless help. This study was supported by the National

Natural Science Foundation of China (No. 31300015) and Natural Science Research

Project of Henan Province (No. 132300410350).

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Mycol. Pap. 163. 302 p.

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based on ITS sequence data. J. Wuhan Bot. Res. 25(3): 239-244.

Doidge EM. 1942. Revised descriptions of South African species of Phyllachora and related genera.

Bothalia 4: 421-463.

Fuckel L. 1870. Symbolae mycologicae. Beitrage zur Kenntniss der rheinischen Pilze. Jahrb.

Nassauischen Vereins Naturk. 23-24. 459 p.

Hennings P. 1902. Fungi javanici novia cl. Prof. Dr. Zimmermann collecti. Hedwigia 41: 140-149.

Hodkinson TR, Chase MW, Lledé MD, Salamin N, Renvoize SA. 2002. Phylogenetics of Miscanthus,

Saccharum and related genera (Saccharinae, Andropogoneae, Poaceae) based on DNA sequences

from ITS nuclear ribosomal DNA and plastid trnL intron and trnL-F intergenic spacers. J. Plant

Res. 115: 381-392. http://dx.doi.org/10.1007/s10265-002-0049-3

Phyllachora jianfengensis sp. nov. (China) ... 1043

Kirk PM, Cannon PF, Minter DW, Stalpers JA. 2008. Ainsworth & Bisby’s dictionary of the fungi.

10th ed. Biddles Ltd.

Liu N, Guo L. 2007. Phyllachora tengchongensis sp. nov. and a new records of Phyllachora

(Phyllachorales) from China. Mycotaxon 100: 251-254.

Parbery DG. 1967. Studies on graminicolous species of Phyllachora Nke. in Fuck. V. A taxonomic

monograph. Austr. J. Bot. 15: 271-375. http://dx.doi.org/10.1071/BT9670271

Pearce CA, Hyde KD. 1994. The genus Phyllachora from Australia: observation

of Phyllachora species from Callistemon. Mycol. Res. 98: 1393-1401.

http://dx.doi.org/10.1016/S0953-7562(09)81069-1

Sawada K. 1959. Descriptive catalogue of Taiwan (Formosan) fungi. Part XI. Special Publ. Coll.

Agric. Natl. Taiwan Univ. 8. 268 p.

Species Fungorum. 2015. http://www.speciesfungorum.org/Names/Names.asp [accessed: March

2015].

Starback K. 1899. Ascomyceten der ersten Regnellschen Expedition. I. Bih. Kongl. Svenska

Vetensk.-Akad. Handl., Afd. 3, 25(1). 68 p.

Sydow H, Sydow P. 1913. Enumeration of Philippine fungi, with notes and descriptions of new

species. Part I: micromycetes. Philipp. J. Sci., C, Bot. 8: 265-285.

Sydow H, Sydow P. 1915. Novae fungorum species - XII. Ann. Mycol. 13: 35-43.

Sydow H, Sydow P. 1917. Beitrag zur Kenntnis der Pilzflora der Philippinen-Inseln. Ann. Mycol.

15: 165-268.

MY COTAXON

http://dx.doi.org/10.5248/130.1045

Volume 130, pp. 1045-1049 October-December 2015

A new species and a new combination in Codinaea

from Brazil

Mayra S. OLIVEIRA', ELAINE MALOSSO? & RAFAEL F. CASTANEDA-RuIZz}

‘Programa de Pés-Graduagao em Biologia de Fungos, Universidade Federal de Pernambuco,

Avenida da Engenharia, s/n Cidade Universitaria, Recife, PE, 50.740-600, Brazil

*Centro de Ciéncias Biolégicas, Departamento de Micologia,

Universidade Federal de Pernambuco, Avenida da Engenharia s/n,

Cidade Universitaria, Recife, PE, 50.740-600, 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: elaine.malosso@ufpe. br

ABSTRACT — Codinaea aquatica sp. nov., a hyphomycete collected on submerged decaying

branches of an unidentified plant, is described and illustrated. It is distinguished by

monophialidic and polyphialidic conidiogenous cells that produce fusiform to navicular

unicellular hyaline conidia with a single unbranched setula at each end, basal setula strongly

curved, apical setula straight or flexuous. Codinaea tropicalis is proposed as a comb. nov.

based on Dictyochaeta tropicalis.

Key worps — freshwater, asexual fungi, systematics

Introduction

Codinaea introduced by Maire (1937) with C. aristata Maire as the type

species is characterized by distinct, single, mononematous or synnematous,

brown to pale brown conidiophores, and an acerose to subulate, septate, dark

brown, thick-walled seta that may occur in some species. The conidiogenous

cells are mono- or polyphialidic, terminal, determinate, or with a few sympodial

extensions and sometimes with enteroblastic percurrent regenerations and with

distinct cylindrical or funnel-shaped flaring, sometimes collapsed collarettes at

the conidiogenous loci and producing unicellular or septate, falcate to lunate

hyaline conidia with a filiform appendage at each end (Almeida & Gusmao 2014,

1046 ... Oliveira, Malosso, & Castafeda-Ruiz

Granados et al. 2014, Hughes & Kendrick 1968, Li et al. 2012, Réblova & Winka

2000, Seifert et al. 2011). For two decades (Gamundi et al. 1977), the genus

Dictyochaeta (Spegazzini 1923) was considered an earlier name for Codinaea

(Maire 1937), but following the molecular analyses of Réblova & Winka (2000),

both genera are currently accepted. Li et al. (2012) recommended that species

with filiform appendages be retained in Codinaea and taxa lacking appendages

be placed in Dictyochaeta.

During a mycological survey of freshwater fungi associated with submerged

plant materials in a river at a Brazilian Atlantic forest a conspicuous fungus was

collected that is described here as a new Codinaea species.

Materials & methods

Individual collections were placed in flasks and plastic bags, taken to the laboratory,

and treated according to Castafieda (2005). Mounts were prepared in polyviny] alcohol-

glycerol (8 g PVA in 100 ml of water, plus 5 ml of glycerol) and in lactic acid (90%),

and measurements were made at a magnification of x1000 under a Nikon Eclipse Ni-U

microscope with bright field optics. Photomicrographs were obtained with the Nikon

microscope with a Nikon digital camera (model DS-Fi2). The holotype was deposited in

the Herbarium of Universidade Federal de Pernambuco, Recife, Brazil (URM).

Taxonomy

Codinaea aquatica R.F. Castafieda, M.S. Oliveira & Malosso, sp. nov. Fie. 1

MycoBAnk MB812851

Differs from all other Codinaea species by its fusiform to navicular conidia with a

strongly curved, shorter basal appendage and a straight or flexuous, longer apical

appendage.

Type Brazil, Pernambuco, Cabo de Santo Agostinho, Refugio de Vida Silvestre, Matas

do Sistema Gurjat, 8°13’S 35°03’W, alt. 85 m., on submerged decaying branches of

unidentified plant in a river, 12.V.2015, coll. M.S. Oliveira (Holotype, URM 87707).

ErymMo_oey: Latin, aquatica, referred to its growing in water.

CoLonliEs on the natural substrate scattered, hairy, black. Mycelium superficial

and immersed, composed of septate, branched, brown, smooth-walled hyphae

2-4 um diam. SETAE absent. ConrpIoPpHoREs distinct, erect, straight or

flexuous, cylindrical, 4-7-septate, brown to dark brown below, pale brown

to brown toward the apex, 200-350 x 4-6 um, smooth. CONIDIOGENOUS

CELLS mono-and polyphialidic, integrated, 40-60 x 6-7 um, terminal, mostly

determinate, but sometimes indeterminate with few sympodial extensions,

brown to pale brown, with 1-2 funnel-shaped collarettes, 2.5-4 um deep.

ConiIpIA acrogenous, fusiform to navicular, sometimes unilateral ventricose,

slightly truncate at the base, obtuse at the apex, unicellular, 25-28 x 10-12 um,

thick-walled, hyaline, smooth, with a filiform appendage at each end: basal

Codinaea sp. & comb. nov. (Brazil) ... 1047

Fic. 1. Codinaea aquatica (ex holotype URM 87707). A. Conidia. B. Conidiogenous cells.

1048 ... Oliveira, Malosso, & Castafeda-Ruiz

appendage strongly curved to forming a 90° angle with the axis of conidial

body, 4.5-6 um long, apical appendage straight or flexuous, 7-8 um long.

Notes: Among all recorded Codinaea and Dictyochaeta species (Index

Fungorum 2015) only Dictyochaeta tropicalis resembles Codinaea aquatica

in the placement and arrangement of the filiform appendage. However,

D. tropicalis [recombined in Codinaea, below] has ellipsoid to drop-shaped

conidia (7.5-9.5 x 3-5 um; Bhat & Kendrick 1993), which cannot be confused

with those of C. aquatica.

Codinaea tropicalis (Bhat & W.B. Kendr.) R.E. Castafieda & Malosso, comb. nov.

MycoBank MB812854

= Dictyochaeta tropicalis Bhat & W. B. Kendr., Mycotaxon 49: 45 (1993).

This species has monophialidic conidiogenous cells with a flared apical

collarette, and its conidia are ellipsoid to drop-shaped, hyaline, and with a

filiform appendage at each end (Bhat & Kendrick 1993). The new combination

is proposed because Dictyochaeta tropicalis has characters that fit the modern

generic concept of Codinaea (Li et al. 2012, Réeblova & Winka 2000, Seifert et

al. 2011).

Acknowledgments

The authors express their sincere gratitude to Dr. Xiu-Guo Zhang and Dr. De-Wei Li

for their critical review of the manuscript. The authors are grateful to the ‘Coordenacao

de Aperfeigoamento de Pessoal de Nivel Superior (CAPES)’ for financial support

through project 88881.062172/2014-01 and the ‘Programa Ciéncia sem Fronteiras.

RFCR is grateful to the Cuban Ministry of Agriculture and ‘Programa de Salud Animal

y Vegetal; project P131LH003033 for facilities. We acknowledge the facilities provided

by Dr. P.M. Kirk and Drs. V. Robert, G. Stegehuis and Arthur Decock through the Index

Fungorum and MycoBank websites. Dr. Lorelei Norvell’s editorial review and Dr. Shaun

Pennycook’s nomenclature review are greatly appreciated.

Literature cited

Almeida DAC, Miller AN, Gusmao LFP. 2014. New species and combinations of conidial

fungi from the semi-arid Caatinga biome of Brazil. Nova Hedwigia 98: 431-447.

http://dx.doi.org/10.1127/0029-5035/2013/0162

Bhat DJ, Kendrick WB. 1993. Twenty-five new conidial fungi from the Western Ghats and the

Adaman Islands (India). Mycotaxon 49: 19-90.

Castaneda-Ruiz RE 2005. Metodologia en el estudio de los hongos anamorfos. 182-183, in: Anais

do V Congresso Latino Americano de Micologia. Brasilia.

Gamundi IJ, Arambarri AM, Giaiotti AL. 1977. Microflora de la hojarasca de Nothofagus dombeyi.

Darwiniana 21: 81-114.

Granados M, Castafieda-Ruiz RF, Castro O, Minter DW, Kendrick 2014. Microfungi from

Costa Rica. A new species and a new combination in Codinaea. Mycotaxon 127: 115-120.

http://dx.doi.org/10.5248/127.115

Codinaea sp. & comb. nov. (Brazil) ... 1049

Hughes SJ, Kendrick WB. 1968. New Zealand fungi 12. Menispora, Codinaea, Menisporopsis. New

Zealand Journal of Botany 6: 323-337. http://dx.doi.org/10.1080/002825X.1968.10428818

Index Fungorum. 2012. http://www.indexfungorum.org/Names/Names.asp. Accession date:

2015-06-10.

Li DW, Kendrick B, Chen JY. 2012. Two new hyphomycetes: Codinaea sinensis sp. nov. and

Parapleurotheciopsis quercicola sp. nov., and two new records from Quercus phillyraeoides leaf

litter. Mycological Progress 11: 899-905. http://dx.doi.org/10.1007/s11557-011-0805-7

Maire R. 1937. Fungi Catalaunici: Series altera. Contributions 4 létude de la flore mycologique de

la Catalogne. Publicaciones del Instituto Botanico, Barcelona 3(4): 1-128.

Réblova M, Winka K. 2000. Phylogeny of Chaetosphaeria and its anamorphs based on morphological

and molecular data. Mycologia 92: 939-954. http://dx.doi.org/10.2307/3761589

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

Biodiversity Series 9. 997 p.

Spegazzini C. 1923. Algunos hongos de Tierra del Fuego. Physis 7: 7-23.

MY COTAXON

http://dx.doi.org/10.5248/130.1051

Volume 130, pp. 1051-1059 October-December 2015

Conidial fungi on Araucaria angustifolia:

Trichoconis foliicola sp. nov. and

two new records from Brazil

SILVANA SANTOS DA SILVA'!, Luis FERNANDO PASCHOLATI GUSMAO! &

RAFAEL FE. CASTANEDA- RUIZ?

‘Universidade Estadual de Feira de Santana, Departamento de Ciéncias Biol6égicas,

Laboratorio de Micologia, Avenida Transnordestina, s/n,

Novo Horizonte, 44036-900, 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

AsstRaAct —Trichoconis foliicola, a new species found on decaying needle-like leaves of

Araucaria angustifolia, is described and illustrated. Trichoconis antillana and T. queenslandica

are newly recorded from Brazil. A key to all Trichoconis species and illustrations of their

conidia are provided.

Key worps —hyphomycetes, leaf litter, taxonomy

Introduction

Trichoconis Clem., typified by T: caudata (Appel & Strunk) Clem., is

characterized by conidiophores that are distinct, colourless, single or branched,

with conidiogenous cells that are polyblastic, sympodial, denticulate, and with

cylindrical denticles that are separating cells in which rhexolytic secession

occurs. The conidia are solitary, fusiform, obclavate, clavate, ellipsoid,

subcylindrical, or navicular, rostrate, apical and lateral, septate, and colourless

(Deighton & Pirozynski 1972). The genus comprises 21 described species

(Index Fungorum 2015; Clements 1909, Pavgi & Singh 1966, Deighton &

Pirozynski 1972, Pirozynski 1974, Hawksworth 1980, Hoog & Oorschot 1985,

Matsushima 1989, 1993, Castafieda-Ruiz & Kendrick 1991, Castaneda-Ruiz

et al. 1997; Baker et al. 2001, Seifert et al. 2011, Brackel 2014). We exclude

1052 ... Silva, Gusmao & Castafieda-Ruiz

two Trichoconis species from our treatment: Trichoconis echinophila (Hoog &

Oorschot 1985) is considered to belong in Pseudotrichoconis (Baker et al. 2001,

Seifert et al. 2011), and Trichoconis indica (Pavgi et al. 1966) was described and

illustrated as having terminal integrated, unilocal conidiogenous cells, lacking

separating cells, and having schizolytic conidial secession, characters that are

unquestionably incompatible with the Trichoconis generic concept (Seifert et

al. 2011).

During research on hyphomycetes occurring on decaying needle-like leaves

of Araucaria angustifolia (Bertol.) Kuntze (Araucariaceae) we noted a unique

specimen of Trichoconis, which we describe here as new. We also present a

synopsis of accepted species and their conidia (Fics 3, 4).

Materials & methods

Samples of decaying needles of A. angustifolia were collected, placed in paper bags,

transported to the laboratory, washed in tap water, and incubated in moist chambers at

25°C for 30 days (Castafieda-Ruiz 2005). Slide mounts were prepared in PVL (polyvinyl

alcohol, lactic acid, and phenol), and micrographs were obtained with an Olympus

microscope BX 51 with differential interference contrast (DIC) DP25. Specimens were

deposited in the Herbarium (HUEFS).

Taxonomy

Trichoconis foliicola S.S. Silva, Gusmao & R.F. Castafieda, sp. nov. Fic. 1

MycoBank MB 812636

Differs from all other Trichoconis spp. by its narrow obclavate 2-septate conidia with

flagelliform and slightly curved to recurved apical cells.

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. 840 m, on decaying needle-like leaves of

Araucaria angustifolia, 14.V.2014, coll. S.S. Silva (Holotype: HUEFS 211338).

ErymMo_oey: Latin, foliicola, growing on leaves.

CoLonies on the natural substrate effuse, hyaline. Mycelium mostly

superficial. Hyphae occasionally branched, smooth, hyaline, 2-3 um wide.

CONIDIOPHORES distinct, single, erect, straight to slightly flexuous, smooth,

1—2-septate, 9-31 x 2.5-3 um. CONIDIOGENOUS CELLS mono- or polyblastic,

integrated, terminal, cylindrical, hyaline, 8-15 x 2.5 um, with conspicuous,

cylindrical denticles, 2.5-4 um long. Conidial secession rhexolytic. CONIDIA

narrowly obclavate, rostrate, 2-septate, smooth, dry, hyaline, 25-50 x 2.5-4 um,

rostrum flagelliform, slightly curved to recurved, 15-25 um long.

Notes: Trichoconis caudata, T. lichenicola D. Hawksw., and T. queenslandica

are somewhat similar to T. foliicola but are easily differentiated as follows:

T. caudata conidia are fusiform, mostly 5-septate, and 27-40 x 6.5-12 um;

Trichoconis foliicola sp. nov. (Brazil) ... 1053

Fic. 1. Trichoconis foliicola (HUEFS 211338): A. Conidiophore and conidia. B. Conidiophores

reduced to conidiogenous cells. C. Conidiogenous cells. D. Conidia.

1054 ... Silva, Gusmao & Castafteda-Ruiz

Fic 2. Trichoconis antillana (HUEFS 211339): A, B. Conidiophore and conidia. C. Conidium.

T. queenslandica (HUEFS 211341): D. Conidiophore and conidia. E. Conidium.

T. lichenicola conidia are fusiform to obclavate, 2—4-septate, and 35-65 x

5.5-7 um; and T: queenslandica conidia are fusiform, mostly 3-septate, and

44-85 x 5-8 um.

Trichoconis foliicola sp. nov. (Brazil) ... 1055

rn)

6 AN,

Fic 3. Conidia of Trichoconis spp.: A. T. africana (Deighton & Pirozynski 1972). B. T: amazonensis

(Matsushima 1993). C. T. angustispora (Deighton & Pirozynski 1972). D. T. antillana (Castaneda-

Ruiz et al. 1997). E. T. appendiculata (Deighton & Pirozynski 1972). F. T. capitata (Pirozynski

1974). G. T. caudata (Deighton & Pirozynski 1972). H. T. englerulae (Deighton & Pirozynski 1972).

I. T. foliicola (this work). J. T. hamata (Deighton & Pirozynski 1972).

20um

Trichoconis foliicola sp. nov. (Brazil) ... 1057

Trichoconis antillana R.F. Castaftieda, W.B. Kendr. & Guarro,

Mycotaxon 65: 103 (1997) Fic. 2A—C

CONIDIOPHORES 20-25 x 3-3.5 um. CONIDIOGENOUS CELLS 15-20 x 3-4

um, denticles 4-5 um long. Conrp1a 45-64 x 3-4 um.

SPECIMENS EXAMINED: BRAZIL, SANTA CATARINA STATE: Urubici, Parque Nacional

de Sao Joaquim, 28°04’S 49°25’ W, 990 m alt., on decaying needle-like leaves of Araucaria

angustifolia, 18.VIII.2014, coll. $.S. Silva (HUEFS 211339). R10 GRANDE DO SUL STATE:

Sao Francisco de Paula, Floresta Nacional de Sao Francisco de Paula, 29°25’S 50°23’W,

alt. 840 m, on decaying needle-like leaves of Araucaria angustifolia, 14.V.2014, coll. S.S.

Silva (HUEFS 211340).

Norte: A new record for Brazil.

Trichoconis queenslandica Matsush., Matsush. Mycol. Mem. 6: 43 (1989) Fic. 2D-E

CONIDIOPHORES 34-67 X 3-4.5 um. CONIDIOGENOUS CELLS 19-30 x 3-4.5

uum, denticles 4.5-10 um long. Conrp1a 42-73 x 4.5-6 um.

SPECIMEN EXAMINED: BRAZIL. SANTA CATARINA STATE: Urubici, Parque Nacional de

Sao Joaquim, 28°04’S 49°25’W, alt. 990 m, on decaying needle-like leaves of Araucaria

angustifolia, 29.V.2014; coll. S.S. Silva (HUEFS 211341).

Norte: A new record for Brazil.

Key to Trichoconis species

Conidiophores and conidia pale olivaceous, obclavate to filiform,

1-10-septate, 30-200 x 3-4.5 UM ....... eee eee eee eee eens T: viridula

Conidiophores andiconidia-colorléss, og Ges <g- (aise hie fed Bec Rac Bs 2

Gonidiawithaostrate-apical cell at Ant Asal Het Meet nett iets Anat 6 Aes 3

Conidiaswitheutkostratedpical.ce lly: i. w5 he Soke Mek es Nake Sadho kt Bou 9

Conidia with hamate rostrum,

l-septate; 20-30: 2-375 UNy, COSTUME PIM by oo. se ests T: hamata

Conidiawithout Wamate LOSthUd Aa, Gt eos aela eae oe eon o ne eons +

Conidia clavate, 1-2-septate, 36-49 x 2.5-3.5 um,

rostrum flagelliform, 17-38 um long ................00005. T. amazonensis

(RS TRE (GRAS Olona BLO (cok emma Laie ds AG aS elise neta Arie wiN. Serratia ie. Seiad 5

Conidia 2-septate, obclavate, 25-50 x 2.5-4 um, rostrum flagelliform,

jee se a U0 ied GG) (1. i ee ree, Cee. Deere. Care Ol T. foliicola

Gorlidigunore than? septate. at tect Meet t Aan ine w eh A ed weet A Meee i, 6

Fic 4. Conidia of Trichoconis spp.: A. T. hibernica (Deighton & Pirozynski 1972). B. T. lichenicola

(Hawksworth 1980). C. T. malloti (Deighton & Pirozynski 1972). D. T. pedicephora (Castanieda-

Ruiz & Kendrick 1991). E. T. physciicola (Brackel 2014). F. T. queenslandica (Matsushima 1989).

G. T. schiffnerulae (Deighton & Pirozynski 1972). H. T. sigmoidea (Deighton & Pirozynski 1972).

I. T. trichiliae (Deighton & Pirozynski 1972). J. T. viridula (Deighton & Pirozynski 1972).

1058 ... Silva, Gusmao & Castafieda-Ruiz

10.

Bi;

t2:

13.

14.

i Foy

16.

17.

18.

19.

Conidia 0-3-septate, mostly 3 septa,

fusiform, 44-85 x 5-8 um, rostrum 17-40 um long......... T. queenslandica

Conidia. sometinies more than-d-septate: 5 A aad an env one ton 7

Coriidiatiotanorée than 4sseplate. fo tell lore Belg nee torneo RS wr RES 8

Conidia 3-5-septate, mostly 5-septate, fusiform, 27-40 x 6.5-12 um,

POSECUIN G59 OF WINTON. Bein Rake ade et tony on Rosen Sakefers todos T. caudata

Conidia 2—4-septate, 35-65 x 5.5-7 um, fusiform to obclavate,

POSEPUT-UiPetOTAOSMIONS 5. rack eons pa a gee «Sp pam re epee T. lichenicola

Conidia 4-septate, obclavate, 50-120 x 4-6 um ..............0005. T. antillana

Conidiawith not:more-thar- one Sepia: 6... ks vse wee eae eel eee 10

Conidiawith more tharronese pines te. <ts. soi aby seats bs irdet abs braeta tts praeta ti greetabecs 11

Conidia navicular, with an apical droplet of mucilage, 1-septate,

LEAZGSRO—7 EN ten hic tata tet edt ia te Piece Piet Biot ee ee 2 T’ capitata

Conidia ellipsoid to suborbicular, 0-1-septate,

VOSA IA at ag tat Ae ee Mee A nee A Anak A siet <2 Ms T. physciicola

Conidia filiform, attenuated at the ends, 3-6-septate,

POSTVORE 2 5E 4 WI ih. cw de geld pees ge keh ooee mh eee dime Sled T. angustispora

Bre sevialirehy av e12 INGL0ig 10 Mh aepraapee mapa re ame NN ene PY aig eee Penal Pena ecmiee Pee & 12

Conidiagwiti not More Wianies 6ePtde, Ge Rl nt il ne inl neha 13

Conidiawaith more than Seba 6! we kes Saket ele Be Cire kM re Rh ae ee 18

Conidial Jetroth 4 Oy hin Sts sect g stig atte s ccc s atsRh g sephaniny omtacitry omehauies cmt 14

Conidial leneth 40g Ao 8s hhass bbe noes nhses aboeadsh Sead heed needs 15

Conidia 21-30 x 3-4 um, 2=3-septate’ oo on eet an eda ede eddies T: hibernica

Conidia 24-38 x 6.5-9.5 um, subcylindrical, clavate-ellipsoid or

obclavater ellipsoid; SSE ptate «ce xy, srece <tc srscd ah: sreh davirden gdrired dates T: englerulae

OH ICIALLEHTOE AD Es Sacre pea Baty prac ak praca pra ab oawn Sat ave Mat an Sat iaw a deen b aw 16

Conidial length, Saori. 2 ato g vdemes dete toms es oe hal oe hae be ye laa ool 17

Conidia 27-40 x 4-6.5 um, obclavate to fusiform,

BESEDUALCR gah, See er wR gS AN NAT rn ae ek, A NTRS CAS SAU RA TE) T: trichiliae

Conidia 20-41 x 5-8 um, obovoid or cylindrical-obovoid,

BESO DUALCS ate eaten tnt, Maas tore ach Sl Ne ee Bel T: schiffnerulae

Conidia 20-50 x 5-9 uum, mostly fusiform, 0-3-septate .............. T. malloti

Conidia 40-72 x 4.5-7 um, fusiform, mostly 3-septate .............. T. africana

Conidiaswithaiotmore-than 4 septa, prs. occ sc arcade Oe une bs die Betas Beas 1g

Conidia 3-9-septate (mostly 5-8-septate), fusiform, sigmoid,

50-806 57M hos whe ¢ob- 63h ced baked b-wadsb.s ad n-baditg T. sigmoidea

Conidia 3—4-septate, fusiform,

apm bocca byrne: OT Pi, Be oases aan Baktin aehies Gakies Gabe «6 T: appendiculata

Conidia 2-4-septate, fusiform or obclavate,

Zi ety ee Sond AL RLS Bese. a ud case ed cette ed coh a Soe ce ie oh «Ha SR er T: pedicephora

Trichoconis foliicola sp. nov. (Brazil) ... 1059

Acknowledgments

The authors express their sincere gratitude to Prof. Bryce Kendrick and Dr. De-Wei

Li for their critical review of the manuscript. The authors are grateful to the ‘Programa

de Pés-Graduacao em Botanica (PPGBot/ UEFS); CNPq for financial support (proc.

141475/2013-7), and ICMBIO for permission to collect in ‘Floresta Nacional de

Sao Francisco de Paula’ and ‘Parque Nacional de Sao Joaquim’ (Number: 42334-1).

LFPG is grateful to CNPq for grant (Proc. 303062/2014-2) and RFCR is grateful to the

“Programa Ciéncia sem Fronteiras” and Cuban Ministry of Agriculture and “Programa

de Salud Animal y Vegetal’, project P131LH003033 for facilities. We acknowledge the

facilities provided by Dr. P.M. Kirk and Drs. V. Robert and A. Decock through the Index

Fungorum and MycoBank websites. Dr. Lorelei Norvell’s editorial review and Dr. Shaun

Pennycook’s nomenclature review are greatly appreciated.

Literature cited

Baker WA, Partridge EC, Morgan-Jones G. 2001. Notes on hyphomycetes. LXXXIV.

Pseudotrichoconis and Rhexodenticula, two new monotypic genera with rhexolytically

disarticulating conidial separating cells. Mycotaxon 79: 361-373.

Brackel WV. 2014. Kommentierter Katalog der flechtenbewohnenden Pilze Bayerns. Bibliotheca

Lichenologica 109. 476 p.

Castaneda-Ruiz RE 2005. Metodologia en el estudio de los hongos anamorfos. 182-183, in: Anais

do V Congresso Latino Americano de Micologia, Brasilia.

Castafieda-Ruiz RE, Kendrick B. 1991. Ninety-nine conidial fungi from Cuba and three from

Canada. University of Waterloo Biology Series 35. 132 p.

Castafeda-Ruiz RF, Kendrick WB, Guarro, J. 1997. Notes on conidial fungi. XIV. New hyphomycetes

from Cuba. Mycotaxon 65: 93-106.

Clements FE. 1909. The genera of fungi. Minneapolis, H.W. Wilson Co. 227 p.

Deighton FC, Pirozynski KA. 1972. Microfungi V. More hyperparasitic hyphomycetes. Mycological

Papers 128. 110 p.

Hawksworth DL. 1980. Notes on some fungi occurring on Peltigera, with a key to

accepted species. Transactions of the British Mycological Society 74: 363-386.

http://dx.doi.org/10.1016/S0007-1536(80)80167-7

Hoog GS de, Oorschot CAN van. 1985. Taxonomy of the Dactylaria complex, VI. Key to the genera

and check-list of epithets. Studies in Mycology 26: 97-124.

Index Fungorum. 2015. Fungal names search. http://www.indexfungorum.org/names/Names.asp

[accessed 11 October 2015].

Matsushima T. 1989. Matsushima mycological memoirs no. 6. Kobe, Matsushima Fungus

Collection.

Matsushima T. 1993. Matsushima mycological memoirs no. 7. Kobe, Matsushima Fungus

Collection.

Pavgi MS, Singh RA, Dular R. 1966. Some parasitic fungi on rice from India. Mycopathologia et

Mycologia Applicata 30: 314-322.

Pirozynski KA. 1974. Meliolina mollis and two new hyperparasites in India. Kavaka 2: 33-41.

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

Biodiversity Series 9. 997 p.

MY COTAXON

http://dx.doi.org/10.5248/130.1061

Volume 130, pp. 1061-1072 October-December 2015

Entyloma scandicis, anew smut fungus on Scandix verna from

Mediterranean forests of Israel

KyRYLO G. SAVCHENKO?*, Lori M. CARRtIs’, LisA A. CASTLEBURY’,

VASYL P. HELUTA?, SOLOMON P. WASSER®* & EVIATAR NEVO*

"Department of Plant Pathology, Washington State University, Pullman, WA 99164, USA

?USDA-ARS, Systematic Mycology & Microbiology Laboratory, USDA-ARS,

10300 Baltimore Ave, Beltsville, MD 20705, USA

°M.G. Kholodny Institute of Botany of the National Academy of Sciences of Ukraine,

2 Tereshchenkivska St., Kyiv 01601, Ukraine

‘Department of Evolutionary & Environmental Biology, University of Haifa,

Mt Carmel, Haifa 31905, Israel

* CORRESPONDENCE TO: kyryll.savchenko@wsu.edu

AsstTRAcT — The morphology and phylogeny of a species of Entyloma on Scandix verna

(Apiaceae, Apioideae, Scandiceae) collected in the Mount Carmel and Lower Galilee regions

of Israel were studied using light microscopy and ITS rDNA sequence analyses. The fungus

differs morphologically from other species on hosts of subfamily Apioideae: E. bupleuri,

E. helosciadiis.l., E. kundmanniae, E. magocsyanum, and E. pastinacae. Molecular phylogenetic

analyses revealed that all specimens of Entyloma from Scandix represent a monophyletic

lineage, sister to E. magocsyanum. As a result, the smut in leaves of Scandix verna is described

and illustrated here as a new species, Entyloma scandicis.

Key worps — Entylomatales, evolution, plant pathogens

Introduction

Entyloma contains more than 170 species of dicotyledon-parasitizing smut

fungi, which are found on all continents except Antarctica (Vanky 2012).

The phylogenetic relationships of Entyloma species based on ITS rDNA were

established by Begerow et al. (2002), who showed that Entyloma evolution has

been driven by its hosts. In a more recent study, both ITS sequence analysis

and morphological data were used to delimit Entyloma spp. on Eryngium

(Savchenko et al. 2014). The integration of molecular phylogenetic analyses,

host plant taxonomy, and morphology provides a natural classification of these

fungi.

1062 ... Savchenko & al.

TABLE 1. GenBank sequences used in this study

SPECIES

Entyloma atlanticum

E. arnicale

E. australe

E. bidentis

E. browalliae

E. calceolariae

E. calendulae

E. carmeli

E. chrysosplenii

E. comaclinii

E. compositarum

E. corydalis

E. costaricense

E. doebbeleri

E. eryngii

E. eryngii-cretici

E. eryngii-plani

E. eschscholziae

E. fergussonii

E. ficariae

E. fuscum

E. gaillardianum

E. guaraniticum

E. hieracii

E. holwayi

E. linariae

E. lobeliae

E. madiae

E. magocsyanum

E. matricariae

E. microsporum

E. polysporum

E. serotinum

E. ranunculi-repentis

GENBANK No.

AY081018

AY854965

AY081019

AY081020

AY081021

AY081022

AY081023

KF310892,

AY081024

AY081025

AY854966

AY081027

AY081028

AY081032

AY854972,

AY081033,

KF310896,

KF310894,

AY081034

KC456226

AY854971

AY081035,

JQ586199

AY081036

AY081037

AY081038

AY081039

AY081040

AY081041

AY081042

AY081043

KF310891

AY081044

AY081045

AY081046

AY081048

AY081047

KF310895

KF310897

KF310895

REFERENCE

Begerow et al., 2002

Boekhout et al., 2006

Begerow et al., 2002

Savchenko et al., 2014

Begerow et al., 2002

Boekhout et al., 2006

Begerow et al., 2002

Begerow et al., 2002;

Boekhout et al., 2006;

Savchenko et al., 2014

Begerow et al., 2002

Genbank, unpublished

Boekhout et al., 2006

Begerow et al., 2002;

Savchenko et al. 2012

Begerow et al., 2002

Savchenko et al., 2014

Begerow et al., 2002

Entyloma scandicis sp. nov. (Israel) ... 1063

Apiaceae harbors ca. twenty Entyloma species (Begerow et al. 2002; Vanky

2012), five of which are known on hosts of subfamily Apioideae: E. bupleuri

Lindr. on Bupleurum (Liro 1904), E. helosciadii Magnus on Helosciadium

(Magnus 1882), E. kundmanniae Malencon & Massenot on Kundmannia

(Guyot et al. 1969), E. magocsyanum Bubak on Tordylium (Bubak 1907), and

E. pastinacae Jaap on Pastinaca (Jaap 1916). Both Entyloma flavum Cif. on

Berula and Sium (Ciferri 1924) and E. oenanthes Maire on Oenanthe (Maire et

al. 1901) are considered conspecific with E. helosciadii s.1. (Reid 1957; Vanky

1994). Species delimitation of Entyloma on Apioideae is based primarily on

host plant taxonomy and secondarily on minor differences in sori and spore

morphologies. The analysis by Savchenko et al. (2014) clustered together

Entyloma species on Apiaceae. That analysis was based mostly on the smut

parasites of Eryngium (subfamily Saniculoideae) and included only one species

(Entyloma magocsyanum) that parasitized a host from subfamily Apioideae.

During a 2011 survey on the diversity of smut fungi in Israel, a few infected

Scandix verna plants were collected in two localities in northern Israel.

Previously, no Entyloma species had been recorded on Scandix. The present

study aims to resolve the specific status of the smut on S. verna through

morphological and ITS sequence analyses and to determine its phylogenetic

affinities within Entyloma.

Materials & methods

Herbarium samples are deposited in Haifa University Herbarium, Haifa, Israel

(HAI); Washington State University Herbarium, Pullman, U.S.A. (WSP); and United

States National Fungus Collection, Beltsville, U.S.A. (BPI). We were not able to include

an Entyloma kundmanniae specimen in our analysis, so the information given on that

species is based on Guyot et al. (1969) and Vanky (2012). Our hologenetype concept

follows Chakrabarty (2010).

Sorus and spore characteristics were studied using dried herbarium material.

Specimens were examined by light microscopy (LM). Pictures of sori were taken with

a Canon Power Shot G10 camera. For LM, small pieces of leaf tissue with sori were

mounted in 90% lactic acid on a microscope slide to which several drops of distilled

water were added after which the slide was heated to the boiling point and cooled. The

softened sori were cut in long pieces and squashed with a lancet, covered with a cover

glass, gently heated to boiling point to eliminate air bubbles, and then examined under

a Carl Zeiss Axiostar™ light microscope at 1000x magnification. LM photographs were

taken with a Canon Power Shot G10 camera. At least 50 spores were measured from

each collection, and the variation is presented as a range, with extreme values given in

parentheses. Mean and standard deviations (SD) calculated from spores measured in all

specimens are provided after the spore size ranges.

For SEM studies, spores were attached to metal stubs by double-sided adhesive

tape and coated with gold. Spore surface ornamentation was observed at 15 kV and

1064 ... Savchenko & al.

photographed with a JEOL JSM-6700F scanning electron microscope with a working

distance of ca. 12-13 mm.

Most sequences were obtained from GenBank (TaBLE 1). Three additional sequences

were generated for this analysis (TABLE 2). Genomic DNA was isolated from sori removed

from herbarium specimens and lysed in 1.5 mL tubes for 1 min using FastPrep*24. Tubes

were incubated in a water bath for 5 hours at 55 °C, and DNA extracted using DNeasy

Plant Mini Kit (QIAGEN) following the manufacturer's instructions.

All amplifications were performed in 20 ul aliquots on an Applied Biosystems*

GeneAmp 9700 thermal cycler. ITS5 or ITS1 were used as the forward primer and ITS4

as the reverse primer (White et al. 1990).

Standard cycling parameters with an annealing temperature of 57 °C were used

for amplification. PCR products were purified with USB ExoSAP-IT according to the

manufacturer's instructions and amplified with respective forward and reverse PCR

primers with the BigDye® Terminator v3.1 Cycle Sequencing Kit. Those products were

sequenced on an ABI PRISM?® 3100 Genetic Analyzer.

The data were initially assembled and edited in Sequencher 4.5 for Windows, and

the consensus sequences were aligned using MAFFTVv6 (Katoh et al. 2002; Katoh & Toh

2008) under the default settings. Ambiguously aligned regions were recorded using

GBlocks 091.b (Castresana 2000) with these options: minimum number of sequences

for a conserved position to 29, minimum number of sequences for a flank position to

29, maximum number of contiguous non-conserved positions to 8, minimum length

of a block to 5 and allowed gap positions to with half, and excluded from the analyses.

PAUPv40b 10 was used for parsimony analysis, with trees inferred using the heuristic

search option with 1000 random sequence additions. Maxtrees were unlimited,

branches of zero length were collapsed, and all multiple parsimonious trees were saved.

Descriptive tree statistics for parsimony (Tree Length [TL], Consistency Index [CI],

Retention Index [RI], Relative Consistency Index [RC], and Homoplasy Index [HI] were

calculated for trees generated under different optimality criteria. Bootstrap analysis (BS)

(Hill & Bull 1993) was based on 1000 replications.

Bayesian analysis using a Monte Carlo Markov Chain (MCMC) technique was

implemented using MrBayes 3.1.2 (Huelsenbeck & Ronquist 2001; Ronquist &

Huelsenbeck 2003). Four MCMC chains were run simultaneously, starting from random

trees, for 1,000,000 generations. Trees were sampled every 100" generation for a total of

10,000 trees. The first 2000 trees were discarded as the burn-in phase of each analysis.

Posterior probabilities (PP) (Rannala & Yang 1996) were determined from a majority-

rule consensus tree generated with the remaining 8,000 trees. This analysis was done

four times starting from different random trees to ensure that trees from the same tree

space were being sampled during each analysis.

Trees were rooted using Entyloma atlanticum as in Begerow et al. (2002).

Results

Morphological analyses

All examined Entyloma specimens (TABLE 2) produced sori in the leaves of

their hosts. The color of sori ranged from white in young sori to pale yellowish

Entyloma scandicis sp. nov. (Israel) ... 1065

TABLE 2. Entyloma specimens examined in this study

SPECIES Host VOUCHER ORIGIN eS ss GENBANK

E. bupleuri Bupleurum subovatum HUJ Israel Chabelska/1942

E. helosciadii Apium nodiflorum BPI 175486 _ Ireland O’Connor/1945

Apium* nodiflorum BPI 175487 — Algeria Maire/1916

Berula angustifolia WSP 34402 Germany Unknown/1900

Helosciadium repens WSP 34403 Algeria Unknown/1904

Oenanthe silaifolia WSP 70797 Romania Vanky1965

E. magocsyanum Tordylium cordatum HAI 4625 Israel Savchenko/2011 =. KF310891

Tordylium maximum WSP 66423 Romania - Vanky/1966

Tordylium maximum BPI 175835 Romania Bubak/1905

Tordylium maximum BPI 175836 Romania Bubak/1905

E. pastinacae Pastinaca opaca BPI 176163 Romania Savulescu &

Rayss/1930

E. scandicis Scandix verna HAI 4799 Israel Savchenko/2011 KF447773

Scandix verna HAI 4805 Israel Savchenko/2011 KF447774

Scandix verna HAI 4806 Israel Savchenko/2011 KF447775

*as Helosciadium

brown in old sori. The spores in all specimens were solitary, in chains, or in

groups (but never crowded) and variable in size within a collection. Asexual

states were present in all species except E. bupleuri. Spore sizes were similar

in all species, but spore wall thicknesses varied from 0.5-1 um in Entyloma

magocsyanum to 1-3 um in E. helosciadii. The color of sori varied from brown

in Entyloma bupleuri to white in E. scandicis (and sometimes E. helosciadii).

Phylogenetic analyses

The ITS alignment included 48 sequences (including three sequences of

Entyloma from Scandix verna) comprising 655 characters (including gaps).

Parsimony analysis revealed that 477 characters are constant and of the variable

characters 77 are parsimony-uninformative and 101 are parsimony informative.

The parsimony analysis yielded 45 equally parsimonious trees, and the strict

consensus tree of all equally parsimonious trees was used.

The different runs of Bayesian phylogenetic analyses yielded consistent

topologies. We present the consensus tree of one run of Bayesian phylogenetic

analyses to illustrate our results (Fic. 1). All analyses clustered together (with

high support: PP = 1.0; BS = 93) the sequences of the smut on Scandix verna,

which formed the sister lineage to Entyloma magocsyanum. The sequences of

the Scandix verna smut from Carmel and Lower Galilee regions were identical

to one another.

1066 ... Savchenko & al.

The phylogenetic relationships among species of Entyloma on Apiaceae

inferred here are congruent with those in the study on Entyloma on Eryngium

(Savchenko et al. 2014).

E. arnicale AY854965

E. australe AY081019

E. compositarum AY854966

E. compositarum AY081026

E. guaraniticum AY081038

E. doebbeleri AY081032

E. comaclinii AY081025

E. zinniae AY081049

E. costaricense AY081028

E. calendulae AY081023

E. diastateae AY081031

E. lobeliae AY081042

E. browalliae AY081021

0.94/-

r E. deliliae AY081030

E. bidentis AY081020

E. dahliae AY081029

E. holwayi AY081040

0.87/-

E. calceolariae AY081022

E. eryngii AY854972

E. eryngii KF310896

Gao) ! E. eryngii KF310897

E. eryngii AY081033

pee eryngii-cretici KF310894

E. eryngii-cretici KF310895

E. eryngii-plani AY081034

4 o/10@ &. carmeli KF310892

E. carmeli KF310893

E. magocsyanum KF310891

10/97 _ E. seandicis KF447773

1.0/9 E. scandicis KF 447774

E. scandicis KF 447775

1.0/90 E. fergussonii AY854971

E. serotinum AY081048

S E. eschscholziae KC456226

E. madiae AY081043

I E. gaillardianum AY081037

pat_|0.99/90 & hieracii AY081039

Y E. matricariae AY081044

1.0/100 E. polysporum AY081046

E. chrysosplenii AY081024

1.0/100r ©: ficariae AY081035

1.0/100 E. ficariae JQ586199

E, linariae AY081041

E. ranunculi-repentis AY081047

E. corydalis AY081027

E. microsporum AY081045

Entyloma on Apiaceae

E. fuscum AY081036

SARI RTOS WAG ws ().{ substitution/site

Fic. 1 Bayesian inference of phylogenetic relationships of Entyloma spp. resulting from the analysis

of ITS nucleotide sequence data. Numbers on branches are estimates for PPs >0.8 from Bayesian

inference (before /) and BS values >70 (after /). The tree was rooted using Entyloma atlanticum.

Entyloma scandicis sp. nov. (Israel) ... 1067

Taxonomy

Entyloma scandicis K.G. Savchenko, Carris, Castl., Heluta, Wasser & Nevo

sp. nov. FIGURE 2

MycoBank MB 811390

Differs from Entyloma helosciadii by its paler spores and its specialization on Scandix.

Type: Israel. Lower Galilee, 2 km E of Kiryat Ata, 32°82’26”N 35°13’90’E, ca. 350 m

asl, on Scandix verna O. Cohen, 15.04.2011, leg. K.G. Savchenko (Holotype, HAI 4799:

GenBank KF447773).

ErymMo.oey: The name of the species refers to the host plant genus, Scandix.

Sori in leaves, in round or elongate to polyangular amphigenous flat scattered

or sometimes gregarious spots, initially white, later pale yellowish brown,

0.2-1 mm diameter or larger when confluent. Spores singular or in small

irregular groups, sometimes in chains, globose, subglobose, ovoid, ellipsoidal

to slightly irregular, (7) 8-11 x 9.5-15 (17) um diameter [av. + SD, 9.8 + 1.6 x

12.6 + 2.5 um], subhyaline. Spore wall uniform, 1.5-2 (2.5) um thick, hyaline

to subhyaline, smooth. Anamorphic state present as fascicles of conidia and

conidiophores, forming the amphigenous, white cover.

ADDITIONAL SPECIMENS EXAMINED: ISRAEL, Harea, Mount Carmel, University of

Haifa campus, 32°75’83”N 35°02’32”E, ca. 430 m a.s.l., on Scandix verna, 11.04.2011,

leg. K.G. Savchenko (HAI 4805; GenBank KF447774); Mount Carmel, Carmel National

Park, 32°75’86’'N 35°02’41”E, ca. 440 m asl, on Scandix verna, 30.03.2011, leg. K.G.

Savchenko (HAI 4806; GenBank KF447775).

ComMENTs - The infected plants were found in March and April in two

localities in northern Israel in different habitats. At Mount Carmel, Entyloma

scandicis occurred on host plants in a shaded forest community under Pinus

halepensis. In the Lower Galilee location, the fungus occurred on host plants

in an open forest in glades between Cupressus sempervirens. Both locations

are typical for Israel’s Mediterranean floristic zone (Zohary 1973). Certainly,

Entyloma scandicis is a Mediterranean forest species, hitherto not known

outside of northern Israel. Entyloma scandicis was not found in steppe and

semi-desert areas within the area of host distribution despite examination of

thousands of host plants.

Discussion

Scandix is a monophyletic genus of the tribe Scandiceae (Downie et al.

1998, 2000; Lee & Downie 2000; Spalik et al. 2001) of which no members were

previously known to host smut fungi (Vanky 2012). Our molecular phylogenetic

analyses and morphological data have helped to resolve the systematic position

of Entyloma on Scandix verna.

1068 ... Savchenko & al.

Entyloma scandicis can be distinguished from E. bupleuri by the sorus color

(yellowish white in E. scandicis, brown in E. bupleuri) and the presence of an

Entylomella-like asexual stage in E. scandicis. The new species can be separated

from Entyloma kundmanniae, E. magocsyanum, and E. pastinacae by thicker

spore walls (1.5-2.5 um in E. scandicis vs. <1.5 um in the other three). In

several aspects, such as in spore dimensions, Entyloma scandicis resembles

E. helosciadii, which differs in its pale to dark yellow spores.

ITS phylogenetic analysis recovered all three Entyloma specimens on Scandix

verna in a strongly supported monophyletic group forming an independent

lineage (Fic. 1). Morphologically there is no difference between the Mt Carmel

and Lower Galilee specimens. As in the molecular phylogenetic analysis of

a smaller sampling of Entyloma species on Apiaceae (Savchenko et al. 2014),

our analysis clustered together all Entyloma species on Apiaceae within the

/apiaceae clade and in sharing a common ancestor with other Entyloma groups

on asterid hosts. Interestingly, E. calceolariae Lagerh., a parasite on species in

the family Calceolariaceae, also clustered within the /apiaceae clade (Fic. 1).

So far, only two species of Entyloma on Apioideae—E. magocsyanum and

E. scandicis—have undergone molecular phylogenetic analyses (Savchenko

et al. 2014). It should be noted that molecular-oriented studies have implied

significant radiation for some Entyloma species (Savchenko et al. 2014; Vanky &

Lutz 2010), which supports the hypothesis that Entyloma species have relatively

narrow host ranges restricted to a single genus or, in some cases, a single host

species (Savchenko et al. 2014; Savile 1947; Vanky 1994, 2009). Additional

sequencing is needed to resolve the taxonomy of Entyloma helosciadii, a

complex of three species known to infect hosts from four different genera in

the tribe Oenantheae (Reid 1957; Vanky 1994). Another taxonomic problem

is the possible conspecificity of the pathogenic Entyloma magocsyanum and

E. pastinacae, both with hosts in tribe Tordyleae and with no diagnostic

morphological differences.

This study shows that genetic divergence, a different host tribe, and different

spore and soral morphologies support Entyloma scandicis on Scandix verna

as a new species. This species fills a gap in the distribution of Entyloma on

representatives of the tribe Scandiceae. Future molecular phylogenetic studies

combined with morphological methods may well reveal higher diversity within

the species of Entyloma on Apioideae.

Acknowledgments

The authors are grateful to Dominik Begerow and Roger G. Shivas for peer reviewing

the manuscript, the curators of herbaria WSP and BPI for allowing us to work with their

collections, and Vitalii Sapsai for help with SEM photography.

Entyloma scandicis sp. nov. (Israel) ... 1069

Fic. 2 Entyloma scandicis (holotype, HAI 4799): A. sori in leaves of Scandix verna; B. spores (LM);

C. spores (SEM). Scale bars: A = 2 mm, B = 20 um, C = 2 um.

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MY COTAXON

http://dx.doi.org/10.5248/130.1073

Volume 130, pp. 1073-1101 October-December 2015

Myxomycetes of Chihuahua (México) 4.

Central plains of the Chihuahuan desert

Marcos LIZARRAGA’, GABRIEL MORENO”, MARTIN ESQUEDA},

CYNTHIA SALAZAR-MARQUEZ? & MARTHA L. CORONADO‘*

' Dpto. Ciencias Quimico Bioldgicas, Instituto de Ciencias Biomédicas,

Univ. Autonoma de Ciudad Juarez, Anillo Envolvente Pronaf y Estocolmo s/n,

Ciudad Juarez, Chihuahua 32300, México

? Dpto. Ciencias de la Vida, Edificio de Biologia, Univ. Alcala, 28805 Madrid, Esparia

> Centro de Investigacion en Alimentacion y Desarrollo,

A.C. Apartado Postal 1735, Hermosillo, Sonora, México

‘Univ. Estatal de Sonora. Apartado Postal 11, Admon. 11, Hermosillo 83000, Sonora, México

* CORRESPONDENCE TO: gabriel. moreno@uah.es

ABSTRACT—Collections of myxomycetes gathered in field and cultivated in moist chamber

cultures from samples collected in 10 localities in the central plains of the Chihuahuan desert

were studied. Of the 37 species identified, eight (Badhamia spinispora, B. verrucospora,

Comatricha ellae, Didymium tehuacanense, Echinostelium paucifilum, Lycogala exiguum,

Physarum apiculosporum, and P. spectabile) represent new records for Chihuahua. Badhamia

verrucospora, Echinostelium paucifilum, and Physarum apiculosporum are recorded for the

first time from Mexico.

Key worps—Amoebozoa, chorology, myxobiota, Myxomycota, SEM, slime moulds,

taxonomy

Introduction

With a total area of 507,000 km’, the Chihuahuan desert is the largest in

North America and extends from New Mexico and Texas in the United States

to north of Oaxaca in Mexico. It ranges in elevation between 1000-2000 m and

has an annual average temperature of 16-18°C. Temperatures lower than 0°C

have been recorded in some northern regions during winter. In summer, the

most important precipitation occurs in the form of monsoons, although not

exceeding 400 mm annually.

The vegetation of the Chihuahuan desert consists mainly of microphyllous

scrubs, with Larrea tridentata (DC.) Coville (Zygophyllaceae), Fouquieria

1074 ... Lizarraga & al.

TABLE 1. Studied localities and vegetation types

LOCALITY LATITUDE N LONGITUDE W ELEVATION VEGETATION

TOWN JUAREZ

31°13’53.1” 106°30°36.8” 1120 m SDV

1. Samalayuca

TOWN ASCENSION

31°36’36.0” 106°47’03.0” 1300 m SDV

2. Ascencion

TOWN GUADALUPE

31°07'54.7” 105°42’47.8” 1100 m MDS

3. El Cuervo

Town AHUMADA

31°07'32.0” 106°29’48.0” 1280 m MDS

4. Candelaria

TOWN OJINAGA

29°14’24.0” 104°26’09.0” 1380 m MDS

5. La Mula

6. Puente Pegiiis I 29°32’28.9” 104°42’51.7” 770m MDS

TOWN MANUEL BENAVIDES

29°05'39.1” 103°52’38.0” 1065 m MDS

7. Camino a San Antonio, km 2

Town DELICIAS

28°06'27.2” 105°24’53.9” 1160 m MDS

8. Parque Industrial

TOWN CAMARGO

27°51'07.1” 104°4.4’04.7” 1265 m MDS

9. Carret. a Ojinaga Km 42

TOWN JIMENEZ

27°24'53.3” 104°53’37.2” 1370 m MDS

10. Establo El Parral

Vegetation: MDS = microphyllous desert scrub; SDV = sandy desert vegetation

splendens Engelm. (Fouquieriaceae), Prosopis glandulosa Torr. (Leguminosae),

and several species of Yucca and Agave (Asparagaceae) among the main

dominants. Especially prominent are a great diversity of small and medium-

sized cacti (Cactaceae), including species of Coryphantha, Cylindropuntia,

Echinocereus, Mammillaria, and Opuntia (Rzedowski 2006).

Despite its considerable biological diversity, only a few studies of its

myxobiota have been conducted, most of which have focused on pine-oak

forest ecosystems (Lizarraga et al. 2004a, 2005a,b, 2006; Moreno et al. 2006a,b;

Salazar-Marquez et al. 2013). Other works on similar desert areas have been

carried out mostly in central and southern Mexico (Mosquera et al. 2003,

Lado et al. 2007, Wrigley de Basanta et al. 2008, Estrada-Torres et al. 2009). In

contrast, few references exist for microphyllous desert scrub (Lizarraga et al.

2003, 2005a,b; Salazar-Marquez et al. 2014) for the more northern Chihuahuan

desert.

During the past decade, the catalog of myxomycetes of the Chihuahua has

been increased from 119 (Lizarraga et al. 2015) to 127 taxa (125 species and 2

Myxomycetes of Chihuahua 4 (Mexico) ... 1075

varieties) due to research by Moreno et al. (2007), Lizarraga et al. (2015), and

Salazar-Marquez et al. (2013, 2014).

Materials & methods

The survey was carried out in the central plains of the Chihuahuan desert. Ten

localities with two types of vegetation were sampled seasonally from spring to winter

2009 (TABLE 1); each sampling effort took a week. The studied specimens were collected

in the field or obtained in moist chamber cultures. Specimens are deposited in the

collection of fungi of the Universidad Estatal de Sonora, Hermosillo, Mexico (UES),

with a duplicate of some samples conserved in the herbarium of the Life Sciences

Department (Botany), University of Alcala, Madrid, Spain (AH). Permanent slides were

prepared with Hoyer’s liquid for microscopic study. Collectors names are abbreviated as

follows: A. Gutiérrez (AG), M. Lizarraga (ML), D. Lopez (DL), D. Saenz (DS), C. Salazar

(CS), and A. Sanchez (AS).

Spores (including surface structures such as spines or warts) were measured with an

oil immersion lens. Light microscopy (LM) was carried out with a Nikon microscope

equipped with an automatic photographic system. SEM micrographs were produced

with a Zeiss DSM-950 microscope. For ultramicroscopic studies, material was

rehydrated in concentrated (28-30%) ammonium hydroxide for 30 min, dehydrated in

aqueous ethanol (70%) for 30 min, fixed for 2 h in pure ethylene glycol dimethyl ether

(= 1,2-dimethoxymethane), and finally immersed in pure acetone for at least 2 h. This

was followed by critical point drying and sputtering with gold-palladium. This technique

allows the use of very little material (ie., part of a single sporocarp or sometimes no

more than a few spores).

Taxonomy

Arcyria cinerea (Bull.) Pers., Syn. Meth. Fung.: 184 (1801).

SPECIMENS EXAMINED: MEXICO, Cuinuanua: Loc. 1, Yucca elata Engelm., 24 July

2009, leg. ML, CS, DL, DS & AG, obtained from moist chamber 12 August 2009 (UES

8250); rat dung, 23 October 2009, leg. ML, CS, AS, AG & DL, obtained from moist

chamber 9 December 2009 (UES 8380); Loc. 3, Tamarix chinensis Lour. (Tamaricaceae),

20 May 2009, leg. ML, CS & DL, obtained from moist chamber 30 June 2009 (UES

7977); Loc. 6, Agave lecheguilla Torr., 23 March 2009, leg. ML & CS, obtained from moist

chamber 20 April 2009 (UES 7561); Euphorbia antisyphilitica Zucc. (Euphorbiaceae), 25

May 2009, leg. ML, CS & DL, obtained from moist chamber 29 May 2009 (UES 7974);

obtained from moist chamber 3 June 2009 (UES 7975, AH 45575). Loc. 10, rabbit dung,

25 July 2009, leg. ML, CS, DL, DS & AG, obtained from moist chamber 5 August 2009

(UES 8255).

OBSERVATIONS — Arcyria cinerea was obtained in moist chamber on different

substrates such as rat and rabbit dung. Its coprophilous habit has been noted by

Eliasson & Lundqvist (1979), Eliasson & Keller (1999), and Krug et al. (2004).

Previously cited for Chihuahua by Lizarraga et al. (2003).

1076 ... Lizarraga & al.

Arcyria denudata (L.) Wettst., Verarb. Pilzfl. Steiermark 1: 9 (1885).

SPECIMEN EXAMINED: MEXICO, Cuinvuanua: Loc. 6, Yucca sp. 23 March 2009, leg. ML

& CS (UES 7562, AH 45576).

OBSERVATIONS — Previously cited for Chihuahua by Lizarraga et al. (2003).

Badhamia affinis Rostaf., Sluzowce Monogr.: 143 (1874).

SPECIMEN EXAMINED: MEXICO, Curnuanua: Loc. 7, Prosopis glandulosa, 26 July

2009, leg. ML, CS, AG, DL & DS (UES 8262, AH 45577).

OBSERVATIONS — See Moreno & Oltra (2010) for a detailed study of Badhamia

affinis, including photomicrographs of the spore ornamentation.

Badhamia gracilis (T. Macbr.) T. Macbr., Myxomycetes: 35 (1934).

Diagnostic characters include dark polygonal (12-14 um diam.) warted

spores and a badhamioid capillitium.

SPECIMENS EXAMINED: MEXICO, Cuinuanua: Loc. 1, rabbit dung, 22 May 2009,

leg. ML, CS & DL, obtained from moist chamber 30 June 2009 (UES 8085); rat dung,

obtained from moist chamber 30 June 2009 (UES 8087); Loc. 2, Yucca elata, 7 February

2009, leg. ML, CS & DS (UES 7690); obtained from moist chamber 25 February 2009

(UES 7691); 22 May 2009, leg. ML, CS & DL (UES 7982); rat dung, obtained from moist

chamber 19 March 2009 (UES 7688); cow dung, 23 July 2009, leg. ML, CS, DL, AG

& DS, obtained from moist chamber 8 September 2009 (UES 8362); Loc. 3, Opuntia

macrocentra Engelm., 20 May 2009, leg. ML, CS & DL (UES 7978, 7979, 7980); 23 July

2009, leg. ML, CS, DL, DS & AG (UES 8266); obtained from moist chamber 3 August

2009 (UES 8268); obtained from moist chamber 8 August 2009 (UES 8267); obtained

from moist chamber 10 August 2009 (UES 8220); obtained from moist chamber 12

August 2009 (UES 8239); Loc. 4, Opuntia macrocentra, 14 February 2009, leg. ML, CS &

DS (UES 7694); obtained from moist chamber 26 February 2009 (UES 7695); obtained

from moist chamber 2 March 2009 (UES 7696); Loc. 5, Opuntia sp., 25 May 2009, leg.

ML, CS & DL, obtained from moist chamber (UES 7992); 25 October 2009, leg. ML, CS,

AS, AG & DL (UES 8379); obtained from moist chamber 6 November 2009 (UES 8384);

obtained from moist chamber 14 November 2009 (UES 8385); obtained from moist

chamber 27 November 2009 (UES 8386, 8387); Loc. 6, Yucca sp., 23 March 2009, leg. ML

& CS, obtained from moist chamber 20 April 2009 (UES 7706, 7707); Agave lecheguilla,

25 May 2009, leg. ML, CS & DL (UES 7988); Loc. 7, Echinocereus enneacanthus Engelm..,

22 March 2009, leg. ML & CS, obtained from moist chamber 14 April 2009 (UES 7702);

Yucca sp., obtained from moist chamber 20 April 2009 (UES 7703); Loc. 8, Opuntia sp.,

23 March 2009, leg. ML & CS, obtained from moist chamber 13 April 2009 (UES 7709);

Loc. 9, Opuntia macrocentra, 25 July 2009, leg. ML, CS, DL, DS & AG, obtained from

moist chamber 22 August 2009 (UES 8313); obtained from moist chamber 29 August

2009 (UES 8316, AH 45578).

OBSERVATIONS — Badhamia gracilis, which is succulenticolous, has been the

most abundant species in both the field and moist chamber cultures in most

studied localities. We cite only a limited number of samples for each locality,

but much more material was deposited in the herbarium. Study of the spore

Myxomycetes of Chihuahua 4 (Mexico) ... 1077

ornamentation under SEM by Moreno & Oltra (2010) demonstrated the

morphological separation of this species from Badhamia melanospora Speg.

Badhamia gracilis was reported on dung by Krug et al. (2004); in this study,

it was observed in rabbit, rat and cow dung.

Previously cited for Chihuahua by Lizarraga et al. (2003) and Salazar-

Marquez et al. (2014).

*Badhamia spinispora (Eliasson & N. Lundq.) H.W. Keller & Schokn.,

Mycologia 81: 632 (1989). Fies 1, 2

Fructificationssessilesubglobosesporocarpstoshortplasmodiocarps, white

to grayish, growing solitary or grouped. Capillitium varying from physaroid

to badhamioid, consisting of irregular filaments and calcareous nodules.

Spores black in mass, light violaceous under LM, globose, subglobose to oval,

10-12 um diam., ornamented by obvious spines.

SPECIMENS EXAMINED: MEXICO, Cuinvuanua: Loc. 1, rat dung, 22 May 2009, leg. ML,

CS & DL, obtained from moist chamber 25 June 2009 (UES 7994); Loc. 2, cow dung, 26

October 2009, leg. ML, CS, AS, AG & DL, obtained from moist chamber 9 December

2009 (UES 8361, AH 45555).

OBSERVATIONS — Badhamia spinispora is a rare species, generally with a

coprophilous habit. The differences between it and Badhamia verrucospora and

Physarum apiculosporum are detailed in the observations under those species.

Previously cited for Sonora by Esqueda et al. (2013).

**Badhamia verrucospora G. Moreno, D.W. Mitch. & Novozh., Bol. Soc. Micol.

Madrid 35: 98 (2011). Figs 3, 4

Fructifications subglobose sporocarps to short plasmodiocarps, in groups,

white, sessile, 0.2-0.5 x 0.2-1 mm. Peridium double, calcareous, with irregular

dehiscence. Capillitium scarce, consisting of filaments connected by irregular

calcareous nodules. Spores (15-)17-18 x 10-12 um diam., black in mass,

purple under LM, subglobose to oval, a broad part of its surface is ornamented

with clearly marked warts, the rest of the spore has a thinner wall and smaller

warts, sometimes with ornamentation almost lacking.

SPECIMENS EXAMINED: MEXICO, CuIHuAHUA: Loc. 1, cow dung, 14 February 2009,

leg. ML, CS & DS, obtained from moist chamber 7 March 2009 (UES 7644, AH 45556);

24 July 2009, leg. ML, CS, DL, DS & AG (UES 8269); obtained from moist chamber

25 August 2009 (UES 8370); obtained from moist chamber 1 September 2009 (UES

8317); obtained from moist chamber 8 September 2009 (UES 8371); Loc. 2, cow dung,

7 February 2009, leg. ML, CS & DS, obtained from moist chamber 9 March 2009 (UES

7641); obtained from moist chamber 18 March 2009 (UES 7642, AH 45578); obtained

from moist chamber 31 March 2009 (UES 7643); rabbit dung, obtained from moist

chamber 1 September 2009 (UES 8318); 22 October 2009, leg. ML, CS, AS, AG & DL,

obtained from moist chamber 9 December 2009 (UES 8517, 8518); rabbit dung, 23 July

1078 ... Lizarraga & al.

2009, leg. ML, CS, DL, DS & AG, obtained from moist chamber 8 September 2009 (UES

8365); Loc. 3, cow dung, 23 July 2009, leg. ML, CS, DL, DS & AG, obtained from moist

chamber 8 September 2009 (UES 8376); Loc. 4, cow dung, 14 February 2009, leg. ML,

CS & DS, obtained from moist chamber 26 March 2009 (UES 7645); Loc. 5, horse dung,

24 May 2009, leg. ML, CS & DL, obtained from moist chamber 30 June 2009 (UES 7995);

Loc. 7, cow dung, 22 October 2009, leg. ML, CS, AS, AG & DL, obtained from moist

chamber 9 December 2009 (UES 8530).

OBSERVATIONS — Badhamia spinispora (Eliasson & Lundqvist 1979; as

Physarum spinisporum), is morphologically similar to P apiculosporum and B.

verrucospora. It is characterized by its globose to broadly oval spores with sharp

spines, while P apiculosporum has oval to ellipsoidal spores that are smooth

and with two apiculi. Badhamia verrucospora spores are similar to those of

Physarum apiculosporum (oval to ellipsoid with two apiculi) but ornamented

by abundant small warts over half the spore surface (Moreno et al. 2011).

Badhamia rhytidosperma H.W. Keller & Schokn., which resembles the three

previous species but with reticulate spores, has not yet been reported from

Mexico, according to the consulted literature.

Comatricha elegans (Racib.) G. Lister, Guide Brit. Mycetozoa, 3rd Ed.: 31 (1909).

Fics 9-11

Sporocarps small (0.5-1 mm tall); columella branched; stipe fibrous; the

capillitium forming an external link with well-defined more or less continuous

meshes; spores 9-10 um diam., warted.

SPECIMEN EXAMINED: MEXICO, Curnuanvua: Loc. 3, Tamarix chinensis, 20 May 2009,

leg. ML, CS & DL, obtained from moist chamber 29 May 2009 (UES 8065, AH 45557).

OBSERVATIONS — A SEM study of Comatricha elegans was carried out by

Lizarraga et al. (2005a).

Poorly known in Mexico (Moreno et al. 2007), C. elegans has been previously

recorded for Chihuahua by Lizarraga et al. (2005b) and Salazar-Marquez et al.

(2014).

*Comatricha ellae Hark., Karstenia 18: 23 (1978). Fic. 12

Sporocarps small (0.5-1 mm tall); sporotheca 0.2-0.4 mm diam.; capillitium

with well-defined meshes mostly into stipe junction; columella percurrent,

spores 9-10 um diam.

Fics 1-8. Badhamia spinispora (AH 45555). 1. Plasmodiocarps. 2. Spore spinulose (SEM).

Badhamia verrucospora (AH 45556). 3. Plasmodiocarps. 4. Spore verrucose (SEM).

Fuligo cinerea (AH 45574). 5. Plasmodiocarps. 6. Spore reticulate (SEM). Physarum

apiculosporum (AH 45566). 7. Plasmodiocarps. 8. Spore smooth (SEM). Scale bars: 1, 3, 5, 7 =

1 mm; 2, 4, 6, 8 = 2 um.

1079

Myxomycetes of Chihuahua 4 (Mexico) ...

1080 ... Lizarraga & al.

SPECIMEN EXAMINED: MEXICO, CuiHuanua: Loc. 1, Artemisia filifolia Torr.

(Asteraceae), 24 July 2009, leg. ML, CS, DL, DS & AG, obtained from moist chamber 5

August 2009 (UES 8307, AH 45558).

OBSERVATIONS — We observed this species frequently in moist chamber

cultures on succulent plant crusts.

Comatricha ellae was cited previously only from Baja California Sur (Moreno

et al. (2007).

Comatricha tenerrima (M.A. Curtis) G. Lister, Guide Brit. Mycetozoa,

4th Ed.: 39 (1919).

Characterized by the acuminate sporothecal apex, sinuous capillitium, and

spores light violaceous under LM and 8-9 um diam., warted.

SPECIMENS EXAMINED: MEXICO, Cuimuaunvua: Loc. 10, Cylindropuntia sp., 21 March

2009, leg. ML & CS, obtained from moist chamber 6 April 2009 (UES 7742, AH 45579);

Koeberlinia spinosa Zucc. (Koeberliniaceae), 25 July 2009, leg. ML, CS, DL, DS & AG,

obtained from moist chamber 10 August 2009 (UES 8229).

OBSERVATIONS —Salazar-Marquez et al. (2014) reported that SEM photographs

show that the spore ornamentation in C. tenerrima is formed by joined bacula

with a characteristic star-shaped morphology.

Previously cited for Chihuahua by Salazar-Marquez et al. (2014) and

Lizarraga et al. (2005b).

Dictydiaethalium plumbeum (Schumach.) Rostaf. ex Lister, Monogr.

Mycetozoa: 157 (1894).

Cultures in moist chambers showing considerable morphological variation;

aethalia small (0.5-1 mm diam.), subglobose to pulvinate.

SPECIMENS EXAMINED: MEXICO, CurHuanua: Loc. 1, Prosopis glandulosa, 22 May

2009, leg. ML, CS & DL, obtained from moist chamber 3 June 2009 (UES 8010); Loc.

3, Larrea tridentata, 24 January 2009, leg. ML, CS & DS, obtained from moist chamber

5 February 2009 (UES 7570); obtained from moist chamber 13 February 2009 (UES

7571); 22 October 2009, leg. ML, CS, AS, AG & DL, obtained from moist chamber 27

November 2009 (UES 8424); obtained from moist chamber 9 December 2009 (UES

8425); Loc. 4, 14 February 2009, leg. ML, CS & DS, obtained from moist chamber 28

February 2009 (UES 7572); obtained from moist chamber 1 April 2009 (UES 7573,

AH 45580); Loc. 7, Porlieria angustifolia (Engelm.) A. Gray (Zygophyllaceae), 22 March

2009, leg. ML & CS, obtained from moist chamber 14 April 2009 (UES 7574); Larrea

tridentata, 26 October 2009, leg. ML, CS, AS, AG & DL, obtained from moist chamber

21 November 2009 (UES 8421); obtained from moist chamber 27 November 2009

(UES 8423); Prosopis glandulosa, obtained from moist chamber 21 November 2009

Fics 9-14. Comatricha elegans (AH 45557). 9. Sporocarp. 10. Capillitium detail forming an

external net and columella. 11. Stalk fibrous. Comatricha ellae (AH 45558). 12. Sporocarp.

Echinostelium arboreum (AH 45560). 13, 14. Sporocarp detail, peridium persistent, membranous,

collar, columella, capillitium, and spores. Scale bars: 9-12 = 100 um; 13, 14 = 10 um.

Myxomycetes of Chihuahua 4 (Mexico) ... 1081

1082 ... Lizarraga & al.

(UES 8422); Loc. 8, 23 March 2009, obtained from moist chamber 4 April 2009 (UES

7576); obtained from moist chamber 20 April 2009 (UES 7685); 24 October 2009, leg.

ML, CS, AS, AG & DL, obtained from moist chamber 9 November 2009 (UES 8417);

obtained from moist chamber 21 November 2009 (UES 8418); Loc. 9, Acacia greggii A.

Gray (Leguminosae), leg. ML & CS, obtained from moist chamber 10 August 2009 (UES

8224); Larrea tridentata, obtained from moist chamber 22 August 2009 (UES 8326); 25

October 2009, leg. ML, CS, AS, AG & DL, obtained from moist chamber 14 December

2009 (UES 8415); obtained from moist chamber 9 December 2009 (UES 8416); Loc.

10, Larrea tridentata, 21 March 2009, obtained from moist chamber 8 April 2009 (UES

7575); Prosopis glandulosa, 22 May 2009, leg. ML, CS & DL, obtained from moist

chamber 3 June 2009 (UES 8009); Larrea tridentata, 25 July 2009, leg. ML, CS, DL, DS

& AG, obtained from moist chamber 8 August 2009 (UES 8287); obtained from moist

chamber 12 August 2009 (UES 8254); obtained from moist chamber 15 August 2009

(UES 8325); 25 October 2009, leg. ML, CS, AS, AG & DL, obtained from moist chamber

6 November 2009 (UES 8419); obtained from moist chamber 9 November 2009 (UES

8420); obtained from moist chamber 14 December 2009 (UES 8427); obtained from

moist chamber, 21 November 2009 (UES 8489).

OBSERVATIONS — We follow the broad species concept of Martin & Alexopoulos

(1969), which accepts a widely variable aethalium size and color.

Previously cited for Chihuahua by Lizarraga et al. (2005a).

Didymium difforme (Pers.) Gray, Nat. Arr. Brit. Pl. 1: 571 (1821).

Spores warted, 13-15 um diam.; sporotheca pulvinate with a crust-like

peridium.

SPECIMEN EXAMINED: MEXICO, Curnuanua: Loc. 4, rabbit dung, 14 February 2009,

leg. ML, CS & DS, obtained from moist chamber 25 March 2009 (UES 7588).

OBSERVATIONS —Eliasson & Lundqvist (1979), Eliasson & Keller (1999) and

Krug et al. (2004) reported this species from dung.

Didymium difforme is rarely cited from Mexico (Moreno et al. 2007).

Previously reported for Chihuahua on rabbit dung (Salazar-Marquez et al.

2014).

Didymium dubium Rostaf., Sluzowce Monogr.: 143 (1874).

SPECIMENS EXAMINED: MEXICO, Cuinuanua: Loc. 1, Prosopis glandulosa, 14

February 2009, leg. ML, CS & DS, obtained from moist chamber 24 February 2009

(UES 7787); obtained from moist chamber 28 February 2009 (UES 7788, 7789, 7790);

Artemisia filifolia, 14 February 2009, leg. ML, CS & DS, obtained from moist chamber

26 February 2009 (UES 7782); cow dung, obtained from moist chamber 26 February

2009 (UES 7783); obtained from moist chamber 28 February 2009 (UES 7784); obtained

from moist chamber 31 March 2009 (UES 7785); rabbit dung, obtained from moist

chamber 18 March 2009 (UES 7786); Yucca sp., 24 July 2009, leg. ML, CS, DL, DS & AG,

obtained from moist chamber 12 August 2009 (UES 8334); Loc. 2, Artemisia filifolia,

7 February 2009, leg. ML, CS & DS, obtained from moist chamber 16 February 2009

(UES 7763); Yucca elata, obtained from moist chamber 16 February 2009 (UES 7776);

Myxomycetes of Chihuahua 4 (Mexico) ... 1083

Prosopis glandulosa, 7 February 2009, leg. ML, CS & DS, obtained from moist chamber

21 February 2009 (UES 7767); rabbit dung, obtained from moist chamber 27 February

2009 (UES 7764); obtained from moist chamber 14 March 2009 (UES 7765); 23 July

2009, leg. ML, CS, DL, DS & AG, obtained from moist chamber 10 August 2009 (UES

8221); Loc. 3, Prosopis glandulosa, obtained from moist chamber 10 February 2009

(UES 7754); Larrea tridentata, 24 January 2009, leg. ML, CS & DS, obtained from moist

chamber 3 February 2009 (UES 7745); Tamarix chinensis, 24 January 2009, leg. ML, CS &

DS, obtained from moist chamber 23 February 2009 (UES 7761); Fouquieria splendens,

20 May 2009, leg. ML, CS & DL, obtained from moist chamber 3 June 2009 (UES 8011);

Loc. 4, Fouquieria splendens, 14 February 2009, leg. ML, CS & DS, obtained from moist

chamber 21 February 2009 (UES 7777); Prosopis glandulosa, 14 February 2009, leg. ML,

CS & DS, obtained from moist chamber 2 March 2009 (UES 7770); Yucca elata, obtained

from moist chamber 3 June 2009 (UES 8020); Larrea tridentata, 24 February 2009, leg.

ML, CS, DL, DS & AG, obtained from moist chamber 10 August 2009 (UES 8223); Loc.

5, Fouquieria splendens, 22 March 2009, obtained from moist chamber 4 April 2009

(UES 7796); Cylindropuntia imbricata (Haw.) RM. Knuth, 24 May 2009, leg. ML, CS

& DL, obtained from moist chamber 29 May 2009 (UES 8032); Prosopis glandulosa,

obtained from moist chamber 29 May 2009 (UES 8034); Loc. 6, Prosopis glandulosa,

23 March 2009, leg. ML & CS, obtained from moist chamber 4 April 2009 (UES 7806);

Fouquieria splendens, obtained from moist chamber 6 April 2009 (UES 7804); Agave

lecheguilla, 26 October 2009, disposed in moist chamber 2.X1.20009, fructifications 14

December 2009 (UES 8462); Loc. 7, Agave lecheguilla, 22 March 2009, leg. ML & CS,

obtained from moist chamber 13 April 2009 (UES 7800); Prosopis glandulosa, 26.V1I.09,

leg. ML, CS, DL, DS & AG, obtained from moist chamber 8 August 2009 (UES 8284);

Larrea tridentata, obtained from moist chamber 21 November 2009 (UES 8447);

Cylindropuntia imbricata, obtained from moist chamber 12 August 2009 (UES 8245);

Loc. 9, Prosopis glandulosa, 22 March 2009, leg. ML & CS, obtained from moist chamber

6 April 2009 (UES 7795); rabbit dung, 22 July 2009, leg. ML, CS, DL, AS & AG, obtained

from moist chamber 15 August 2009 (UES 8335); Loc. 10, rabbit dung, 21 March 2009,

leg. ML & CS, sporocarps obtained 13 April 2009 (UES 7794); Acacia constricta A. Gray

(Leguminosae), 23 May 2009, leg. ML, CS & DL, obtained from moist chamber 3 June

2009 (UES 8025).

OBSERVATIONS — Didymium dubium exhibits great morphological variation

in the sporocarps but can be recognized by spores 12-14 um diam., with small

bacula that form a characteristic reticle easily visible under SEM (Moreno et

al. 2001). It was frequently collected in the studied area; thus, only samples of

different localities and habitats are cited.

Previously recorded for Chihuahua by Lizarraga et al. (2003) and Salazar-

Marquez et al. (2014).

Didymium mexicanum G. Moreno, Lizarraga & Ilana, II Int. Congr. Syst. Ecol.

Myxomycetes: 56 (1996).

= Didymium umbilicatum D. Wrigley, Lado & Estrada, Mycologia 100: 922 (2008).

SPECIMENS EXAMINED: MEXICO, CurHuauua: Loc. 6, Yucca sp., 23 March 2009, leg.

ML & CS (UES 7577); leg. ML, CS, DL, DS & AG, obtained from moist chamber 22

August 2009 (UES 8337); 26 October 2009, leg. ML, CS, AS, AG & DL, obtained from

1084 ... Lizarraga & al.

moist chamber 9 November 2009 (UES 8455); Agave lecheguilla, obtained from moist

chamber 27 November 2009 (UES 7578, 7579, 7580), obtained from moist chamber

4 April 2009 (UES 7581); obtained from moist chamber 10 April 2009 (UES 7582);

obtained from moist chamber 14 April 2009 (UES 7802); obtained from moist chamber

20 April 2009 (UES 7803, 7812); obtained from moist chamber 1 May 2009 (UES 7583);

25 May 2009, leg. ML, CS & DL (UES 8027, 8028); obtained from moist chamber 25

June 2009 (UES 8030); obtained from moist chamber 30 June 2009 (UES 8031); 26 July

2009, leg. ML, CS, DL, DS & AG (UES 8286); 26 October 2009, leg. ML, CS, AS, AG &

DL, obtained from moist chamber 27 November 2009 (UES 8459); obtained from moist

chamber 18 December 2009 (UES 8463, 8465); obtained from moist chamber 5 January

2010 (UES 8466).

OBSERVATIONS — Moreno et al. (2012) included samples from Chihuahua

in their recent macro- and microscopical (including SEM) examinations of

Didymium mexicanum and D. umbilicatum that confirmed these species are

synonyms.

In Mexico, this species is known from Baja California (Moreno et al. 1997,

2012; Lizarraga et al. 2004b), Chihuahua (Moreno et al. 2012; Salazar-Marquez

et al. 2014), Hidalgo (Wrigley de Basanta et al. 2008, as D. umbilicatum),

Oaxaca (Wrigley de Basanta et al. 2008, as D. umbilicatum; Estrada-Torres

et al. 2009, as D. umbilicatum), Puebla (Wrigley de Basanta et al. 2008, as

D. umbilicatum; Estrada-Torres et al. 2009, as D. umbilicatum), and Querétaro

(Wrigley de Basanta et al. 2008, as D. umbilicatum). It has also been cited from

Argentina (Lado et al. 2011), Guatemala (Wrigley de Basanta et al. 2008, as

D. umbilicatum), and Russia (Novozhilov et al. 2006). It has been collected

from Spain (Murcia), Kazakhstan (West Kazakhstan region), and United States

(Arizona and New Mexico) (Discover Life 2014).

Didymium squamulosum (Alb. & Schwein.) Fr., Symb. Gasteromyc. 3: 19 (1818).

SPECIMENS EXAMINED: MEXICO, Cuinuanua: Loc. 3, Larrea tridentata, 24 January

2009, leg. ML, CS, DS, obtained from moist chamber 25 March 2009 (UES 7585); 20

May 2009, leg. ML, CS & DL, obtained from moist chamber 15 June 2009 (UES 8038,

AH 45583); obtained from moist chamber 25 June 2009 (UES 8040); obtained from

moist chamber 30 June 2009 (UES 8039, AH 45584); Larrea tridentata, 22 October 2009,

leg. ML, CS, AS, AG & DL, obtained from moist chamber 27 November 2009 (UES

8440); obtained from moist chamber 9 December 2009 (UES 8441); Loc. 4, Yucca elata,

14 February 2009, leg. ML, CS & DS, obtained from moist chamber 18 March 2009

(UES 7586, AH 45582); 20 March 2009 (UES 7813); Loc. 6, Yucca sp., 23 March 2009,

leg. ML & CS, obtained from moist chamber 20 April 2009 (UES 7686, AH 45581); Loc.

9, Xanthium sp. (Asteraceae), 22 March 2009, obtained from moist chamber 20 April

2009 (UES 7687).

OBSERVATIONS — Didymium squamulosum is a cosmopolitan species (Martin

& Alexopoulos 1969). Previously cited for Chihuahua by Lizarraga et al. (2003)

and Salazar- Marquez et al. (2014).

Myxomycetes of Chihuahua 4 (Mexico) ... 1085

Po Pe

Fics 15-24. Didymium tehuacanense (AH 45559). 15. Sporocarp. 16. Peridium simple and fragile.

17, 18. Columella detail: cylindrical, calcareous, whitish, with ramified and spinose apex. 19.

Peridium with starry crystals. 20-22. Capillitium filamentous with abundant nodules (SEM). 23,

24. Spore (SEM). Scale bars. 15-18 = 0.25 mm; 19-22 = 5 um; 23, 24 = 2 um.

*Didymium tehuacanense Estrada, D. Wrigley & Lado, Fungal Diversity 36: 32

(2009). Fries 15-24

Fructifications stipitate, gregarious, 0.5-1 mm height (Fic. 15). Sporotheca

globose to subglobose of 0.3-0.5 mm diam. Peridium simple, iridescent to

1086 ... Lizarraga & al.

grayish, membranous, fragile, persistent on the base of the sporotheca like a

collar and covered by starry crystals of whitish calcium carbonate, of bigger

size than spores (7-22 um diam.) Stipe 0.3-0.5 x 0.2-0.4 mm, cylindrical,

calcareous, whitish to dark according to the presence of calcium carbonate,

wider and darker at the base. Columella 0.2 x 0.1 mm, prominent, calcareous,

cylindrical, whitish, with the apex branched and spiny. Hypothallus discoid,

membranous. Capillitium formed by thin filaments of 1 um diam., with

thickening or nodules. Spores 8-10 um diam., dark in mass, violaceous under

LM, with a paler area, ornamented with thick warts. Spore ornamentation is

formed by irregularly distributed bacula that occasionally join to form short

crests as can be seen under SEM.

SPECIMENS EXAMINED: MEXICO, CurHuanua: Loc. 6, Agave lecheguilla, next to

Didymium mexicanum, 23 March 2009, leg. ML & CS (UES 7577); 25 May 2009, leg. ML,

CS & DL (UES 8036); 26 July 2009, leg. ML, CS, DL, DS & AG (UES 8285, AH 45559).

OBSERVATIONS — Didymium tehuacanense is characterized by a calcareous

stipe and columella, cylindrical columella with a ramified apex and a capillitium

with calcareous thickenings.

The above specimens represent a new record for Chihuahua. Previously cited

from Oaxaca and Puebla (Estrada-Torres et al. 2009), where it was described as

a species new to science.

Echinostelium apitectum K.D. Whitney, Mycologia 72: 954 (1980).

Fructifications stalked, 190-230 um tall; body spore-like, globose. Stalk

120-210 um long, filled with granular matter, tapered. Spores 9-11 um diam.,

subglobose, uniform wall, hyaline, smooth to weakly warted.

SPECIMENS EXAMINED: MEXICO, Cuinvuanua: Loc. 3, Artemisia filifolia, 24 January

2009, leg. ML, CS & DS, obtained from moist chamber 16 February 2009 (UES 7822);

Loc. 7, Prosopis glandulosa, 26 July 2009, leg. ML, CS, DL, DS & AG, obtained from moist

chamber 8 August 2009 (UES 8297); Chilopsis linearis (Cav.) Sweet (Bignoniaceae), 24

May 2009, leg. ML, CS & DL, obtained from moist chamber 29 May 2009 (UES 8050).

OBSERVATIONS — Echinostelium apitectum can be confused with fructifications

of E. colliculosum, which differs by its spores with articulated surfaces and

smaller (70-150 um tall) fructifications (Whitney 1980; Lado & Pando 1997).

Infrequently collected in Mexico, E. apitectum is reported for Baja California,

Chihuahua, Sonora, and Tlaxcala (Moreno et al. 2007).

Echinostelium arboreum H.W. Keller & T.E. Brooks, Mycologia 68: 1207 (1977)

Fics 13, 14

Peridium persistent, membranous, with a collar; sporotheca 40-50 um

diam.; columella cylindrical <25 um tall, without a spore-like body; capillitium

Myxomycetes of Chihuahua 4 (Mexico) ... 1087

well developed in our sample, dichotomously branched, not anastomosed.

Spores 7-8 um diam., globose to subglobose, hyaline, smooth to rough and

without articulated surfaces.

SPECIMEN EXAMINED: MEXICO, Cuinvuanua: Loc. 2, Artemisia filifolia, 22 October

2009, leg. ML, CS, AS, AG & DL, obtained from moist chamber 14 November 2009 (UES

8471, AH 45560).

OBSERVATIONS —Echinostelium arboreum is known from Baja California,

Chihuahua, and Yucatan (Moreno et al. 2007).

Echinostelium colliculosum K.D. Whitney & H.W. Keller, Mycologia 72: 641 (1980).

SPECIMENS EXAMINED: MEXICO, Cuinuanua: Loc. 1, Artemisia filifolia, 23 October

2009, leg. ML, CS, AS, AG & DL, obtained from moist chamber 6 November 2009 (UES

8481); cow dung, 14 February 2009, leg. ML, CS & DS, obtained from moist chamber

28 February 2009 (UES 7839); Prosopis glandulosa, 14 February 2009, leg. ML, CS &

DS, obtained from moist chamber 24 February 2009 (UES 7830); Artemisia filifolia, 14

February 2009, leg. ML, CS & DS, obtained from moist chamber 19 February 2009 (UES

7828); Loc. 2, Artemisia filifolia, 7 February 2009, leg. ML, CS & DS, obtained from

moist chamber 13 February 2009 (UES 7821); Prosopis glandulosa, 7 February 2009, leg.

ML, CS & DS, obtained from moist chamber 16 February 2009 (UES 7823); Yucca elata,

23 July 2009, leg. ML, CS, DL, DS & AG, obtained from moist chamber 3 August 2009

(UES 8288); Loc. 3, Prosopis glandulosa, 24 January 2009, leg. ML, CS & DS, obtained

from moist chamber 5 February 2009 (UES 7834); Larrea tridentata, obtained from

moist chamber 6 November 2009 (UES 8477); Prosopis glandulosa, obtained from moist

chamber 4 February 2009 (UES 7836. Koeberlinia spinosa, 20 May 2009, leg. ML, CS &

DL, obtained from moist chamber 29 May 2009 (UES 8041); Loc. 4, Prosopis glandulosa,

14 February 2009, leg. ML, CS & DS, obtained from moist chamber 24 January 2009 (UES

7824); cow dung, 14 February 2009, leg. ML, CS & DS, obtained from moist chamber 5

March 2009 (UES 7837); Yucca elata, obtained from moist chamber 23 February 2009

(UES 7827); Larrea tridentata, obtained from moist chamber 26 February 2009 (UES

7838); Loc. 5, Prosopis glandulosa, 25 July 2009, leg. ML, CS, DL, DS & AG, obtained

from moist chamber 29 May 2009 (UES 8051); Larrea tridentata, obtained from moist

chamber 8 August 2009 (UES 8293); Cylindropuntia imbricata, obtained from moist

chamber 10 August 2009 (UES 8218); Loc. 6, Viguiera stenoloba S.F.Blake (Asteraceae),

23 March 2009, leg. ML & CS, obtained from moist chamber 4 April 2009 (UES 7843);

Loc. 7, Acacia greggii, 26 July 2009, leg. ML, CS, DL, DS & AG, obtained from moist

chamber 3 August 2009 (UES 8296); Cylindropuntia imbricata, 22 March 2009, leg. ML

& CS, obtained from moist chamber 4 April 2009 (UES 7840); Porlieria angustifolia,

obtained from moist chamber 4 April 2009 (UES 7841); Viguiera stenoloba, obtained

from moist chamber 4 April 2009 (UES 7842); 26 October 2009, leg. ML, CS, AS, AG

& DL, obtained from moist chamber 6 November 2009 (UES 8484); Loc. 8, Larrea

tridentata, next to Dictydiaethalium plumbeum, 23 March 2009, leg., ML & CS, obtained

from moist chamber 4 April 2009 (UES 7576); 24 October 2009, leg. ML, CS, AS, AG &

DL, obtained from moist chamber 9 November 2009 (UES 8473); Porlieria angustifolia,

23 March 2009, leg. ML & CS, obtained from moist chamber 4 April 2009 (UES 7844);

Viguiera stenoloba obtained from moist chamber 6 April 2009 (UES 7831); Yucca sp., 24

October 2009, leg. ML, CS, AS, AG & DL, obtained from moist chamber 6 November

1088 ... Lizarraga & al.

2009 (UES 8472); obtained from moist chamber 9 November 2009 (UES 8474); Loc. 9,

Prosopis glandulosa, 25 July 2009, obtained from moist chamber 8 August 2009 (UES

8291); Acacia greggii, obtained from moist chamber 10 August 2009 (UES 8231); Loc.

10, Acacia greggii, 22 May 2009, leg. ML, CS & DL, obtained from moist chamber 29 May

2009 (UES 8046); Larrea tridentata, 25 July 2009, leg. ML, CS, DL, DS & AG, obtained

from moist chamber 8 August 2009 (UES 8292).

OBSERVATIONS — Echinostelium colliculosum is very difficult to distinguish

from E. coelocephalum T.E. Brooks & H.W. Keller, since both develop stalked

fructifications with a spore-like body on the stipe apex and have globose spores

with articulated surfaces. We can differentiate them, because E. coelocephalum

forms small fructifications 40-70 um height and very thickened articulated

surfaces, while E. colliculosum presents larger fructifications of 70-150 um, and

less thickened articulated surfaces (see Whitney 1980).

Echinostelium colliculosum has not been reported as coprophilous by

Eliasson & Lundqvist (1979), Eliasson & Keller (1999), or Krug et al. (2004).

Previously cited from Chihuahua by Lizarraga et al. (2003) and Salazar-

Marquez et al. (2014).

**Echinostelium paucifilum K.D. Whitney, Mycologia 72: 974 (1980). | Fics 25-27

Sporocarps dispersed to gregarious, pinkish brown, stalked, 200-330 um

tall. Stalk 150-210 um long, filled with granular matter, wider at base with

5-6 um diam., reducing gradually until the apex to 2-3 um diam. Peridium

fleeting, persisting at base like a collar ca. 4 um diam. Columella cylindrical.

Capillitium rudimentary or barely developed. Spores 10-12 um diam., free,

globose to subglobose, hyaline, warted, with one or several protoplasts.

SPECIMENS EXAMINED: MEXICO, Cuinvuanua: Loc. 3, Artemisia filifolia, 24 January

2009, leg. ML, CS & DS, obtained from moist chamber 27 January 2009, 16 February

2009 (UES 7822); Loc. 8, Acacia constricta, 23 May 2009, leg. ML, CS & DL, obtained

from moist chamber 29 May 2009 (UES 8044, 8045, AH 45561).

OBSERVATIONS — Echinostelium paucifilum is similar to E. minutum de

Bary, which differs in its larger sporocarps (250-500 um) and smaller spores

(6-8 um) with articulated surfaces (Whitney 1980).

These collections are new records for Mexico and the Neotropics (Lado &

Wrigley de Basanta 2008).

Fuligo cinerea (Schwein.) Morgan, J. Cincinnati Soc. Nat. Hist. 19: 33 (1896).

Fics 5, 6

SPECIMENS EXAMINED: MEXICO, Cuinuanua: Loc. 2, cow dung, 22 May 2009, leg.

ML, CS & DL, obtained from moist chamber 11 June 2009 (UES 8053, AH 45599); Loc.

4, rabbit dung, 14 February 2009, leg. ML, CS & DS, obtained from moist chamber 27

Myxomycetes of Chihuahua 4 (Mexico) ... 1089

Fics 25-27. Echinostelium paucifilum (AH 45561). 25. Sporocarp. 26. Spores. 27. Collar, columella

and spore. Scale bars. 25= 50 um; 26, 27 = 10 um.

March 2009 (UES 7589, AH 45574); Loc. 7, above ground, 26 October 2009, leg. ML,

CS, AS, AG & DL (UES 8486).

OBSERVATIONS — Fuligo cinerea generally fruited on herbivore dung following

cultivation in moist chamber; macroscopically confused with Badhamia

verrucospora, B. spinispora, and Physarum apiculosporum but distinguished

from those three species by its spores that are oval to subglobose, warted, and

subtly reticulated (Fic. 6). Small fructifications that are convoluted or gyrose

on dung could resemble Physarum gyrosum Rostaf., but the characteristic

F. cinerea spores clearly identify it, as noted by Eliasson & Lundqvist (1979)

from collections from Tenerife (Canary Islands, Spain) on rabbit dung. We

have collected them on rabbit and cow dung.

Previously cited for Chihuahua by Lizarraga et al. (2003) and Salazar-

Marquez et al. (2014).

1090 ... Lizarraga & al.

Fuligo septica (L.) FH. Wigg., Prim. Fl. Holsat.: 112 (1780).

Fructifications showed a whitish aethalium which coincides with Fuligo

septica var. candida (Pers.) R.E. Fr.; spores globose to subglobose with thin

warts, 9-11 um diam., slightly larger than the size (7-9 um) cited in the

literature.

SPECIMEN EXAMINED: MEXICO, Cuinvuanua: Loc. 7, above-ground, 26 October 2009,

leg. ML, CS, AS, AG & DL (UES 8486, AH 45565).

OBSERVATIONS — ‘This species is widely distributed in Mexico (Moreno et al.

2007).

Licea biforis Morgan, J. Cincinnati Soc. Nat. Hist. 15: 131 (1893).

Sporocarps large, sessile, yellowish brown to dark brown, with a longitudinal

centerline dehiscence; spores globose to subglobose, yellow in mass, subtly

warted, 10-11 um diam.

SPECIMENS EXAMINED: MEXICO, Cuinuanvua: Loc. 3, Tamarix chinensis, 24 January

2009, leg. ML, CS & DS, obtained from moist chamber 5 February 2009 (UES 7596,

AH 45597); Acacia greggii, obtained from moist chamber 12 August 2009 (UES 8242);

Prosopis glandulosa, 24 January 2009, leg. ML, CS & DS, obtained from moist chamber

26 February 2009 (UES 7602); Loc. 6, Viguiera stenoloba, 23 March 2009, obtained from

moist chamber 20 April 2009 (UES 7610); Loc. 7, Porlieria angustifolia, 22 March 2009,

obtained from moist chamber 13 April 2009 (UES 7606); Acacia greggii, obtained from

moist chamber 8 August 2009 (UES 8300); Loc. 10, Cylindropuntia sp., 21 March 2009,

leg. ML & CS, obtained from moist chamber 8 April 2009 (UES 7603, 7604); Larrea

tridentata, obtained from moist chamber 8 April 2009 (UES 7605).

OBSERVATIONS — Licea biforis has been cultivated in moist chambers over

different tree barks and bushes, including succulent desert plants such as

members of the Cactaceae and Liliaceae.

Two species have been described with a succulenticolous habit similar to

Licea biforis: Licea succulenticola (Mosquera et al. 2003) and Licea capacia Oltra

& G. Moreno (Moreno & Oltra 2014).

Licea biforis has been previously cited for Chihuahua and Sonora (Moreno

et al. 2007).

Licea denudescens H.W. Keller & T.E. Brooks, Mycologia 69: 668 (1977).

SPECIMENS EXAMINED: MEXICO, Curnuanua: Loc. 2, 7 February 2009, leg. ML, CS

& DS, obtained from moist chamber 13 March 2009 (UES 7614); obtained from moist

chamber 23 February 2009 (UES 7717); 22 October 2009, leg. ML, CS, AS, AG & DL,

obtained from moist chamber 9 December 2009 (UES 8338); Loc. 3, Prosopis glandulosa,

24 January 2009, leg. ML, CS & DS, obtained from moist chamber 16 February 2009

(UES 7612); obtained from moist chamber 23 February 2009 (UES 7718); obtained from

moist chamber 28 February 2009 (UES 7613, AH 45596); 22 October 2009, leg. ML, CS,

Myxomycetes of Chihuahua 4 (Mexico) ... 1091

AS, AG & DL, obtained from moist chamber 21 November 2009 (UES 8488); obtained

from moist chamber 27 November 2009 (UES 8502); obtained from moist chamber 9

December 2009 (UES 8503); Loc. 7, Acacia greggii, 26 July 2009, leg. ML, CS, DL, DS &

AG, obtained from moist chamber 29 August 2009 (UES 8340); 26 October 2009, leg.

ML, CS, AS, AG & DL, obtained from moist chamber 9 December 2009 (UES 8500);

Chilopsis linearis, 26 July 2009, leg. ML, CS, DL, DS & AG, obtained from moist chamber

29 August 2009 (UES 8341); Fouquieria splendens, 26 October 2009, leg. ML, CS, AS, AG

& DL, obtained from moist chamber 27 November 2009 (UES 8498); Cylindropuntia sp.,

obtained from moist chamber 9 December 2009 (UES 8501).

OBSERVATIONS — Recently described for Chihuahua by Salazar-Marquez et al.

(2014), and previously from Puebla (Estrada-Torres et al. 2009) and Sonora

(Esqueda et al. 2013).

Licea kleistobolus G.W. Martin, Mycologia 34: 702 (1942).

SPECIMENS EXAMINED: MEXICO, Cuinuanua: Loc. 1, 14 February 2009, leg. ML, CS

& DS, obtained from moist chamber 25 February 2009 (UES 7617); obtained from moist

chamber 26 February 2009 (UES 7619); obtained from moist chamber 27 February

2009 (UES 7618); obtained from moist chamber 2 March 2009 (UES 7621, AH 45595);

obtained from moist chamber 12 March 2009 (UES 7620); 23 July 2009, leg. ML, CS,

DL, DS & AG, obtained from moist chamber 5 August 2009 (UES 8302); obtained from

moist chamber 8 August 2009 (UES 8303); Prosopis glandulosa, obtained from moist

chamber 8 August 2009 (UES 8304); Loc. 2, Artemisia filifolia, 7 February 2009, leg. ML,

CS & DS, obtained from moist chamber 12 February 2009 (UES 7616); obtained from

moist chamber 16 February 2009 (UES 7714); Loc. 3, Prosopis glandulosa, 24 January

2009, leg. ML, CS & DS, obtained from moist chamber 22 February 2009 (UES 7615);

23 July 2009, leg. ML, CS & DL, DS & AG, obtained from moist chamber 8 August

2009 (UES 8301); Ibidem, Tamarix chinensis, 25 October 2009, leg. ML, CS, AS, AG

& DL, obtained from moist chamber 21 November 2009 (UES 8495); Loc. 6, Viguiera

stenoloba, 25 May 2009, leg. ML, CS & DL, obtained from moist chamber 29 May 2009

(UES 8058); Yucca sp., 26 July 2009, obtained from moist chamber 22 August 2009

(UES 8345); Loc. 9, Acacia greggii, 25.X.20009, leg. ML, CS, AS, AG & DL, obtained

from moist chamber 14 November 2009 (UES 8493); obtained from moist chamber 21

November 2009 (UES 8494); Loc. 10, Larrea tridentata, 25 October 2009, obtained from

moist chamber 21 November 2009 (UES 8496).

OBSERVATIONS — Previously cited from Chihuahua by Lizarraga et al. (2003)

and Salazar- Marquez et al. (2014).

Licea succulenticola Mosquera, Lado, Estrada & Beltran-Tej., Anales Jard. Bot.

Madrid 60: 4 (2003).

SPECIMENS EXAMINED: MEXICO, Cuinvuanua: Loc. 6, Agave lecheguilla, 23 March

2009, leg. ML & CS (UES 7622, AH 45594; UES 7638 next to Perichaena vermicularis).

OBSERVATIONS — Recently described from Chihuahua by Salazar-Marquez et

al. (2014). It might be widely distributed in Mexico but has been unobserved

because of its small size.

1092 ... Lizarraga & al.

*Lycogala exiguum Morgan, J. Cincinnati Soc. Nat. Hist. 15: 134 (1893).

Aethalium small (2-4 mm diam.); peridium ornamented by outgoing

reddish brown warts which form a compartmented structure that gives a false

pluricellular appearance under LM; spores 5-6 um diam.; capillitium abundant,

2-10 um diam., filamentous, tubular, with sinuous walls.

SPECIMEN EXAMINED: MEXICO, Curmuanua: Loc. 6, Yucca sp., 23 March 2009, leg.

ML & CS (UES 7623, AH 45593).

OBSERVATIONS — See Hooff (2014) for diagnostic differences among species of

Lycogala Adans.

Lycogala exiguum has been reported for Jalisco, Nuevo Leon, Quintana Roo,

Tabasco, Veracruz, and Yucatan (Moreno et al. 2007).

Perichaena pachyderma D.W. Mitch., G. Moreno & Lizarraga, Bol. Soc. Micol.

Madrid 35: 104 (2011). Figs 28-33

Fructifications sessile, globose to subglobose, solitary to gregarious.

Sporotheca 0.1-0.3 mm diam., dark yellow in youth, to dark brown when

maturing. Peridium simple, 3-4.5 um thick, externally covered by granular

matter; the internal surface presents small warts which binding with a reticulated

appearance (not noted by Mitchell et al. (2011) in his type description).

Capillitium not observed. Spores 10-11 um diam., orange-yellow in mass, light

yellow under LM, globose, subtly spinulose. Spore ornamentation is formed by

irregular distributed bacula.

SPECIMENS EXAMINED: MEXICO, Curnuanua: Loc. 3, cow dung, 24 January 2009, leg.

ML, CS & DS, obtained from moist chamber 13 February 2009 (UES 7591); obtained

from moist chamber 17 February 2009 (UES 7592); Loc. 5, 23 March 2009, leg. ML &

CS, obtained from moist chamber 6 April 2009 (UES 7594, AH 45562); Loc. 6, 23 March

2009, obtained from moist chamber 6 April 2009 (UES 7595); Loc. 9, 24 May 2009, leg.

ML, CS & DL, obtained from moist chamber 29 May 2009 (UES 8057).

OBSERVATIONS — Fructifications cultivated in moist chamber over herbivore

dung were similar to Licea tenera E. Jahn (studied by Martin & Alexopoulos,

1969), but L. tenera has smooth spores and our collections have spinulose or

verrucose ornamentation. The specimens also resemble Perichaena cf. liceoides

Rostaf. as described by Eliasson & Lundqvist (1979) but which has a poorly

developed capillitium, absent in our collections.

Although it is very possible that this coprophilous species has a wider

distribution, it is only known from the United States (New Mexico) and Mexico

(Baja California and Chihuahua) (Mitchell et al. 2011).

Perichaena quadrata T. Macbr., N. Amer. Slime-Moulds: 184 (1899). Figs 34-42

Fructifications gregarious (rarely solitary), rounded to pulvinate, convex,

0.3-0.5 cm diam., reddish brown to more or less blackish when maturing,

Myxomycetes of Chihuahua 4 (Mexico) ... 1093

31. Peridium thick (SEM). 32. Peridium internal surface detail (SEM). 33. Spore (SEM). Scale bars:

28-31 = 0.1 mm; 31 = 5 um; 32 = 1 um; 33 = 2 um.

dehiscence circumscissile and separating the peridium like a convex

operculum. Peridium internal surface warted, with the warts joining to form

sometimes anastomosing sinuous structures. Capillitium abundant, filiform,

1.5-2 um diam.; under SEM, the ornamentation seen as thick isolated more

or less abundant warts and a reticle full of variably wide meshes. Spores 9-11

um diam., yellowish, spinulose (LM); under SEM, ornamentation is formed by

uniformly distributed bacula and a weakly coralloid apex.

SPECIMENS EXAMINED: MEXICO, CurHuanua: Loc. 2, Yucca elata, 7 February 2009,

leg. ML, CS & DS (UES 7626); cow dung, 22 May 2009, leg. ML, CS & DL, obtained from

moist chamber 30 June 2009 (UES 8066); Loc. 4, Tamarix chinensis, 14 February 2009,

leg. ML, CS & DS, obtained from moist chamber 25 March 2009 (UES 7627, AH 45563);

Loc. 6, Agave lecheguilla, 26 October 2009, leg. ML, CS, AS, AG & DL (UES 8511, AH

45564); Loc. 8, Yucca sp., 25 July 2009, leg. ML, CS, AG, DL & DS, obtained from moist

chamber 22 August 2009 (UES 8354).

1094 ... Lizarraga & al.

Fics 34-42. Perichaena quadrata (AH 45563). 34. Sporocarps gregarious. 35. Peridium, internal

surface detail (SEM). 36-40. Variable capillitium reticulate ornamentation (SEM). 41, 42. Spore

ornamentation (SEM, detail). Scale bars: 34 = 1 mm; 35-40, 42 = 1 um; 41 = 2 um.

OBSERVATIONS — Perichaena quadrata has been confused or synonymized

with P. depressa Lib., which presents larger sporocarps (0.5-1.2 cm diam.), also

with a circumscissile peridium but flat apex; capillitium 2-2.5(-3) um diam.,

Myxomycetes of Chihuahua 4 (Mexico) ... 1095

filiform, and without a reticulated ornamentation, as observed by Keller &

Eliasson (1992), whose concept we follow.

Rammeloo (1981) examined under the SEM two African species close to

P. quadrata that fructify over different substrata: P areolata Rammeloo (leaves),

and P. dictyonema Rammeloo (crusts and wood debris); P dictyonema is

morphologically very similar to P quadrata, and they might represent the same

species.

Perichaena quadrata has been recorded from Puebla (Estrada-Torres et al.

2009) and Chihuahua (Lizarraga et al., 2015). It was cited as coprophilous by

Eliasson & Lundqvist (1979), Eliasson & Keller (1999), and Krug et al. (2004).

Previously cited from Chihuahua by Lizarraga et al. (2015).

**Physarum apiculosporum Hark., Karstenia 18: 24 (1978). Fics 7, 8

Plasmodiocarps gregarious, sessile, pulvinate, sinuous, curved or reticulated,

0.3-0.6 mm wide x 4.5 mm long. Peridium greyish to white, double, with

the two layers closely associated, outer layer with abundant lime; inner layer

thin, membranous. Dehiscence irregular. Capillitium formed by scarce thin

filaments, white, rounded to angular lime nodes. Spores black in mass, reddish

brown, 10-14 x 8-10 um, ellipsoid to oval, smooth with an apiculus and a

longitudinal ridge by LM; spore surface smooth under SEM.

SPECIMEN EXAMINED: MEXICO, CurHuauua: Loc. 7, cow dung, 26 October 2009, leg.

ML, CS, AS, AG & DL, obtained from moist chamber 9 December 2009 (UES 8530, AH

45566).

OBSERVATIONS — Physarum apiculosporum is characterized by forming

plasmodiocarps and spores oval to ellipsoid, smooth, with two pointed

ends, one at each ending (Hark6nen 1978, Moreno et al. 2011). This species

may be confused with Badhamia verrucospora; both share habit and spore

ornamentation, but B. verrucospora differs in warted spores that are strongly

marked over half of the surface, while the remainder is smooth, even under

SEM (Moreno et al. 2011).

Physarum apiculosporum is a new record for Mexico and the Neotropics

(Lado & Wrigley de Basanta 2008).

Physarum decipiens M.A. Curtis, Amer. J. Sci. Arts, Ser. 2, 6: 352 (1848).

SPECIMENS EXAMINED: MEXICO, Cuinvuanua: Loc. 1, Prosopis glandulosa, 14 February

2009, leg. ML, CS & DS, obtained from moist chamber 21 February 2009 (UES 7661);

obtained from moist chamber 28 February 2009 (UES 7662); obtained from moist

chamber 6 March 2009 (UES 7663); Loc. 2, 7 February 2009, leg. ML, CS & DS, obtained

from moist chamber 14 March 2009 (UES 7659); Loc. 3, Prosopis glandulosa, 24 January

2009, leg. ML, CS & DS, obtained from moist chamber 2 March 2009 (UES 7658); Loc.

1096 ... Lizarraga & al.

4, Larrea tridentata, 14 February 2009, 16 February 2009, leg. ML, CS & DS, obtained

from moist chamber 18 March 2009 (UES 7660); Fouquieria splendens, 26 July 2009, leg.

ML, CS, DL, DS & AG, obtained from moist chamber 10 August 2009 (UES 8236); Loc.

7, Viguiera stenoloba, 22 March 2009, leg. ML & CS, obtained from moist chamber 20

April 2009 (UES 7664); Acacia greggii, 24 May 2009, leg. ML, CS & DL, obtained from

moist chamber 11 June 2009 (UES 8074); Acacia greggii, 26 July 2009, leg. ML, CS, DL,

AG & DS, obtained from moist chamber 8 August 2009 (UES 8215); obtained from

moist chamber 10 August 2009 (UES 8235); 26 October 2009, leg. ML, CS, AS, AG &

DL, obtained from moist chamber 6 November 2009 (UES 8515); obtained from moist

chamber 27 November 2009 (UES 8529, AH 45592); Loc. 10, Prosopis glandulosa, 26

July 2009, leg. ML, CS, DL, DS & AG, obtained from moist chamber 8 September 2009

(UES 8366, AH 45591).

OBSERVATIONS — Physarum decipiens is morphologically highly variable.

Previously reported from Chihuahua (Lizarraga et al. 2003), Oaxaca, Puebla

(Estrada-Torres et al. 2009), Sonora (Esqueda et al. 2013), and Tlaxcala

(Rodriguez-Palma et al. 2002).

Physarum leucophaeum Fr., Symb. Gasteromyc. 3: 24 (1818).

SPECIMENS EXAMINED: MEXICO, CuIHuAHUA: Loc. 1, cow dung, 14 February 2009,

leg. ML, CS & DS, obtained from moist chamber 14 March 2009 (UES 7665); rabbit

dung, obtained from moist chamber 2 April 2009 (UES 7653, AH 45567); 22 May 2009,

leg. ML, CS & DL, obtained from moist chamber 22 June 2009 (UES 8092); Loc. 3,

Prosopis glandulosa, 20 May 2009, leg. ML, CS & DL, obtained from moist chamber 25

June 2009 (UES 8091, AH 45568); Loc. 6, Prosopis glandulosa, 25 June 2009, obtained

from moist chamber 3 June 2009 (UES 8080, AH 45590); Loc. 8, Cylindropuntia

imbricata, 25 July 2009, leg. ML, CS, DL, DS & AG, obtained from moist chamber 5

August 2009 (UES 8211).

OBSERVATIONS — Physarum leucophaeum is very close to P. robustum and

P. notabile T. Macbr., two species that appear confused in the literature. We

follow Nannenga-Bremekamp (1991) for distinguishing P leucophaeum from

P. robustum: P. leucophaeum presents a capillitium with very few calcium

carbonate nodules, which are small and round, while P robustum has a

capillitium with bigger and abundant fusiform calcium carbonate nodules that

generally join at sporotheca center to form a whitish pseudocolumella (see

photographs in Poulain et al. 2011). Our specimens were collected on cow and

rabbit dung. This coprophilous habit was not indicated in Eliasson & Lundqvist

(1979), Eliasson & Keller (1999), and Krug et al. (2004). Previously cited from

Chihuahua by Lizarraga et al. (2003).

Physarum pusillum (Berk. & M.A. Curtis) G. Lister, Monogr. Mycetozoa, 2nd Ed.:

64 (1911).

SPECIMENS EXAMINED: MEXICO, Curmuanva: Loc. 1, cow dung, obtained from moist

chamber 30 June 2009 (UES 8086); Loc. 2, Yucca elata, 7 February 2009, leg. ML, CS

Myxomycetes of Chihuahua 4 (Mexico) ... 1097

& DS, obtained from moist chamber 24 March 2009 (UES 7670); obtained from moist

chamber 24 March 2009 (UES 7669); 23 July 2009, leg. ML, CS, DL, DS & AG, obtained

from moist chamber 15 August 2009 (UES 8360); Loc. 3, Tamarix chinensis, 24 January

2009, leg. ML, CS & DL, obtained from moist chamber 25 February 2009 (UES 7650);

obtained from moist chamber 2 March 2009 (UES 7651); Larrea tridentata, obtained

from moist chamber 3 March 2009 (UES 7646); Koeberlinia spinosa, 14 February 2009,

leg. ML, CS & DS, obtained from moist chamber 30 June 2009 (UES 8081); Loc. 4, Larrea

tridentata, leg. ML, CS & DS, obtained from moist chamber 18 March 2009 (UES 7652);

Loc. 5, Cylindropuntia imbricata, 22 May 2009, obtained from moist chamber 11 June

2009 (UES 8073); Loc. 7, Chilopsis linearis, 24 May 2009, leg. ML, CS & DL, obtained

from moist chamber 15 June 2009 (UES 8094); Loc. 8, Opuntia sp., 24 October 2009, leg.

ML, CS, AS, AG & DL (UES 8378); inflorescence of Yucca sp., 23 May 2009, obtained

from moist chamber 29 May 2009 (UES 8071); obtained from moist chamber 3 June

2009 (UES 8096, AH 45587); obtained from moist chamber 22 June 2009 (UES 8093);

Loc. 10, Koeberlinia spinosa, 25 July 2009, obtained from moist chamber 22 August 2009

UES 8363); Acacia constricta, obtained from moist chamber 8 September 2009 (UES

8364).

OBSERVATIONS — Physarum pusillum is frequently observed in moist chamber

cultures. Previously reported for Chihuahua by Lizarraga et al. (2003).

Physarum robustum (Lister) Nann.-Bremek., Proc. Kon. Ned. Akad. Wetensch.,

C 76: 484 (1973).

SPECIMENS EXAMINED: MEXICO, CurHuanua: Loc. 4, Tamarix chinensis, 22 May

2009, leg. ML, CS & DL, obtained from moist chamber 30 June 2009 (UES 8084, AH

45569); Loc. 6, Prosopis glandulosa, 26 July 2009, leg. ML, CS, DL, DS & AG, obtained

from moist chamber 10 August 2009 (UES 8234, AH 45570).

OBSERVATIONS — For Physarum robustum we follow the concept of Nannenga-

Bremekamp (1991). Differences with closely related species like P album (Bull.)

Chevall., P leucophaeum, and P. leucopus Link have been described for Sonora

(Moreno et al. 2004) and Chihuahua (Lizarraga et al. 2015).

*Physarum spectabile Nann.-Bremek., Lado & G. Moreno, Proc. Kon. Ned. Akad.

Wetensch., C 87: 91 (1984).

SPECIMENS EXAMINED: MEXICO, Cuinvuanua: Loc. 6, Agave lecheguilla, 23 March

2009 (UES 7668); Yucca sp., 23 March 2009 (UES 7667, AH 45571).

OBSERVATIONS — Physarum spectabile is a common succulenticolous species.

Recorded for Oaxaca and Puebla by Estrada-Torres et al. (2009)

Physarum straminipes Lister, J. Bot. 36: 163 (1898). Fics 43, 44

Fructifications whitish, stipitate with a false stalk, which is an extension of

a whitish to yellowish hypothallus; capillitium with whitish nodules, globose,

more abundant at the center of the sporocarp; spores polygonal, dark, with

scarce and thick spines of irregular distribution (see SEM).

1098 ... Lizarraga & al.

PSs = : Ley

Fics 43, 44. Physarum straminipes (AH 45572). Spores (SEM). Scale bars = 2 um.

SPECIMEN EXAMINED: MEXICO, Curnuanva: Loc. 9, rabbit dung, 22 March 2009, leg.

ML & CS, obtained from moist chamber 1 May 2009 (UES 7671, AH 45572).

OBSERVATIONS — Physarum straminipes was not noted as coprophilous by

Eliasson & Lundqvist (1979), Eliasson & Keller (1999), or Krug et al. (2004).

Previously reported for Baja California, Sonora (Moreno et al. 2007), and

Querétaro (Tapia et al. 2008).

Protophysarum phloiogenum M. Blackw. & Alexop., Mycologia 67: 33 (1975).

SPECIMENS EXAMINED: MEXICO, Chihuahua: Loc. 1, Atriplex canescens (Pursh) Nutt.

(Amaranthaceae), obtained from moist chamber 6 March 2009 (UES 7684); 24 July

2009, leg. ML, CS, DL, DS & AG, obtained from moist chamber 12 August 2009 (UES

8238); Loc. 3, Tamarix chinensis, 24 January 2009, leg. ML, CS & DS, obtained from

moist chamber 3 February 2009 (UES 7673); obtained from moist chamber 4 February

2009 (UES 7674); obtained from moist chamber 5 February 2009 (UES 7675); obtained

from moist chamber 11 February 2009 (UES 7676); Atriplex canescens, 22 October 2009,

leg. ML, CS, AS, AG & DL, obtained from moist chamber 9 November 2009 (UES 8532);

obtained from moist chamber 14 November 2009 (UES 8533); obtained from moist

chamber 21 November 2009 (UES 8522); Loc. 4, Atriplex canescens, 14.11.2209, leg. ML,

CS & DS, obtained from moist chamber 23 February 2009 (UES 7677); obtained from

moist chamber 2 March 2009 (UES 7678); obtained from moist chamber 11 March 2009

(UES 7679); 22 May 2009, leg. ML, CS & DL, obtained from moist chamber 29 May 2009

(UES 8097); obtained from moist chamber 1 June 2009 (UES 8098); obtained from moist

chamber 3 June 2009 (UES 8099); obtained from moist chamber 11 June 2009 (UES

8100); obtained from moist chamber 15 June 2009 (UES 8101); decomposing wood,

obtained from moist chamber 3 June 2009 (UES 8102); Yucca elata, 14.11.2209, leg. ML,

CS & DS, obtained from moist chamber 27 February 2009 (UES 7680); obtained from

moist chamber 2 March 2009 (UES 7681); rabbit dung, obtained from moist chamber

20 March 2009 (UES 7682).

Myxomycetes of Chihuahua 4 (Mexico) ... 1099

OBSERVATIONS — Protophysarum phloiogenum appears frequently on such

substrates as Atriplex canescens, Tamarix chinensis, Yucca elata, and even on

rabbit dung when cultured in moist chambers. A complete study of this species,

including its variable spore ornamentation, was carried out by Castillo et al.

(1998). Previously cited for Baja California, Chihuahua (Moreno et al. 2007),

and Sonora (Esqueda et al. 2013).

Trichia agaves (G. Moreno, Lizarraga & Illana) Mosquera, Lado, Estrada & Beltran-

Tej., Cuad. Trab. Fl. Micol. Ibér. 16: 82 (2001).

SPECIMEN EXAMINED: MEXICO, Cuinuanua: Loc. 6, Agave lecheguilla, leg. ML, CS,

DL, DS & AG, 26 July 2009 (UES 8214, AH 45573).

OBSERVATIONS — ‘This species was described by Moreno et al. (2000) from

several collections from Mexico and Spain on members of the Cactaceae and

Liliaceae. It is frequently collected in desert areas and was recently reported for

Cuba (Camino et al. 2008).

Acknowledgements

We wish to express our gratitude to Mr. A. Priego and Mr. J. A. Pérez of the Electron

Microscopy Service of the University of Alcala de Henares for their invaluable help with

the SEM. We also thank L. Monje and A. Pueblas of the Department of Drawing and

Scientific Photography at the Alcala University for their help in the digital preparation

of photographs and to Dr. J. Rejos, curator of the AH herbarium, for his assistance

with the specimens examined in the present study. We want to express our gratitude to

S.L. Stephenson, J.R. Deschamps, and A. Hladki for reviewing the manuscript.

M. Lizarraga and M. Esqueda thank CONABIO for supporting project GT016.

Salazar-Marquez thanks CONACYT for the support to perform master studies.

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MY COTAXON

http://dx.doi.org/10.5248/130.1103

Volume 130, pp. 1103-1110 October-December 2015

Three Trichoderma species associated with

wood discoloration in South Korea

SEOKYOON JANG, YEONGSEON JANG, GYU-HYEOK KIM & JAE-JIN K1m*

Division of Environmental Science & Ecological Engineering,

College of Life Sciences & Biotechnology, Korea University

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

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

ABSTRACT — Three Trichoderma species previously recorded as new records from South

Korea, T: dorotheae, T. gamsii, and T. koningiopsis, are described and illustrated.

KEY WORDS — ascomycetes, Hypocrea, phylogeny, taxonomy

Introduction

Trichoderma Pers. [= Hypocrea Fr.] is a cosmopolitan genus of fungi

(Hypocreaceae, Ascomycota) comprising more than one hundred species

(Druzhinina et al. 2006), which can be isolated from soil, wood, and various

other substances, even including other fungi (Druzhinina et al. 2011).

In South Korea, about 20 Trichoderma species have been reported (Lee et al.

2003; Park et al. 2005, 2006; Cho et al. 2007; Huh et al. 2011). Ten Trichoderma

species isolated from wood were identified by Huh et al. (2011), who examined

the culture characteristics and analyzed the ITS and TEF sequences of the

isolates; they reported three species—Trichoderma dorotheae, T. gamsii, and

T: koningiopsis—as new records from South Korea but provided no microscopic

descriptions. Here we confirm the identifications of these Trichoderma records

through macro- and microscopical observations and an expanded phylogenetic

analysis using translation elongation factor-la (TEF) gene region sequences.

Materials & methods

Analysis of phenotype

Six isolates of the three Trichoderma species were obtained from the Korea

University Culture Collection, Seoul, Republic of Korea (KUC). Cultures used for

1104... Jang & al.

micromorphology were grown on corn meal dextrose agar (CMD: cornmeal 20 g,

glucose 20 g, agar 18 g, distilled water 1000 ml), potato dextrose agar (PDA: Difco™

potato dextrose agar 39 g, distilled water 1000 ml) or Synthetic Nitrogenpoor or Nutrient

poor agar (SNA: sucrose 0.2 g, glucose 0.2 g, KNO, 1 g, KH,PO, 1 g, MgSO,-7H,O 0.5

g, NaCl 0.5 g, agar 12 g, distilled water 1000 ml). The fungi were cultured at 25 or 30°C

for 7d because their optimal growth temperatures were placed at 25-30°C (Huh et al.

2011). Microscopic characters were observed with an Olympus BX51 light microscope.

Pictures of conidiophores and conidia were taken using the same microscope mounted

with an Olympus DP20 microscopic camera. Measurements were made from 3% KOH

mounts. At least 30 units of each parameter were measured for each collection. To make

reliable data, 5% of the measurements from each end of the range were removed. The

isolates were deposited at the National Institute of Biological Resources, Incheon, South

Korea (NIBR).

Phylogenetic analysis

The TEF region was used for phylogenetic analysis of the Trichoderma species

(Samuels et al. 2006). For three isolates (KUC1747, KUC5027, and KUC5063), TEF

sequences were obtained from Huh et al. (2011). For the other three isolates (KUC3025,

KUC5028, and KUC5064), DNA samples were extracted and the TEF region was

amplified according to Huh et al. (2011). The TEF sequences were proofread and aligned

with selected GenBank reference sequences using MAFFT 7.130 (Katoh & Standley

2013) and modified manually with MacClade 4.08 (Maddison & Maddison 2005).

Datasets were tested by MrModeltest 2.3 using the AIC criteria with default options

(Nylander 2004). The HKY + G model was chosen under the AIC criteria as a result

of the test. Bayesian analysis was performed with MrBayes 3.2.1 (Ronquist et al. 2012).

A phylogenetic tree was created according to Jang et al. (2012).

Taxonomy

Trichoderma dorotheae Samuels & Dodd, Stud. Mycol. 56: 112 (2006) Fic. 1a-c,j,k

Conidiophores lacking a discernible main axis and branches not obviously

paired or unilaterally branched, re-branching to form second branches, which

terminate in slightly divergent whorls of 3-4 phialides; conidiophores often

arising from pustules, 2.0-2.5 um wide. Phialides lageniform, swollen in the

middle, 5.5-10.5 um long, 2.5-3.5 um at the widest point. Conidia broadly

ellipsoidal, smooth, 3.2-4.0 x 2.2-3.5 um.

CULTURE CHARACTERISTICS — Optimum temperature for growth on PDA

and SNA 25°C. Colonies grown on SNA at 25°C remaining sterile. A white

mycelium covering the 9 cm diam Petri plate at 25°C after 1 wk in darkness;

No pigment diffusing through the agar; no distinctive odor. Colonies grown

on PDA at 25°C in darkness forming conidia only after 96 h. A dense white

mycelium covering the 9 cm diam Petri plate at 25°C after 1 wk in darkness;

conidia moderate, bluish green. No pigment diffusing through the agar; no

Trichoderma spp. discoloring wood (Korea) ... 1105

Fic. 1. Trichoderma dorotheae: growth on (a) PDA; (b) CMD; (c) SNA. Trichoderma gamsii:

growth on (d) PDA; (e) CMD; (f) SNA. Trichoderma koningiopsis: growth on (g) PDA; (h) CMD;

(i) SNA. [All cultures in 9-cm-diam Petri dishes after 96 h at 25°C]. Trichoderma dorotheae:

(j) conidiophores; (k) conidia. Trichoderma gamsii: (1) conidiophores; (m) conidia. Trichoderma

koningiopsis: (n) conidiophores; (0) conidia. Scale bar = 10 um.

distinctive odor. Colonies grown on CMD at 25°C in darkness filling the Petri

plate within 1 wk, colonies remaining sterile.

DISTRIBUTION — New Zealand, Australia (Samuels et al. 2006), and South

Korea.

SPECIMEN EXAMINED: KOREA, JEOLLABUK-DO, Iksan-si, a log of Pinus radiata, August

2008, Jae-Jin Kim (NIBR-FG0000120470, culture KUC5027, GenBank HM534661).

REMARKS — The TEF sequences of T: dorotheae KUC5027 (503 bp) and other

T. dorotheae strains were monophyletic with high posterior probability support

(PP = 1). Trichoderma dorotheae can cause light green discoloration, but wood-

discoloration rate was low (Huh et al. 2011). The Korean isolate of T: dorotheae

has a relatively high antagonistic ability against other wood-damaging fungi, so

it has potential as a biological control agent (Lee et al. 2012).

1106... Jang & al.

Trichoderma gamsii Samuels & Druzhin., Stud. Mycol. 56: 168 (2006) Fie. 1d-f,l,m

Conidiophores more or less uniformly branched for a short distance,

branches often arising from swollen nodes on the conidiophore, a single

phialide forming at the tip, 2.0-3.0 um wide. Phialides lageniform, at most

slightly swollen in the middle, 7.0-10.5 um long, 2.5-3.0 um at the widest point,

1.5-2.5 um at the base; Intercalary phialides common. Conidia ellipsoidal,

smooth, 3.3-3.9 x 2.0-2.8 um.

CULTURE CHARACTERISTICS Optimum temperature for growth on PDA and

SNA 25-30°C; after 72 h on PDA colony radius reaching 50-52 mm, on SNA

colony radius 39-42 mm. On PDA at 25°C and 30°C after 1 wk in darkness

typically a dense white mycelium covering the 9 cm diam Petri plate; colonies

remaining sterile. More or less strong coconut-like odor. On SNA at 25°C and

30°C covering the 9 cm diam Petri plate after 1 wk in darkness; conidia forming

abundantly, grayish green, in broad sheets and sometimes confluent pustules

sporadically produced, 1-2 mm diam. No pigment diffusing through the agar,

no distinctive odor.

DISTRIBUTION — Australia, Central Russia, Italy, U.S.A. (Jaklitsch et al.

2006), and South Korea.

SPECIMEN EXAMINED: KOREA, JEOLLABUK-DO, Iksan-si, a log of Pinus radiata, August

2008, Jae-Jin Kim (NIBR-FG0000120471, culture KUC1747, GenBank HM534663).

REMARKS — The most conspicuous characters of T! gamsii were its coconut-

like odor and abundant aerial mycelia on CMD or PDA. As observation of

conidiophores of T. gamsii on CMD or PDA was obscured by their aerial

mycelia, most measurements were determined on SNA.

The macro- and micromorphological features of the Korean isolate agreed

well with the protologue description, except for the protologue conidia being

longer ((3.2—)4.0-5.0(-6.2) um; Jaklitsch et al. 2006). The TEF sequences of

T. gamsii KUC1747 (594 bp) and other T. gamsii strains were monophyletic

with high support (PP = 1).

Trichoderma gamsii can cause green discoloration on wood surfaces with

high wood-discoloration rate (Huh et al. 2011). On the other hand, T’ gamsii

has strong antagonistic properties against wood-rotting fungi and sap-staining

fungi (Lee et al. 2012). Trichoderma gamsii KUC1747 produces 6-pentyl-

a-pyrone that is a known antagonistic metabolite against sap-staining fungi

(Hong et al. 2014).

Trichoderma koningiopsis Samuels, C. Suarez & H.C. Evans, Stud. Mycol. 56: 117

(2006) Fic. 1g-i,n,o

Conidiophores 2.5-3.5 um wide, branches formed from the interior of

pustules, the first branches rebranching or making phialides directly, the

Trichoderma spp. discoloring wood (Korea) ... 1107

second branches tending to be paired; all fertile branches terminating in a

whorl of phialides. Phialides lageniform, held in whorls of 2-5, sometimes

several phialides arising from the same point and crowded, 5.5-7.5 um long,

2.5-3.0 um at the widest point. Conidia broadly ellipsoidal, smooth, 2.8-3.6 x

2.6-3.0 um.

CULTURE CHARACTERISTICS Optimum temperature for growth on PDA

and SNA 25-30°C. Colonies grown on PDA in darkness sometimes forming

conidia after 48 h at 25 and 30°C in a concentric ring. A dense white mycelium

covering the 9 cm diam Petri plate at 25°C and 30°C after 1 wk in darkness;

conidia moderate, sometimes abundant at 30°C from some isolates. No

pigment diffusing through the agar; rarely a faint coconut odor detected.

Colonies grown on SNA in darkness for 96 h producing abundant aerial

mycelium; conidia tending to be uniformly dispersed in the aerial mycelium

in 1-2 broad concentric rings, sometimes forming cottony pustules. Conidial

masses dark green. No pigment diffusing through the agar; no distinctive odor.

Colonies grown on CMD in darkness a dense white mycelium covering the

9-cm-diam Petri plate within 1 wk at 25 °C. Conidial production restricted

near the inoculated spot of the colony with a tendency to form highly compact

to cottony, 1-2 mm diam pustules; Often long, entirely fertile branches visible

in the pustules. Rarely a faint coconut odor detected.

DISTRIBUTION — Brazil, Canada, Cuba, Ethiopia, Ecuador, Germany,

Ghana, Peru. Puerto Rico, U.S.A. (Samuels et al. 2006), Japan (Yabuki et al.

2014), and South Korea.

SPECIMENS EXAMINED: KOREA, SEOUL, Seongbuk-gu, Anam-dong, Korea University,

chromated copper arsenate treated wood of Pinus radiata, 2003, Jae-Jin Kim (NIBR-

FG0000120472, culture KUC3025, GenBank KJ652555); Mapo-gu, Sangam-dong,

creosote contaminated soil, May 2007, Jae-Jin Kim (NIBR-FG0000120474, culture

KUC5063, GenBank HM534662; NIBR-FG0000120475, culture KUC5064, GenBank

KJ652554). GYEONGGI-DO, Gwangmyeong-si, creosote treated wood, 6 November

2007, Jae-Jin Kim (NIBR-FG0000120473, culture KUC5028, GenBank KJ652556)

REMARKS — The colony morphology and microscopic features of the Korean

isolates agreed well with the protologue description, except for the protologue

conidia being longer ((3.0—)3.5-4.5(-6.2) um; Samuels et al. 2006).

The TEF sequences of T: koningiopsis KUC3025 (604 bp), KUC5028 (580

bp), KUC5063 (555 bp), and KUC5064 (553 bp) were placed inside the

T. koningiopsis clade with high support (PP = 1). Trichoderma koningiopsis

KUC5028, KUC5063 and KUC5064 were placed in the same subclade with

high support (PP = 1). However, T. koningiopsis KUC3025 was clustered in

another subclade with T. koningiopsis GJS 06-219 with high support (PP = 1).

This intraspecific genetic variation was not associated with any phenotypic

variation.

1108 ... Jang & al.

4, Trichoderma koningiopsis GJS 97-273 (FJ463277)

Trichoderma koningiopsis KUC5063 (HM534662)

1+ Trichoderma koningiopsis KUC5028 (KJ652556)

Trichoderma koningiopsis KUC5064 (KJ652554)

Trichoderma koningiopsis AN143 (HQ292992)

Trichoderma koningiopsis UNISS 17b-36a (EF488124)

4 Trichoderma koningiopsis KUC3025 (KJ65255)

1 Trichoderma koningiopsis GJS 06-219 (FJ463276)

oF Trichoderma koningiopsis DAOM 179516 (EU280012)

0.98 Trichoderma koningiopsis GJS 04-11 (DQ289009)

Trichoderma koningiopsis GJS 04-379 (DQ289001)

Hypocrea koningii GJS 89-122 (AY376045)

47 Trichoderma dorotheae GJS 99-194 (DQ284974)

1 1 Trichoderma dorotheae GJS 99-202 (DQ307536)

Trichoderma dorotheae KUC5027 (HM534661)

1 Trichoderma taiwanense GJS 95-93 (DQ284973)

Trichoderma caribbaeum GJS 97-3 (DQ284977)

Trichoderma dingleyae GJS 99-105 (DQ289008)

067 Hypocrea intricata GJS 02-78 (EU248630)

Trichoderma ovalisporum GJS 06-31 (FJ463280)

0.96r— Trichoderma gamsii PPRC $19 (FJ436186)

0.7 Trichoderma gamsii UNISS 17-16 (EF488118)

! Trichoderma gamsii GJS 92-60 (DQ307529)

Trichoderma gamsii UNISS 17-26 (EF488119)

1+ Trichoderma gamsii KUC1747 (HM534663)

Trichoderma gamsii 05-111 (DQ841722)

Trichoderma gamsii UNISS 14-16 (EF488115)

Trichoderma neokoningii GJS 04-216 (DQ841718)

0.86 4p Hypocrea viridescens GJS 05-464 (DQ841714)

1 Hypocrea viridescens CBS119321 (DQ672610)

gee Hypocrea rufa GJS 94-10 (DQ307506)

Hypocrea vinosa GJS 99-183 (DQ841719)

Trichoderma samuelsii S398 (JN715654)

0.96 Hypocrea hispanica S423 (JN715658)

0.88

0.59

0.77

Trichoderma austrokoningii CTR 85-57 (DQ307564)

0.1

Fic. 2. 50% majority-rule consensus tree of Trichoderma dorotheae, T. gamsii, T. koningiopsis, and

allied species. Strains analyzed in this study are in bold font. GenBank accession numbers are in

parentheses. Posterior probabilities >0.50 are shown above branches.

Even though T. koningiopsis caused green discoloration on wood surfaces,

its wood-discoloration rate was low (Huh et al. 2011). Trichoderma koningiopsis

KUC5063 showed strong antagonistic properties against wood-damaging fungi

(Lee et al. 2012).

Trichoderma spp. discoloring wood (Korea) ... 1109

Phylogeny

The Bayesian tree of TEF region sequences was constructed to confirm

the morphological identification. The aligned dataset of TEF contained 35

sequences and 600 characters. The three species are placed into Viride-Koningii

section (JJaklitsch 2011).

Trichoderma gamsii was grouped with T. neokoningii Samuels & Soberanis

(Fic. 2). Our phylogenetic result was similar to the study by Jaklitsch et al.

(2006).

Trichoderma koningiopsis was monophyletic with T. koningii Oudem.

[= Hypocrea koningii Lieckf. et al.],and T: dorotheae was placed ina monophyletic

group with T. caribbaeum Samuels & Schroers, T. dingleyae Samuels & Dodd,

and T’ taiwanense Samuels & M.L. Wu. Again, the T’ koningiopsis and

T. dorotheae clades grouped together. However, phylogenetic trees in Jaklitsch

(2011) showed that T: koningiopsis and T. dorotheae were monophyletic, but not

with T. koningii. Trichoderma koningii was placed in the basal lineage below the

two species (Jaklitsch 2011).

Acknowledgments

This research was supported by Basic Science Research Program through the

National Research Foundation of Korea (NRF) funded by the Ministry of Education

(NRF-2013R1A1A2A10011390). This work was also supported by the project on

survey and excavation of Korean indigenous species of NIBR under the Ministry of

Environment, Republic of Korea, and by a Korea University Grant. We are grateful

to Dr. Seonju Marincowitz and Dr. Toru Okuda for their valuable suggestions on the

manuscript.

Literature cited

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soil-borne fungi on Mount Halla, Korea, Canadian Journal of Forest Research 37: 371-382.

http:/dx.doi.org/10.1139/x06-226

Druzhinina IS, Kopchinskiy AG, Kubicek CP. 2006. The first 100 Trichoderma species characterized

by molecular data. Mycoscience 47: 55-64. http:/dx.doi.org/10.1007/s10267-006-0279-7

Druzhinina IS, Seidl-Seiboth V, Herrera-Estrella A, Horwitz BA, Kenerley CM, Monte E, Mukherjee

PK, Zeilinger S, Grigoriev IV, Kubicek CP. 2011. Trichoderma: the genomics of opportunistic

success. Nature Reviews Microbiology 9: 749-759. http:/dx.doi.org/10.1038/nrmicro2637

Hong J-H, Lee J, Min M, Ryu S-M, Lee D, Kim G-H, Kim J-J. 2014. 6-Pentyl-a-pyrone as an anti-

sapstain compound produced by Trichoderma gamsii KUC1747 inhibits the germination of

ophiostomatoid fungi. Holzforschung 68: 769-774. http://dx.doi.org/10.1515/hf-2013-0171

Huh N, Jang Y, Lee J, Kim G-H, Kim J-J. 2011. Phylogenetic analysis of major molds inhabiting

woods and their discoloration characteristics. Part 1. Genus Trichoderma. Holzforschung 65:

257-263. http:/dx.doi.org/10.1515/HE2011.018

Jaklitsch WM. 2011. European species of Hypocrea part II: species with hyaline ascospores. Fungal

Diversity 48: 1-250. http:/dx.doi.org/10.1007/s13225-011-0088-y

1110... Jang & al.

Jaklitsch WM, Samuels GJ, Dodd SL, Lu B-S, Druzhinina IS. 2006. Hypocrea rufa/Trichoderma

viride: a reassessment, and description of five closely related species with and without warted

conidia. Studies in Mycology 55: 135-177. http:/dx.doi.org/10.3114/sim.2006.56.04

Jang Y, Lee SW, Jang S, Lim YW, Lee JS, Kim J-J. 2012. Four unrecorded wood decay fungi from

Seoul in Korea. Mycobiology 40: 195-201. http:/dx.doi.org/10.5941/MYCO.2012.40.3.195

Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment software version 7:

improvements in performance and usability. Molecular Biology and Evolution 30: 772-780.

http:/dx.doi.org/10.1093/molbev/mst010

Lee S, Hong S-B, Kim C-Y. 2003. Contribution to the checklist of soil-inhabiting fungi in Korea.

Mycobiology 31: 9-18. http://dx.doi.org/10.4489/MYCO.2003.31.1.009

Lee J, Huh N, Hong JH, Kim BS, Kim G-H, Kim J-J. 2012. The antagonistic properties of

Trichoderma spp. Inhabiting woods for potential biological control of wood-damaging fungi.

Holzforschung 66: 883-887. http:/dx.doi.org/10.1515/HF.2011.187

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

Version 4.08. Sunderland, MA: Sinauer Associates.

Nylander JAA. 2004. MrModeltest v2. Program distributed by the author. Evolutionary Biology

Centre, Uppsala University.

Park MS, Seo GS, Bae KS, Yu SH. 2005. Characterization of Trichoderma spp. associated with green

mold of oyster mushroom by PCR-RFLP and sequence analysis of ITS Regions of rDNA. Plant

Pathology Journal 21: 229-236. http:/dx.doi.org/10.5423/PPJ.2005.21.3.229

Park MS, Bae KS, Yu SH. 2006. Two new species of Trichoderma associated with green mold of

oyster mushroom cultivation in Korea. Mycobiology 34: 111-113.

http://dx.doi.org/10.4489/MYCO.2006.34.3.111

Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Hohna S, Larget B, Liu L,

Suchard MA, Huelsenbeck JP. 2012. MrBayes 3.2: efficient Bayesian phylogenetic

inference and model choice across a large model space. Systematic Biology 61: 539-542.

http://dx.doi.org/10.1093/sysbio/sys029

Samuels GJ, Dodd SL, Lu B-S, Petrini O, Schroers H-J, Druzhinina IS. 2006. The Trichoderma

koningii aggregate species. Studies in Mycology 56: 67-133.

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Yabuki Y, Duncan I, Okuda T. 2014. Comparative study reveals unique features of the

mycobiota in peat soils samples from Japan and Scotland. Mycoscience 55: 168-176.

http://dx.doi.org/10.1016/j.myc.2013.08.002

MY COTAXON

http://dx.doi.org/10.5248/130.1111

Volume 130, pp. 1111-1116 October-December 2015

Terriera fici sp. nov. on Ficus vasculosa

from Hainan Province, China

YUAN Wu", SHI-JUAN WANG”, YAN-QIONG MENG’,

YAN-PING TANG? & YING-REN LIN”

"School of Life Science & ? School of Forestry & Landscape Architecture,

Anhui Agricultural University, West Changjiang Road 130, Hefei, Anhui 230036, China

*CORRESPONDENCE TO: yingrenlin@yahoo.com

ABSTRACT — Terriera fici, a new species that develops on leaves of Ficus vasculosa, was

collected in 2014 from Jianfengling National Forest Park of Hainan Province, China.

Description, illustration, and comments are given for this fungus. The type collection is

deposited in the Reference Collection of Forest Fungi of Anhui Agricultural University,

China (AAUF).

Key worps — Rhytismataceae, Rhytismatales, Moraceae, morphology, taxonomy

Introduction

Eriksson (1970) erected the monotypic genus Terriera B. Erikss. for T. cladophila

(Lév.) B. Erikss.; Minter (1996) provided a detailed description of the type

species. Terriera species are distributed worldwide and associate with both

angiosperms and gymnosperms. IndexFungorum (2015) lists 27 specific

and two varietal epithets for the genus, of which SpeciesFungorum (2015)

accepts 24 species in Terriera.

Recently, during investigations of the Rhytismatales from Jianfengling

National Forest Park of Hainan Province, China, a new Terriera species was

discovered.

Materials & methods

The study was based on collections made in 2014 from Hainan, China. Specimens

were rehydrated in water for 15 min and ascomata and conidiomata were sliced into

10-15 um thick sections using a freezing microtome (Jinhua, China). The structure of

“Yuan Wu and Su1-JUAN WANG contributed equally to this work.

1112 ... Wu, Wang & al.

the fruiting bodies was observed in 0.1% (w/v) cotton blue in phenol glycerin. The color

of the internal structures of the fruiting bodies was observed in water. Measurements

and drawings of asci, ascospores, paraphyses, conidiogenous cells and conidia were

made from squash mounts in 5% KOH solution. Line and point integrated illustrations

of internal structures of ascomata were prepared using a Panasonic XSJ-2 microscope

drawing device. Photographs were taken using a Sony DSC-TX7C camera and a

digital camera connected to an Olympus CX31RTSF microscope. The type specimen is

deposited in the Reference Collection of Forest Fungi of Anhui Agricultural University,

Hefei, China (AAUF).

Taxonomy

Terriera fici Y.R. Lin & Yuan Wu, sp. nov. Fics 1, 2

MycoBAnk MB812746

Differs from Terriera nitens by its larger subrounded ascomata, its much thinner

excipulum, its well developed subhymenium consisting of textura angularis and

intricata, its septate paraphyses branching 1-2 times, its somewhat long-stalked asci,

and the absence of trichogynes in its conidiomata.

Type: China, Hainan, Jianfengling National Park, Tianchi, alt. 880 m, on dead leaves of

Ficus vasculosa Wall. ex Mig. (Moraceae), 18 June 2014, Y.R. Lin, S.J. Wang & Y.E Xu

2804 (Holotype, AAUF 68912).

ETyMoLoey: fici, referring to the host genus.

Cotontgs on both sides of dead leaves, forming distinct subround to irregular,

yellowish to gray-white bleached spots 5-12 mm diam. that tend to coalesce

into larger irregular shapes.

ZONE LINES somewhat frequent, gray-black or black, thin, surrounding or

partly surrounding the pale areas.

ConIDIOMATA developing on both sides of fallen leaves, principally on

the lower side of the leaf, scattered, occasionally coalescent. In surface view,

conidiomata 90-200 um diam., rounded or subrounded, somewhat raising

the substratum surface, dark brown to gray-black, discharging spores through

an conspicuous central ostiole or split. In vertical section, conidiomata

subepidermal, lenticular. UPPER waLL 10-18 um thick, black-brown but

roughly black near the opening, consisting of 1.5—-4 um diam., thick-walled

angular cells. BASAL WALL dark brown, 10-20 um thick, composed of 2-3 um

diam. angular cells. SUBCONIDIOGENOUS LAYER 4—8 um thick, comprising

nearly colorless, thin-walled angular cells. CONIDIOGENOUS CELLS 9-14 x

2-3 um, subcylindrical, tapering towards the apex, proliferating sympodially

and percurrently. Conrp1a 4—5 x 1—1.2 um, cylindrical or subclavate, hyaline,

straight or slightly curved.

Ascomarta in similar positions to conidiomata on the substratum, scattered

to clustered. In surface view, ascomata 360-520 um diam., rounded or

Terriera fici sp. nov. (China) ... 1113

Fic. 1. Terriera fici (holotype AAUF 68912) on Ficus vasculosa: A. Fruiting bodies on leaf.

B. Detail of ascomata and conidiomata. C. Ascoma in median vertical section. D. Upper portion

of hymenium. E. Ascoma in section, showing strongly carbonized and brittle tissue of covering

stromatic extension. F Subhymenium and basal stroma. G. Asci and paraphyses. H. Ascospores.

I. Conidioma in median vertical section. Scale bars: A = 20 mm; B = 8 mm; C = 50 um; D, H = 20 um;

E, E.1 = 30 pm; G = 15 um.

1114 ... Wu, Wang & al.

Fic. 2. Terriera fici (ex holotype AAUF 68912): A. Portion of ascoma in median vertical section.

B. Asci, paraphyses, and ascospores. C. Conidioma in median vertical section. Scale bars: A = 20

um; B = 15 um; C = 10 um.

subrounded, black, slightly shiny, with conspicuous edge, moderately raising

the surface of the leaf, opening by a single longitudinal split extending 2/3—4/5

the length of the ascoma. In median vertical section, ascomata subepidermal.

COVERING STROMA 18-25 um thick, composed of a black-brown textura

angularis with thick-walled cells 3—5.5 um diam., connecting to the basal

stroma. Along the edge of the ascomatal opening, there is a 8-15 um thick,

flat extension adjacent to the covering stroma, which is comprised of strongly

carbonized and brittle tissue with no obvious cellular structure. ExcIPULUM

very poorly developed, arising from the inner layer of the basal stroma,

consisting of hyaline textura porrecta. BASAL STROMA 15—22 um thick, dark

brown to black, composed of (3—)4—6 rows of 3.5—6 um diam., angular, thick-

Terriera fici sp. nov. (China) ... 1115

walled cells. A layer of 10-30 um thick textura prismatica, comprised of nearly

colorless or light brown cells 2.5—4 um diam. occurs between the covering

and basal stromata. Subhymenium well developed, 15-20 um thick, strongly

concave to form a cup shape, consisting of hyaline textura angularis and

intricata. PARAPHYSES 100-120 x 1—1.2 um, filiform, septate, mostly branching

1-2 times in the upper portion, sometimes slightly tapered at the branched

apices, covered in ac. 1 um thick gelatinous matrix. Asci ripening sequentially,

90-115 x 4—5.5 um, narrow cylindrical, apex rounded or subacute, without

circumapical thickening, not bluing in iodine, discharging spores through a

small apical pore, 8-spored, stalk 20-25 um in length. Ascosporgs arranged in

a fascicle, 65-80 x 0.8—1 um, filiform, colorless, aseptate, rounded to obtuse at

the apex, slightly tapered towards the rounded or subacute base, hyaline, with a

c. 0.5 um thick gelatinous sheath.

Host, HABITAT, & DISTRIBUTION: Producing conidiomata and ascomata

on fallen leaves of Ficus vasculosa. Known only from the type locality, Hainan

Province, China.

CoMMENTSs: Terriera fici is very similar to T’ nitens Y.R. Lin in the ascomatal

openings, the appearance of the conidiomata, and the ascal and ascospore

shapes. However, T! nitens differs in its suborbicular or broadly elliptical smaller

ascomata (240-320 x 190-250 um), a well developed excipulum (12-16 um

thick near the opening), a much thinner subhymenium (5—9 um) consisting of

textura porrecta, unbranched aseptate paraphyses, slightly short-stalked asci,

and trichogynes present in the conidiomata (Chen et al. 2014).

Terriera minor (Tehon) P.R. Johnst. is distinguished from the new species by

the absence of zone lines and asexual conidiomata, oblong to oblong-elliptic

ascomata, a flat or slightly concave subhymenium, much wider asci (6—7 um),

paraphyses extending 20-30 um beyond asci, and slightly larger ascospores

(70-100 x 1.5—2 um) (Johnston 1989, as Lophodermium minus).

Terriera fici also resembles Lophodermium illiciicola S.J. Wang et al.,

which is distinguished by smaller ascomata (280-360 x 260-300 um), absent

conidiomata, a thinner subhymenium (10-15 um thick) consisting of textura

angularis-porrecta, and unbranched paraphyses markedly longer than the asci

(Wang et al. 2006).

Acknowledgements

We are grateful to Prof C.L. Hou (Capital Normal University, China) and Prof M.

Ye (Hefei University of Technology, China) for invaluable suggestions and critically

reviewing the manuscript, to Mr Y.F. Xu for the field investigations. This study was

supported by the National Natural Science Foundation of China (No. 31270065,

31500019).

1116 ... Wu, Wang & al.

Literature cited

Chen L, Minter DW, Wang SJ, Lin YR. 2014 [“2013”]. Two new species of Rhytismataceae on

fagaceous trees from Anhui, China. Mycotaxon 126: 109-120. http://dx.doi.org/10.5248/126.109

Eriksson B. 1970. On Ascomycetes on Diapensales and Ericales in Fennoscandia. Symbolae

Botanicae Upsalienses 19(4): 1-71.

IndexFungorum. 2015. www.indexfungorum.org (viewed online on 16 June 2015).

Johnston PR. 1989. Lophodermium (Rhytismataceae) on Clusia. Sydowia 41: 170-179.

Minter DW. 1996. Terriera cladophila. IMI Descr. Fungi & Bact. no. 1296.

SpeciesFungorum. 2015. www.speciesfungorum.org (viewed online on 16 June 2015).

Wang SJ, He YE, Ye GB, Lin YR. 2006. A new species and a new Chinese record of Rhytismataceae

[in Chinese]. Mycosystema 25(1): 1-5.

MY COTAXON

http://dx.doi.org/10.5248/130.1117

Volume 130, pp. 1117-1124 October-December 2015

Six new Russula records from Turkey

Hasan HUSEYIN DOGAN” & OYKUM OzTURK?

' Selcuk University, Faculty of Science, Biology Department, Konya, Turkey

? Hacettepe University, Faculty of Science, Biology Department, Ankara, Turkey

* CORRESPONDENCE TO: hhuseyindogan@yahoo.com

Asstract — During field studies in 2012-13, six Russula species from the Samanli

Mountains region were identified as new records for Turkey: R. clariana, R. faginea,

R. insignis, R. odorata, R. sericatula, and R. velutipes. Descriptions of the taxa along with their

distinct features are given and a key to the species is provided.

Key worps — Basidiomycota, Russulaceae, Turkish mycobiota

Introduction

There have been many studies of the diversity of macrofungi in Turkey.

Sesli & Denchev (2008) noted that 2158 macrofungi species were recorded for

Turkish mycota. Subsequent studies by Sesli & Kobayashi (2014), Giingor et

al. (2014), Solak et. al (2014a,b), Sesli (2014), Uzun et al. (2014), and Akata et

al. (2014) have increased that number to 2191. Here we record six additional

Russula spp. collected in Samanli Mountains, Turkey

Samanl: Mountains lie southeast of the Marmara Region and stretch between

Bozburun at the edge of Armutlu Peninsula in the west and Geyve Strait

formed by Sakarya River in the east. A close look on the natural vegetation of

the area highlights kermes oak (Quercus coccifera L.), holly oak (Quercus ilex

L.), and bay laurel (Laurus nobilis L.) as the main shrubs and ligneous plants

in the maquis formation of Samanli Mountains up to 500-600 m. Hawthorn

(Crataegus oxyacantha L.) and boxwood (Buxus sempervirens L.) are also seen

in patches among maquis elements. Main ligneous plants in the forest cover of

the area are pedunculate oak (Quercus robur L.), oriental beech (Fagus orientalis

Lipsky), Uludag fir (Abies bornmuelleriana Mattf.), chestnut (Castanea sativa

Mill.), black pine (Pinus nigra subsp. pallasiana (Lamb.) Holmboe, stone

pine (Pinus pinea L.), Turkish pine (Pinus brutia Ten.), common hornbeam

1118 ... Dogan & Oztiirk

(Carpinus betulus L.), and Scots pine (Pinus sylvestris L.). Also seen in patches

are groups of oriental plane (Platanus orientalis L.), maple (Acer platanoides L.),

and white poplar (Populus alba L.). The area is located in the Mediterranean

climatic zone, and the annual rainfall varies between 400mm and 1200mm in

different regions (Akman, 1999).

Materials & methods

The macrofungi were collected in the provinces Bursa, Yalova, Kocaeli during 2012-

13. Important macroscopical features and ecological information were noted in the field,

and digital images were taken of the specimens in their habitat. Collected specimens

dried in dehydrators after each study day; the dried materials were put into plastic bags

to bring them to the fungarium in good condition for further analysis.

Micromorphological characters were examined using a Leica DM3000 light

microscope and photographed digitally. Specimen tissues were examined in Melzer’s

reagent, 5% KOH, and SBA (sulfobenzaldehyde), and the pileus context was spot-tested

with FeSO, and phenol. At least 20 spores per specimen were measured. The specimens

were identified according to Galli (2003), Sarnari (1998, 2006), and Kranzlin (2005).

The specimens are kept in the Fungarium of Selcuk University, Konya, Turkey (SELUK).

Taxonomy

Russula clariana Kuyper & Vuure, Persoonia 12: 452. 1985. FIGS 1A, 2A

PitEus 40-70 mm across, hemispherical when young, later plane, violet,

purple, usually light olive color in the center, margin short striate. LAMELLAE

white, narrowly attached. StrpE 30-50 x 10-20 mm, cylindrical to slightly

clavate, white. Spores 7—8.5 x 6—7.5 lum, subglobose, with solitary warts.

Basip1A 35-55 x 10-15 um, clavate, with 4 sterigmata. CHEILOCYSTIDIA and

PLEUROCYSTIDIA 45-85 x 10-15 Um, fusiform to clavate, with gray-black

contents in SBA.

MACROCHEMICALS: FeSO, negative, phenol light rose.

SPECIMENS EXAMINED: TURKEY, Bursa: Mahmudiye, Taslitarla, 40°36’09”N

29°46'04”E, 947 m, on soil under Fagus orientalis, 4 Jun 2013, leg. HH. Dogan & O.

Oztiirk, HHD 10308, 10319, 10453 (SELUK).

Russula faginea Romagn., Russules Eur. Afr. Nord: 681. 1967. FIGS 1B, 2B,C

PrLeus 40-80 mm across, convex to plane, wine-red to pink, usually whitish

yellow tones in the center. LAMELLAE white, narrowly attached. STrpE 30—70 x

10-25 mm, cylindrical, white when young, later yellowing to browning. SPORES

7-10 x 6—-8.5 um, subglobose to elliptical, with distant warts. Bastp1A 45—60 x

10-15 um, clavate, with 4 sterigmata. CHEILOCYSTIDIA and PLEUROCYSTIDIA

45-85 x 11-14 um, fusiform, obtuse or with a prominent projection, with

gray-black contents in SBA.

Russula species new for Turkey... 1119

rat

FiGuRE 1. Basidiocarps: A, Russula clariana; B, R. faginea; C, R. insignis; D, R. odorata;

E, R. sericatula; F, R. velutipes.

MACROCHEMICALS: FeSO, green, phenol reddish brown.

SPECIMEN EXAMINED: TURKEY, YALOva: Canakpinar, 40°32’40”N 29°11'34”E, 700 m,

on soil under Fagus orientalis, 7 Jun 2013, leg. HH. Dogan & O. Oztiirk HHD10509

(SELUK).

1120 ... Dogan & Oztiirk

Russula insignis Quél., C.R. Ass. Frang. Av. Sci. 16: 588. 1888. FIGS 1C, 3A,B

PitEus 40-60 mm across, plane, funnel-shaped later, dark grey-brown to

brown. LAMELLAE white, turning yellow or brown later, notched. Stipe 30—50

x 10-15 mm, cylindrical, white, turning yellow with age. SporEs 6.5—8.5 x

5—6.5 tum, subglobose to elliptical, with elongated warts. BAstp1a 40—50 x 8-15

um, clavate, with 4 sterigmata. CHEILOCYSTIDIA and PLEUROCYSTIDIA 40-80 x

6—15 um, clavate to fusiform, with faintly gray- black contents in SBA.

MACROCHEMICALS: FeSO, pale pink, phenol wine red.

SPECIMENS EXAMINED: TURKEY, Bursa: Gemlik, Haydariye, District No. 250,

40°30’27”N 29°09’44’E, 740 m, on soil under Quercus coccifera and Fagus orientalis, 3

Jun 2013, leg. H.H. Dogan & O. Oztiirk HHD10239, 10241 (SELUK).

Russula odorata Romagn., Bull. Soc. Linn. Lyon 19: 76. 1950. FIGS 1D, 2D-F

PitEus 30-60 mm across, plane, carmine to light red, entire pileus often

strongly fading to white. LaMELLAE whitish when young after pale yellow,

narrowly attached. Stipe 25-50 x 5-10 mm, cylindrical, white and somewhat

yellowing from the base upward. Spores 6.5—-8.5 x 5.5—-7 um, subglobose

to elliptical, with connected warts. Basip1a 35-45 x 10-15 um, clavate.

CHEILOCYSTIDIA and PLEUROCYSTIDIA 45—75 x 10-15 um, fusiform, apically

with a small knob, with gray-black contents in SBA.

MACROCHEMICALS: FeSO, pink, phenol wine brown.

SPECIMEN EXAMINED: TURKEY, Sakarya: Akyazi, near Keremali Forester Cottage,

40°39’10’N 30°43’38”E, 972 m, on soil under Fagus orientalis, 11 Nov 2012, leg. H.H.

Dogan & O. Oztiirk HHD8660 (SELUK).

Russula sericatula Romagn., Bull. Soc. Linn. Lyon 27: 287. 1958. FIGS 1E, 3F,G

Pireus 50-80 mm across, plane, later infundibuliform, violet-brown,

cinnamon-brown. LAMELLAE white, narrowly attached. Stipe 50—70 x 10—20

mm, cylindrical, white, turning yellow or brown with age. SpoREs 7—9.5 x 6-8

um, subglobose, with elongated warts. Basrp1a clavate, 40-50 x 10-15 um,

with 4 sterigmata. CHEILOCYSTIDIA and PLEUROCYSTIDIA 50—70 x 10-15 Um,

fusiform, most with a small apical projection, with gray-black contents in SBA.

MACROCHEMICALS:: FeSO, light pink, phenol wine brown.

SPECIMEN EXAMINED: TURKEY, KOCAELI: Golciik, Menekse Yaylasi, District No: 13,

40°35’01”N 29°54’48’E, 890 m, on soil under Fagus orientalis, 5 Jun 2013, leg. H.H.

Dogan & O. Oztiirk HHD10398 (SELUK).

Russula velutipes Velen., Ceské Houby 1: 133. 1920. FIGS 1B, 3C-E

PILEus 30-70 mm, convex to plane, pink to red, yellowish toward to center.

LAMELLAE white when young, later pale cream-yellow, narrowly attached.

STIPE 40—60 x 10—25 mm, clavate, white. SPoREs 6.5—8 x 5—6.5 um, elliptical,

Russula species new for Turkey ... 1121

FIGURE 2. Russula clariana: A, pleurocystidia and basidiospores. R. faginea: B, basidia;

C, pleurocystidia and basidiospores. R. odorata: D, basidia;. E, cheilocystidia; F, basidiospores.

with elongated warts. Basrp1a 35—50 x 5—10 um, clavate, with (2—)4 sterigmata.

CHEILOCYSTIDIA 40—70 x 5—15 um, fusiform, most obtuse. PLEUROCYSTIDIA

40-70 x 5-15 um, fusiform most with an apical projection which is sometimes

forked, with faintly gray-black contents in SBA.

1122 ... Dogan & Oztiirk

FiGuRE 3. Russula insignis: A, pleurocystidia; B, basidiospores. R. velutipes: C, basidia (2-4

sterigmata); D, cheilocystidia; E, basidiospores. R. sericatula: F, pleurocystidium; G, basidiospores.

MACROCHEMICALS: FeSO, pink, phenol purple brown.

SPECIMEN EXAMINED: TURKEY, Bursa: Mahmudiye, Tasliyayla, 40°36’09”N

29°46'04”E, 947 m, on soil under Fagus orientalis, 4 Jun 2013, leg. HLH. Dogan & O.

Oztiirk HHD10314 (SELUK).

Russula species new for Turkey ... 1123

Discussion

Sesli & Denchev (2008) cite 108 Russula taxa as previously recorded for the

Turkish mycobiota; six additional species are recorded here for the first time.

The examined specimens do not significantly differ morphologically from the

species descriptions in the literature. The simple key to the six identified species

indicates the diagnostic differences among the species.

La akesQ seaction nésative.. 0 Tait re, Ore ei esl ose loa lead R. clariana

ESOP TEACH OM DSH YG eM ie cee: taba i ees na ee ee IS A cy Mi Z

2 FeSO s reaction light todark greens, ..g..45 0044 0n yen gnwar ganna» R. faginea

Hes@)s React Ori lig lite punks 10; PINKS SAN SMa An aad Maat aad an ee eee ee 3

3. Pileus rose, light pink, entire pileus whitening with age.............. R. odorata

Entite. pileus Mot wititenite With Agee A. ARPS cen vet reise h tre pas ee eal wt eld tke 4

# \UPhenol-reactionewine=ted sheesh ee Rade Sateen Baden take wri ades oats R. insignis

Phenel réaction notwinesred... 2 ty 2 3 iy 24,5 by eae By eh By ey ba eh eB ee Bt ae 5

5." SPiléus purple-brown. 24.0 2t¢s cert ce ee hes eh 4 ehoa ee eoe eee oe R. sericatula

ileus git Rtas 7 5. ten) ep ates fete fap cates Ee sce! Paeoegl spccaeg ayes hee R. velutipes

Russula odorata has typically fruity odor and the pileus of our specimen was

almost white, as it was old. Russula insignis is known as obligately mycorrhizal

with Quercus species, and we did collect our specimen in an oak forest.

A mycorrhizal association with Fagus and 2- to 4-spored basidia the important

diagnostic characters for R. velutipes, which we observed in our specimen.

Russula faginea can be easily distinguished with its dark green FeSO, reaction.

In contrast, R. clariana is typical with its negative FeSO, reaction. Russula

sericatula can be distinguished from the other species by its larger spores and a

pileus that yellows toward the disc.

Acknowledgments

The authors are indebted to the Scientific & Technical Research Council of Turkey

(TUBITAK TBAG 112T136) and the Scientific Research Projects (BAP) Coordinating

Office (BAP/13401072) at Selcuk University for their financial support of the current

work. We would like to thank Mitko Karadelev (Ss “Cyril and Methodius” University,

Macedonia) and Yusuf Uzun (Yiiziincii Yil University, Turkey) for reviewing this article.

We also especially appreciate the help of Adapazari and Bursa Regional Directorate of

Forestry and Zekeriya Beyazli (Chief of Akyazi Forest Management Department) and

Turgut Keskin (Chief of Bursa Non-Wood Forest Products and Services Department)

for logistic support in collecting the specimens around the study area.

Literature cited

Akman Y. 1999. iklim ve Biyoiklim, Kariyer Matbaacilik Ltd.Sti., Ankara.

Akata I, Uzun Y, Kaya A. 2014. Macromycetes determined in Yomra (Trabzon) district. Turkish

Journal of Botany 38: 999-1012. http://dx.doi.org/10.3906/bot-1309-22

1124 ... Dogan & Oztiirk

Galli R. 2003. Le Russule. 2a edizione. Medial s.r.l, Milano.

Giingor Yaratankul M, Giingor H, Solak MH. 2014. New Crepidotus (Fr.) Staude record for Turkish

mycota. Biological Diversity and Conservation (BioDiCon) 7(2): 127-128.

Kranzlin F. 2005. Fungi of Switzerland. Vol. 6. Verlag Mykologia, Luzerne.

Sarnari M. 1998. Monografia illustrata del genere Russula in Europa, vol. 1. A.M.B. Fondazione

centro studi micologici, Vicenza.

Sarnari M. 2006. Monografia illustrata del genere Russula in Europa, vol. 2. Fondatione Centro

Studi Micologici, Trento.

Sesli E. 2014. Studies on new fungal records for Turkish Mycota from Trabzon. Turkish Journal of

Botany 38: 608-616. http://dx.doi.org/10.3906/bot-1306-15

Sesli E, Denchev CM. 2008. Checklists of the myxomycetes, larger ascomycetes, and larger

basidiomycetes in Turkey. (accessed February 2015):

http://www.mycotaxon.com/resources/checklist/sesli-v106-checklist.pdf

Sesli E, Kobayashi T. 2014. A new record for the Turkish mycota: Inocybe phaeodisca Kithner var.

phaeodisca. Biological Diversity and Conservation (BioDiCon) 7(1): 44-46.

Solak MH, Alli H, Isiloglu M, Giingor H, Kalmis E. 2014a. Contributions to the macrofungal

diversity of Kilis Province. Turkish Journal of Botany 38: 180-185.

http://dx.doi.org/10.3906/bot-1301-19

Solak MH, Alli H, Isiloglu M, Giingor H, Kalmis E. 2014b. Contributions to the macrofungal

diversity of Antalya Province. Turkish Journal of Botany 38: 386-397.

http://dx.doi.org/10.3906/bot-1302-15

Uzun Y, Acar I, Akcay ME, Akata I. 2014. Additions to the Turkish discomycetes. Turkish Journal

of Botany 38: 617-622. http://dx.doi.org/10.3906/bot-1307-53

MY COTAXON

http://dx.doi.org/10.5248/130.1125

Volume 130, pp. 1125-1133 October-December 2015

Dictyosporium amoenum sp. nov.

from Chapada Diamantina, Bahia, Brazil

CAROLINA RIBEIRO SILVA’, Luis FERNANDO PASCHOLATI GUSMAO”?*

& RAFAEL F. CASTANEDA-RuIz?

‘Departamento de Micologia, Universidade Federal de Pernambuco, 50670-420, Recife, Brazil

*Departamento de Ciéncias Biologicas, Laboratorio de Micologia,

Universidade Estadual de Feira de Santana, Avenida Transnordestina s/n,

Novo Horizonte, 44036-900, Feira de Santana, 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 — Dictyosporium amoenum, a new asexual fungus collected on leaves of

Calophyllum brasiliense in the Brazilian semiarid Caatinga region, is described and illustrated.

It is distinguished by digitate complanate brown conidia composed of four parallel compact

rows of cells, each row with a globose hyaline mucous apical appendage. Conidial illustrations

and a table comparing all Dictyosporium species are provided.

KEY worps — conidial fungi, leaf litter, taxonomy

Introduction

During several mycological surveys of conidial fungi on decaying leaves

of Calophyllum brasiliense in the Chapada Diamantina, Bahia, Brazil, a

conspicuous fungus was collected that is clearly related to Dictyosporium

Corda, introduced by Corda (1836) with D. elegans Corda as the type species.

The genus comprises 47 accepted species distinguished by conidiomata that

are sporodochial, scattered, compact pulvinate, and olivaceous to dark brown.

Conidiophores are inconspicuous and the conidiogenous cells are monoblastic,

discrete, cylindrical, doliiform or inflated, and determinate. Conidial secession

is rhexolytic. The conidia are solitary, digitate, complanate to complex

subcylindrical, multiseptate and comprising several rows of cells that are

brown, pale brown or yellowish brown, and sometimes with mucilaginous,

1126 ... Silva, Gusmao & Castafieda-Ruiz

clavate, globose, filiform to tubular-capitate, hyaline appendages or tunica at

the apex of or (rarely) lateral to some or all rows of cells.

Materials & methods

Samples of leaves of C. brasiliense were placed in paper bags, taken to the laboratory,

and treated according to Castafieda-Ruiz (2005). Mounts were prepared in PVL

(polyvinyl alcohol and lactic acid) and measurements were taken at x1000. Micrographs

were obtained with an Olympus microscope (model BX51) equipped with bright field

and Nomarski interference optics. The holotype is deposited in the Herbarium of

Universidade Estadual de Feira de Santana, Bahia, Brazil (HUEFS).

Taxonomy

Dictyosporium amoenum C.R. Silva, Gusmao & R.F. Castafieda, sp. nov. Fie. 1

MycoBank MB812865

Differs from all other Dictyosporium spp. by its conidia comprising four vertical rows of

cells, each row with a globose mucilaginous hyaline apical appendage.

Type: Brazil, Bahia State, Piataé, Chapada Diamantina, Serra da Tromba, 13°05’S 41°51’W,

alt. 1263 m, on decaying leaves of Calophyllum brasiliense Cambess. (Calophyllaceae),

31.VII.2014, coll. C.R. Silva (Holotype, HUEFS 211336).

EryMo_oey: Latin, amoenus, meaning pleasant, delightful, agreeable.

CONIDIOMATA on the natural substrate sporodochial, pulvinate, scattered,

mucilaginous, pale brown. CONIDIOPHORES inconspicuous. CONIDIOGENOUS

CELLS monoblastic, cylindrical, discrete determinate. CONIDIA solitary, digitate,

complanate, 20-30 x 12.5-15 um, smooth, brown, composed of four rows of

cells compacted together, parallel, 5-7-septate, slightly constricted at septa,

smooth, brown, all with a globose, mucilaginous, hyaline apical appendage,

10-17.5 um wide

Note: Among 47 described Dictyosporium species, only seventeen have been

recorded with conidial appendages: D. alatum Emden, D. aquaticum Abdel-

Aziz, D. bulbosum Tzean & J.L. Chen, D. canisporum L. Cai & K.D. Hyde,

D. digitatum J.L. Chen et al., D. freycinetiae McKenzie, D. gauntii Bhat &

B. Sutton, D. hughesii McKenzie, D. inflatum (Matsush.) K.L. Pang et al.,

D. manglietiae Kodsueb & McKenzie, D. musae Photita, D. nigroapice Goh

et al., D. strelitziae Crous & A.R. Wood, D. tetraploides L. Cai & K.D. Hyde,

D. tetraseriale Goh et al., D. thailandicum M.]J. D’souza et al., and D. zhejiangense

Wongs. et al. All these taxa differ from D. amoenum in the number of rows of

cells and in the number, shape, and size of apical appendages on their conidia.

(TABLE 1, Fics 2-4; data were obtained and conidial drawings adapted from

Arambarri et al. 2001, Cai & Hyde 2007, Cai et al. 2003a,b; Castafieda-Ruiz et

al. 1997; Crous et al. 2009; Crous et al. 2011, Goh et al. 1999; Hu et al. 2010,

Dictyosporium amoenum sp. nov. (Brazil) ... 1127

10 pm

Fic. 1. Dictyosporium amoenum (ex HUEFS 211336). Conidia.

Liu et al. 2015; Kirschner et al. 2013; Kodsueb et al. 2006; McKenzie 2008, 2010;

and Photita et al. 2002, Whitton et al. 2012, Wongsawas et al. 2009, Zhang 2009,

and Zhao & Zhang 2003).

1128 ... Silva, Gusmao & Castafieda-Ruiz

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Fic. 2. Conidia of Dictyosporium species: A. D. acroinflatum (Whitton et al. 2012); B. D. alatum

(Goh et al. 1999); C. D. amoenum (this paper); D. D. aquaticum (Liu et al. 2015); E. D. australiense

(Goh et al. 1999); F. D. biseriale (Hu et al. 2010); G. D. brahmaswaroopii (Goh et al. 1999); H. D.

bulbosum (Castafieda-Ruiz et al. 1997); I. D. campaniforme (Goh et al. 1999); J. D. canisporum

(Cai et al. 2003a); K. D. cocophilum (Goh et al. 1999); L. D. digitatum (Goh et al. 1999); M. D.

dkagarwalii (Manoharachary et al. 2007); N. D. elegans (Goh et al. 1999); O. D. foliicola (Goh et al.

1999); P. D. freycinetiae (McKenzie 2008); Q. D. gauntii (Goh et al. 1999); R. D. giganticum (Goh et

al. 1999); S. D. heptasporum (Goh et al. 1999).

Dictyosporium amoenum sp. nov. (Brazil) ... 1129

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Fic. 3. Conidia of Dictyosporium species: A. D. hughesii (McKenzie 2010); B. D. inflatum (Kirschner

et al. 2013); C. D. lakefuxianense (Cai et al. 2003b); D. D. manglietiae (Kodsueb et al. 2006); E. D.

meiosporum (Liu et al. 2015); F. D. micronesiacum (Goh et al. 1999); G. D. musae (Photita et al.

2002); H. D. nigroapice (Goh et al. 1999); I. D. oblongum (Goh et al. 1999); J. D. pandani (Whitton

et al. 2012); K. D. polystichum (Goh et al. 1999); L. D. rhopalostylidis (McKenzie 2010); M. D.

schizostachyfolium (Goh et al. 1999); N. D. sinensis (Zhang 2009); O. D. stellatum (Crous et al 2011);

P. D. strelitziae (Crous et al 2009); Q. D. subramanianii (Goh et al. 1999); R D. taishanense (Zhao

& Zhang 2003).

1130... Silva, Gusmao & Castafieda-Ruiz

Fic. 4. Conidia of Dictyosporium species: A. D. tetraploides (Cai et al. 2003a); B. D. tetraseriale

(Goh et al. 1999); C. D. tetrasporum (Cai & Hyde 2007); D. D. thailandicum (Liu et al. 2015); E.

D. tnlakhanpalii (Manoharachary et al. 2007); F. D. toruloides (Goh et al. 1999); G. D. triramosum

(Arambarri et al. 2001); H. D. triseriale (Goh et al. 1999); I. D. yunnanense (Cai et al. 2003b); J. D.

zeylanicum (Goh et al. 1999); K. D. zhejiangense (Wongsawas et al. 2009).

TABLE 1. Comparison of conidia in Dictyosporium species

SPECIES

D. amoenum

D. acroinflatum

D. alatum

D. aquaticum

D. australiense

D. biseriale

D. brahmaswaroopii

D. bulbosum

D. campaniforme

D. canisporum

D. cocophilum

D. digitatum

D. dkagarwalii

D. elegans

D. foliicola

D. freycinetiae

D. gauntii

D. giganticum

D. heptasporum

D. hughesii

D. inflatum

D. lakefuxianense

D. manglietiae

D. meiosporum

D. micronesiacum

D. musae

D. nigroapice

D. oblongum

D. pandani

D. polystichum

D. rhopalostylidis

D. schizostachyfolium

D. sinense

D. stellatum

D. strelitziae

D. subramanianii

D. taishanense

D. tetraploides

D. tetraseriale

CONIDIAL SIZE (1m)

20-30 x 12-15

28-52 x 23-35

26-32 x 15-25

60-85 x 20-30

36-43 x 11-12

22-30 x 9-10

17-24 x 12.5-19

27-46 x 11-30

22-40 x 20-30

27-47 x 20-25

53-76 x 19-22

47-77 x 22-39

30-40 x 9.5-11.5

40-80 x 24-31

34-56 x 20-38

27-40 x 5-6

40-50 x 18-25

105-121 x 25-32

50-80 x 20-30

33-50 x 23-32

20-50x5-8(-10)

15-22x 10-16.5

22-28 x 12.5-18

17-28 x 6-8.5

20-30 x 10-12

45-65 x 20-27

28-41 x 15-20

30-50 x 12-30

22-48 x 14-28

26-34 x 23-34

28-48 x 20-29

15-17 x 11-12

50-130 x 20-35

50-175 x 27.5-52

30-55 x 20-23

33-42 x 16-21

27-43 x 15-30

52-72 x 18-26.5

24-40 x 14-20

TABLE 1, concluded on p. 1132

Dictyosporium amoenum sp. nov. (Brazil) ... 1131

No. OF

ROWS

(5-)7-9

5-6

4-7

5-7

4-6

3-7

(3-)5(-7)

REFERENCE

this paper

Whitton et al. (2012)

Goh et al. (1999)

Liu et al. (2015)

Goh et al. (1999)

Hu et al. (2010)

Goh et al. (1999)

Castafieda-Ruiz et al. (1997)

Goh et al. (1999)

Cai et al. (2003a)

Goh et al. (1999)

Manoharachary et al. (2007)

Goh et al. (1999)

McKenzie (2008)

Goh et al. (1999)

McKenzie (2010)

Kirschner et al. (2013)

Cai et al. (2003b)

Kodsueb et al. (2006)

Liu et al. (2015)

Goh et al. (1999)

Photita et al. (2002)

Goh et al. (1999)

Whitton et al. (2012)

Goh et al. (1999)

McKenzie (2010)

Goh et al. (1999)

Zhang (2009)

Crous et al (2011)

Crous et al (2009)

Goh et al. (1999)

Zhao & Zhang (2003)

Cai et al. (2003a)

Goh et al. (1999)

1132 ... Silva, Gusmao & Castafieda-Ruiz

TABLE 1, concluded

SPECIES CONIDIAL SIZE (1m) ‘ee REFERENCE

D. tetrasporum 23.5-40 x 16-21.5 4 Cai & Hyde (2007)

D. thailandicum 15-35 x 14.5-21 5 Liu et al. (2015)

D. tnlakhanpalii 80-120 x 25-36 (4-)6(-8) Manoharachary et al. (2007)

D. toruloides 38-56 x 25-32 6-8 Goh et al. (1999)

D. triramosum 40-60 x 10-13.5 (2-)3 Arambarri et al. (2001)

D. triseriale 26-32 x 16-18 3 Goh et al. (1999)

D. yunnanense 25-45 x 22-38 (5-)6(-7) Cai et al. (2003b)

D. zeylanicum 26-40x 13-25 5 Goh et al. (1999)

D. zhejiangense 25-35 x 17-24 (4-)5 Wongsawas et al. (2009)

Acknowledgments

The authors express their sincere gratitude to Dr. Xiu Guo Zhang and Dr. De-Wei Li

for their critical review of the manuscript and Lucas Cardoso Marinho for his adaptations

of the original drawings. The authors thank the Programa de Pés-Graduag¢ao em Biologia

de Fungos (PPGBF/ UFPE), ‘Programa de pesquisa em Biodiversidade do Semiarido

(PPBIO semi-arid/ Ministry of Technology and Science): C.R. Silva and L.EP. Gusmao

extend their gratitude to CNPq for financial support (Proc. 132415/2013-5). The authors

thank the support provided by ‘Programa Ciéncia sem Fronteiras. RFCR is grateful to

Cuban Ministry of Agriculture and “Programa de Salud Animal y Vegetal’, project

P131LH003033 for facilities. We acknowledge the assistance provided by Dr. P.M.

Kirk and Drs. V. Robert and A. Decock through the Index Fungorum and MycoBank

websites. Dr. Lorelei Norvell editorial and Dr. Shaun Pennycook nomenclatural reviews

are greatly appreciated.

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Argentina. Mycotaxon 78: 185-189.

Cai L, Hyde KD. 2007. Anamorphic fungi from freshwater habitats in China: New species of

Dictyosporium tetrasporum and Exserticlava yunnanensis spp. nov., and two new records

for Pseudofuscophialis lignicola and Pseudobotrytis terrestris, Mycoscience 48: 290-296.

http://dx.doi.org/10.1007/S10267-007-0369-1

Cai L, Zhang K, McKenzie EHC, Hyde KD. 2003a. New species of Dictyosporium and Digitodesmium

from submerged wood in Yunnan, China. Sydowia 55: 129-135.

Cai L, Zhang K, McKenzie EHC, Lumyong S, Hyde KD. 2003b. New species of Canalisporium and

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Castaneda-Ruiz RF. 2005. Metodologia en el estudio de los hongos anamorfos. 182-183, in: Anais

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Dictyosporium amoenum sp. nov. (Brazil) ... 1133

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TI, Carnegie AJ, Hardy GESJ, Hiscock N, Hiiberli D, Jung T, Louis-Seize G, Okada G,

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Chen J, Chethana KWT, Singtripop C, Dai DQ, Dai YC, Daranagama DA, Dissanayake AJ,

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Pergoh D, Pinruan U, Senanayake IC, Somrithipol S, Suetrong S, Tanaka K, Thambugala KM,

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K, Zeng XY, Abdel-Aziz FA, Adam¢cik S, Bahkali AH, Boonyuen N, Bulgakov T, Callac P,

Chomnunti P, Greiner K, Hashimoto A, Hofstetter V, Kang JC, Lewis D, Li XH, Liu XZ, Liu ZY,

Matsumura M, Mortimer PE, Rambold G, Randrianjohany E, Sato G, Sri-Indrasutdhi V, Tian

CM, Verbeken A, Brackel Wv, Wang Y, Wen TC, Xu JC, Yan JY, Zhao RL, Camporesi E. 2015.

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23-28.

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MY COTAXON

http://dx.doi.org/10.5248/130.1135

Volume 130, pp. 1135-1139 October-December 2015

Synchaetomella aquatica sp. nov.

from submerged leaves from Brazil

PATRICIA OLIVEIRA Fiuza', LuIs FERNANDO PASCHOLATI GUSMAO?*

& RAFAEL F. CASTANEDA-RUIZ?

"Departamento de Ciéncias Biolégicas, Laboratério de Micologia,

Universidade Estadual de Feira de Santana, Avenida Transnordestina s/n,

Novo Horizonte, 44036-900, Feira de Santana, 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.

*CORRESPONDENCE TO: lgusmao@uefs. br

ABsTRACT — Synchaetomella aquatica, a new microfungus collected on the submerged

decaying leaves of Calophyllum brasiliense, is described and illustrated. It is distinguished by

conidiogenous cells that are synnematal, determinate conidiomata, monophialidic, discrete,

and terminal and solitary falcate unicellular hyaline conidia.

KEY worDs — asexual fungi, systematics, freshwater fungi

Introduction

During the studies of saprobic conidial fungi colonizing submerged leaves

of Calophyllum brasiliense in the Brazilian semiarid Caatinga biome, an

interesting fungus belonging to the genus Synchaetomella was collected. Critical

morphological observation of the specimen revealed it to be an undescribed

species, which we describe here as S. aquatica.

Materials & methods

During several expeditions between November 2013 to May 2014 through “Serra

da Tromba” in the semiarid region northeastern Brazil, samples of submerged leaves of

Calophyllum brasiliense were stored in litter bags, taken to the laboratory, and processed

according to Castafieda Ruiz (2005). Mounts were prepared in PVL (polyvinyl alcohol

and lactic acid) and measurements were made at a magnification of x1000. Micrographs

were obtained with an Olympus microscope (model BX51) with bright field and

Nomarski interference optics. The type specimen of the new species is deposited in the

Herbarium of “Universidade Estadual de Feira de Santana” (HUEFS).

1136 ... Fiuza, Gusmao & Castafieda-Ruiz

Taxonomy

Synchaetomella aquatica Fiuza, Gusmao & R.F. Castafieda, sp. nov. Figs 1, 2

MycoBank MB812900

Differs from all other Synchaetomella spp. by its exclusively terminal conidiogenous

cells.

Type: Brazil, Bahia, Piata, Chapada Diamantina, Serra da Tromba, 13°07’S 41°50’W,

on decaying submerged leaves of Calophyllum brasiliense Cambess. (Calophyllaceae),

11.X1.2013, coll. P.O. Fiuza (Holotype, HUEFS 209015).

EryMo_oey: Latin, aquatica, referred to its growing in water.

On the natural substratum mycelium partly immersed and partly superficial.

Hyphae septate, branched, smooth, pale brown, 2-3 um diam. CONIDIOMATA

synnematal, determinate, scattered, erect, cylindrical, dark brown, 60-150

um tall, 25-30 um wide. Stipe composed of parallel hyphal filaments

(conidiophores), multi-septate, straight, cylindrical, closely packed at the base

and slightly loosely and branched towards the apex, brown below, brown or pale

brown above, smooth, 1.5-3 um diam. CONIDIOGENOUS CELLS monophialidic,

discrete, terminal, determinate, brown to pale brown, 15-45 x 1.5-2.5 um

with a narrow, slightly obscure, sub-cupulate collarette with a distinct, very

narrow periclinal channel, 1-2.5 um deep. Conrpia solitary, acrogenous,

falcate, unicellular, 8-18 x 1.5-2 um, smooth, hyaline, accumulating in a white

mucilaginous mass.

Note: Decock et al. (2005) introduced the synnematous genus Synchaetomella

Decock & Seifert, typified by S. lunatospora Decock et al. and characterized by

phialidic, terminal and intercalary conidiogenous cells and falcate, 0-1-septate,

hyaline conidia. The second species, S. acerina (Seifert & Louis-Seize 2012), also

has intercalary and terminal conidiogenous cells, but the conidia are unicellular,

allantoid to ellipsoidal. Synchaetomella aquatica is clearly differentiated by the

terminal subulate conidiogenous cells.

Acknowledgments

The authors express their sincere gratitude to Dr. Xiu-Guo Zhang and Dr. De-Wei Li

for their critical review of the manuscript. The authors are grateful to Dr. Keith Seifert

for his commentaries and opinion. POF thanks the ‘Coordenacao de Aperfeigcoamento

de Pessoal de Nivel Superior” (CAPES) and the ‘Programa de Pds-graduacao em

Botanica PPGBot/UEFS. The authors are grateful to the “Programa de Pesquisa em

Biodiversidade” — (PPBio Semi-arid/MCTI/CNPq) for financial support. The authors

thank the support provided by “Programa Ciéncia sem Fronteiras’. LFPG is grateful

Fic. 1. Synchaetomella aquatica (ex holotype HUEFS 209015). A, B. conidia; C, D. conidiogenous

cells and conidia. Scale bars: A, C, D = 10 um, B = 20 um.

Synchaetomella aquatica sp. nov. (Brazil) ... 1137

1138 ... Fiuza, Gusmao & Castafieda-Ruiz

Fic 2. Synchaetomella aquatica (ex holotype HUEFS 209015).

A. conidia; B. conidiogenous cells and conidia; C. synnema.

Scale bars: A, B = 10 um; C = 25 um.

Synchaetomella aquatica sp. nov. (Brazil) ... 1139

to CNPq for grant (Proc. 303062/2014-2) and RFCR is grateful to Cuban Ministry of

Agriculture and “Programa de Salud Animal y Vegetal’, project P131LH003033 for

facilities. We acknowledge the facilities provided by Dr. P.M. Kirk and Drs. V. Robert

and A. Decock through the Index Fungorum and MycoBank websites. Dr. Lorelei

Norvell’s editorial review and Dr. Shaun Pennycook’s nomenclature review are greatly

appreciated.

Literature cited

Castaneda-Ruiz RE 2005. Metodologia en el estudio de los hongos anamorfos. 182-183, in: Anais

do V Congresso Latino Americano de Micologia. Brasilia.

Decock C, Delgado RG, Seifert K. 2005. Taxonomy and phylogeny of Synchaetomella lunatospora, a

new genus and species of synnematous fungi from Southeast Asia. Antonie van Leeuwenhoek

88: 231-240. http://dx.doi.org/10.1007/s10482-005-7210-7

Seifert KA, Louis-Seize G. 2012. Synchaetomella acerina Seifert, sp. nov. (Fungal Planet 150).

Persoonia 29: 192-193. http://dx.doi.org/10.3767/003158512X661589

MY COTAXON

http://dx.doi.org/10.5248/130.1141

Volume 130, pp. 1141-1152 October-December 2015

Tuber petrophilum, a new truffle species from Serbia

MiIrROLJUB MILENKOVIC!, TINE GREBENC’,

MIROSLAV MARKOVIC? & BORIS IVANCEVIC*

"Institute for Biological Research “Sinisa Stankovic”

Bulevar despota Stefana 142, RS-11060 Belgrade, Serbia

Slovenian Forestry Institute, Vecna pot 2, SI-1000 Ljubljana, Slovenia

*Banatska 34, RS-26340 Bela Crkva, Serbia

‘Natural History Museum, Njegoseva 51, RS-11000 Belgrade, Serbia

* CORRESPONDENCE TO: i.boris@beotel.net

AsstRaAct — Tuber petrophilum sp. nov., within the Tuber melanosporum lineage, is described

from Mount Tara (western Serbia) based on morphological and ITS molecular data. It is

recognizable by its minute ascomata that produce ovoid to ellipsoid to subfusiform spores

bearing aculeate ornamentation. Among black truffles, the new species is distinguished by its

irregularly roundish to subglobose ascomata not exceeding 1.6 cm in diameter, with a basal

depression or cavity and peridium surface which appears as a thin semi-transparent layer

while fresh. The species forms a monophyletic well-supported clade in Maximum Likelihood

ITS phylogeny, closely related to Tuber brumale aggr. The distinctive feature of the new species

lies in its specific and unique microhabitat, limited to humus-rich substrata accumulated

as soil pockets in limestone rocks, commonly 20—100 cm above the continuous forest soil

terraces. The species description is supplemented with macro- and micro-photographs, and a

key to the species of the T. melanosporum lineage.

Key worps — biodiversity, ecology of truffles, hypogeous fungi, Tuberaceae

Introduction

On several occasions between 2004 and 2014, the first author with associates

explored hypogeous fungi in the Tara National Park on Mount Tara, which

is located in western Serbia on the edge of northeastern belt of the Dinaric

Alps. This area is a significant refugium for many endemic and relict plant

species (Stevanovic et al. 1995; Cvjeticanin & Novakovi¢ 2007, 2010). Apart

from several hypogeous species found in quite usual habitats that were already

known from other areas of Serbia, a few black truffles were discovered in an

unusual and atypical habitat: cavities, crevices, and recesses in limestone rocks.

1142 ... Milenkovié & al.

These specimens were readily distinguishable by a unique aroma, ascomatal

size, and structure that did not correspond to any previously described species.

This paper aims to (1) describe a new species based on both morphological

and molecular data, (2) ascertain the ecology of the species, and (3) provide an

identification key and a phylogram to the Tuber melanosporum lineage.

Materials & methods

SAMPLING SITES AND COLLECTION TREATMENT. — Our morphological examination and

description of Tuber petrophilum is based on eleven collections comprising 26 ascomata

from five localities in western Serbia on Mount Tara during September and October,

2004-14. All sites are situated within a narrow belt, a few hundred meters wide, along

the local Mitrovac-Kaluderske Bare road. The distance between the two furthest sites is

approx. 6.7 km. Ascomata were discovered by a trained dog. Vouchers are conserved in

the fungal reference collection of the Natural History Museum, Belgrade, Serbia (BEO),

the fungal reference collection of Ljubljana University, Slovenia (LJU), and the private

herbarium of M. Markovic, Bela Crkva, Serbia. Ascomata were preserved by drying or

by ethanol-glycerin fixative (70% ethanol to which 15% of glycerol was added). Sampling

was done in representative forest communities in a limestone-dominated area where

hypogeous fungal biodiversity was completely unknown. Climate data are available for

the nearby meteorological station at Mitrovac (1082 m asl). Over the last 25 years, the

yearly average temperature at the station was 5 °C and mean annual precipitation was

980 mm.

Description of hues uses the Pantone colour standard (Eiseman & Lawrence 1990).

The letter “P” followed by the number in parentheses represents the Pantone code.

Microscopic characters of Tuber petrophilum were examined in both fresh and dry

material with a Leica Leitz DMRB microscope equipped with a Leica DFC320 digital

microscope camera and Leica DMLS microscope equipped with Leica DFC295 digital

microscope camera. Spores were measured in 5% KOH in a bright field microscopy

using Zeiss Axio Imager.Z2 microscope (100-1000x magnification) with the automated

measuring function in ZEN pro 2011 software. Spores considered to be not full-size,

anomalous, or aberrant were excluded. The length and width of full-sized spores were

measured in side view. Ornamentation was excluded from spore measurements and

measured separately. The spore size quotient Q represents the length : width ratio range

determined from all measured ascospores. The mean quotient Q_ was calculated as

arithmetic average of the Q values from all ascospore measured. Twenty spores were

measured for each collection.

As truffles are known for their specific soil requirements, several basic soil

characteristics at the three most productive localities (Dolak [the holotype site],

Tisovo brdo, and Rustine), were analyzed. About 0.5 L of soil was taken around the

ascomata and analyzed for humus, phosphorus (P,O,) and potassium (K,O) content,

and pH. Standard laboratory analyses of collected soil samples included the Kotzman

method for humus content, measurement of pH by placing a glass electrode in 1M

KCL solution, Egner-Rhim Al flame photometry method for potassium, Egner-Rhim

Al colorimetry method for phosphorus, and determination of calcium carbonate by

Tuber petrophilum sp. nov. (Serbia) ... 1143

Scheibler volumetric method (Tan 1998). Analyses were performed at the Institute for

Soil Science (Belgrade, Serbia).

DNA EXTRACTION, PCR AMPLIFICATION AND SEQUENCING. — DNA was extracted

from four dried glebal samples from a mature and sufficiently large Tuber petrophilum

ascoma using a Qiagen DNeasy Plant Mini Kit. DNA amplification using general ITS

primers ITS1f combined with ITS4 (White et al. 1990, Gardes & Bruns 1993) followed

the modified protocol in Grebenc et al. (2009). Prior to sequencing, products were

cleaned from the gel using the Promega Wizard SV Genomic DNA Purification System.

Both strands were sequenced separately at Macrogen Korea (Seoul, Korea). All samples

were sequenced in both directions using the same primers as in the amplification.

Sequencher 5.1 was used to assemble the consensus sequence from the two strands of

the ITS nrDNA of each isolate. Complete ITS sequences were deposited in GenBank

under accession numbers HG810883-810886.

PHYLOGENETIC ANALYSES. — All obtained consensus sequences were aligned

with a selection of Tuber sequences from the International Nucleotide Sequence

Databases using the stand-alone freeware version of the MAFFT programme

(http://align.bmr.kyushu-u.ac.jp/mafft/software/) with the E-INS-i aligning strategy

(Katoh et al. 2005). Reddellomyces sp. (GenBank: HM485336 and HM485337) was

used as outgroup as a close Tuber relative from the southern hemisphere with a basal

position according to Bonito et al. (2013). The MEGA 5.10 package was used to

construct Maximum Likelihood (ML) phylogenetic reconstruction based on a Tamura-

3-parameter substitution model with Gamma distributed rates among sites including

invariant sites. In the dataset gaps / missing data were partially deleted and 95% site

coverage cutoff and a strong branch swap filter were applied. 2000 bootstrap replicates

were run. The Maximum Parsimony (MP) reconstruction and branch support was

based on a MEGA 5.10 incorporated Subtree-Pruning-Regrafting MP search method

with 2000 replicates, gaps / missing data were partially deleted, and a 95% site coverage

cutoff was applied. MEGA 5.10 package was used also to annotate the final tree and

collapse branches at species level.

Taxonomy

Tuber petrophilum Milenkovic¢, P. Jovan., Grebenc, Ivanéevi¢ & Markovic,

sp. nov. PLATE 1

MycoBAnk MB812245

Differs from all other species of the Tuber melanosporum lineage by its considerably

smaller mature ascomata, its reddish-black peridium with a thin semi-transparent

layer when fresh, its spores with shorter ornamentation, and its distinctive habit in

accumulated soil pockets in rocks.

Type: Serbia, Bajina Basta, Tara National Park, Mitrovac, Dolak, 43.924810°N

19.462610°E, 1015 m asl, under Fagus sylvatica & Abies alba, in humus-rich substrate

accumulated as soil pockets in limestone rocks, commonly 20-100 cm above the

continuous forest soil terraces, 5 ascomata, 28 Sep 2004, leg. M. Milenkovic (Holotype,

BEO 20600).

1144 ... Milenkovié & al.

ETryMOLoGy: in reference to the specific habitat of the species - petrophilum (Gr. nétpa

= stone, rock; pidoc = who love).

Ascomara Strictly hypogeous, solitary or in small groups, rather small, ranging

from 8-16 mm in diam., nearly globose to irregularly subglobose, with basal

depression or cavity, firm.

PeripiuM reddish-black (P 19-1015), when fresh, surface appears as a thin

semi-transparent layer, turning to non-transparent and completely black when

dried, pseudoparenchymatous, total thickness 150-450 um (average 320 um),

with melanised outer layer, 20-200 um, without hyphal outgrowth. Warts

angular, flattened and depressed at the apex, 0.6—1.2 mm diam., 0.3—0.7 mm

high.

GLEBA solid, whitish at first, becoming brown (P 18-1137) and finally dark

brown (coffee brown; P 18-1154), at maturity with wide, infrequently (1-3x)

branched white veins, which radiate from the base and terminate close to

the peridium. Asci from fresh ascomata subglobose to irregular with well-

developed stalk, (1-)2-4(-6, extremely rarely)-spored, 40-50 x 55-80 um,

spores when inside asci randomly disposed and loosely packed. AscosporREs

22.7—42.5 x 13.1-26 um, symmetrical to asymmetrical, ovoid to ellipsoid to

subfusiform, Q = 1.37—2.29, Q. = 1.68 (SD = 0.16); at first hyaline to bright

brownish (P 15-1225), later brown (P 16-1422/17-1430) to dark brown

(P 17-1422) (resembling T: melanosporum Vittad.); ornamented with straight

or sometimes slightly curved, concolorous, dense, separate spines, 2-4(-4.5)

um high and 0.4-0.6 um diam., 1-2.5 um diam. at the base.

Opovur of mature ascomata intensive and specific, pleasant, resembling a

mixture of the aromas of Tuber borchii Vittad. and Tuber brumale Vittad.

FLAVOR not observed.

ADDITIONAL SPECIMENS EXAMINED: SERBIA, Bajina BaStTA, Tara National Park,

Mitrovac, Dolak, 43.924810°N 19.462610°E, 1015 m asl, under Fagus sylvatica & Abies

alba, 3 ascomata, 12 Oct. 2014, leg. M. Milenkovic & B. Ivanéevic (BEO 20606, 20607,

20608); Mitrovac, Tisovo brdo-Lokvica, 43.932980°N 19.448070°E, 1084 m asl, under

E sylvatica, A. alba, and Picea abies, 2 ascomata, 29 Sep. 2004, leg. M. Milenkovic (BEO

20601); 4 ascomata, 8 Oct. 2010, leg. M. Milenkovic (LJU GIS TUBPET/xx1010A, B, C,

D; GenBank HG810883-810886); Mitrovac, Tisovo brdo, c. 43.927220°N 19.443649°E,

1075 m asl, under F. sylvatica, A. alba & P. abies, 1 ascoma, 29 Sep. 2004, leg. M.

Milenkovi¢ (herb. Markovic); Mitrovac, Children’s resort, 43.922180°N 19.421160°E,

1062 m, under F sylvatica & P. abies, 3 ascomata, 5 Oct. 2006, leg. M. Milenkovi¢ (herb.

Markovic); 1 ascoma, 11 Oct. 2014, leg. M. Milenkovic & B. Ivanéevic (BEO 20605);

Sljivovica, Kovaci, north slopes, 43.917050°N 19.491500°E, 1111 m, under EF sylvatica

& A. alba, 2 ascomata, 9 Oct. 2010, leg. M. Milenkovic (herb. Markovic); 2 ascomata, 11

Oct. 2014, leg. M. Milenkovic & B. Ivancevic (BEO 20603, 20604); Sljivovica, Rustine,

43.912210°N 19.502850°E, 1029 m, under E sylvatica, A. alba & P. abies, 2 ascomata,

18 Oct. 2010, leg. M. Milenkovi¢ (herb. Markovic); 1 ascoma, 11 Oct. 2014, leg. M.

Milenkovi¢ & B. Ivanéevi¢ (BEO 20602).

Tuber petrophilum sp. nov. (Serbia) ... 1145

25 um

— |

PLaTE 1. Tuber petrophilum: A. Fresh ascoma (BEO 20604). B, C. Fresh ascomata and cross section

(B: BEO 20600: C: BEO 20608). D-F. Asci with spores from fresh ascomata (D, F: BEO 20608;

E: BEO 20600).

1146 ... Milenkovié & al.

Hasitat — All ascomata were found in humus-rich substrata accumulated as

soil pockets in limestone rocks, 5-12 cm below the surface of the substratum

and commonly 20-100 cm above the continuous forest soil terraces, formed

on limestone mountain slopes of Mount Tara. The forest type is the continental

montane forest dominated with Fagus sylvatica L., Abies alba Mill., and Picea

abies (L.) H. Karst. forming an association Piceo-Abietetum Colié 1965 (Tomié¢

2006, Tomi¢ & Rakonjac 2011) at altitudes from 1000-1100 m asl.

PHENOLOGY — September to October.

DISTRIBUTION — Known only from the type locality and its environs in

Serbia.

Discussion

Tuber P. Micheli ex EH. Wigg. is among the best studied hypogeous genera

in Europe (Hall et al. 2003). Until recently, about 32 different Tuber species had

been described in Europe (Ceruti et al. 2003) and at least 180 species estimated

worldwide (Bonito et al. 2010). Tuber petrophilum, unlike other hypogeous

fungi, inhabits accumulated soil pockets in rocks, partly isolated from the

surrounding continuous soil layer.

A combination of macro- and microscopic characters together with its unique

ecology separate Tuber petrophilum from all other taxa in the T. melanosporum

lineage (Riousset et al. 2001, Bonito et al. 2013), and its mature ascomata are

considerably smaller than any other closely related species. Molecular data

support its position in the T’ melanosporum lineage and significantly support

its separation from close relatives T: brumale aggr. and T: pseudoexcavatum

Y. Wang et al. Spore size range and ornamentation size are key microscopic

characters that distinguish it from species in the T: melanosporum lineage.

Tuber petrophilum forms a separate, well supported clade in the nrITS1-

5.8S-ITS2-based phylogeny (PLATE 2). The closest ITS-based relatives are the

European Tuber brumale aggr., T. pseudoexcavatum (Asia), T: regimontanum

Guevara et al. (Central America), and T. melanosporum (Europe). We have

observed a persistent presence of a c. 300-bp intron in the ITS1 region

of T: petrophilum and T. brumale aggr. but not in other species in the

T. melanosporum lineage. Excluding the intron, T. petrophilum shared 82-85%

sequence similarity with other lineages within the T: brumale aggr. and <82%

similarity with other species in the T: melanosporum lineage.

The ecology of T: petrophilum is strikingly distinct within the T. melanosporum

lineage. While other species are found primarily in Mediterranean or

Continental climates with both hot summer and moderate winter periods

(Montecchi & Sarasini 2000, Riousset et al. 2001), T: petrophilum was discovered

in a colder, montane climate at higher elevations and on different soil types. In

Tuber petrophilum sp. nov. (Serbia) ... 1147

Tuber brumale (pan-European lineage

100/98 (p e 9e)

100/100

T. brumale group A

| Tuber brumale A (Eastern European lineage) (Merényi et al. 2013)

95/84 ‘—{ Tuber brumale group B - Pannonian basin s.! —_T. brumale agg.

Tuber petrophilum HG810886

Tuber petrophilum HG810883

99/94 Tuber petrophilum HG810885

Tuber petrophilum HG810884

-———_———« Tuber pseudoexcavatum GU979043, GU979046

64/59

84/- (100/100

97/97

Tuber regimontanum £U375838

—— Tuber melanosporum FM205579, FM205580

Tuber formosanum F3176917, FJ176919 T. melanosporum lineage

aa (Riousset et al. 2001)

Tuber indicum 3Q639001, AF300823

| Tuber rufum group

-——______—=ag "small white truffles" group (N. America)

98/89

57/-

~«g "small white truffles" group (Europe and Asia)

4 Tuber regianumM 3Q288915, JQ288912

| Tuber excavatum / fulgens agg.

Tuber macrosporum FM205592, FM205634

88/61

81/- —— Tuber magnatum_ FM205529, FM205528

|__scseasug) Tuber aestivum group

100/100f Tuber lacunosum 3N392221

-_____ Tuber gennadii 3N392204, JN392205

|____100/100_ Reddellomyces sp. (outgroup) HM485337, HM485336

0.1

PLATE 2. nr ITS1-5.8S-ITS2 phylogenetic reconstruction of the genus Tuber with a particular focus

on the T. melanosporum lineage, with T. petrophilum as a neighbouring clade to T. brumale aggr.

and T: pseudoexcavatum. The phylogenetic reconstruction is based on 140 sequences selected

from well characterised collections deposited in the International Nucleotide Sequence Databases.

Values for ML and MP branch supports are given for nodes with 250 bootstrap support.

particular, pH and humus contents deviate from requirements and ecological

limits of other European species in the T: melanosporum lineage (Callot et

al. 1999, Riousset et al. 2001). Site conditions for T. petrophilum are unique:

its small size permits it to fruit well in small, humus-rich, and slightly acidic

accumulations of soil in rock pockets that are physically separated from nearby

soils. All specimens collected at different sites in the course of ten years were

found in such pockets (cavities) or crevices in larger and smaller limestone

rocks jutting from the soil, commonly 20-100 cm above the surrounding

soil (PLATE 3). No ascomata were collected in the nearby continuous soil

1148 ... Milenkovié & al.

PLATE 3. Tuber petrophilum: Typical microhabitat inside humus rich soil pocket in limestone rocks.

A-C. Red arrows indicate the exact location where ascomata were unearthed. D. The substrate

inside the hole is visible after removing the litter. E, F The cavity containing ascomata.

layer, despite careful searching with the aid of a trained dog. In this unique

microhabitat, humus mixed with eroded stone particles form a black, loose and

well-aerated substratum. Chemical analysis of the T: petrophilum substratum

confirmed high humus content (11.8-13.6%), with phosphorus measured

at 5.15-11.70 mg/100 g and potassium at 24.35-34.06 mg/100 g. The pH of

the substratum ranged from 6.3 to 7.0. This indicates that T: petrophilum is

Tuber petrophilum sp. nov. (Serbia) ... 1149

a highly demanding species with regard to the habitat requirements, which

include presence of adequate rock cavities with a humus-rich substratum

and simultaneously accessible to mycorrhizal roots of an appropriate tree

species, which we assume to be Fagus sylvatica. Fructification appears limited

to years with sufficient precipitation throughout the year, as mature ascomata

were found in September and October (until snowfall) only in years without

prolonged drought. The sporadic and irregular fruiting of only a small number

of ascomata is probably caused by the combination of the karstic habitat and

climatic conditions.

Tuber petrophilum has been found in only a few localities within a very small

area, suggesting that it is a very rare species. Although Fagus sylvatica along

with Abies alba stands are not rare in Serbia and throughout the Dinarides,

here in combination with karst terrain, rocks, and soil a unique habitat was

formed. Sites are near the road, in a National Park zone with the lowest regime

of protection where forest exploitation is allowed, and felling was launched

in the immediate vicinity of where the new species was found. On that

basis, T: petrophilum is clearly threatened given its small known population

and endangered habitat. Research on distribution, population trends, and

ecological requirements of the species is needed, as well as protection measures

and monitoring for the known population and sites.

The small size of the T: petrophilum ascomata and its relatively scarce

fruiting is unlikely to establish the species as commercially or gastronomically

significant, despite the high quality of its aroma.

Key to the species of Tuber melanosporum lineage

The T. melanosporum lineage differs from other lineages in the genus

Tuber by black colour of the mature ascomata with spores bearing more or

less pronounced warts or aculei.

1. Mature spores light brown to hazel brown, spines 3-6 um long, thin;

gleba with few or infrequent branching veins............... 0... e cece eee eee 2

Mature spores hazel brown to dark brown (when mature),

spines 1.5-8 um long, variably thick; gleba with few or many thin veins....... 4

2. Spores 33-55(-62) x 23-31 um, Q = 1.3-2.0, spines 2-5 um long, thin;

1-4 spores per ascus; gleba solid, dark brown;

described from Central America (Guevara et al. 2008) ........ T. regimontanum

Spores generally smaller, 1-6 spores per ascus, Q not exceeding 1.8 .......... 3

3. Spores 25-31 x 16.5-18.5 um, pan-European distribution,

ITS-supported clade (Merényi et al. 2014) ................ T: brumale Group A

Spores 25-34 x 15.5-19.5 um, Pannonian basin (Europe) distribution,

ITS-supported clade (Merényi et al. 2014) ................ T. brumale Group B

1150 ... Milenkovié & al.

4. Spores dark brown, opaque; ascomata prominent, roundish to irregular,

lobed,-niever- with basal ‘depression: oF Cavity. alo ak! eek! ede, eoeatindes orion 5

Spores brown to dark brown; ascomata with cavity or basal depression

emenoing front: thie daasal Oust oti, chun. habe vas abd brani abs besal a Wi brdnh ab brody s be doula bes brs 6

5. Spores (20-)25-55 x 14-35 um, spines 2—4(-6) um long, thin,

without wide base; 1-6 spores per ascus, Q = 1.4—2.1; gleba blackish brown

with purple tinges, firm, with well-defined veins, reddening;

aroma pleasant and characteristic, Mediterranean Europe

Giiroduced-wonld wide ha peels etc Pacey unseat Rawal pesos T: melanosporum

Spores 15-35(-40) x 10-30(-35) um, spines robust, curved, 3-5 um long,

with wide base and forming incomplete meshes of a reticulum; 1-6 spores

per ascus; gleba blackish brown, compact, veins well defined, infrequent, thin;

described from Asia. [Species an aggregate of several phylogenetic species

(Ghenmetal AOTT i) . ei to ie Mie eg Ms. oe T. indicum Cooke & Massee

6. Spores 12.5-25 x 10-18 um, spines 3 um long, truncate; gleba dark brown

to black, with white to yellowish brown or purplish veins; described from

Fabaceae and Lauraceae forests in southeast Asia (Huang et al. 2009)

NERO ek Rates wh Bal wh Ais ee Bog Tale Es ee T: formosanum H.T. Hu & Y. Wang

Spores >23 um long, brown to dark brown; ascomata from native forests

in Europe or (when from Asia) then ascomata deeply excavated;

ALGMaiiitenise, Pleagarit ha Rr Pease rk oie tae ele toh oat wa vial wa etl heed 7

7. Spores 23-42.5 x 13-26 um, spines straight or slightly curved,

2-4(-4.5) um long; (1-)-4(-6) spores per ascus; gleba dark brown

with wide, 1-3x branched white veins that radiate from the base and end

close to peridium; aroma pleasant resembling mixture of T: borchii and

T: brumale; described from the eastern edge of Dinaric mountains

GAVE SOLD Ia) Breer cttw cyte oth te tctac bel ahead tries vas hers covcithanaied! Mena T. petrophilum

Spores (23—)24—28(-35) x 16-19(-22) um, spines prominent,

5-8 um long; 1-8 spores per ascus; gleba grey-brown with

white sinuous veins; aroma intense; Asia, but introduced

WD ELCITCLOS ot of oct oder a Ho arava oe a. ea ne erg oy Past ah hESC a We bantlagri bsoe T. pseudoexcavatum

Acknowledgements

The authors express their sincere gratitude to Dr Ivana Kugan, Dr David Minter

and Mr Neven Mato¢ec for their very valuable suggestions and critical pre-submission

review of the manuscript, Mr Predrag Jovanovic for assistance in field research from

2004 to 2010, Dr Mirjana Zdravkovic for soil analyses, Dr Dmitar LakuSi¢ for laboratory

resources, Ms Vojislava Tasi¢ for translation and Dr Marjan Niketi¢ for technical support.

The partner from the Slovenian Forestry Institute was co-financed by the Ministry of the

Higher Education, Science and Technology through Research Programme P4-0107 and

by EUFORINNO 7" FP EU Infrastructure Program (RegPot No. 315982).

Tuber petrophilum sp. nov. (Serbia) ... 1151

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MY COTAXON

http://dx.doi.org/10.5248/130.1153

Volume 130, pp. 1153-1164 October-December 2015

Pouzarella alissae, a new species

from northwestern California, United States

Davip L. LARGENT* & SARAH E. BERGEMANN?

' Biological Sciences, Humboldt State University, 1 Harpst St, Arcata CA 95521, USA

? Evolution and Ecology Group, Biology Department, Middle Tennessee State University,

PO Box 60, Murfreesboro TN 37132, USA

* CORRESPONDENCE TO: mrp@humboldt1l.com

ABSTRACT —Pouzarella alissae sp. nov. is known only from north coastal California. It is

characterized by the evanescent silvery-white layer of fibrils and squamules initially covering

the pileus and stipe surface, the dark greyish brown to dark brown innately fibrillose pileus

and stipe, a pileipellis of entangled hyphae with cylindro-clavate terminal cells, a suprapellis

with cytoplasmic pigments, a subpellis with encrusted pigments, heterogeneous lamellar

edges, lageniform to rostrate-ventricose leptocystidia, cheilocystidia and pleurocystidia,

abundant aborted basidia, and basidiospores averaging <11 «1m in length.

Key worps —Basidiomycota, Entolomataceae, phylogeny, taxonomy

Introduction

The name Pouzarella is applied to fungi in the Entolomataceae that possess

stipitate-pileate basidiomata with the following features: basidiome typically

mycenoid with a strigose stipe base, pileipellis disc composed of erect to

semi-erect multicellular hyphae, tramal hyphae with encrusted pigments,

nodulose heterodiametric 5-9-angled basidiospores, and the absence of clamp

connections. Phylogenetically, these fungi form a well-supported clade (He

et al. 2013), and Pouzarella Mazzer is applied as a generic concept (Mazzer

1976; Karstedt et al. 2007; Baroni et al. 2008, 2012; Horak 2008) or is treated

as Entoloma subg. Pouzarella (Mazzer) Noordel. (Manimohan et al. 2006,

Noordeloos 2004, He et al. 2013). We treat Pouzarella as a genus because all

species form a monophyletic group with distinctive morphological characters.

Although Pouzarella species are distributed worldwide, they are encountered

most frequently in mesic areas that receive >20 in. annual rainfall (Mazzer

1976). In North America, Pouzarella is common in the eastern United States

1154 ... Largent & Bergemann

and Canada, but is rare in the west where it is restricted to a narrow coastal

region extending from southern California to southern British Columbia

(Largent 1994, Mazzer 1976).

Of the fifty-nine Pouzarella species reported worldwide in Index Fungorum,

four have been described from western North America: P fernandae (Romagn.)

Largent and P fulvostrigosa (Berk. & Broome) Mazzer from Washington,

P. araneosa (Quél.) Mazzer from California, and P. versatilis (Gillet) Mazzer

from California, Oregon, and Washington (Largent 1994, Mazzer 1976). In this

report, we describe a third and new species from California, P alissae.

Materials & methods

Macromorphological and micromorphological features

Techniques and equipment for collecting and describing basidiomata in the field

and GPS coordinates have been described in Largent et al. (2011a,b), while techniques

for color descriptions using Kornerup & Wanscher (1978) and factors determined from

mathematical analyses in the descriptions are covered in Largent et al. (2013a,b). Dried

specimens were examined microscopically with a research-grade trinocular Nikon

Eclipse Ci-L compound microscope. Microphotographs were made using a Lumenera

Infinity 2-5 CCD digital camera, and all microscopic measurements were obtained

using Infinity Analyze software 6.5.0.

DNA sequences and phylogenetic analyses

Sequences of P. alissae were obtained from the mitochondrial small subunit ribosomal

RNA (mtSSU), the nuclear large subunit ribosomal RNA (LSU), and second largest

subunit of RNA polymerase II (RPB2) following protocols described in Largent et al.

(2011b). Sequences were edited using Sequencher 4.2.2 (Gene Codes, USA) and aligned

using MAFFT v. 7 (Katoh & Standley 2013). Thirty-eight sequences were generated

for this phylogenetic analysis along with sequences obtained from GenBank including

those for the majority of Pouzarella species and from species from other representative

clades as defined in Baroni & Matheny (2011) (TABLE 1).

TABLE 1. Collections used in the phylogenetic analyses.

New sequences generated for this study are shown in bold.

Square brackets enclose GenBank names differing from those in Fic. 1.

Taxa COLLECTION GENBANK ACCESSION NUMBERS

IDENTIFIER mtSSU LSU RPB2

Alboleptonia angustospora MCA1978 GU384583 GU384609 GU384632

[Alboleptonia aff. sericella]

Alboleptonia sericella 10 GQ289330 GQ289190 GQ289261

[Entoloma sericellum]

Catathelasma imperiale 11CA001A KR869913 = -KR869941 KC816816

Claudopus minutoincanus DLL9871 HQ731511 HQ731514. HQ731517

Claudopus parasiticus 10617 TJB KR233902 KR233872 KR233924

Claudopus viscosus DLL9788 HQ31513 HQ31516 HQ31518

Clitocella fallax [Rhodocybe fallax] 256680KM KR869909 = AF261283 KC816937

Clitopilopsis hirneola

Clitopilus apalus

Entocybe haastii [Entocybe sp.]

Entocybe trachyospora

[Rhodocybe trachyospora]

Entoloma abortivum

E. albidoquadratum

E. bloxamii

E. caespitosum

E. changchunense

E. crassicystidiatum

E. discoloratum

E. fragilum

E. furfuraceum

E. indoviolaceum

E. myrmecophilum

E. omiense

E. pallidocarpum

E. praegracile

E. procerum

E. prunuloides

E. readiae

E. rhodopolium

E. rugosiviscosum

E. sericatum

E. sinuatum

E. subaraneosum

E. aff. subsinuatum [E. sinuatum]

E. tenuissimum

E. undatum

E. valdeumbonatum

E. violaceotinctum

E. violaceovillosum

E. yunnanense

Inocephalus hypipamee

I. “lactifluus”

I. plicatus

I. virescens

Lepista nuda

Leptonia ambigua

L. boardinghousensis

L. omphalinoides

L. odorifera

L. poliopus

Pouzarella alissae sp. nov. (U.S.A.) ... 1155

REH8490

26394WAT

DLL10087

5856 TJB

5772 TJB

219

GDGM 27564

HMJAU 3886

GDGM 28821

GDGM 27357

DLL10217

MCA2415

GDGM 28818

i13

231

GDGM 27563

GDGM 28828

GDGM 29251

DLL9676

KA12-1534

GDGM 28823

5349 TJB

GDGM 28813

GDGM 28814

189

DLL10088

GDGM 28815

DLL10071

7962 TJB

DLL9691

DLL9772

11CA041

DLL9872

DLL10086

DLL9673

6534 TJB

DLL10209a

GU384587

KR869907

JQ793645

GU384605

KR233897

GQ289291

GQ289294

JQ993070

JQ993061

JQ993058

JQ993056

JQ793646

KJ021690

JQ993062

GQ289312

GQ289314

JQ993067

JQ993074

JQ993072

GQ289323

GQ289324

GQ289326

GQ289327

JQ793647

GQ289329

GQ289333

KR233896

JQ993059

JQ993060

GQ289342

GQ289343

JQ793643

GQ289345

JQ993057

JQ624604

JQ624605

KR233889

KJ021692

JQ756399

JQ756398

JQ756400

KR233899

JQ756402

GU384611

KR869936

JQ793652

GU384629

KR233869

GQ289151

GQ289154

JQ410327

JQ993095

JQ291567

JQ291569

JQ793653

KJ021700

JQ993094

GQ289172

GQ289174

JQ410330

JQ410331

JQ320129

GQ289183

GQ289184

GQ289186

GQ289187

JQ793654

GQ289189

GQ289193

KJ523137

JQ291568

KR233868

JQ993097

JQ993096

GQ289202

GQ289203

JQ793650

GQ289205

JQ320128

JQ624609

AF261304

JQ624610

KR233859

KJ021705

JQ756414

JQ756413

JQ756416

KR233870

JQ756418

KC816904

KC816906

JQ793659

GU384658

KR233926

GQ289223

GQ289226

JQ993078

JQ993083

JQ993085

JQ793660

KJo21694

JQ993084

GQ289243

GQ289245

JQ993079

JQ993080

JQ993077

GQ289254

GQ289255

GQ289257

GQ289258

JQ793661

GQ289260

GQ289264

AY691891

JQ993086

JQ993087

GQ289270

GQ289271

JQ793657

GQ289273

JQ624616

JQ624617

KR233918

KJ136110

JQ756429

JQ756428

JQ756431

KR233927

JQ756433

1156 ... Largent & Bergemann

L. serrulata [Entoloma serrulatum]

L. “subdecurrentiba” [L. sp.]

L. tjallingiorum [Entoloma tjallingiorum]

L. trichomata

L. umbraphila

Mycena aff. pura

Nolanea cetrata

N. cf. conferenda [Entoloma conferendum]

N. hebes [Entoloma hebes]

N. sericea

N. strictior var. isabellina

LE. strictius var. isabellinus]

Panellus stipticus

Paraeccilia sericeonitida

[Entoloma sericeonitidum|

Pouzarella albostrigosa

P. alissae

P. araneosa [|Entoloma araneosum|

P. debilis

P. farinosa

P. fusca

P. lageniformis

P. lasia

P. farinosa

P. nodospora

P. pamiae

P. parvula

P. pilocystidiata

P. setiformis

Rhodocybe roseiavellanea

R. spongiosa

Rhodophana nitellina

Tricholoma flavovirens

Trichopilus porphyrophaeus

[Entoloma porphyrophaeum]

163

MCA1486

243

11CA006

DLL9793

11CA007

DLL9531

DLL9527

7710 TJB

11CA052

7144 TJB

DLL9641

DLL9663

DLL10490

DLL9784

DLL9900

DLL9934

DLL9623

DLL9895

DLL9662

DLL9670

DLL9729

DLL9807

DLL9811

DLL9900

DLL9934

5716 TJB

DLL9794

DLL9808

DLL9834

DLL9901

DLL9848

DLL9809

DLL9810

8130 TJB

MCA2129

7861 TJB

11CA038

113

GQ289332

GU384589

GQ289337

KR233873

JQ756409

KR869914

KF738927

GQ289300

GQ289310

KR233882

GU384594

KR869915

EF421098

HQ876557

HQ876558

KT074359

GQ289293

HQ876550

HQ876537

HQ876538

HQ876549

HQ876545

HQ876551

HQ876552

HQ876553

HQ876555

HQ876556

HQ876537

HQ876538

KT074358

HQ876539

HQ876540

HQ876541

HQ876546

HQ876542

HQ876547

HQ876548

KR869901

GU384604

KR869900

KJ021691

GQ289322

GQ289192

GU384623

GQ289197

KR233873

JQ756424

KR869942

KF738942

GQ289160

GQ289170

KR233851

GU384618

KR869943

AF261315

HQ876535

HQ876536

KT074357

GQ289153

HQ876528

HQ876515

HQ876516

HQ876527

HQ876523

HQ876529

HQ876530

HQ876531

HQ876533

HQ876534

HQ876515

HQ876516

KT074356

HQ876517

HQ876518

HQ876519

HQ876524

HQ876520

HQ876525

HQ876526

KR869930

GU384628

KR869929

KJ021704

GQ289182

GQ289263

GU384635

GQ289267

KR233903

JQ756440

KC816995

KF771346

GQ289231

GQ289241

KR233913

GU384641

KC816996

EF421016

HQ876513

HQ876514

KT074361

GQ289225

HQ876506

HQ876494

HQ876495

HQ876505

HQ876501

HQ876507

HQ876508

HQ876509

HQ876511

HQ876512

HQ876494

HQ876495

KT074360

HQ876496

HQ876497

HQ876498

HQ876499

HQ876503

HQ876504

KC816982

GU384657

KC816959

KC816997

GQ289253

Pouzarella alissae sp. nov. (U.S.A.) ... 1157

100 i ceponla eelulata 4 n006

titi F eptonia trichomata

0.07 substitutions/site Entoloma praegracile GDGM 29254

Leptonia “subdecurrentiba” MCA1486

100 Entoloma caespitosum GDGM 27564

Alboleptonia angus shore MCA1978

Leptonia poliopus DLL10209a

86 4100 Inocephalus hypipamee DLL10071

Entoloma omiense GDGM 27563

Leptonia umbraphila DLL9793 '

100 Inocephalus “lactifluus” 7962 TJB

use Inocephalus virescens DLL9772

os Inocephalus plicatus DLL9691

28 Entoloma procerum 70 .

Entoloma albidoquadratum i4

Leptonia omphalinoides DLL9673

Alboleptonia sericella 10

Trichopilus porphyrophaeus 113

Leptonia boardinghousensis DLL10086

400 ntoloma undatum 18

97 Claudopus minutoincanus DLL9871

tof 29 Entoloma abortivum 9/72 TJB

Paraeccilia sericeonitda 7144 TJB

100 Claudopus viscosus DLL9788

Claudopus parasiticus 10617 TJB

100 Leptonia tjallingiorum 243

00 Leptonia ambigua DLL9872

Entoloma indovidlaceum i13

Nolanea strictior var. isabellina 7710 TJB

89 Nolanea cf. conferenda 6

86 Nolanea cetrata DLL9531

too Nolanea sericea DLL9527

jo0_|82 Entoloma valdeumbonatum 189

Nolanea hebes 46

Nolanea readiae 102 :

gor Pouzarella lasia DLL9807

Pouzarella lasia DLL9662

yr Pouzarella lasia DLL9811

Pouzarella lasia DLL9670

a4 Pouzarella lasia DLL9729

Tibor Pouzarella albostrigosa DLL9663

Pouzarella albostrigosa DLL9641

69 Pouzarella fusca 623

Pouzarella debilis DLL9784

1o0_,Pouzarella setiformis DLL9809

Pouzarella setiformis DLL9810

Entoloma changchunense HMJAU 3886

1o0_, Entoloma crassicystidiatum GDGM 28821

Entoloma crassicystidiatum GDGM 27357

Entoloma furfuraceum GDGM 28818

Entoloma yunnanense GDGM 28815

i100; Pouzarélla farinosa DLL9900

486 100 Pouzarella farinosa DLL9934

100 r Entoloma tenuissinum GDGM 28813

Entoloma tenuissimum GDGM 28814

Pouzarella pamiae DLL9834

100 7 100 Pouzarella pamiae DLL9794

Pouzarella pamiae DLL9808

100 -- Pouzarella lageniformis DLL9895

98 Pouzarella parvula DLL9901

100 ———. Pouzarella pllocystidiata DLL9848

Entoloma subaraneosum KA12-1534

Entoloma subaraneosum GDGM 2882

-Pouzarella alissae DLL10490

Pouzarellaaraneosail4

: — Entoloma violaceovillosum.

baa ~ Pouzarella nodospora 5/16

n og Entoloma aff. subsinuatum 5349 TJB

99 L Entoloma sinuatum 50

eS Entoloma pallidocarpum GDGM 28828

99 Entoloma rhodopolium 8

goL- Entoloma myrmecophilum 231

Entoloma seficatum 28 |

Leptonia odorifera 6534 TJB

s3— Entoloma prunuloides 40

- Entoloma bloxamii 219

95 28 Entoloma rugosiviscosum DLL9676

Entoloma discoloratum DLL10217

Entoloma violaceotinctum DLL10088

ee Entoloma fragilum MCA2415

100-— Entocybe haastii DLL10087

Entocybe traehyospola 5856 TJB

92 100 litocella fallax OKM25668

is —_— Clitopilus apalus 26394WAT

99 Clitopilopsis hirneola REH8490

75 Rhodocybe sponaisd MCA2129

hodocybe roselavellanea 8130 TJB

—— Rhodophana nitellina 7861 TJB

87 Tricholoma flavovirens 11CA038

Lepista nuda 11CA041

100 Mycena aff. pura 11C A007

ue - - Panellus stipticus 11CA052

Catathelasma imperiale 11CA001A

eljoseznod

Figure 1. Maximum-likelihood topology, highlighting the sub-clade within Pouzarella containing

the new species, Pouzarella alissae. Each sequence is labeled with the GenBank-listed isolate identifier.

Branches with =70% support from 1000 rapid bootstraps are shown. Catathelasma imperiale, Lepista

nuda, Mycena aff. pura, Panellus stipticus, and Tricholoma flavovirens were used as the outgroup.

1158 ... Largent & Bergemann

After exclusion of the RPB2 introns and mtSSU hypervariable regions and manual

optimization, alignment lengths were 592 bp (mtSSU), 1498 bp (LSU), and 1078 bp

(RPB2). A Maximum Likelihood (ML) analysis based on a concatenated dataset with

1000 replicates and specifying a GTRGAMMA model using RAXML-HPC2 v. 8.1.11

(Stamatakis 2014) was carried out with a partitioned dataset for each RPB2 codon (three

partitions) and separate partitions for mtSSU and LSU. One thousand bootstrap (BS)

replicates were generated for assessment of branch supports.

Phylogeny

Monophyly of Pouzarella is well supported (BS = 100, Fic. 1), and Pouzarella

alissae clusters with other representatives of P. sect. Versatiles (P. araneosa and

Entoloma subaraneosum) with significant support (BS = 99, Fic. 1). These

three species share similar features including a fibrillose pileus and stipe and

thin-walled hymenial cystidia. Additionally, all three species have dark brown

basidiomata, evanescent silvery-white coverings on stipe and pileus, appressed

fibrillose pileipelli that become subglabrous, intracellular pigments in the pileus

suprapellis, hyphae with encrusted pigments in the pileus subpellis and trama,

and lageniform to rostrate-ventricose leptocystidioid cheilocystidia. They

cluster within a larger clade with other members of Pouzarella sect. Pouzarella

(P. nodospora (G.F. Atk.) Mazzer and Entoloma violaceovillosum Manim. &

Noordel.), but without support (Fic. 1). Whether this lack of support for the

morphologically based infrageneric classification is due to a lack of adequate

taxon sampling or gene resolution will require further research.

Taxonomy

Pouzarella alissae Largent & Bergemann, sp. nov. PLaTEs 1, 2

MycoBank MB 812300

Differs from Pouzarella araneosa by its dark brown pileus, shorter basidiospores, shorter

cheilocystidia, and the presence of pleurocystidia.

Type — United States, California, Humboldt County, Prairie Creek Redwoods State

Park, Intersection of Big Tree Trail and Prairie Creek Highway, in duff beneath Acer

circinatum and Polystichum munitum, within 20 m of 41°22’33.15”N 124°00’51.66”W,

62 m elev., 22 November 2014, DL Largent 10490 (Holotype, HSC; GenBank KT074359

(mtSSU), KT074357 (LSU), KT074361 (RPB2)).

EryMoLocy — named for Alissa Allen, who first discovered this species.

BASIDIOMATA with the pileus and stipe initially covered with silvery-whitish

evanescent layer of fibrils that forms squamules over the pileus surface, small

pointed squamules on the pileus margin, and longitudinally arranged appressed

fibrils on the stipe. When fresh, the stipe fibrils can be rubbed off revealing a

dark brownish fibrillose layer on the stipe surface. In older basidiomata, this

layer first disappears from the pileus before eventually disappearing on the

Pouzarella alissae sp. nov. (U.S.A.) ... 1159

PLaTE 1. Pouzarella alissae (HSC DLL10490, holotype): A. basidiomata (Photo by Noah Segal);

B. cross section through strigose stipe base; C. longitudinal section through strigose stipe base.

Scale bars: A = 8 mm; B, C = 20 um.

stipe. In silica gel-dried basidiomata, this layer is absent when sections of the

basidiomata are viewed under the compound microscope.

Piteus 8-30 mm broad, 2-7 mm high, conic to campanulate, becoming

convex to broadly convex, then plane, and finally uplifted; initially lacking an

umbo or obscurely umbonate, broadly umbonate when mature; opaque (not

1160 ... Largent & Bergemann

translucent and thus not translucent-striate), dull, scurfy or tomentulose on disc,

innately fibrillose elsewhere; margin incurved then decurved and eventually

plane; color overall dark brownish grey (4-5F2-3) fading to dark brown

(5F3-4); flesh <1.5 mm above the stipe, pale greyish brown. LAMELLAE 6-12

mm long, 1.25-4 mm deep, initially adnexed and sigmoid and later narrowly to

broadly adnate (at times with a small subdecurrent tooth), subdistant to distant,

moderately broad, pale greyish-tan when young, maturing dark greyish-brown

(5F2) with smoky greyish brown (5-7F5) spots and blotches, when fully mature

with a pinkish cast from spores; edge fairly thick, at first concolorous and

then developing whitish areas with spore maturation; lamellulae 3-4 between

lamellae in two series [one always long (4-8 mm) and one always short

(1-2.5 mm)]. Stipe 33-88 mm long, apex 1-2.5 mm diam. and base 1.25-4.5

mm diam., cylindrical, equal when young and enlarged at the base when

mature, very finely pruinose at the apex and innately and appressed-fibrillose

elsewhere, in very scattered places the fibrils separating and becoming matted

in clumps; hairs or setae absent; basal area decidedly strigose up to 7-8 mm

from substrate, brownish with reddish tones; basal tomentum also present,

whitish (in some basidiomata developing orangish areas). TasTE and ODOR

not distinctive. SPORE DEPOSIT pinkish to dingy salmon-tan.

BASIDIOSPORES with 5-6 distinct angles in side and profile views,

heterodiametric in side, profile, dorsal, and ventral views, 9-12 x 6-8 um

(m = 10.2 + 0.8 x 7.2 + 0.5 um; E = 1.2-1.8; Q= 1.4 + 0.1; n/1 = 40). Basrp1a

cylindro-clavate, 34-47 x 8-13 um (m = 41.2 + 3.5 x 9.8 + 1.5 um; E = 3.2-6.0;

Q =4.3 + 0.7; n/1 = 10), 4-sterigmate; aborted basidia abundant on the lamellar

edge and face, wall thickening at first after which the entire contents become

reddish brown in 10% NH,OH or 3% KOH, cylindro-clavate, 21-39 x 4-11 um

(m= 29.9+4.2 x 6.5 + 1.6 um; E = 3.1-8.3; Q = 4.8 + 1.3; n/1 = 20). LAMELLAR

EDGE heterogeneous. CHEILOCYSTIDIA (leptocystidia) thin-walled, when

present scattered (but often absent on some lamellae), colorless or staining

reddish brown in alkali solutions, versiform (cylindro-clavate, clavate,

obclavate), 34-62 x 10.5-16.4 um or lageniform with a short to medium neck

or rostrate-ventricose, 46-656 x 13-23.5 um (including the neck or rostrum),

rostrum typically short to medium (rarely longer), 3.1-7.0(-17.5) x 3.0-3.5

(-6.0) um (n/1 = 4)). PLeuRocystip1a colorless, uncommon and very

scattered, obclavate, acuminate-ventricose, lageniform, or rostrate-ventricose,

40-42.5 x 12-17 um (including rostrum, which is 6.4-7.0 um long and 2.7-7.0

um diam.). LAMELLAR TRAMAL HYPHAE subparallel, 125-564 x 9.2-42 um

(E = 9.2-19.1; n/1 = 10). PILEIPELLIS composed of entangled hyphae, erect and

resembling a trichodermium on the disc, semi-erect around the disc, becoming

Pouzarella alissae sp. nov. (U.S.A.) ... 1161

2 we,

)

—~ °

hy,

PLATE 2. Pouzarella alissae (HSC DLL10490, holotype): A. pileipellis radial section from disc;

B. stipitipellis with caulocystidium (longitudinal section from mid-stipe); C. basidium (white

arrow), aborted basidium filled with dark pigment (black solid arrow), and cheilocystidia (dotted

arrows); D. basidium (white arrow) vs. aborted basidium (dotted arrow); E. basidiospores.

Scale bars: A, B = 12 um; C = 15 um; D = 10 um; E=7 um.

1162 ... Largent & Bergemann

prostrate towards the margin; PILEOCYSTIDIA (terminal cells) on or around the

disc cylindro-clavate with a rounded apex 93-184 x 9-18 um (m = 131.9 + 31.1

x 12.6 + 2.4 um; E = 8.6-14.3; Q = 10.6 + 2.1; n/1 = 11), towards and at the

margin/edge longer and tapered, 166-218 x 14-17 um. PILEAL TRAMAL

HYPHAE subparallel and entangled, 49-310 x 3-30.5 um (m = 152.2 + 71.6

x 13.7 + 9.4 um; E = 4.9-46.3; Q = 15.9 + 12.3; n/1 = 13). STIPITIPELLIs at

the apex composed of hymenial elements (= pruinose), below the apex hyphae

primarily prostrate, 2-4 cells deep but occasionally out-turned in some

areas; caulocystidia (terminal cells) cylindro-clavate and similar in shape

to the pileocystidia, 67-136 x 9-14 um (m = 89.4 + 24.4 x 11.2 + 1.9 um;

E = 5.8-12.2; Q = 8.2 + 2.5; n/1 = 6). STIPE TRAMAL HYPHAE Nearly parallel,

somewhat entangled, 9-67 um diam. (n/1 = 7). OLEIFEROUS HYPHAE rare in

the pileal trama. BRILLIANT GRANULES scattered to somewhat abundant in

the basidia. Liporp GLOBULES absent. PIGMENTs brownish, cytoplasmic and

parietal on the apical 1-4 cells in the pileus and stipe suprapelli, encrusted as

rings in the pileus and stipe subpelli, and decidedly encrusted on all tramal

hyphae. Clamp connections absent.

ECOLOGY & DISTRIBUTION — Solitary, in clumps or loose clusters in wet

duff under Acer circinatum Pursh and Polystichum munitum (Kaulf.) C. Presl in

Prairie Creek Redwoods State Park.

ComMENTs — Morphologically, P alissae, P araneosa, P celata Mazzer,

P. versatilis, and Entoloma subaraneosum Xiao L. He & T:H. Li are placed in

Pouzarella sect. Versatiles Mazzer because of their innately fibrillose pileus

and stipe, thin-walled hymenial cystidia, and basidiospores that average <13

um in length. Pouzarella araneosa and E. subaraneosum can be differentiated

from P alissae by their longer spores (9.5-15 um for P araneosa; 9.5-13 um for

E. subaraneosum), longer cheilocystidia (45-100 um) with much longer necks,

and lack of pleurocystidia (He et al. 2013; Mazzer 1976; Noordeloos 1992,

2004).

In North America, P. celata, described from Michigan, and P. versatilis,

described from Europe (Noordeloos 1979, 1992, 2004; as Entoloma versatile

(Gillet) M.M. Moser) and reported from western North America including

California (Mazzer 1976) and Washington (Largent 1994), are similar in

size and also produce dark brown colored basidiomata. Both species can be

distinguished from P. alissae by their abundant and longer cheilocystidia and

pleurocystidia and absence of silvery white evanescent fibrils and squamules on

the pileus. Pouzarella celata also differs by its longer (11-14 um) basidiospores

and glabrous pileus surface that is streaked by innate silky grey fibrils while

P. versatilis is distinguished by its abundant pleurocystidia, olivaceous green

Pouzarella alissae sp. nov. (U.S.A.) ... 1163

tints or olivaceous brown pileus color (Noordeloos 2004, Largent 1994), and

the metallic luster of the dried pileus (Mazzer 1976).

Pouzarella araneosa, reported from California under Acer circinatum,

consisted of one basidiome with a missing stipe base (Largent 1994). The

collection differs from P. alissae and P. araneosa by the absence of a silvery

fibrillose layer, its medium dark brown (5-6F4) pileus, its translucent-striate

pileal margin, and its broader and longer cheilocystidia; until more material

is obtained, the diagnosis of the collection formerly reported as P. araneosa

remains doubtful.

Acknowledgments

Materials required to complete this manuscript were supported by the Largent

family trust, and we are particularly grateful for the support of Pamela Largent. The

DNA sequences generated in this study are based upon work supported by the National

Science Foundation under Grant No. DRI 0922922 awarded to Dr. Sarah Bergemann.

Comments by the two reviewers, Dr. Timothy J. Baroni and Dr. Joseph F. Ammirati,

and by the Nomenclature Editor, Dr. Shaun Pennycook, were also helpful. We wish to

thank especially Noah Siegel for allowing us to use his illustration of P alissae in the field

and to see his description of the species in his forthcoming book.

Literature cited

Baroni TJ, Matheny PD. 2011. A re-evaluation of gasteroid and cyphelloid species of

Entolomataceae from Eastern North America. Harvard Papers in Botany 16: 293-310.

http://dx.doi.org/10.3100/0.25.016.0205

Baroni TJ, Cantrell SA, Perdomo-Sanchez OP, Lodge DJ. 2008. New species of Pouzarella

(Entolomataceae, Agaricales) from the Dominican Republic and Jamaica. North American

Fungi 3: 241-260. http://dx.doi.org/10.2509/naf2008.003.007 16

Baroni TJ, Albert6 E, Niveiro N., Lechner B. 2012. New species and records of Pouzarella

(Agaricomycetes, Entolomataceae) from northern Argentina. Kurtziana 37(1): 41-63.

He XL, Li TH, Xi PG, Jiang ZD, Shen YH. 2013. Phylogeny of Entoloma s.l. subgenus

Pouzarella with descriptions of five new species from China. Fungal Diversity 58: 227-243.

http://dx.doi.org/10.1007/s13225-012-0212-7

Horak E. 2008. Agaricales of New Zealand 1: Pluteaceae-Entolomataceae. ‘The fungi of New

Zealand, vol. 5. Fungal Diversity Press, Hong Kong

Karstedt FE, Capelari M, Stiirmer SL. 2007. A new combination and new records of Pouzarella

(Agaricales, Entolomataceae) from Brazil. Mycotaxon 102: 147-153.

Katoh K, Standley DM. 2013. MAFFT Multiple sequence alignment software version 7:

improvements in performance and usability. Molecular Biology and Evolution: 30: 772-780.

http://dx.doi.org/10.1093/molbev/mst010

Kornerup A, Wanscher JH. 1978. Methuen handbook of colour, 3rd ed. Richard Clay Ltd:

Chichester, Sussex.

Largent DL. 1994. Entolomatoid fungi of the western United States and Alaska. Mad River Press

Inc: Eureka, California.

Largent DL, Abell-Davis SE, Cummings GA, Ryan KL, Bergemann SE. 2011a. Saxicolous

species of Claudopus (Agaricales, Entolomataceae) from Australia. Mycotaxon 116: 253-264.

http://dx.doi.org/10.5248/116.253

1164 ... Largent & Bergemann

Largent DL, Bergemann SE, Cummings GA, Ryan KL, Abell-Davis SE, Moore S. 2011b. Pouzarella

(Agaricales, Entolomataceae) from New South Wales (Barrington Tops National Park) and

northeastern Queensland. Mycotaxon 117: 435-483. http://dx.doi.org/10.5248/117.435

Largent DL, Bergemann SE, Abell-Davis SE, Kluting KL, Cummings GA. 2013a. Three new

Inocephalus species with cuboid basidiospores from New South Wales and Queensland,

Australia. Mycotaxon 123: 301-309. http://dx.doi.org/10.5248/123.301

Largent DL, Bergemann SE, Abell-Davis SE, Kluting KL, Cummings GA. 2013b. Five Leptonia

species from central New South Wales and Queensland, Australia. Mycotaxon 125: 11- 35.

http://dx.doi.org/10.5248/125.11

Manimohan P, Noordeloos, ME, Dhanya AM. 2006. Studies in the genus Entoloma (Basidiomycetes,

Agaricales) in Kerala State, India. Persoonia 19(1): 45-93.

Mazzer SJ. 1976. A monographic study of the genus Pouzarella. Bibliotheca Mycologica 46. 191 p.

Noordeloos ME. 1979. Entoloma subgenus Pouzaromyces emend. Persoonia 10: 207-243.

Noordeloos ME. 1992. Entoloma s.l. Fungi Europaei vol. 5. Ed. Candusso: Alassio, Italy.

Noordeloos ME. 2004. Entoloma s.1. Fungi Europaei vol. 5a. Ed. Candusso: Alassio, Italy.

Stamatakis A. 2014. RAXML version 8: a tool for phylogenetic analysis and post-analysis of large

phylogenies. Bioinformatics 30: 1312-1313. http://dx.doi.org/10.1093/bioinformatics/btu033

MY COTAXON

http://dx.doi.org/10.5248/130.1165

Volume 130, pp. 1165-1169 October-December 2015

The discovery of Syncephalis obliqua

(Zoopagomycotina, Zoopagales) in the Neotropics

ROGER FAGNER RIBEIRO MELO, LEONOR COSTA MAIA &

ANDRE LUIZ CABRAL MONTEIRO DE AZEVEDO SANTIAGO

Universidade Federal de Pernambuco, Departamento de Micologia, Centro de Ciéncias Biol6gicas,

Av. da Engenharia, s/n, 50740-600, Recife, Pernambuco, Brazil

* CORRESPONDENCE TO: rogerfrmelo@gmail.com

AspsTRACT — During an investigation on coprophilous fungi from Brazil, several

sporangiophores of a mycoparasitic fungus were recovered from horse dung collected in

Recife, Pernambuco, Northeastern Brazil, and identified as Syncephalis obliqua. ‘This is the

first record of the species in the Neotropics, as well as in a host growing on herbivore dung,

and its second world record. A detailed description and high quality images are provided,

along with an identification key to all Syncephalis species recorded in Brazil.

Key worps — Piptocephalidaceae, taxonomy

Introduction

Syncephalis was first described by van Tieghem & Le Monnier (1873),

and c. 60 species are accepted (Ho & Benny 2008, Kirk et al. 2008). The

genus includes haustorial mycoparasites of Mortierellales, Mucorales, or (less

commonly) ascomycetes, forming coenocytic aerial hyphae in or over the

host, merosporangiophores with basal rhizoids, and usually with apical fertile

vesicles bearing several one- to multi-spored merosporangia. Zygospores, when

present, are globose to subglobose, ornamented, with apposed suspensors,

possibly adorned with a multi-lobed vesicle (Benny 2011). Most records are

associated with mucoraceous hosts in soil and dung (Santiago et al. 2010).

In Brazil, only seven species have previously been recorded: S. aggregata

A.L. Santiago & Benny, S. asymmetrica Tiegh. & G. Le Monn., S. clavata

H.M. Ho & Benny, S. cornu Tiegh. & G. Le Monn., S. penicillata Indoh,

S. sphaerica Tiegh., and S. tengi S.H. Ou (Trufem 1984, Trufem & Viriato 1985,

Melo et al. 2011).

1166 ... Melo, Maia & Santiago

Syncephalis obliqua was described by Ho & Benny (2008) parasitizing

a Mucor species from soil samples collected in Taiwan. Here we present the

second world record of S. obliqua, identified as a mycoparasite of coprophilous

ascomycetes on herbivore dung from Brazil and recorded for the first time in

the Neotropics.

Materials & methods

Samples of cattle, goat and horse dung collected in clean plastic bags on the campus

of Universidade Federal Rural de Pernambuco, Recife (8°00’54”S 34°56’59”W) were

taken to the laboratory and incubated in moist chambers (9 cm diam. Petri dishes

with a moistened disk of filter paper) at room temperature (28 + 2°C) for at least 20

days under alternating natural light and dark periods. The specimens were observed

directly from the substrata under a Leica EZ4 stereomicroscope; sporangiophores were

mounted in slides with tap water, lactophenol plus cotton blue, or Polyvinyl-Lacto-

Glycerol and examined with the aid of a Leica DM500 light microscope. Specimens

were identified based on Benny (2011), Ho & Benny (2008), and Santiago et al. (2011).

A careful literature review and survey of Brazilian herbaria were performed in order

to access information regarding older Syncephalis records. High quality images were

captured with a QImaging QColor 3 digital camera mounted on an Olympus BX51

compound microscope using differential interference or phase contrast microscopy.

Permanent slides were deposited in Herbario Padre Camille Torrend, Departamento

de Micologia, Universidade Federal de Pernambuco, Recife, Brazil (URM). Additional

information regarding deposit records can be accessed at the “INCT - Herbario Virtual

da Flora e dos Fungos” database website (http://inct.florabrasil.net/).

Taxonomy

Syncephalis obliqua H.M. Ho & Benny, Bot. Studies 49: 45 (2008). PLATE 1

SOMATIC HYPHAE hyaline, 1.5-2 um wide, immersed in substrata or on host

hyphae, weakly cyanophilous. SPORANGIOPHORES numerous, isolated, erect,

simple, cylindrical, hyaline, smooth to slightly punctate, arising from the host

hyphae or from the dung, broader at the base, 650-710 um long, 15-17.5 um

diam., narrowing gradually towards the apex, 7.5-10 um diam. just below the

vesicle. RHIZoIDs well developed, hyaline to weakly pigmented, branching to

form horizontal primary rhizoids that branch once or twice, septate. APICAL

VESICLES globose to obovoid, hyaline, persistent or collapsing, cyanophilous,

62.5-77.5 x 52.5-62.5 um, with about half of its apical surface covered by

merosporangia, leaving conspicuous denticles on the surface after release.

MEROSPORANGIA 4-6-spored, pedunculate, unbranched, thin-walled, hyaline,

cylindrical, obliquely placed on the vesicle, usually detaching in mounts.

Merosportes cylindrical to doliiform, rounded on both ends or eventually

with one end truncate and the other rounded, hyaline, smooth, 5.5-6.5 x

2.5-3 um. ZYGOSPORES not observed.

Syncephalis obliqua new to Brazil ... 1167

PLATE 1. Syncephalis obliqua (URM 87589a): A. sporangiophore growing on host immersed on

dung; B. apical vesicle of a young sporangiophore, strongly cyanophilous, showing the obliquely

placed merosporangia on the left; C. apical vesicle in a mature sporangiophore, with several visible

multispored merosporangia; D. sporangiophore after merosporangia release, showing conspicuous

denticles on the upper half of the vesicle; E. rhizoids; F. released merospores. Scale bars: A = 250 um;

B= 10 um; C = 50 um; D, E = 25 um; F = 15 um.

1168 ... Melo, Maia & Santiago

Hasirat: Parasitic on Ascobolus immersus Pers. (Pezizales) and Zopfiella

longicaudata (Cain) Arx (Sordariales) growing on horse dung.

MATERIAL EXAMINED: BRAZIL. PERNAMBUCO, Recife, Dois Irmaos, Universidade

Federal Rural de Pernambuco, [X.2011, R.ER. Melo s.n. (URM 87588); XI.2014, R.ER.

Melo s.n. (URM87589a-f).

DISTRIBUTION: Brazil and Taiwan. This is the first record from Brazil.

Notes: The main distinguishing feature of S. obliqua is the oblique placement

of the merosporangia on the apical vesicle. It differs from S. plumigaleata by

the larger merospores (6.5-10 x 3.5-4.5 um; Embree 1965) and from S. fuscata

(which produces similar obliquely placed merosporangia) in spore shape,

ornamentation, and size (Kuzuha 1973). Our Brazilian specimens show a close

morphological similarity with the descriptions of the Taiwanese material by Ho

& Benny (2008). However, some merospores are slightly truncate at one end,

which is not noted in the original description. Most specimens observed on

incubated dung produced several sporangiophores (usually more than 100 per

5 g of dung) and persisted for 4-5 weeks. Although some sporangiophores arose

from Ascobolus immersus apothecia and Zopfiella longicaudata cleistothecia

growing on dung, clear signs of direct parasitism were not observed.

Key to Syncephalis spp. recorded from Brazil

I, “Sporangiophores:singleystronglycurved s.. 2.1.55 eae sk she nee ee eos 8 S. cornu

Sporangiophores single or in clusters, erect or slightly curved ............... 2

2. Sporangiophores with irregular swellings, rarely branched,

FORTIES -ACTSE-TULES. Ot Rn bea ora gett goat eter tet aee ys SAREE al cts S. aggregata

Sporangiophores without swellings, unbranched, single .................... 3

3. Vesicle truncate; merosporangia cylindrical at the base and

tinilateralys branched err caren fiz sla) 5 tte peta dpa paint Epes aes S. penicillata

Vesicle and merosporangial base not as above............... cece eee eee 4

4. Merosporangia branched once at the base, the first spore being cordate,

Here Stil ar aS ape ie le, hs ty hwtiard Si bewnedc Barents « ceased Steere Rats ft Beets 5

Meéesosporanciaunbranched hi. 1 ha's 00 gests 8 Sesh 8 heh 8 Ses 8 beaks tales acted 6

5. Vesicle asymmetric; merospores 4-8 x 2-4um................ S. asymmetrica

Vesicle symmetric; merospores 7.5-12.5 x 3.5-5 UM ....... eee eee eee S. clavata

6. | Merosporangia obliquely placed at the vesicle...................00. S. obliqua

Merosporangia equally placed at the vesicle......... 0... eee eee eee ee 7

7. Merosporangia 2-8 spored; merospores 6-8 x 8 um long .............. S. tengi

Merosporangia 3-4 spored; merospores 5-5.1 x 11-12 um long..... S. sphaerica

Syncephalis obliqua new to Brazil ... 1169

Acknowledgments

The authors wish to acknowledge José Ivanildo de Souza (Instituto de Botanica,

Sao Paulo, Brazil) and Merlin White (Boise State University, Idaho, USA) for

presubmission reviews of the manuscript. Acknowledgments are also due to Conselho

Nacional de Desenvolvimento Cientifico (CNPq) for providing a PhD scholarship to

R.ER. Melo and grant support to L.C. Maia

Literature cited

Benny GL. 2011. Syncephalis. http://zygomycetes.org/index.php?id=157 [updated 18 May 2011].

Embree RW. 1965. A new species of Syncephalis (Mucorales). American Journal of Botany 52:

737-741. http://dx.doi.org/10.2307/2446595

Ho HM, Benny GL. 2008. A new species of Syncephalis from Taiwan. Botanical Studies 49: 45-48.

Kirk PM, Cannon PF, Minter DW, Stalpers JA. 2008. Dictionary of the fungi. CABI: Wallingford

(United Kingdom).

Kuzuha S. 1973. Notes on Japanese Syncephalis. Transactions of the Mycological Society of Japan

14; 237-245.

Melo RER, Santiago ALCMA, Maia LC. 2011. Syncephalis clavata (Zoopagales, Zygomycetes), a first

record from the neotropics. Mycotaxon 116: 113-116. http://dx.doi.org/10.5248/116.133

Santiago ALCMA, Benny GL, Maia LC. 2011. Syncephalis aggregata, a new species from the

semiarid region of Brazil. Mycologia 103: 135-138. http://dx.doi.org/10.3852/10-049

Tieghem P van, Le Monnier G. 1873. Recherches sur les Mucorinées. Annales des Sciences

Naturelles, Botanique, 5e Sér., 17: 261-399.

Trufem SFB. 1984. Mucorales do Estado de Sao Paulo. 4. Espécies coprofilas. Rickia 11: 65-75.

Trufem SFB, Viriato A. 1985. Mucorales do Estado de Sao Paulo. 6. Mucoraceae coprofilas. Rickia

E2? 113—123,

MY COTAXON

http://dx.doi.org/10.5248/130.1171

Volume 130, pp. 1171-1183 October-December 2015

Two new records of Agaricus spp. from Ethiopia

REDIET SITOTAW”?, Y. LY’, T.-Z. WEI’, D. ABATE? & Y.-J. YAO”

"Microbial, Cellular and Molecular Biology Department, Addis Ababa University,

PO. Box 1176, Addis Ababa, Ethiopia

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

Beijing 100101, China

* CORRESPONDENCE TO: redietsitotaw7@gmail.com, yaoyj@im.ac.cn

ABSTRACT — Agaricus xanthodermus and A. xanthodermulus are recorded as new to Ethiopia

based on morphological and molecular data. A description of A. campestris (previously

reported from Ethiopia) is also provided. The specimens were collected from Menagesha

and Holeta sites located in the Wolmera District of Oromia Special Zone surrounding Addis

Ababa. Morphological identification of the specimens was supported by nrDNA ITS sequence

analysis. All three species are described in detail and illustrated based on the collections from

Ethiopia.

Key worps — Agaricaceae, Basidiomycota, taxonomy, highland, mycota

Introduction

Agaricus L. isa large and important genus encompassing many edible and few

poisonous mushrooms, with possibly 200-400 (Bas 1991, Kirk et al. 2008, Zhao

et al. 2011) to possibly over 400 (Thongklang et al. 2014) species worldwide.

Agaricus species are generally distinguished from other members of the family

Agaricaceae by their small to large basidiomata, white or brown pileus, annulus

and central stipe, free lamellae that are whitish or pinkish when young and

chocolate-brown to dark brown when mature, and smooth dark basidiospores

(Cappelli 1984). Studies have shown that the genus is monophyletic (Geml et

al. 2004) and contains eight recognized sections (Parra 2008), some of which

have been phylogenetically reconstructed, e.g., A. sect. Bivelares (Kauffman)

L.A. Parra and A. sect. Xanthodermatei Singer (Challen et al. 2003, Kerrigan

et al. 2005, Thongklang et al. 2014). Agaricus species are generally found in

pastures among grass and in mixed forests (Bas 1991).

1172 ... Sitotaw & al.

TABLE 1. ITS sequences of Agaricus and outgroup species used in the molecular

analysis.

SPECIES

Agaricus aff. endoxanthus

. aridicola

. arvensis

. augustus

benesii

. bernardii

. bitorquis

bohusii

. bresadolanus

. brunneolus

. californicus

ise tec hk he EP ha he eet

. campestris

A. caribaeus

A. cupreobrunneus

A. fissuratus

A. gennadii

A. langei

A. laskibarii

A. moelleri

A. parvitigrinus

A. pseudolutosus

A. silvaticus

A. viridopurpurascens

A. xanthodermus

A. xanthodermulus

VOUCHER/STRAIN

ZRL3095

CA101

CA640

CA590

LAPAG283

strain ARP173

strain RWK1462

LAPAGS531

strain CA177

CA490

strain RWK 1936

strain W1H

Strain B9

strain H2

strain AGRK111

HMAS272461

HMAS272462

F2530

LAPAG 322

strain CA 87

strain WC777

CA387

strain WC784

LAPAGI41

LAPAGI15

strain CA31

strain CA156

strain CA157

strain CA158

LAPAG77

LAPAG341

Horak68/79

strain CA15

strain CA161

strain W31

strain CA236

strain CA6

HMAS272456

HMAS272457

strain CA160

strain CA174

strain CA204

ORIGIN

Thailand

France

USA

Czech Republic

France

USA

China

India

China

Ethiopia, Menagesha

Ethiopia, Holeta

Martinique

Spain

USA

Denmark

France

USA

France

USA

France

Spain

France

New Zealand

France

England

France

Ethiopia, Menagesha

Ethiopia, Holeta

France

GENBANK*

JF691554

JF797195

JF797194

JE797193

JE797179

AF432880

AF432898

JF797180

DQ185570

JF797203

DQ182510

DQ182533

JX434655

KM609406

FJ223222

KP229419

KP229420

JF727856

JQ824136

DQ182532

AY484683

JF797188

AY484699

JE797181

AY943975

AY899263

AY899264

AY899266

AY899267

JE727868

JF797178

JF514525

AY899271

AY899272

DQ182534

DQ185564

DQ185563

KP229414

KP229415

AY899273

AY899274

AY899276

Agaricus spp. new for Ethiopia ... 1173

TABLE 1, concluded

(A. xanthodermulus) LIP CA160 France NR119528

strain CA188 France AY899275

HMAS272459 Ethiopia, Holeta KP229416

HMAS272460 Ethiopia, Menagesha KP229417

A. xanthosarcus Goossens5415 Belgium JF514523

Hymenagaricus ardosiicolor LAPAF9 Togo JF727840

Heinemannomyces splendidissimus ecv3586 Thailand HM488760

* Sequences produced in this study in bold.

Pegler (1977) noted that agaric mycota in East Africa is extremely rich both

in the range of genera and in the number of species. However, large parts of this

region remain mycologically unexplored. Although Ethiopia is known to have

highly diverse flora and fauna (Friis & Sebsebe 2001) but the diversity, ecology,

and distribution of macrofungi is so far poorly explored.

Macrofungal diversity in general and Agaricus species in particular have

not been a focus of taxonomic investigation in Ethiopia. Only a few reports

on macrofungi in Ethiopia have been published (Abate 1995, 1999). Abate

(1999), who cited Agaricus campestris as the dominant mushroom in the

grazing areas on the highland (2000-3000 m) plateau in southern (Bale),

central (Debreberhan), and northwestern (Gojam) Ethiopia, appearing in a

large numbers during the middle of the rainy season (July and August) did

not provide a detailed morphological description or any molecular data of the

species.

As the first report of the on-going work on macrofungal diversity in

Ethiopia, we present detailed morphological descriptions and molecular

analyses of three Agaricus species including two new records, A. xanthodermus

and A. xanthodermulus, for Ethiopia in addition to the previously cited

A. campestris. This report will provide a baseline for future taxonomic,

ecological, and economic studies on Ethiopian Agaricus species.

Materials & methods

Collection sites

Specimens were collected from Menagesha and Holeta, Wolmera district, in

the Oromia Special Zone surrounding Addis Ababa, the capital of Ethiopia, during

June-September, 2012-2013. Menagesha (9°02’N 38°35’E, 2500-3300 m a.s.l.)

comprises forests and grazing areas. Holeta (9°03’N 38°30’E, 2391 m a.s.l.) contains

mainly settlement and grazing areas.

Morphological observation

Distinguishing macroscopic features and microhabitat were noted for each specimen

in the field. Microscopical observations were made from dried specimens mounted in

5% KOH and stained with 1% aqueous Melzer’s reagent and Congo red. At least 20

1174 ... Sitotaw & al.

basidiospores were measured from each specimen. Chemical testing involved the

application of 20% KOH on the pileus surface, with negative = no colour change and

positive = change to yellow or orange. Dried specimens are deposited in the Department

of Microbial, Cellular and Molecular Biology, Addis Ababa University, Ethiopia, and

duplicates are preserved in the Fungarium, Institute of Microbiology, Chinese Academy

of Sciences, Beijing, China (HMAS). The specimens were identified morphologically

according to Pegler (1977), Pegler & Piearce (1980), Singer (1986), and Callac &

Guinberteau (2005). Abbreviations: RSK = Rediet Sitotaw Kebede.

DNA extraction, PCR amplification and sequencing

Total genomic DNA was extracted from dried specimens using the modified CTAB

method described by Yao et al. (1999). The fungal universal primer pairs ITS5/ITS4 were

used to amplify the internal transcribed spacer (ITS) region and LROR/LRS to amplify

the nuclear larger subunit RNA (LSU) region (White et al. 1990). The PCR reaction

mixture was comprised 25 uL Taq PCR MasterMix, 0.5 uL of 10 uM each primer, 1 uL

diluted DNA template, and RNase-Free water to bring the total volume to 50 uL. The

PCR conditions were as follows: 94 °C for 5 min, followed by 35 cycles of 95 °C for

Imin, 53 °C for Imin, 72 °C for 1 min and final extension step of 72 °C for 10 min ona

GenAmp PCR System 9700 thermocycler (Vers. 3.03).

PCR products were sequenced from both directions using the same primers on an

Applied Biosystems 3730 Analyzer™ by the Beijing Genomics Institute (Beijing, China).

Sequence ends were manually edited and assembled using DNAStar Lasergene SeqMan™

II vers. 6.1. Sequences were then aligned using Clustal W algorithm (Thompson et al.

1994) and manually edited with BioEdit vers. 7.1.9 (Hall 1999).

Phylogenetic analysis

ITS sequences from the Ethiopian Agaricus collections were aligned with 43 closely

related ITS sequences from GenBank representing the eight Agaricus sections (Zhao et

al. 2011) (TaBLE 1). Hymenagaricus ardosiicolor and Heinemannomyces splendidissimus

were selected as outgroup taxa since these species consistently formed a sister clade to

the monophyletic Agaricus (Zhao et al. 2011). The datasets were analysed using PAUP

vers. 4.0b8 (Swofford 2002). Maximum parsimony (MP) analyses were conducted with

1000 replicates of random sequence addition followed by tree-bisection reconnection

branch swapping. All characters were equally weighted, and gaps were treated as missing

data. Bootstrap proportions (BP) were calculated using analyses of 1000 replicates with

five replicates of random sequence addition.

Results of phylogenetic analyses

ITS sequences from the six Ethiopian specimens were submitted to GenBank

as KP229414-229417, 229419, and 229420. (Analysis of the LSU sequences also

obtained from the same specimens and deposited in GenBank as KP331526-

KP331530 will be presented in a future publication.)

A total of 49 ITS sequences (TABLE 1) were aligned for the phylogenetic

analysis. The dataset generated a MP tree with 709 total characters, of which 439

were constant and 185 parsimony informative sites. Our ITS sequence analysis

Agaricus spp. new for Ethiopia ... 1175

supported the eight major Agaricus sections, and MP analyses confirmed the

placement of our six sequences in two well-defined sections (FIGURE 1).

A. xanthodermus KP229415

A. xanthodermus KP229414

A. xanthodermus AY899271

A. xanthodermus AY899272

A. xanthodermus DQ182534

A, xanthodermus DQ185563

A. xanthodermus DQ185564

$2 A. moelleri AY899263

A. moelleri AY899264

100 A. xanthosarcus JF514523

A. aff. endoxanthus JF691554

A. xanthodermulus KP229416

A. xanthodermulus KP229417

A, xanthodermulus AY899273

A, xanthodermulus AY899274

A. xanthodermulus AY899275

sect. Xanthodermatei

xanthodermulus AY899276

xanthodermulus NR 119528

— A, laskibarii AY943975

oP A, californicus DQ182510

99 | A. parvitigrinus AY899266

A. parvitigrinus AY 899267

A. caribaeus JF727856

A. campestris KP229419

A. campestris KP229420

A. campestris DQ182533

A. campestris FJ223222

A, campestris JX434655 sect. Agaricus

A. campestris KM609406

100, A. cupreobrunneus DQ18253

A, cupreobrunneus JQ8&24136

A. langei AY 484699

A. langei JF797181

A. fissuratus AY 484683

A. arvensis JF797194

A. augustus JF797193

A, viridopurpurascens JF514525

A, pseudolutosus JF727868

sect. Arvensis

100 A. brunneolus JF797203 at a =

A. aridicola JF797195

A. bresadolanus DQ185570 I sect. Spissicaules

100 A. silvaticus JF797178

L | L A. benesii JF797179 sect. Sanguinolenti

58 A. bohusii JF797180

89 A. bernardii AF432880 sect Chitonioides

A. gennadii JF797188

A. bitorquis AF432898 J sect. Bivelares

He. splendidissimus HM488760 4

Hy. ardosiicolor JF727840 ene

— 5 changes

FiGuRE 1. MP tree based on nrDNA ITS sequences. The species in bold were sequenced by the

authors. Bootstrap numbers greater than 50% are included above branches; analysis performed in

PAUP* using 1000 bootstrap replicates.

1176 ... Sitotaw & al.

Two sequences (KP229419, KP229420) fell within A. sect. Agaricus,

where they grouped with A. campestris sequences retrieved from GenBank

with 88% bootstrap support. The other four sequences fell within A. sect.

Xanthodermatei, two (KP229416, KP229417) grouping with A. xanthodermulus

with 94% bootstrap support for the clade (containing A. xanthodermulus and

A. laskibarii) and two (KP229414, KP229415) grouping with A. xanthodermus

with 83% bootstrap support for the clade (containing A. xanthodermus and

A. moelleri).

Taxonomy

Agaricus campestris L., Sp. Pl. 2: 1173 (1753). Fic. 2

Piteus 4-8 cm diam., at first subglobose to hemispherical, becoming convex,

broadly convex to plano-convex, sometimes slightly umbonate; surface white

to cream-color, sometimes slightly ochraceous, with grayish to pale ochraceous

fibrils or fibrillose scales, dry, sometimes silky; margin incurved at first, finally

expanding to decurved, with remnants of partial veil attached. LAMELLAE free,

<5 mm wide, initially pinkish, then pinkish brown to cinnamon-brown, finally

dark brown, crowded, with lamellulae. STIPE 3-8 x 0.5-1(-1.5) cm, central,

cylindrical or slightly tapering toward base; surface white and silky above

the annulus, white to brownish with whitish appressed fibrils below; solid to

soft, fibrous. CONTEXT white or whitish, fleshy, soft. ANNULUs thin, white,

membranous, superior. ODoR and TASTE pleasant. BASIDIOSPORE DEPOSIT

dark brown. CHEMICAL REACTION 20% KOH negative.

BASIDIOSPORES 5-8.5 x 3.5-5.5 um, avl x avw = 6.5 x 4.5 um, Q = 1.4-1.5,

avQ = 1.45, ellipsoid to ovoid, pale brown to brown, inamyloid, smooth, thick-

walled. Basip1A 18-24 x 6-8 um, clavate, 4-spored, thin-walled, subhyaline to

brownish. LAMELLA EDGE heterogeneous, with sterile cells, 14-22 x 5-7.5 um,

clavate, thin-walled, subhyaline to brownish. CHEILOCysTIDIA not found.

SUBHYMENIUM <10 um wide; hyphae narrow, branched, hyaline, thin-walled,

2-5 um diam. HYMENOPHORAL TRAMA regular, 80-120 um wide, hyphae

hyaline, thin-walled, 4.0-20 um diam. PILEIPELLIs a radially aligned repent

epicutis; hyphae 3.5-10 um diam., cylindrical, branched, hyaline to subhyaline,

thin-walled. PrILEus TRAMA hyphae 4.5-20 um diam., cylindrical to inflated,

hyaline, thin-walled. ANNULUs hyphae 3.5-8.5 um diam., cylindrical, hyaline

to subhyaline, thin-walled. CLAMP CONNECTIONS absent.

HasitTat: Scattered in pastureland.

SPECIMENS EXAMINED: ETHIOPIA, Oromia SpEcIAL ZONE, Wolmera District,

Holeta, on grazing land, 26 June 2012, RSK32 (HMAS 272461; GenBank KP229419);

Menagesha, Menagesha suba forest, 10 July 2013, RSK33 (HMAS 272462; GenBank

KP229420).

Agaricus spp. new for Ethiopia ... 1177

FIGURE 2. Agaricus campestris:

A. basidiomata (HMAS 272461); B. basidiomata (HMAS 272462);

C. basidiospores (HMAS 272461); D. cystidia (HMAS 272462).

REMARKS— Although A. campestris was first reported from Ethiopia by Abate

(1999), we have included this species to provide detailed morphological

and molecular data from Ethiopian specimens. As noted by Abate (1999),

A. campestris was very common on the grazing area of the highland plateau

of Ethiopia and we found it to be fairly common and widespread in the

grazing area (above 2300 m) right after the beginning of the rainy season. Our

A. campestris specimens share similar morphological traits of the A. campestris

complex: a white, subglobose to broadly convex pileus, initially pinkish then

pinkish brown and finally dark brown lamellae, a membranous white annulus,

a negative KOH reaction, and a mild taste and odor (Pegler 1977, Mitchell &

Walter 1999, Kerrigan et al. 2005).

Agaricus xanthodermulus Callac & Guinb., Mycologia 97: 421 (2005). FIG. 3

Piteus 5-7 cm diam., cuboidal at first, then conico-convex to broadly

convex, finally expanding to plano-convex, slightly umbonate at center; surface

1178 ... Sitotaw & al.

FIGURE 3. Agaricus xanthodermulus:

A. basidiomata (HMAS 272459); B. basidiomata (HMAS 272460);

C. basidiospores (HMAS 272459); D. basidia (HMAS 272460).

white to grayish, becoming yellow when rubbed, sometimes squamulate,

dry; margin incurved at first, then decurved. LAMELLAE free, <5 mm wide,

pinkish at first, then pinkish brown to brown, finally dark brown, crowded,

with lamellulae. Stipe 4-8 x 0.5-1 cm, central, cylindrical with slightly bulbous

base; surface white to whitish, becoming yellow when rubbed; solid at first,

then soft, finally hollow, and fibrous in old specimen. CONTEXT white or

whitish, becoming light yellow when cut, fleshy. ANNULUS white to whitish,

membranous, superior. BASIDIOSPORE DEPOSIT dark brown. TasTE and ODOR

unpleasant. CHEMICAL REACTION with 20% KOH positive.

BASIDIOSPORES 5-7 X 4-5.5 um, avl x avw = 6.0 x 4.7 um, Q = 1.25- 1.27,

avQ = 1.26 ellipsoid, pale brown to brown, inamyloid, smooth, thick-

walled. Bastp1a 18-27 x 6-8 um, clavate, 4-spored, thin-walled, subhyaline

to brownish. LAMELLA EDGE heterogeneous, with sterile cells, 15-24 x 5-7

um, clavate, thin-walled, subhyaline to brownish. CHEILOCYSTIDIA rare,

Agaricus spp. new for Ethiopia ... 1179

18-27 x 7-11 um, clavate to pyriform, thin-walled, hyaline. SuBHYMENIUM

<12 um wide; hyphae narrow, branched, hyaline, thin-walled, 3.5-5 um diam.

HYMENOPHORAL TRAMA regular, 80-120 um wide; hyphae hyaline, thin-

walled, 5-20 um diam. PILEIPELLIs a repent radially aligned epicutis; hyphae

thin-walled, 4-10 um diam., cylindrical, branched, hyaline to subhyaline.

PILEUS TRAMA hyphae 5-20 um diam., cylindrical to inflated, hyaline, thin-

walled. ANNULUS hyphae 4-9 um diam., cylindrical, hyaline to subhyaline,

thin-walled. Clamp CONNECTIONS absent.

Hasirat: Solitary, grazing land.

SPECIMENS EXAMINED: ETHIOPIA, Oromia SprEcIAL ZONE, Wolmera District,

Holeta, on grazing land, 10 July 2013, RSK12 (HMAS 272459; GenBank KP229416);

Menagesha, Menagesha suba forest, 10 July 2013, RSK12 (HMAS 272460; GenBank

KP229418).

REMARKS—Agaricus xanthodermulus, described recently by Callac &

Guinberteau (2005) from France, differs from other members in A. sect.

Xanthodermatei by its small size (maximum cap diameter 6 cm) and larger

spores (6.7-7.8 x 4.5-5.5 um). The Ethiopian material generally agreed with

the original description, although its slightly larger pileus and slightly smaller

spores brought it closer to the type specimen of A. xanthodermus (with a

6.0-12 cm diam. pileus and 4.0-5.5 x 3.0-4.0 um spores; Callac & Guinberteau

2005). Kerrigan et al. (2005) suggested that A. xanthodermulus might

represent a small form of A. xanthodermus. The ITS sequences show only two

nucleotide differences (at positions 120 and 537) between our collections and

A. xanthodermulus from France.

Agaricus xanthodermus Genev., Bull. Soc. Bot. Fr. 23: 32 (1876) FIG. 4

PILEus 6-12 cm diam., at first subglobose or hemispherical to campanulate

then expanding to convex or broadly convex to finally almost applanate; surface

at first whitish to grayish, then grayish to pale grayish brown and grayish brown

at center, becoming yellow when rubbed, with pale gray to grayish brown

fibrillose squamules, dry; margin incurved at first, then decurved, finally

sometimes undulating; with remnants of partial veil attached. LAMELLAE free,

<4 mm wide, at first white to pinkish, then pinkish brown to brown, finally

dark brown; crowded, with lamellulae. Stipe 8-13 x 0.5-1.3 cm, central,

cylindrical with bulbous base; surface white to whitish, and brownish at base,

becoming yellow when rubbed, with whitish to brownish fibrils at the lower

part; solid to soft, fibrous. CONTEXT white or whitish, becoming yellowish to

yellow when cut, fleshy, firm. ANNULUS white to whitish, thin, membranous,

superior. BASIDIOSPORE DEPOSIT chocolate brown to blackish brown. TASTE

and Opor unpleasant. CHEMICAL REACTION with 20% KOH positive.

1180 ... Sitotaw & al.

FIGURE 4. Agaricus xanthodermus (HMAS 272456):

A. basidiomata; B. basidiospores; C. cheilocystidia; D. basidia.

BASIDIOSPORES 4-5.5 x 3-4 um, avl x avw = 4.5 x 3.5 um, Q = 1.3-1.4,

avQ = 1.35 ellipsoid, pale brown to brown, smooth, thick-walled, inamyloid.

Basip1a 15-25 x 6-8 um, clavate, 4-spored, thin-walled, subhyaline to

brownish. LAMELLA EDGE heterogeneous, with sterile cells, 14-22 x 4-7 um,

clavate, thin-walled, subhyaline to brownish. CHEILOCYSTIDIA rare, 18-30 x

6-11 um, pyriform, thin-walled, hyaline. SuUBHYMENIUM <12 um wide; hyphae

narrow, branched, hyaline, thin-walled, 2.5-5 um diam. HyMENOPHORAL

TRAMA regular, 100-120 um wide; hyphae hyaline, thin-walled, 4-25 um

diam. PILEIPELLIS a radially aligned repent epicutis; hyphae thin-walled,

4.0-10 um diam., cylindrical, branched, hyaline to subhyaline. PILEUs TRAMA

hyphae 5-20 um diam., cylindrical to inflated, hyaline, thin-walled. ANNULUS

hyphae 4-10 um diam., cylindrical, hyaline to subhyaline, thin-walled. CLamp

CONNECTIONS absent.

HasiTatT: Growing in groups on grazing land and on soil with plant litter;

in semi-open middle-aged mixed forest.

SPECIMENS EXAMINED: ETHIOPIA, OROMIA SPECIAL ZONE, Wolmera District, Holeta,

on grazing land, 10 July 2013, RSK5 (HMAS 272456; GenBank KP229414); Menagesha,

Menagesha suba forest, 10 July 2013, RSK7 (HMAS 272457; GenBank KP229415).

Agaricus spp. new for Ethiopia ... 1181

REMARKS—Agaricus xanthodermus is characterized by its strong and rapid

yellow coloration when cut, phenolic odor, cylindrical stipe (often with a small

basal bulb), negative Schaffer reaction, and positive KOH reaction. Both macro-

and micro-morphological characters observed in the Ethiopian collections

were similar to those in the original description. The fungus was common

and abundant on grazing land and on litter in semi-open mixed forest in the

study sites starting from end of June to the beginning of August. However,

ITS sequence analyses showed the clade including the Ethiopian sequences

with GenBank sequences of A. xanthodermus and A. moelleri had only 83%

support (Fic. 1). Comparisons of ITS sequences between our collections and

A. xanthodermus from GenBank indicated three nucleotide differences (at

positions 334, 630, and 665). More extensive analyses may reveal the true link

among the sequences within the cluster.

Discussion

Morphological observation and molecular analysis placed our Ethiopian

collections within two sections, Agaricus sect. Agaricus and A. sect.

Xanthodermatei.

Agaricus sect. Xanthodermatei, represents a monophyletic clade comprising

species allied to the type species of the section, A. xanthodermus (Kerrigan

et al. 2005), known to produce one or more toxic compounds (Wood et al.

1998, Jovel et al. 1996) that can cause gastrointestinal distress leading to violent

vomiting. Species in the section are generally considered inedible or not fit

for human consumption. The toxicity experienced in Ethiopia after ingestion

is reflected by “Dem Astefy, the local Amharic name for these mushrooms

meaning ‘causes vomiting of blood:

In contrast, the type species of A. sect. Agaricus, A. campestris, is an

excellent edible mushroom and abundant at the beginning of the rainy season,

but the edibility of this mushroom is not recognized within the Ethiopian

community. This might be due to its similarity to the toxic species in A. sect.

Xanthodermatei. The confusability of the mushrooms could lead to mushroom

poisoning, leading the local community to shun all the mushrooms growing

in the wild and become mycophobic. This in spite of the fact that species of

A. sect Xanthodermatei can usually be distinguished from similar mushrooms

(like A. campestris) by their tendency to stain and bruise yellow and by the

characteristic phenolic odor.

The high Agaricus species diversity of Agaricus underscores the need for

further study of the genus; we hope that many more reports on Agaricus species

will follow.

1182 ... Sitotaw & al.

Acknowledgements

The authors are grateful to Prof. Anthony J.S. Whalley and Dr. Jie Chen for serving as

pre-submission reviewers and for their valuable comments and suggestions. The authors

would like to acknowledge the financial support of Addis Ababa University, Wollega

University, and Organization for Women in Science for Developing World (OWSD).

Rediet Sitotaw Kebede is a recipient of the OWSD postgraduate fellowship to study in

YJY’s laboratory for her PhD degree at the Institute of Microbiology, Chinese Academy

of Sciences.

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MY COTAXON

http://dx.doi.org/10.5248/130.1185

Volume 130, pp. 1185-1189 October-December 2015

Phoma candelariellae sp. nov.,

a lichenicolous fungus from Turkey

ZEKIYE KOCAKAYA’, MEHMET GOKHAN HALIcI” & MuSTAFA KOCAKAYA'

' Department of Organic Agriculture, Bogazlryan Vocational School, University of Bozok,

66400, Yozgat, Turkey

? University of Erciyes, Faculty of Science, Department of Biology, 38039, Kayseri, Turkey

* CORRESPONDENCE TO: mghalici@gmail.com

ABSTRACT — Phoma candelariellae is described from the apothecia of Candelariella aurella

in the Central Anatolia region of Turkey. The new species is distinguished among the

lichenicolous Phoma species by its clustered conidiomata arising in groups and its subglobose

conidia. It is also the first Phoma species reported on Candelariella.

Key worps — _ biodiversity, coelomycetes, mitosporic fungi, Phaeosphaeriaceae,

Dothideomycetes

Introduction

Phoma Sacc. is a large fungus genus with approximately 220 species, most

of which are plant pathogens, endophytes, or saprophytes (Boerema et al.

2004). Twenty-four species in this genus, however, are obligately lichenicolous

(Hawksworth 1981, Hawksworth & Cole 2004, Diederich et al. 2007,

Brackel 2008, Kondratyuk et al. 2010, Lawrey et al. 2012, Halici et al. 2014).

Keys to lichenicolous Phoma species have been provided by Hawksworth

(1981), Hawksworth & Cole (2004), and Diederich et al. (2007). Recent

molecular analyses place the genus in the Phaeosphaeriaceae, Pleosporales,

Dothideomycetes (Lawrey et al. 2012).

Halic1 (2008) published a key to the lichenicolous fungi of Turkey covering 117

taxa. Of the 190 lichenicolous species reported from Turkey (Halici et al. 2012,

2014), only three lichenicolous Phoma species have been reported: P. peltigerae

(P. Karst.) D. Hawksw. on Peltigera spp. (Candan et al. 2010; Halici et al. 2012);

P. caloplacae D. Hawksw. [= Diederichomyces caloplacae; Trakunyingcharoen et

al. 2014] on the apothecia of Caloplaca stillicidiorum (Hawksworth 1981); and

P recepii Halici & Candan on the apothecia of Caloplaca cerina and C. monacensis

1186 ... Kocakaya, Halici & Kocakaya

(Halici et al. 2014). A new species, described here as P. candelariellae, represents

the fourth lichenicolous Phoma species from Turkey.

Material & methods

The holotype and paratype specimens of the new species are deposited in Erciyes

University Herbarium Kayseri, Turkey (ERC). Sections were prepared by hand and

examined in I [Lugol’s iodine (MERCK 9261) with (KI) and without (I) pre-treatment

with 10% KOH], 10% KOH, cotton blue, and water. Conidia and pycnidia were measured

in water; measurements are presented as (min-) X-sd - X - X+sd (-max), where ‘min

and ‘max’ represent the extreme values, ‘X’ the arithmetic mean, and ‘sd’ the standard

deviation. The microphotographs were taken with a Leica DFC 420 digital microscope

camera with a c-mount interface and a 5 megapixel CCD.

Taxonomy

Phoma candelariellae Z. Kocakaya & Halici, sp. nov. FIGURE 1

MycoBAank MB 812271

Differs from all other lichenicolous Phoma species by its combination of clustered

conidiomata arising in groups, subglobose conidia, and host genus Candelariella.

Type: Turkey, Eskisehir, Sivrihisar, northwest of Sivrihisar, 39°27'16’N 31°31'36’E,

alt. 1260 m, on apothecia of Candelariella aurella (Hoftm.) Zahlbr., 22 July 2012, M.G.

Halic1 MGH3.274 (Holotype, EUH).

EryMo.ocy: The epithet refers to the host lichen genus Candelariella.

Conidiomata pycnidial, arising in groups on the apothecia of the host

lichen (Candelariella aurella), at first more or less immersed but at maturity

becoming partially erumpent, clustered, black in macroscopic view, in

section pale golden brown in lower parts and dark brown in the upper

parts, covered by a hyaline gelatinous sheath, subglobose to almost globose,

(72—)80—98.5-117(-129) x (69-)74-89-103(-111) um (n = 20), ostiolate,

ostiole c. 12 um diam.; pycnidial wall 9-14 um thick, composed of 2—5 layers of

pseudoparenchymatous cells, mainly polyhedral but some globose, outer cells

dark brown, about 4—6 x 6—7 um, inner ones paler, 4-7 um wide. Cells near

the ostiole have a darker brownish tinge. Conidiogenous cells lining the inner

wall of the pycnidial cavity, short ampulliform to subglobose, hyaline, smooth-

walled, 4-7 x 3-5 um, conidiogenesis enteroblastic. Conidia abundantly

produced, arising singly, subglobose to globose, hyaline, generally 1-guttulate,

sometimes one additional small guttule present, simple, smooth-walled,

with a gelatinous sheath, (3.5—)4.5-5—-5.5(-6) x (3—)3.5-3.9-4.5(—5.4) um;

I/b = (0.9-)1.1-1.3-1.5(-1.7) (n = 100).

ADDITIONAL SPECIMEN EXAMINED: TURKEY, Kitanya, Ilica, north of Yazlica Village,

39°36’41”N 30°03'03”E, alt. 1050 m, on apothecia of Candelariella aurella, 29 June 2012,

M.G. Halic1 MGH3.331 (EUH).

Phoma candelariellae sp. nov. (Turkey) ... 1187

Fic. 1. Phoma candelariellae (holotype, ERC MGH 3.274): A, infected apothecial discs of the

host lichen, Candelariella aurella, showing conidiomata arising in groups; B, conidiomata in the

hymenium of the host; C, closer view of a conidioma; D, conidia.

ECOLOGY & DISTRIBUTION: Phoma candelariellae is currently known from only

two localities in the northwest part of the Central Anatolia Region, Turkey, on

the apothecia of Candelariella aurella on calcareous rocks at an 1000-1300 m

elevation. As the host lichen occurs on calcareous rocks throughout the northern

hemisphere, P candelariellae may have a potentially much wider distribution.

The species seems to be pathogenic, as the infected apothecial discs of the host

discolour and become blackish; ascospore production is apparently inhibited

in the infected parts of the hymenium, until eventually the entire hymenium of

an infected apothecium is destroyed.

Notes: Phoma candelariellae is the only Phoma species known to grow on

Candelariella. Among the previously described lichenicolous Phoma species,

only P. caloplacae, P. lobariae Diederich & Etayo, P puncteliae Diederich

1188 ... Kocakaya, Halici & Kocakaya

TABLE 1. Comparison of lichenicolous Phoma species with subglobose conidia

CONIDIOMATA CONIDIOGENOUS CONIDIA REFERENCE

SPECIES

(um) CELLS (ttm) (um)

P. caloplacae 50-130 5-6 4-7 Hawksworth (1981)

P. candelariellae 72-129 x 69-111 4-7 x 3-5 3.5-6 x 3-5.4 This paper

P. lobariae 50-100 4-6 x 1.7-3.5 3-4 x 2.5-3 Etayo & Diederich

(1995)

P. physciicola 100-150 4-7 4-6 x 2.5-4 Hawksworth (1981)

P. puncteliae 40-60 2.5-5 x 3.5-4.5 2.5-3 x 2-2.4 Lawrey et al. (2012)

& Lawrey [= Xenophoma puncteliae; Trakunyingcharoen et al. 2014], and

P. physciicola Keissl. have subglobose conidia. These species all grow on

different lichen hosts and differ from P. candelariellae in various morphological

characters (TABLE 1). Phoma caloplacae differs from P. candelariellae by its

conidiomata arising singly in the host apothecia and its slightly larger conidia

(Hawksworth 1981, Halici et al. 2014). Phoma lobariae, described on the older

parts of the thallus of Lobaria pulmonaria, also has conidiomata arising singly

and slightly smaller conidia and conidiomata (Etayo & Diederich 1995). Phoma

physciicola, reported on Physciaceae, Parmeliaceae, and Baeomyces rufus,

produces conidiomata that sometimes arise in small groups on the apothecia

or thalli of the host lichens but is distinguished by its more ellipsoid conidia

(Keissler 1911, Hawksworth 1981, Diederich et al. 2007). Phoma puncteliae,

growing on the thalli of Punctelia rudecta, has obviously smaller conidia and

conidiomata (Lawrey et al. 2012).

Acknowledgements

The manuscript was reviewed by Kerry Knudsen (USA and Czech Republic) and

Wolfgang von Brackel (Germany). This study was financially supported by FDK-2014-

5259 coded Erciyes University project. The samples were collected during the project

supported by Tibitak (111T927 coded project).

Literature cited

Boerema GH, Gruyter J de, Noordeloos ME, Hamers MEC. 2004. Phoma identification manual.

Differentiation of specific and infra-specific taxa in culture. CABI Publishing, Wallingford, UK.

http://dx.doi.org/10.1079/9780851997438.0000

Brackel W von. 2008. Phoma ficuzzae sp. nov. and some other lichenicolous fungi from Sicily, Italy.

Sauteria 15: 103-120.

Candan M, Halic1 MG, Ozdemir Tiirk A. 2010. New records of peltigericolous fungi from Turkey.

Mycotaxon 111: 149-153. http://dx.doi.org/10.5248/111.149

Diederich P, Kocourkova J, Etayo J, Zhurbenko M. 2007. The lichenicolous Phoma species

(coelomycetes) on Cladonia. Lichenologist 39: 153-163.

http://dx.doi.org/10.1017/S0024282907006044

Phoma candelariellae sp. nov. (Turkey) ... 1189

Etayo J, Diederich P. 1995. Lichenicolous fungi from the western Pyrenees, France and Spain

I. New species of deuteromycetes. 205-221, in: FJA Daniels et al. (eds). Flechten Follmann:

contributions to lichenology in honour of Gerhard Follmann. Cologne.

Halici MG. 2008. A key to the lichenicolous Ascomycota (including mitosporic fungi) of Turkey.

Mycotaxon 104: 253-286.

Halici MG, Candan M, Ozdemir Tiirk A. 2012. A key to the peltigericolous fungi in Turkey.

Mycotaxon 119: 277-289. http://dx.doi.org/10.5248/119.277

Halici MG, Candan M, Giillii M, Ozcan A. 2014. Phoma recepii sp. nov. from the Caloplaca cerina

group in Turkey. Mycotaxon 129(1): 163-168. http://dx.doi.org/10.5248/129.163

Hawksworth DL. 1981. The lichenicolous coelomycetes. Bulletin of the British Museum (Natural

History), Botany 9. 98 p.

Hawksworth DL, Cole MS. 2004. Phoma fuliginosa sp. nov., from Caloplaca trachyphylla

in Nebraska, with a key to the known lichenicolous species. Lichenologist 36: 7-13.

http://dx.doi.org/10.1017/S0024282904013982

Keissler K von. 1911. Zwei neue Flechtenparasiten aus Steiermark. Hedwigia 50: 294-298.

Kondratyuk S, Karnefelt I, Goward T, Galloway D, Kudratov I, Lackovi¢ova A, Lisicka E, Guttova

A. 2010. Diagnoses of new taxa. 435-445, in: AM Oksner (ed.). Flora li8aynikiv Ukraini y

dvoch tomach, Tom 2, Vypusk 3. Kiiv, Naukovo Dumka.

Lawrey JD, Diederich P, Nelsen MP, Freebury C, Van den Broeck D, Sikaroodi M, Ertz D. 2012.

Phylogenetic placement of lichenicolous Phoma species in the Phaeosphaeriaceae (Pleosporales,

Dothideomycetes). Fungal Diversity 55: 195-213. http://dx.doi.org/10.1007/s13225-012-0166-9

Trakunyingcharoen T, Lombard L, Groenewald JZ, Cheewangkoon R, To-anun C, Alfenas AC,

Crous PW. 2014. Mycoparasitic species of Sphaerellopsis, and allied lichenicolous and other

genera. IMA Fungus. 5(2): 391-414. http://dx.doi.org/10.5598/imafungus.2014.05.02.05

MY COTAXON

http://dx.doi.org/10.5248/130.1191

Volume 130, pp. 1191-1202 October-December 2015

Neoalbatrellus subcaeruleoporus sp. nov. (Scutigeraceae)

from western North America

SERGE AUDET’” & BRIAN S. LUTHER?

'1264, Gaillard, Québec, G3] 1J9, Canada

? PSMS Office, Center for Urban Horticulture, University of Washington,

Box 354115, Seattle, Washington 98195, U.S.A.

* CORRESPONDING AUTHOR: serge.au@videotron.ca

AsstRACT —Neoalbatrellus subcaeruleoporus is described as a new species. Its systematic

position is supported by molecular and morphological analyses, and a key to the three

Neoalbatrellus species is provided. A revised key to American and European species of Scutiger

sensu lato (Albatrellopsis, Albatrellus, Laeticutis, Neoalbatrellus, Polypus, Polyporoletus,

Polyporopsis, Scutiger s.s., Xanthoporus, and Xeroceps) is appended.

Keyworops —Albatrellaceae, caeruleoporus, correct family, genetic, Russulales

Introduction

The genus Neoalbatrellus was proposed by Audet (2010) and is distinguished

from Albatrellus by the hymeniderm and the blue, black and brown colors of the

basidiomata. Two species have been placed in Neoalbatrellus: N. caeruleoporus

and N. yasudae (Audet 2010). Their trophic status is poorly understood

and specific ectomycorrhizal host trees have not been confirmed, although

N. caeruleoporus is often found under Tsuga (Gilbertson & Ryvarden 1986).

The presence of extracellular laccase has been reported in basidiomata of

N. caeruleoporus (Marr et al. 1986). The closest genus to Neoalbatrellus is

Albatrellopsis, based on molecular (ITS) and the dimeric meroterpenoid

pigment, grifolinone B (Zhou & Liu 2010). The correct family for these genera

is Scutigeraceae Bondartsev & Singer ex Singer 1969, a name that has priority

over Albatrellaceae Nuss 1980 (Audet 2010: 439).

In this paper a new species, Neoalbatrellus subcaeruleoporus, is described

from western North America. In the past it has been confused with

N. caeruleoporus due to similarity in basidioma color, but the two species differ

both morphologically and genetically.

1192 ... Audet & Luther

TABLE 1: Species names, vouchers, and GenBank accession numbers of the sequences

used in the phylogenetic analysis.

REVISED NAME DEPOSITED NAME VOUCHER ITS

i i : SEQUENCE

Albatrellopsis flettii Albatrellus flettii : MICH AHS82164 : AY621802

Neoalbatrellus caeruleoporus — : Albatrellus caeruleoporus : MICH : AY963565

: K.A. Harrison 8825

Neoalbatrellus Albatrellus caeruleoporus OSC 66097 ' KC985126

subcaeruleoporus

Neoalbatrellus : Albatrellus caeruleoporus ? DAVEP 28304 : KC862265

subcaeruleoporus

Neoalbatrellus yasudae Albatrellus yasudae HKAS 357766 (Zheng 2006)

Materials & methods

This study is based on collections of Neoalbatrellus from western and eastern North

America. The specimens were studied macroscopically, microscopically, and genetically.

Color terms for basidiomata beginning with a capital letter (e.g. Cadet Gray) follow

Ridgway (1912). Unless otherwise specified, the macroscopic description is based on

Luther's notes from fresh material

Microscopic features were described from dried specimens (with herbarium

acronyms following Thiers 2013). Basidiospores and basidia were observed using

bright field microscopy at a magnification of 1250x. Melzer’s reagent or IKI was used

for microscopic observations of basidiospores. Basidiospores were measured in profile

view with the extreme values indicated in parentheses. The number of measurements is

cited as n = x/y, indicating x measurements of spores from y specimens. 100 spores were

measured for all collections of N. subcaeruleoporus studied. The following abbreviations

are used: Q = the quotient of spore length and spore width in any one spore,

indicated as a range of variation in n spores measured; L_ = mean spore length (um),

W., = mean spore width (um), Q. = quotient of mean spore length and mean spore

width (L_/W,, ratio).

Hyphae, basidia, and spores were observed in cotton blue in lactic acid to verify the

presence of gloeoplerous hyphae and cyanophilous (CB+) elements. The cyanophilous

thin-walled hyphae appear blue in mass.

The protocols for DNA extraction and amplification of the ITS1+5.8S+ITS2 region

in N. subcaeruleoporus were done by R.S. Winder and M. Gordon, who provided two

subcaeruleoporus sequences. Winder slightly modified the method of Kranabetter et al.

(2013) by not soaking the sample overnight but by freezing a 50 mg portion of the sample

in liquid N and grinding in a sterile mortar and pestle before adding the CTAB buffer.

For PCR amplification, the universal primer pair ITS5/ITS4 was used (White et al. 1990;

Gardes & Bruns 1993); the PCR product was sequenced by the Genome Sequencing

and Genotyping Platform (CHUL Medical Research Centre (Quebec, Canada) using

capillary electrophoresis on an ABI 3730/XL Analyzer (Applied Biosystems). Gordon

extracted DNA from 50 mg of dried mushroom using the UltraClean DNA Isolation

Neoalbatrellus subcaeruleoporus sp. nov. (Canada & United States) ... 1193

A flettii AY621802

A. caeruleoporus AY963565 Ja

N. subcaeruleoporus KC985126

N. subcaeruleoporus KC862265

A. yasudae HKAS357766 Ic

0.03

FiGurRE 1: rDNA ITS1+5.8S+ITS2 strict consensus tree as generated using MrBayes (Huelsenbeck

& Ronquist 2001; 1,000,000 generations). A: Neoalbatrellus caeruleoporus; B: Neoalbatrellus

subcaeruleoporus; C: Neoalbatrellus yasudae; outgroup: Albatrellopsis flettii.

Kit (MoBio Laboratories, Inc.) and amplified the ITS region following a standard PCR

protocol using primers ITS1f and ITS4 with an anneal temperature of 58°C. The ITS

amplicon was sequenced at Eurofins MWG Operon (operon.com).

The sequences used in the phylogenetic analysis are presented in TaBLE 1. Albatrellus

flettii |= Albatrellopsis flettii] served as outgroup.

Taxonomy

The phylogeny inferred from the ribosomal ITS1-5.8S-ITS2 sequences from

three samples labeled Albatrellus caeruleoporus and one each of A. flettii and

A. yasudae is shown in Fic. 1. One A. caeruleoporus sequence (AY963565)

from eastern North America formed a separate clade (A). Two caeruleoporus

sequences (KC985126, KC862265) from British Columbia and California

formed a distinct and strongly supported clade (B). The sequence labeled

A. yasudae (HKAS357766) from China formed a third clade (C) separated

from the two A. caeruleoporus clades. The samples in clade B are considered

as representing an independent taxon, described here as Neoalbatrellus

subcaeruleoporus. We obtained nearly a complete ribosomal ITS sequence from

our N. subcaeruleoporus holotype and alignment supports it as a new species.

1194 ... Audet & Luther

Neoalbatrellus subcaeruleoporus Audet & B.S. Luther, sp. nov. FIGS 2A, 3

MycoBank MB801480

Differs from Neoalbatrellus caeruleoporus by its paler blue fresh basidiomata, its

gloeoplerous hyphae, its smaller basidia and basidiospores, and its western North

American distribution.

Type: USA. Washington state, Yakima Co., Bumping Lake Road, in William O. Douglas

Wilderness across the footbridge from Soda Springs Campground, a.s.l. 918 m, in

conifer duff and forest litter directly under Alnus sp., with Abies grandis and Pseudotsuga

menziesii nearby, 7 October 1995, coll. B.S. Luther 1995-107-1, det. S. Audet (Holotype,

WTU-F-50696).

EryMo_oey: from the bluish colors shared with Neoalbatrellus caeruleoporus.

PILEUs 3-6.5 cm wide x 3-5.5 mm thick, umbonate to convex, irregularly plane

or more frequently depressed, orbicular, flabelliform, dimidiate or reniform,

firm when fresh, drying hard and brown, black laccate, or greenish-brown and

sometimes resinous reddish in areas; surface irregular, dry, glabrous to finely

rugose or subtomentose; color a Pale Glaucous Blue at first, later Cadet Gray

to Light Mineral Gray with margin often paling to Pale Smoke Gray to Light

Olive Gray, when drying reddish undertones developing with ochraceous-

orange or orangish areas showing in pileal cracks or where damaged (rodent

bites, etc.); MARGIN uniform, convoluted or irregular and inrolled, with lobes

or undulations and splits (clefts), concolorous to generally much paler than

the pileus, with orangish or ochraceous tints in age, surface glabrous and soft

or breaking up into minute scales that are darker than the underlying tissue

and slightly roughened, with an appendiculate, sterile edge, when mature

prominently incurved or slightly involute and somewhat even; CONTEXT <1

cm thick, white at first, slowly Pale Ochraceous Buff along margins, then

OQOQQQD a

MHOQQQ,__*

FiGuRE 2. Basidiospores (Scale bar = 9.2 um).

A. Neoalbatrellus subcaeruleoporus (holotype). B. Neoalbatrellus caeruleoporus (isotype).

Neoalbatrellus subcaeruleoporus sp. nov. (Canada & United States) ... 1195

FTE TET ULL CAAA AI EUR MAPA LLP SALT OS HOU AY ULL UGE UEHUDUOCLNURUGSAGRH VL UQQUTVQGRO WLLL

be | "2 '5| la '5| '6| 7] 'B '9| Mola a2" a3 ala! i

Figure 3. Neoalbatrellus subcaeruleoporus (WTU-F-50695).

Fresh basiodiomes (one cut to show context).

Light Pinkish Cinnamon or Light Vinaceous Cinnamon; when dried cinnabar

necropigments light, becoming darker after extended storage; oDoR sweetly

fungoid or not distinctive; TASTE mild and slightly sweetish to slightly peppery.

Pores 1-3 per mm (3-6 per mm in dried OSC 66097), orbicular, elongate to

slightly angular, pruinose inside, with orange-pinkish necropigments; near the

stipe uniform, <1 mm wide, concolorous with or paler than the pileus, smaller

at the growing margin, uniformly pale bluish and with pale ochraceous or pale

orangish to rusty areas at maturity or when injured/bruised, very pale when

dry. Tuses decurrent, shallow, 1-2 mm deep, dissepiment walls paler (very

pale when dry), fimbriate or granulose. STIPE 3-6 x 1-2 cm, central to strongly

eccentric, tapering noticeably toward the base and widening/enlarging toward

the pileus, somewhat irregular to slightly twisted, curved or bent, surface

smooth to innately fibrillose streaked or with very fine scaly patches, generally

sub-cylindric or slightly flattened, fasciculate; surface color concolorous

with the pileus, paler and often with very slight pale pinkish overcast tones,

or becoming slightly darker or grayer toward the extreme base or below, but

paler and almost whitish near the base where starting to taper and beneath

where forest duff has adhered to the base, in age with orangish or ochraceous

tints; when dried resinous reddish-brown in some areas; context solid in cross-

section, pallid when freshly cut, turning pale pinkish very slowly.

1196 ... Audet & Luther

HYPHAL SYSTEM monomitic. PILEIPELLIS hymeniderm, hyphae in a tight

palisade covered with a brownish resin, narrowly clavate with ends sometimes

inflated to 7.1 um diam., refractive, hyaline to yellowish in IKI, inamyloid with

weakly cyanophilous walls. StrpITIPELLIs hyphae similar. CONTEXTUAL HYPHAE

2.8-11.3 um diam. (larger hyphae often ramified and with variable diameters),

simple-septate, hyaline, thin-walled, inamyloid, walls weakly cyanophilous;

gloeoplerous hyphae occasional, ramified and flexuous, refractive and pale

yellow in IKI, intense blue in cotton blue (CB+, cyanophilous). TRAMAL

HYPHAE 2.8-3.5 um diam., simple-septate, hyaline, thin-walled, inamyloid,

walls weakly cyanophilous, often flexuous and ramified, sometimes with

perpendicular branching before or near septa; gloeoplerous hyphae refractive

and yellow in IKI, intense blue (cyanophilous) in cotton blue. BAasrp1a 25-33

x 6-7 um, narrowly clavate, wall weakly cyanophilous, contents blue with

yellowish brown inclusions in cotton blue; sterigmata four, straight or slightly

curved, <4.8 um long. BAsip1osporEs amygdaliform, ellipsoid to subglobose,

hyaline, thin-walled, smooth, inamyloid, weakly to moderately cyanophilous,

sometimes with a large brownish-yellow guttule in cotton blue, with a relatively

long apiculus, (3.3—)3.6—4.7(-5.1) x (2.8-)2.9-3.7(-3.9) um, n = 100/4; L. x

W,, = 4 x 3.2 um; Q = 1.1-1.4(-1.5); Q. = 1.3; half of the spores with a flat to

slightly convex adaxial side.

MACROCHEMICAL REACTIONS on context of fresh pileus stipe context: KOH

(3%): Pale Pinkish Cinnamon to Light Pinkish Cinnamon, darkening the longer

it sits in the reagent. NH,OH (30%): very pale or similar to KOH reactivity.

FERRIC SULFATE: a distinctive reddish-brown exudate that completely darkens

the solution around the fresh sample. TINCTURE OF GUAIAC RESIN: —. PHENOL:

-. ANILINE: -. PDAB(4-dimethyl amino benzaldehyde): + (striking bright

turquoise after 5 minutes).

ADDITIONAL SPECIMENS EXAMINED (all as Albatrellus caeruleoporus): U.S.A.

WASHINGTON STATE: King Co., Lake Quinault, Rain Forest Mushroom Festival,

18 October 2008, Steve Trudell (WTU-F-13070); Pierce Co., Mt. Rainier National

Park, Carbon River area, 15 October 1987, B.S. Luther 1987-1015-1 (WTU-F-50684);

Skamania Co., Gifford Pinchot National Forest, Curly Creek Falls Trailhead, 5 October

2008, W. Foster SKAWAO001 (WTU-F-13071); Yakima Co., Bumping Lake Road,

William O. Douglas Wilderness from Soda Springs Campground, 1 October 1994,

B.S. Luther 1994-101-1 (WTU-F-50695); 23 September 1995, B.S. Luther 1995-923-1

(WTU-F-50681). OREGON: Lane Co., JM Honeyman Park, 12 November 1992, George

Barron (DAOM 215968); CALIFORNIA: Humboldt Co., Redwood National Park

(“Prairie Creek”), UTM N: 415268, UTM E: 4582445 zone 10, 12 December 1997, T.

O'Dell 4550 & M. Madsen (OSC 66097). CANADA. BRITISH COLUMBIA: (Mainland)

N of Clearwater, near Spahats, 10 September 1982, T. Goward (DAOM 194800); Mt.

Elphinstone, Roberts Creek, 29 September 2000, Paul Kroeger (DAOM 229493);

Vancouver Island, just west of Sooke, close to Juan de Fuca Strait, second growth, 28

Neoalbatrellus subcaeruleoporus sp. nov. (Canada & United States) ... 1197

October 2010, P. Hutchinson & K. Trim (DAVFP 28293); Sooke, Otter Point Road, 22

November 2011, K. Trim (DAVFP 28304).

EcoLocy & DIsTRIBUTION— Arising from deep in forest duff during mid-

September to mid-December. Associated with Abies grandis, Pseudotsuga

menziesii, Alnus sp., Picea sitchensis, Sequoia sempervirens, Tsuga heterophylla.

It fruited in an arc around the base of Alnus sp. but conifers were also close

by and mixed in. Rare in western North America in northwestern California,

central and southwestern British Columbia, western Oregon, and northwestern

and central Washington on the east slopes of the Cascades.

OTHER SPECIMENS EXAMINED: Neoalbatrellus caeruleoporus:s CANADA. QUEBEC:

Quebec, 1999, det. R. Labbé (QFB-8563); Richmond County, Kingsbury, 1984, det. R.

Cauchon (QFB-16558L); Beauce, Sainte-Clothilde, late August 2011, coll. M. Nadeau,

det. R. Labbé (WTU-F-50683); Nova Scotia: Annapolis Co., Bishop Mt. Road, 19

October 1969, coll. & det. K.A. Harrison (MICH 00073662; KAH 8825). U.S.A. NEw

YORK STATE: near Oneonta, 1963, det. S. Smith (QFB-199L,); Duchess County, Copake,

October, leg. & det. C.H. Peck (NY 00730549, isotype of Polyporus caeruleoporus).

REMARKS: Neoalbatrellus subcaeruleoporus is considered rare and has been listed

(as Albatrellus caeruleoporus) for protection and received special conservation

status in the United States (Molina 2008). Also as A. caeruleoporus, it has been

cited as a possible indicator of western old growth forests and considered in

forest management plans (Ginns 1997).

A detailed comparison of Neoalbatrellus subcaeruleoporus and N. caeruleoporus

is presented in TABLE 2.

The pores of N. subcaeruleoporus are somewhat smaller and orbicular

to angular with a thicker wall than those of N. caeruleoporus. Our

N. subcaeruleoporus basidiospore measurements are slightly smaller than those

given by Ginns (1997, as A. caeruleoporus). The dull colors (bluish-gray, with a

tinge of orange) not seen in the very young, actively growing basidiomata and

appearing only in fully mature specimens, are typical of basidiomata collected

late in the season.

It is interesting to note that N. yasudae is characterized by basidiospore

sizes intermediate (4-5.5 x 3.5-5 um; Zheng & Liu 2008) between

N. subcaeruleoporus (3.6-4.7 x 2.9-3.7 um; TABLE 2) and N. caeruleoporus

(4.6-5.6 x 3.8-4.3 um; TABLE 2) but shares with N. subcaeruleoporus a pileipellis

covered with a brownish resinous substance (as seen under the microscope)

and a pale hymenophore. These morphological distinctions are supported by

ITS sequence analysis.

Concerning macrochemical reactions, ferric sulfate in solution is not as

reactive as ferrous sulfate. The strong reaction of the N. subcaeruleoporus context

in ferric sulfate suggests that ferrous sulfate will likely have a stronger reaction

1198 ... Audet & Luther

TABLE 2: Comparison between Neoalbatrellus subcaeruleoporus

and N. caeruleoporus

CHARACTER N. subcaeruleoporus N. caeruleoporus

CAP, DRY Becoming brown or brown to Becoming brownish,

black, with tubes and context over : with the entire basidiome

time turning very slowly cinnabar : eventually often turning

orange : cinnabar orange

CAP, FRESH : <6 cm in diameter : $15 cm in diameter '

Light blue to pale blue, often Grayish blue to violaceous,

glaucous with reddish orange- : in age tan to light ochraceous

reas where damaged, with age, : brown?

or in cracks

CONTEXT, IN HERBARIUM : White to pale orange (when : Typically pale orange

donne nr ieee ee ee Bae a oae este Cee et Oar a)

CONTEXT, FRESH : White to pale wood brown; slowly: White ?

: discoloring afterafewhoursin — :

open air to pale ochraceous buff,

light pinkish cinnamon, or light

vinaceous cinnamon

: 2-5 mm long '

TUBES, FRESH Pye a os ghee enh me on. tel TE Eee SY

: Concolorous with the cap or paler, : Grayish blue ?

: becoming orangish or rusty

irregularly or where damaged

: Without lateral stipe With lateral stipe when young

STIPE, FRESH SN Ne ME ZAG, LS eee er a eM 3A. Aceh eet ened Ee Eee,

Concolorous with the pileus or Deep indigo-blue !

paler; context bruising orange-

brown

KOH ON CONTEXT Pale or light pinkish cinnamon, Not discoloring (2%) ”

darkening over time (3%)

TINCTURE OF GUAIAC RESIN _ Negative : Slowly positive blue reaction (230

(ON CONTEXT) i : minutes) when tincture replenished

SYRINGALDAZINE (laccase test) Not tested : Reddish (positive: 0.5/4) *

GLOEOPLEROUS HYPHAE Scattered in context (trama) * : Not seen ?

aa, i Ruane asin Pane eater tage = tances ta cate ae eS at 8

“BASIDIOSPORE APICULUS "Relatively long, tapered? “Small, but obvious?

ek aes a Nis) AN SRC ry Crna tieek india hae a ee a ea a ee tae We ee

3.6-4.7x2.9-37um, -4.6-5.6 x 3.8-4.3 um,

: n= 100/4 : n=40/2

i (DAVFP 28293; OSC 66097; : (NY 00730549;

WTU-E-50696; WTU-E-50684) : MICH 00073662)

' Overholts (1953); * Ginns (1994); > Ginns (1997, as Albatrellus caeruleoporus); * Marr et al. (1986).

Neoalbatrellus subcaeruleoporus sp. nov. (Canada & United States) ... 1199

on N. subcaeruleoporus than on N. caeruleoporus. It is interesting to note that

N. caeruleoporus gives a slow positive reaction with an alcoholic solution of

guaiac resin, but N. subcaeruleoporus does not. The positive reaction with this

reagent simply shows the presence of extracellular phenol-oxidases (tyrosinase,

laccase, and peroxidase) and is not specific for a particular enzyme (Gilbertson

et al. 1975). This reaction is consistent with a study by Marr et al. (1986) who

show a positive laccase reaction with syringaldazine for N. caeruleoporus.

Key to species of Neoalbatrellus

1. Pileipellis with densely agglutinated pyriform cells ................ N. yasudae

Pileipellis without densely agglutinated pyriform cells ...................0.. 2

2. Basidioma pale blue; western North American distribution;

Spotesd.0- 4a OSS ain eee Te Ge Sate bee hts N. subcaeruleoporus

Basidioma blue; eastern North American distribution;

spores 4.6-5.6 X 3.8-4.3 UM ..... eee cece eee eee ee N. caeruleoporus

Summary

The genus Neoalbatrellus has an additional species, N. subcaeruleoporus. It

is separated from N. caeruleoporus by its pale blue basidioma, shorter basidia

and basidiospores, lack of a phenol-oxidase reaction, and a western North

American distribution, as well as by its DNA sequences.

Acknowledgements

We thank and express our gratitude to the following individuals or herbaria for

contributing to this study: the staff from Louis-Marie Herbarium at Laval University

(Québec, Canada) for significant assistance (loans, material support, and more); the

curators of NY, QFB, MICH, WTU, OSC, DAVFP, and DAOM for loans or photos of

specimens; new genetic sequences from Dr. Richard S. Winder (and his technician,

Grace Ross) and Mr. Matthew Gordon, useful information from Huan-Di Zheng’s

doctoral thesis (2006) provided by Fugiang (Michael) Yu (Kunming Institute of

Botany, China); Jean Bérubé for help in extracting and aligning DNA from the

N. subcaeruleoporus holotype; Huan-Di Zheng, Yu-Cheng Dai, Lorelei Norvell, Joseph F.

Ammnirati, Jim Ginns, Christian Schwarz, Pierre-Arthur Moreau, and Roland Labbé for

information or documentation; Kevin Trim for giving his N. subcaeruleoporus collection

for DNA extraction; and Michel Nadeau and Roland Labbé (Quebec) for supplying fresh

N. caeruleoporus collections. Finally, we want to thank Jim Ginns (Penticton, British

Columbia) and Joseph F Ammirati (Seattle, Washington) for providing constructive

reviews.

1200 ... Audet & Luther

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Albatrellus, Polyporoletus, Scutiger et description de six nouveaux genres. Mycotaxon 111:

431-464. http://dx.doi.org/10.5248/111.431

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

to the identification of mycorrhizae and rusts. Molecular Ecology 2: 113-118.

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

Gilbertson RL, Ryvarden L. 1986. North American polypores. Fungiflora: Oslo (Norway). 435 p.

Gilbertson RL, Lombard FF, Canfield ER. 1975. Gum guaiac in field tests for extracellular phenol

oxidases of wood-rotting fungi and other basidiomycetes. Madison, Wis.: U.S. Dept. of

Agriculture, Forest Service, Forest Products Laboratory. 23 p.

Ginns J. 1994. Albatrellus (Fungi: Basidiomycota) in Michigan. Michigan Botanist 33: 75-90.

Ginns J. 1997. The taxonomy and distribution of rare or uncommon species of Albatrellus in

western North America. Canadian Journal of Botany 75: 261-273.

Huelsenbeck JP, Ronquist FR. 2001. MrBayes: Bayesian inference of phylogeny. Biometrics 17:

754-755.

Kranabetter JM, de Montigny L, Ross G. 2013. Effectiveness of green-tree retention in the

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Marr CD, Grund DW, Harrison KA. 1986. The taxonomic potential of laccase and tyrosinase spot

tests. Mycologia 78: 169-184. http://dx.doi.org/10.2307/3793162

Molina R. 2008. Protecting rare, little known, old-growth forest associated fungi in the Pacific

Northwest USA: a case study in fungal conservation. Mycological Research 112: 613-638.

http://dx.doi.org/10.1016/j.mycres.2007.12.005

Overholts LO. 1953. The Polyporaceae of the United States, Alaska and Canada. Ann Arbor.

University of Michigan Press. 466 p.

Ridgway R. 1912. Color standards and color nomenclature. Washington, D.C., published privately

(by the author). 43 p. + 53 color pls.

Thiers B. [continuously updated]. Index Herbariorum: A global directory of public herbaria and

associated staff. New York Botanical Garden's Virtual Herbarium.

http://sweetgum.nybg.org/ih/ [accessed February 2013].

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

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

Methods and Applications. Academic Press, Inc., New York.

Zheng HD. 2006. Studies on the taxonomy and phylogeny of Albatrellaceae Nuss (Polyporales,

Basidiomycetes, Basidiomycota) (in Chinese). PhD dissertation, Department of Biogeography

& Ecology, Kunming Institute of Botany, the Chinese Academy of Sciences, Kunming, Yunnan,

PR. China. 220 p.

Zheng HD, Liu PG. 2008. Additions to our knowledge of the genus Albatrellus (Basidiomycota) in

China. Fungal Diversity 32: 157-170.

Zhou ZY, Liu JK. 2010. Pigments of fungi (macromycetes). Natural Product Reports 27(11):

1531-1570. http://dx.doi.org/10.1039/c004593d

Neoalbatrellus subcaeruleoporus sp. nov. (Canada & United States) ... 1201

Appendix 1. Key to American and European species of Scutiger s. lat.

10.

BE:

13.

14.

(From Audet 2010; revised by Serge Audet & Georges Fannechere)

Clamp connections on contextual generative hyphae numerous .............. 2

Clamp connections on contextual generative hyphae absent or very rare ..... 15

Spores SIP (S14) uimlong? 25 obey a tee yh ee eee cnn ey eeinny tinge ee eee as 3

spores stiallemorequalto: 7 wir TON os, 6. os 5 <0 tse aan on ate nh mk aptate ae ageees 10

Spares without goule wall, tin. sa a. babs prake a Ws reed a be brat te prada groeea Ms greet te oes 4

Spores-with-double wall nc 32 echo eh sie h sete) etre S wie bh nines Recetas Steg 7

Spores not primarily tear-drop shaped io wae was Denes ease aes eae 5

Spores: primarily teat-drop shaped: ...<0. 4 .tes4 ens tmae eins et tines Ble nde na 6

Pileus brown (fresh) or dark violet (dried) ................ Xeroceps skamania

Pileus yellow (fresh) or pinkish white to pale red (dried) .... Xeroceps yunnanensis

Pileus greenish olive to milk chocolate brown ................. Scutiger ellisii

Pilewsiclarke br wars. tS 28 rah ek rs OR oe oe te ce Ph ark il Scutiger pes-caprae

Fiyphalisystern Gimitice 2 Ac ctait ast cit attin deena toesd aces Polyporopsis mexicana

Ebyphalesy stent fAOMONIEIG stots. c. ata. oe nea ae tore ene ae eee hea. espns 8

Spores .¢..9,6—12,6 wmediams 25.2.5 ae wee as" Polyporoletus sublividus s. lat.

SROPeR CAs 2 IS Iiiara tit sree Soke te ees LeRoy Wee Wey Sera a LED 9

Basidiomes solitary and with bulbous stipe ............ Polyporoletus bulbosus

Basidiomes caespitose and with no bulbous stipe....... Polyporoletus sylvestris

Hymenophore entirely yellow when young ............. 00. s eee cence eee 11

Hymenophore not entirely yellow when young .....................00 000 12

Some thick-walled hyphae (often >0.5 tum) near the surface from stipe base;

spores small (3.5—4.5 x 2.5-3 um); pores small (4-6 per mm)

4 paptcreed o Bow tex Mere o Dito Ms co Mew coc Scr « Nacatins): Lysol Xanthoporus peckianus

No thick-walled hyphae near the surface from stipe base;

spores larger (4—5.6 x 3—4 tum); pores larger (3-5 per

DUEELE) pee Mey 2m Slee A ge et eR ee Re Ee ee Xanthoporus syringae

Spores elongated to cylindrical, 4.8-6.4 x 2-2.5um ........ Albatrellus pilosus

Sporesmotewnd real ki AM OM Bt AN taal Nace Neal Att Meee Nee ae: 13

Spinose hyphae present on the mycelium and covering the stipe base;

Sporeswe ak bat lOle. 16 hata Ma a eieleton snatore wnt oa Aaa ee eae ema ego 14

WNotspinosé-hyphae; sporesamyloidorgote\ iar Sas yaa eae eke te 15

Pileus variably colored when fresh (chamois, apricot, rarely brownish) but not

TRG 8 here Ba Ba hb Rca coto 8 tawicese ecw? ewan « Neowin Albatrellopsis confluens

Pileus partly or entirely blue when fresh................... Albatrellopsis flettii

1202 ... Audet & Luther

boy

16.

18.

19;

20.

ZI,

23,

Pileussurtace laccates oa 8 eat a ee ae i ie ae Albatrellus arizonicus

Pies sutiece non laiceate < ey oe hey ce key cers oe ey chee one ens eee has 16

Pileus with black squamules and resinous dark zone

betweetitti bes andicOntext. 2. e es ate ee te ae Albatrellus tianschanicus

Prleusswithout black’ squat less sek, Ss cecats fe pacts cporscete fore deere blo ered Ege ae Em 17

Basidiome with numerous petaloid pilei from common base;

pileus coldentwirer Tesh ss!) TRF Rls net BR gr lier grid Polypus dispansus

Basidiome with solitary pileus or confluent.............. 0.0... e eee eee eee 18

Majotity.of spores longer than 5.5 qMy 058. boa tom ied. Laeticutis cristata

Majorityrotsporesshortetet hain 5,5: Utvie yb Ree eed or RP ee RRS po ARS TERS 19

With hymeniderm; basidiome blueswhenmtteshs 3 tek ee es 20

Without hymeniderm; basidiome not blue when fresh ...................4. 21

Occurring in western North America;

spores 3.6-4.7 X 2.9-3.7 UM «2... cx cence ee Neoalbatrellus subcaeruleoporus

Occurring in eastern North America;

spores 4.6—-5.6 X 3.8-4.3 pm .....-.. 200. enue Neoalbatrellus caeruleoporus

Spores inamyloid; associated with Tsuga, Picea, or Abies ................04 22

Spores distinctly amyloid; associated with especially Pinus or Picea .......... 23

Spores 5-5.6 x 3.6—4.2 um; under conifers, especially Tsuga and Picea;

on west coast of North America...................00. Albatrellus avellaneus

Spores 4.0-4.8 x 3.2—3.8 tum; under conifers, especially Picea and Abies;

WAS CEST Ce tacce iw cine Basie Reldos ash dll sme ls ex eedlsenn Albatrellus ovinus

Under Pinus; basidiome yellow or orange where bruised or with age;

taste bitter Or mild svi h cavet cavet.ca vets yeh se veins Albatrellus subrubescens

Under Picea; basidiome yellow where bruised; taste mild .... Albatrellus citrinus

MY COTAXON

http://dx.doi.org/10.5248/130.1203

Volume 130, pp. 1203-1208 October-December 2015

New Acrocordia and Candelariella records for Turkey

ZEKIYE KOCAKAYA! & MEHMET GOKHAN HALICI”

‘Department of Organic Agriculture, Bogazliyan Vocational School, University of Bozok,

66400 Yozgat, Turkey

University of Erciyes, Faculty of Science, Department of Biology, 38039 Kayseri, Turkey

* CORRESPONDENCE TO: mghalici@gmail.com

ABSTRACT — Acrocordia subglobosa, Candelariella faginea, and C. subdeflexa are reported for

the first time from Turkey. Comments on habitats, substrata, and key anatomical features are

provided for each species.

Key worps — Ascomycota, lichens, lichenized fungi, biodiversity

Introduction

Turkish lichenology has a relatively short history compared to Europe and

America. In the last 30 years, however, numerous papers have been published,

including checklists of several regions in Turkey (e.g., John 2007, Halici &

Aksoy 2009, Kinalioglu 2010a, Senkardesler 2011, Yazici et al. 2013, Kocakaya

et al. 2014). About 1300 species of lichenized fungi are known from Turkey,

but unfortunately there is no checklist of the whole country. At least 2000

lichenized fungi are expected to occur in the country, considering its size,

phytogeographical diversity, and different habitats (Halici et al. 2007). It is clear

that the lichen biota is still far from being completely known in Turkey and that

taxonomic studies focusing on genera or species complexes are needed.

The genus Candelariella includes species with 8- or polyspored asci of the

Lecanora-type, biatorine or lecanorine apothecia, and a secondary chemistry

with pulvinic acid and derivatives (Westberg 2005). Approximately 50

Candelariella species are known worldwide (Westberg 2004), and in Turkey

15 species—C. aggregata M. Westb., C. antennaria Rasanen, C. athallina

(Wedd.) Du Rietz, C. aurella (Hoffm.) Zahlbr., C. coralliza (Nyl.) H. Magn., C.

kuusamoensis Rasanen, C. lutella (Vain.) Rasanen, C. medians (Nyl.) A.L. Sm.,

C. plumbea Poelt & Vézda, C. reflexa (Nyl.) Lettau, C. rosulans (Mull. Arg.)

1204 ... Kocakaya & Halici

Zahlbr., C. unilocularis (Elenkin) Nimis, C. viae-lacteae G. Thor & V. Wirth,

C. vitellina (Hoftm.) Mull. Arg., C. xanthostigma (Pers. ex Ach.) Lettau—have

been reported (Breuss & John 2004; Candan & Tiirk 2008; Giiveng et al. 2006;

Halici & Aksoy 2009; Halici & Gtiveng 2008; Halici et al. 2013, 2014; Yazici et

al. 2012). Here we report two new Candelariella species for Turkey.

The genus Acrocordia is characterized by cylindrical asci with an apparently

unique apical structure and by uniseriate ellipsoid ascospores with a warted

perispore (Smith et al. 2009). Five species—Acrocordia cavata (Ach.) R.C.

Harris, A. conoidea (Fr.) Korb., A. gemmata (Ach.) A. Massal., A. macrospora

A. Massal., A. salweyi (Nyl.) A.L. Sm.)—have been recorded in Turkey (Dogru

& Giiven¢g 2007, Giiveng et al. 2006, Kinalioglu 2010b, Ozdemir Tiirk &

Giiner 1998, Yazici & Aslan 2006, Yazici et al. 2007). Here we report one new

Acrocordia species for Turkey.

Material & methods

The newly recorded specimens are deposited in Erciyes University Herbarium

Kayseri, Turkey (EUH). They were examined by standard microscopic techniques.

Hand-cut sections were studied in water, potassium hydroxide (KOH), and Lugol’s

solution (I). Measurements were made in water. Ascospores were measured from five

different ascoma for each species. The measurements are given as minimum-—maximum

from N measurements. The descriptions summarized below for each species are based

the Turkish specimens.

Taxonomy

Acrocordia subglobosa (Vézda) Poelt & Vézda, Biblioth. Lichenol. 9: 11 (1977)

FIGS 1A,B

A detailed description is provided by Poelt & Vézda (1977).

Thallus immersed to poorly developed, whitish. Perithecia 0.3-0.7 mm

diam. (N = 10); involucrellum incurved around the exciple and continuous

below, black, globose. Asci cylindrical, 120-156 x 10-14 um. (N = 15),

8-spored, ascospores uniseriately arranged in the asci. Hamathecium of

pseudoparaphyses that are not appreciably widened at the tips, 1.2-1.4 um

diam. Ascospores colourless, simple, 1-septate and with a warted perispore,

broadly ellipsoid, 12-18 x 7—8.5 um (N = 20).

SPECIMEN EXAMINED: TURKEY, ZONGULDAK, Kozlu, North of Eregli-Akcakoca

highway, 41°25’26’N 31°43’11’E, alt. 0-10 m, coastal rocks, 27 July 2012, M.G. Halici

( ERC MGH 4652).

The specimen was collected from coastal calcareous rocks in Turkey’s western

Black Sea Region; its ascospores are slightly longer and wider than those cited

in the literature (Poelt & Vézda 1977, Smith et al. 2009, Pykala 2008).

Acrocordia & Candelariella spp. new for Turkey... 1205

ae SI MO

Aa > + / F _

MS MPAA, oe +

Sa A fal, se

eS y ¥ | #” A .

pe ; ex A nF 1% ;

Fic. 1. Acrocordia subglobosa: A, thallus; B, ascus, ascospores, and pseudoparaphyses. Candelariella

faginea: C, thallus; D, paraphyses and polyspored asci. Candelariella subdeflexa: E, thallus;

F, paraphyses and 8-spored asci.

1206 ... Kocakaya & Halici

Acrocordia subglobosa is very similar to A. salweyi, differing mainly in

its smaller perithecia and ascospores (Smith et al. 2009), and is known on

calcareous rocks at 500 m from Scotland, Central Europe, Finland, and Ukraine

(Smerechynska 2005, Pykala 2008, Smith et al. 2009). New to Turkey.

Candelariella faginea Nimis, Poelt & Puntillo, Nova Hedwigia 49: 276 (1989)

FIGS 1C,D

A detailed description is provided by Nimis et al. (1989).

Thallus areolate to subsquamulose; areoles grayish green in the center but

yellowish green towards the margins; with blastidia. Apothecia common,

lecanorine, disc greenish yellow and margin yellow, 0.2—0.4 mm diam. (N = 8).

Hymenium colorless, 70-80 um tall. Paraphyses simple. Asci clavate,

polyspored, 65-80 x 20-29 um (N = 10). Ascospores simple, narrowly ellipsoid,

13-16 x 4-6 um (N = 15). Spot tests K—, KC-, C-.

SPECIMEN EXAMINED: TURKEY, Izmir, Bornova, northeast of Sancakhi, 38°31/13”N

27°11'35’E, alt. 700 m, on bark of Quercus sp., 30 June 2012, M.G. Halici (ERC CAN

0,212),

Candelariella faginea has hitherto been reported on bark of deciduous trees

(usually Fagus). The Turkish specimen was collected on Quercus bark in

the Aegean part of Turkey where a Mediterranean climate prevails. It was

accompanied by Caloplaca monacensis (Leder.) Lettau, Lecidella elaeochroma

(Ach.) M. Choisy, Lecanora hagenii (Ach.) Ach., Physconia sp., and Rinodina sp.

Candelariella faginea differs from C. xanthostigma by its blastidiate thallus

and areoles that are almost squamulose. Candelariella coralliza differs from

C. faginea by its thallus composed of coralloid cylindrical granules forming

compact cushions and also by substrate and general ecology (Nimis et al. 1989).

This species is mainly known from southern Europe (Italy, Spain, Greece)

and Ukraine (Aragon & Martinez 2002, Khodosovtsev 2005, Spribille et al.

2006). New to Turkey.

Candelariella subdeflexa (Nyl.) Lettau, Hedwigia 52: 196 (1912) FIGS 1E,F

A detailed description is provided by Westberg (2007).

Thallus squamulose, distinct, surface dirty gray, smooth. Apothecia

biatorine, scattered, pale yellow to greenish yellow, 0.3—0.5 mm diam. (N = 10).

Hymenium colorless, 50—60 um tall. Paraphyses simple. Asci clavate, 8-spored,

38-45 x 13-19 um (N = 15). Ascospores simple, narrowly ellipsoid, 9-4 x 4-5.5

um (N = 20). Spot tests K—, KC-, C-.

SPECIMEN EXAMINED: TURKEY, Sivas, Gemerek, Sizir, 39°18’30"N 35°59’ 25”E, alt.

1250 m, on bark of Quercus sp., 3 October 2010, M.G. Halici & M. Kocakaya (ERC CAN

0.049).

Acrocordia & Candelariella spp. new for Turkey ... 1207

The Turkish specimen was collected from Central Anatolia on the bark of

Quercus. According to Westberg (2007), C. subdeflexa is a corticolous species

growing mainly on the trunks of various deciduous trees such as Populus,

Quercus, Fraxinus, and Juniperus and is characterized by its biatorine apothecia

and gray squamulose thallus. The Turkish specimen agrees well with the

description given in Westberg (2007).

This species has a bipolar distribution and is known from North America,

Europe, North Africa, and New Zealand (Poelt & Vézda 1977; Nimis 1993;

Galloway & Hafellner 2002; Westberg 2004, 2007; Westberg & Clerc 2012).

New to Turkey.

Acknowledgements

The manuscript was reviewed prior to submission by Lidia Yakovchenko (Russia)

and Martin Westberg (Sweden). Alexander Khodosovtsev (Ukraine) helped us identify

Candelariella faginea. This study was financially supported by FDK-2014-5259 coded

Erciyes University project.

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Lichenologist 34: 81-88. http://dx.doi.org/10.1006/lich.2001.0357

Breuss O, John V. 2004. New and interesting records of lichens from Turkey. Osterreichische

Zeitschrift fir Pilzkunde 13: 281-294.

Candan M, Turk A. 2008. Lichens of Malatya, Elazig and Adiyaman provinces (Turkey). Mycotaxon

105: 19=22;

Dogru Z, Giiveng $. 2007. Lichenized and lichenicolous fungi from Bursa province new to Turkey.

Mycotaxon 102: 389-394.

Galloway DJ, Hafellner J. 2002. Additional lichen records from New Zealand 37, Candelariella

subdeflexa (Nyl.) Lettau. Australasian Lichenology 51: 33-35.

Giiveng $, Oztiirk S, Aydin S. 2006. Contributions to the lichen flora of Kastamonu and Sinop

Provinces in Turkey. Nova Hedwigia 83: 67-98.

http://dx.doi.org/10.1127/0029-5035/2006/0083-0067.

Halici MG, Aksoy A. 2009. Lichenized and lichenicolous fungi of Aladaglar National Park (Nigde,

Kayseri, Adana) in Turkey. Turkish Journal of Botany 33: 169-189.

Halici MG, Giiveng $. 2008. Lichens from Mediterranean Region of Turkey. Cryptogamie,

Mycologie 29: 95-106.

Halic1 MG, Hawksworth D, Aksoy A. 2007. Contributions to lichenized and lichenicolous fungal

biota of Turkey. Mycotaxon 102: 403-414.

Halici1 MG, Kocakaya M, Kilig E. 2013. New Candelariella records for Turkey. Mycotaxon 121:

313-318. http://dx.doi.org/10.5248/121.313

Halici MG, Kocakaya M, Kirts Z. 2014. Lichenized and lichenicolous fungi of Bakirdag (Kayseri,

Adana). Acta Botanica Hungarica 56(3-4): 319-332.

http://dx.doi.org/10.1556/ABot.56.2014.3-4.8

John V. 2007. Lichenological studies in Turkey and their relevance to environmental interpretation.

Bocconea 21: 85-93.

1208 ... Kocakaya & Halici

Khodosovtsev AE. 2005. Pog Candelariella (Candelariaceae, Lecanorales). (Ora Ykpauupi. Hopoctu

Cucrematuxn Husuimx Pacrenun, Tom 39.

Kinalioglu K. 2010a. New and interesting records of lichens from Turkey. Mycotaxon 114: 85-90.

http://dx.doi.org/10.5248/114.85

Kinalioglu K. 2010b. Epiphytic and saxicolous lichens of the District Centre of Arakli and

in vicinity (Trabzon, Turkey). Pakistan Journal of Biological Sciences 13: 588-595.

http://dx.doi.org/10.3923/pjbs.2010.588.595

Kocakaya M, Halici MG, Aksoy A. 2014. Lichenized and lichenicolous fungi of Gevne Valley

(Konya, Antalya). Turkish Journal of Botany 38: 358-369.

http://dx.doi.org/10.3906/bot- 1303-29

Nimis PL. 1993. The lichens of Italy—an annotated catalogue. Museo Regionale di Scienze Naturali

Monografie 12. 897 p.

Nimis PL, Poelt J, Puntillo D. 1989. Candelariella faginea spec. nov. (Lichenes, Candelariaceae)

eine bemerkenswerte neue Art einer schwierigen Gattung aus Siideuropa. Nova Hedwigia

49 (3-4): 274-480.

Ozdemir Tiirk A, Giiner H. 1998. Lichens of the Thrace Region of Turkey. Turkish Journal of

Botany 22: 397-407.

Poelt J, Vezda A. 1977. Bestimmungsschliissel europadischer Flechten. Erganzungsheft I. Bibliotheca

Lichenologica 9. 258 p.

Pykala J. 2008. Additions to the lichen flora of Finland. II. Graphis Scripta 20: 19-27.

Senkardesler A. 2011. Das die Turkei betreffende lichenologische Schrittum ic¢in ek kayitlar. Turk

Liken Toplulugu Biilteni 9: 9-10.

Smerechynska TO. 2005. New for Ukraine lichen species from the Medobory Nature Reserve.

Ukrainian Botanical Journal 62: 719-725.

Smith CW, Aptroot A, Coppins BJ, Fletcher A, Gilbert OL, James PW, Wolseley PA. 2009. The

lichens of Great Britain and Ireland. The British Lichen Society, London.

Spribille T, Schultz M, Breuss O, Bergmeier E. 2006. Notes on the lichens and lichenicolous fungi

of western Crete (Greece). Herzogia 19: 125-148.

Westberg M. 2004. Candelariella. 46-53, in: TH Nash et al. (eds.), Lichen Flora of the Greater

Sonoran Desert Region, Vol. 2. Lichens Unlimited, Tempe, Arizona.

Westberg M. 2005. The lichen genus Candelariella in western North America, Doctoral thesis,

Lund University. S6dra Sandby.

Westberg M. 2007. Candelariella (Candelariaceae) in western United States and northern

Mexico: the species with biatorine apothecia, Bryologist 110: 365-374.

http://dx.doi.org/10.1639/0007-2745(2007)110[365:CCIWUS]2.0.CO;2

Westberg M, Clerc P. 2012. Five species of Candelaria and Candelariella (Ascomycota, Candelariales)

new to Switzerland. MycoKeys 3: 1-12. http://dx.doi.org/10.3897/mycokeys.3.2864

Yazici K, Aslan A. 2006. Four new lichens from Turkey. Mycotaxon 95: 315-318.

Yazici K, Aptroot A, Aslan A. 2007. Lichen biota of Zonguldak, Turkey. Mycotaxon 102: 257-260.

Yazici K, Aptroot A, Aslan A. 2012. Candelariella, Ochrolechia, Physcia, and Xanthoria species new

to Turkey. Mycotaxon 119: 149-156. http://dx.doi.org/10.5248/119.149

Yazici K, Aslan A, Aptroot A. 2013. New lichen records from Turkey. Bangladesh Journal of Plant

Taxonomy 20(2): 207-211.

MYCOTAXON

http://dx.doi.org/10.5248/130.1209

Volume 130, pp. 1209-1211 October-December 2015

BOOK REVIEWS AND NOTICES:

ABSTRACT—Books reviewed include Distribution, ecology & status of 51 macromycetes

in Europe (Fraiture & Otto 2015).

FUNGAL CONSERVATION

Distribution, ecology & status of 51 macromycetes in Europe. Results of the

ECCF Mapping Programme. By A. Fraiture & P. Otto (eds), 2015. Scripta

Botanica BEtaica 53. <http://shopbotanicgarden.weezbe.com> ISBN 978-9-082-

35255-9; 247 p, numerous maps and illustrations. Price € 25.00.

One of the main obstacles for fungal conservation is knowledge about

distribution, ecology, life history, and changes in occurrence over an entire

area. This is particularly well understood by the European Council for the

Conservation of Fungi, which has worked for the last decade and a half on

gathering that type of information for 50 species of easily recognizable

basidiomycetes and ascomycetes that have been considered threatened in

several countries in Europe, representing different vegetation types and

distribution patterns. The final list covers 51 species, because it became clear

that Poronia punctata records represented two species, P. punctata and Perici,

and both are included in the final list. Another criterion was that at least two of

those 50 species should occur in each country.

National coordinators gathered the data concerning these species, and the

two editors, with help from mapmakers and others, compiled all data (including

those from the literature) in the present publication. The maps use a UTM grid

on which the dots represent areas up to 2500 km’.

The introduction gives an overview of the various problems that must

be overcome in such a collaborative project concerning the occurrence of

* Book reviews or books for consideration for coverage in this column should be sent to the

Editor-in-Chief at editor@mycotaxon.com or 6720 NW Skyline, Portland OR 97229 USA.

1210 ... Mycotaxon 130(4)

ephemeral basidiomes, discusses distribution patterns among species linked

to vegetation, climate, and host trees, and provides a list of the contributing

countries and their mycological coordinators.

The main part of the book gives the information about the species. For each

species, the accepted name, notes on nomenclature, an abstract, geography,

nutritional mode and host, habitat, phenology and frequency, and threats and

conservation status are given. Also presented are photos of the fungi and a

distribution map comparing data before and after 1970, and in some cases a

map of the host tree.

In other words, a wealth of information is presented so that distribution

patterns independent of the inventory efforts emerge, and generalizations can

be made beyond the separation of northern and southern species.

Changes in distribution are not easily inferred from the maps, but a few

species, especially some hydnaceous fungi, do show a distinct decrease in

records over at least part of their area. Decline is also very apparent for Poronia

punctata, although it maintains a stronghold in those countries where horses

are used for nature management purposes.

This is not the first publication on macromycete distribution in Europe;

Lange (1974) compiled data during an earlier similar initiative that did not

focus on threatened fungi but was similarly aimed at producing information

on fungi throughout Europe. Her publication also covered 50 species, ten of

which are included in the 2015 work. Increase in mycological activities in

the Mediterranean countries since 1974 is very obvious in maps of Amanita

caesarea and Panaeolus (Annellaria) semiovatus.

Of course, there are mistakes and things that could have been done better.

For instance, I miss a map showing the coverage of national mapping and

recording schemes (before and after 1970) that would be particularly insightful

for indicating distribution patterns within Russia. There are several small

mistakes in the nomenclature and references (e.g., Berkeley and Brome instead

of Berkeley and Broome; omission of the combination authority for Craterellus

melanoxeros; and misspelling of an author’s name, Nieves-Rivera). The text

would have been easier to read if a native English speaker had reviewed it. Some

species represent a group of species — this has been recognized for Pisolithus

arrhizus, but it is also the case for Battarrea phalloides (Martin et al. 2013), and

Sarcosphaera coronaria (compare nrITS data in Genbank). And, the occurrence

of European species in North America and elsewhere outside Europe should

be critically approached. The western North American “Entoloma bloxamii,”

which has been shown to differ from the European species, has recently been

described as a separate species, E. medianocte (Morgado et al. 2013; Schwarz

Book reviews & notices... 1211

2015), while the ITS sequences of Suillus umbonatus from western North

America are identical to those of European S. flavidus.

However, these are all small comments on a project that has to be fully

applauded. We hope the project will be followed up in Europe and serve as an

inspiration for similar work on other continents. In a divided Europe, this is

also a sign of hope that collaboration and cooperation are both possible and

deliver results.

Lastly, I hope that the data will serve as a baseline for conservation of fungi

on a larger scale than that of country or region, and that it will show politicians

that fungi, just like animals and plants, need protection.

Lange L. 1974. The distribution of macromycetes in Europe. Dansk Botanisk

Arkiv 30 (1): 1-105.

Martin MP, Rusevska K, Duefias M, Karadelev M. 2013. Battarrea phalloides in

Macedonia: genetic variability, distribution and ecology. Acta Mycologica 48:

113-122.

Morgado LN, Noordeloos ME, Lamoureux Y, Geml J. 2013. Multi-gene

phylogenetic analyses reveal species limits, phylogeographic patterns, and

evolutionary histories of key morphological traits in Entoloma (Agaricales,

Basidiomycota). Persoonia 31: 159-178.

Schwarz C. 2015. Nomenclatural novelties. Index Fungorum no. 220.

ELSE C. VELLINGA

861 Keeler Avenue

Berkeley CA 94708-1323, USA

Book ANNOUNCEMENT

Keys to Lichens of North America. Revised and expanded. By I.M. Brodo, 2016. Yale

University Press, P.O. Box 209040, New Haven, CT 06520-9040, USA; <sales.press@yale.edu>.

ISBN 978-0-300-19573-6; 424 p., 13 color + 33 black & white illustrations. Price US$ 29.95.

MYCOTAXON

http://dx.doi.org/10.5248/130.1213

Volume 130, pp. 1213-1214 October-December 2015

Regional annotated mycobiotas new to the MYCOTAXON website

ABSTRACT — Mycotaxon is pleased to announce four new species distribution lists to our

‘web-list’ page covering gasteroid agaricomycetes in Argentina (by Hernandez Caffot et al.),

Hymenochaetaceae in Northeast Brazil (by Lira et al,), corticioid fungi in the Canary Islands

(by Beltran-Tejera & al.), and lichenicolous fungi in Burdur Province (by Yazici & Etayo).

This brings to 120 the number of free access mycobiotas now available on the MycoTaxon

website: http://www.mycotaxon.com/resources/weblists.html

SOUTH AMERICA

Argentina

Maria L. HERNANDEZ CAFFOT, XIMENA A. BROIERO, MARIA E. FERNANDEZ,

LEDA SILVERA RuIZ, ESTEBAN M. CRESPO & EDUARDO R. NOUHRA. Gasteroid

mycobiota (Agaricales, Geastrales and Phallales) from Espinal forests in

Argentina. 20 pp.

ABSTRACT—Sampling and analysis of gasteroid agaricomycete species

(Phallomycetidae and Agaricomycetidae) associated with relicts of native Espinal

forests in the southeast region of Cordoba, Argentina, have identified twenty-nine

species in fourteen genera: Bovista (4), Calvatia (2), Cyathus (1), Disciseda (A),

Geastrum (7), Itajahya (1), Lycoperdon (2), Lysurus (2), Morganella (1), Mycenastrum

(1), Myriostoma (1), Sphaerobolus (1), Tulostoma (1), and Vascellum (1). The gasteroid

species from the sampled Espinal forests showed an overall similarity with those

recorded from neighboring phytogeographic regions; however, a new species of

Lysurus was found and is briefly described, and Bovista coprophila is a new record

for Argentina.

Brazil

CARLA R. S. DE LIRA, GEORGEA S. NOGUEIRA-MELO, LEIF RYVARDEN & TATIANA

B. GIBERTONI. A checklist of Hymenochaetaceae from Northeast Brazil. 9 pp.

ABSTRACT—This study is an update of the available data about species of

Hymenochaetaceae from Northeast Brazil. This revision is based on literature and

herbaria revision besides recent field trips in the Atlantic Rain Forest and Caatinga

biomes. Currently, there are 64 species and 12 genera of Hymenochaetaceae in Brazil.

Among them, Coltricia fragilissima is reported for the first time in this country, while

eight species are new to the Northeast region, 11 to the Brazilian semiarid and 22 to

the Northeastern States.

1214 ... New regional mycobiotas online

EUROPE

Portugal & Spain (Iberian Peninsula)

ESPERANZA BELTRAN-TEJERA, J. LAURA RODRIGUEZ-ARMAS, M. TERESA TELLERIA,

MARGARITA DUENAS, IRENEIA MELO, ISABEL SALCEDO & J. CARDOSO.

Corticioid fungi of the western Canary Islands. Chorological additions.

74 pp.

ABSTRACT—he diversity of corticioid fungi from the western Canary Islands

is presented. Almost 200 species were found in 32 sites, including 63 species

reported for the first time from the archipelago. Seventy-six genera are

represented; the most common genera are Hyphodontia, Trechispora, Peniophora,

Botryobasidium, Hyphoderma, Tubulicrinis and Sistotrema. Half of the genera are

represented by a single species. Samples were collected from 32 plant species of

which 22 are endemic to the Macaronesian bioregion. Although found in just 9

pine-dominated forest sites, Pinus canariensis was host to the most collections

and species.

MiIp-EASstT

Turkey

KENAN YAZICI & JAVIER Etayo. The lichenicolous fungi of Burdur province

(Turkey). 9 pp.

ABSTRACT — In the course of studies of lichens and bryophytes of Burdur province,

Turkey, 42 species of lichenicolous fungi belonging to 20 genera in Ascomycota

and Basidiomycota have been identified. Three of them, Dactylospora parellaria,

Didymellopsis perigena, and Zwackhiomacromyces constrictocarpus, are new to

Turkey in particular and Asia in general; short descriptions and distributional

data are presented. In addition, the Turkish material of Z. constrictocarpus,

hitherto only known from the type collection in Spain, represents the second

record of this species.

MY COTAXON

http://dx.doi.org/10.5248/130.1215

Volume 130, p. 1215 October-December 2015

NOMENCLATURAL NOVELTIES AND TYPIFICATIONS

PROPOSED IN MYCOTAXON 130(4)

Acaulospora reducta Oehl, B.T. Goto & C.M.R. Pereira [MB812332], p. 986

Codinaea aquatica R.F. Castaneda, M.S. Oliveira & Malosso [MB812851], p. 1046

Codinaea tropicalis (Bhat & W.B. Kendr.) R.E Castafteda & Malosso [MB812854],

p. 1048

Dictyosporium amoenum C.R. Silva, Gusmao & R.E. Castaneda [MB812865], p. 1126

Entyloma scandicis K.G. Savchenko, Carris, Castl., Heluta, Wasser & Nevo

[MB811390], p. 1067

Hymenoscyphus aurantiacus H.D. Zheng & W.Y. Zhuang [FN570094], p. 1026

Hymenoscyphus macrodiscus H.D. Zheng & W.Y. Zhuang [FN570095], p. 1028

Hymenoscyphus ginghaiensis H.D. Zheng & W.Y. Zhuang [FN570096], p. 1030

Hymenoscyphus tetrasporus H.D. Zheng & W.Y. Zhuang [FN570098], p. 1031

Hymenoscyphus yui H.D. Zheng & W.Y. Zhuang [FN570099], p. 1033

Kroswia epispora H.J. Liu & Chao Li [MB811551], p. 952

Lecanicillium uredinophilum M.J. Park, S.B. Hong & H.D. Shin [MB814832], p. 1002

Matsushimiella paraensis J.S. Monteiro, Gusmao & R.F. Castafieda [MB810689], p. viii

[validation of Matsushimiella paraensis J.S. Monteiro, Gusmao & R.F. Castaneda,

Mycotaxon 130: 312 (2015), nom. inval., ICN (2012) Art. 42.1]

Neoalbatrellus subcaeruleoporus Audet & B.S. Luther [MB801480], p. 1194

Phoma candelariellae Z. Kocakaya & Halici [MB812271], p. 1186

Phyllachora jianfengensis Na Liu & M.Z. Li [FN570152], p. 1040

Pouzarella alissae Largent & Bergemann [MB812300], p. 1158

Pyramidospora quadricellularis M.S. Oliveira, Malosso & R.F. Castaneda [MB812756],

p-973

Synchaetomella aquatica Fiuza, Gusmao & R.F. Castafieda [MB812900], p. 1136

Terriera fici Y.R. Lin & Yuan Wu [MB812746], p. 1112

Tetrolylea Cantillo, R.F. Castaheda & Gusmao [MB812409], p. 978

Tetrolylea pleiomorpha Cantillo, R.E. Castafieda & Gusmao [MB812410], p. 978

Trichoconis foliicola S.S. Silva, Gusmao & R.F. Castaneda [MB812636], p. 1052

Tuber petrophilum Milenkovic, P. Jovan., Grebenc, Ivanéevic & Markovic [MB812245],

p. 1143